- Email: [email protected]
AvGp50, a predominantly axonally expressed glycoprotein, is a member of the IgLON's subfamily of cell adhesion molecules (CAMs)
Molecular Brain Research 44 Ž1997. 273–285
Research report
AvGp50, a predominantly axonally expressed glycoprotein, is a member of the IgLON’s subfamily of cell adhesion molecules ž CAMs/ K.A. Hancox a , A.A. Gooley b, P.L. Jeffrey b
a,)
a DeÕelopmental Neurobiology, Children’s Medical Research Institute, Westmead, NSW, Australia Macquarie UniÕersity Center for Analytical Biotechnology, Macquarie UniÕersity, North Ryde, NSW, Australia
Accepted 6 August 1996
Abstract We have previously reported a 50 kDa glycoprotein ŽAvGp50. expressed specifically in the chick nervous system wHancox, K.A., Sheppard, A.M. and Jeffrey, P.L., Characterisation of a novel glycoprotein ŽAVGP50. in the avian nervous system, with a monoclonal antibody, DeÕ. Brain Res., 70 Ž1992. 25–37x, and we present its molecular characterization. A PCR fragment was generated following sequencing of peptide and N-terminal fragments derived from purified AvGp50. A 1.58 kb clone ŽpUEX762. containing the 5X-UTR, the entire coding sequence and a short 3X-UTR was then isolated from a chick embryonic day 18 forebrain library. The deduced amino acid sequence encodes a 338 amino acid peptide containing a 31 amino acid signal peptide at the N-terminal and a 19 amino acid phosphatidylinositol glycan linkage sequence at the C-terminal. The mature protein contains three C2-immunoglobulin-like domains and a glycosyl phosphatidylinositol anchor and shares significant homology to other members of the immunoglobulin superfamily, including neural cell adhesion molecule ŽN-CAM., myelin-associated glycoprotein ŽMAG. and the Drosophila protein Amalgam. AvGp50 exhibits highest sequence identity to a recently classified subgroup of the immunoglobulin superfamily ŽIgLONs – immunoglobulin LAMP, OBCAM and neurotrimin – classified by Pimenta et al. wPimenta, A.F., Zhukareva, V., Barbe, M.F., Reinoso, B.S., Grimley, C., Henzel, W., Fischer, I. and Levitt, P., The limbic system-associated membrane protein is an Ig superfamily member that mediates selective neuronal growth and axon targeting, Neuron, 15 Ž1995. 287–297x, comprising the opioid binding cell adhesion molecule ŽOBCAM., neurotrimin and the limbic system-associated membrane protein ŽLAMP. suggesting that AvGp50 is a member of this subgroup. AvGp50 is expressed predominantly on the cell surface of axons, in particular Purkinje cell and granule cell axons in the cerebellum. In cerebellar and forebrain neuronal cultures, protein expression is exclusively located at the cell surface. Despite its cell surface localization, AvGp50 does not directly influence the outgrowth of neurons from explant cultures from ED8 to ED10 chick forebrain, prompting the suggestion that AvGp50 may act in later maturational events. Keywords: AvGp50; Axonally expressed; Immunoglobulin superfamily; Opioid binding cell adhesion molecule ŽOB-CAM.; Limbic system-associated membrane protein ŽLAMP.; Neurotrimin
1. Introduction The formation of the nervous system depends on the correct expression of a specific subset of the large number of cell surface proteins and extracellular matrix molecules, which interact to control neuronal migration, axonal extension, fasciculation and synapse formation. Numerous cell adhesion molecules ŽCAMs., including members of the immunoglobulin superfamily, have been shown to be involved in many aspects of nervous system development ) Corresponding author. Children’s Medical Research Institute, Locked Bag 23, Wentworthville, NSW 2145, Australia. Fax: q61 Ž2. 687-2120; E-mail: [email protected]
including cell-cell and cell-substrate adhesion w1,10,11,14,16x, axonal outgrowth and target recognition w18,27,28x, fasciculation w20,21,30–32,39x, synaptogenesis and migration w21,25,35,36x. The majority of immunoglobulin superfamily members are attached to the membrane via a single transmembrane spanning domain which is hydrophobic, however, an increasing number, initially represented by Thy-1, have been shown to be linked to the membrane via a phosphatidylinositol glycan moiety w5,16,28,29,40,44,45x. Previously a monoclonal antibody MabSA1.7 had been used to characterize the avian neuronal specific glycoprotein, AvGp50 w23x. AvGp50 is expressed at high levels in the chick forebrain, cerebellum and tecta, with slightly
0169-328Xr97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 6 . 0 0 2 2 8 - 8
274
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
lower levels detected in the retina and spinal cord. The protein is expressed at low levels until embryonic day 18 when expresssion is upregulated in all neuronal tissues. Immunohistochemically, the protein has been shown to localize to areas of interneuronal contact, for example, the molecular layer of the cerebellum and both the inner and outer plexiform layers of the retina. It is absent from cell bodies and is only expressed on neuronal processes. By Western blot analysis, AvGp50 was shown to be enriched in both plasma membranes and synaptic plasma membranes. The pattern of expression and cellular localization makes AvGp50 a potential candidate for functional involvement in the development of the chick nervous system. In this present study we have determined the molecular characteristics of AvGp50. A specific PCR clone was generated between peptide fragments derived from AvGp50. This was then used to obtain a near full length cDNA clone for AvGp50. Its deduced amino acid sequence revealed that AvGP50 is significantly homologous Ž21–25%. to members of the immunoglobulin superfamily including N-CAM, MAG and the Drosophila cell adhesion molecule amalgam. In particular AvGp50 is 56% identical to the opiate binding cell adhesion molecule OB-CAM w26,34x and 50% identical to the protein neurotrimin w40x. Together with the limbic system-associated membrane protein ŽLAMP. w29x, with which AvGp50 shares it highest sequence identity Ž90%., AvGp50 forms a specific subset of the immunoglobulin superfamily known as the IgLONs Žimmunoglobulins LAMP, OB-CAM and neurotrimin w29x..
2. Materials and methods 2.1. Protein purificationr protein sequencing Immunoaffinity purified AvGp50 was prepared as previously described w23x. Briefly, a deoxycholate ŽDOC. extract of 1-day-old chick brains was passed over an MabSA1.7 monoclonal antibody immunoaffinity column. Purified protein was eluted in 500 mM diethylaminer0.5% Žwrv. deoxycholate pH 11.5, immediately neutralized with solid glycine and then dialysed against Tris-buffered saline ŽTBS. pH 7.4 for 48 h at 48C. Protein sequencing was carried out by two independent laboratories. Macromolecular Resources, Colorado, USA: 1–2 nmol AvGp50 was resuspended in 1% Žwrv. sodium dodecyl sulfate ŽSDS. and digested with 10% Žwrw. trypsin. The resulting pellet was resuspended in 70% Žvrv. trifluoroacetic acid, incubated for 1 h and then diluted dropwise with water until fully resuspended. The peptide fragments were separated on a reverse phase-HPLC column and sequenced on a Applied Biosystems 473A protein sequencer using standard Edman chemistry. Macquarie UniÕersity Center for Analytical Biotechnol-
ogy, Macquarie UniÕersity, Australia: For peptide fragments, AvGp50 was reduced and alkylated with vinylpyridene, digested with 2.5% Žwrw. Endoproteinase Lys-C ŽBoehringer Mannheim. for 2 = 2 h at 378C in 25 mM Tris-HClr0.1 mM ethylenediaminetetraacetic acid ŽEDTA.r0.025% Žwrv. SDS pH 8.6. The peptide fragments were separated on a C18 reversed-phase HPLC column and sequenced on an Applied Biosystems 470A protein sequencer using standard Edman chemistry. For N-terminal sequence, AvGp50 was electrophoresed on SDS-PAGE, transferred to PVDF ŽBio-Rad. nylon membrane and directly sequenced using a MilliGen 6600 Prosequencer using standard Edman chemistry. 2.2. PCR and library screening Oligonucleotides were designed from protein sequence using chick preferred codon usage w3x. PCR was performed using a Hybaid OmniGene thermocycler in 500 mM KClr100 mM Tris-HClr4.5 mM magnesium chloride pH 8.4 and 1 mgrml gelatin. Oligonucleotide primers were added to a final concentration of 50 m M. Single stranded cDNA was reverse transcribed from total adult forebrain RNA using 200 U Moloney murine leukemia virus ŽMMLV. reverse transcriptase w42x, 50–100 ng of the resulting cDNA was added per PCR reaction. The reaction mix was denatured for 5 min at 948C, Taq ŽBoehringer Mannheim. added and PCR products amplified by 30 cycles of 948C, 1 min Ždenature.; 608C, 1 min Žanneal.; 728C, 1 min Žextend. with a final extension of 5 min at 728C. PCR products of interest were purified using QIAEX resin ŽBresatec. according to the manufacturer’s specifications and subcloned into pGEM-T ŽPromega. for sequencing. Sequencing was performed on an Applied Biosystems 373A automated DNA sequencer using the Applied Biosystems dyedeoxy terminator reactions kit. A 600 bp fragment ŽpGM276r261-5. generated between two of the peptide fragments was used to screen approximately 200 000 colonies from an embryonic day 18 forebrain oligo dT primed pUEX-1 library constructed as previously described w12x. Colony screening was performed as described by Grunstein and Hogness w22x. Lifts were prehybridized in a buffer containing 6 = SSC Ž20 = is 3 M NaClr300 mM sodium citrate pH 7.0.r50 mM sodium di-hydrogen phosphater5 = Denharts Ž1% Žwrv . Ficollr1% Žwrv. polyvinylpyrrolidoner1% Žwrv. BSA. and 10% Žwrv. dextran sulfate at 608C for 6 h and hybridized overnight in fresh buffer containing pGM276r261-5 labeled with w a- 32 PxdCTP at approximately 1.5 = 10 6 countsrml of hybridization solution. The lifts were washed twice with 0.5 = SSCr0.1% Žwrv. SDS for 1 h and twice with 0.1 = SSCr0.1% Žwrv. SDS for 45 min at 608C before being autoradiographed on Kodak X-OMAT X-ray film. Positive colonies were further purified by a second round of screening.
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
2.3. Northern analysis Total mRNA was purified from 1-day-old chick cerebellum and forebrain using TRIzol total RNA isolation reagent ŽLife Technologies. and resuspended in 0.1% Žwrv. SDS. Poly Aq RNA was purified from total RNA using PolyA Tract mRNA Isolation Systems kit ŽPharmacia.. For analysis, samples were electrophoresed on a 1% agaroserformaldehyde gel and blotted onto nylon filters overnight in 20 = SSC by the method of Southern w38x. Hybridization was carried out as described for library screening except that the temperature was increased to 658C and washes were 2 = 0.5 = SSCr0.1% Žwrv. SDS for 30 min and 1 = 0.1 = SSCr0.1% Žwrv. SDS for 30 min. 2.4. In situ hybridization Sense and anti-sense digoxygenin RNA probes were transcribed from linearized pGM276r261-5 using SP6rT7 Digoxigenin ŽDIG . Transcription kit ŽBoehringer Mannheim.. Tissue for in situ hybridization was fixed in 4% Žwrv. paraformaldehyde in PBS, pH 7.4 overnight at 48C washed and dehydrated, cleared in 3 successive changes of xylene and then infiltrated with paraffin wax at 658C. Sections Ž5 m m. were collected onto HClrethanol cleaned and baked slides subbed with 1 mgrml poly-Llysine ŽSigma.. Sections were dewaxed with histolene, hydrated in decreasing concentrations of ethanol, washed in 2 = SSC and prehybridized for 2 h at room temperature in a buffer containing 50% Žvrv. deionized formamider4 = SSCr1= Denhardt’srsonicated herring sperm DNA Ž0.5 mgrml.ryeast tRNA Ž0.25 mgrml. and 10% Žwrv. dextran sulfate. DIG-labeled probe was added at a concentration of 2 mgrml in fresh buffer and incubation was overnight at 558C in a humid chamber. Slides were blocked and then incubated with anti-DIG alkaline phosphatase conjugated secondary antibody ŽBoehringer Mannheim. diluted 1:500. Color was developed using a BCIPrNBT detection kit ŽLife Technologies. in 100 mM Tris-HClr100 mM NaClr50 mM MgCl, pH 9.5. Slides were dehydrated, cleared in histolene and mounted in Entellan. 2.5. Immunofluorescence Cultures were fixed in 4% Žwrv. paraformaldehyde in PBS for 30 min and then blocked with 2% Žvrv. cold water fish skin gelatin ŽCWFSG.r0.5% Žwrv. bovine serum albumin ŽBSA.. Cells were incubated with either MabSA1.7 or anti-neurofilament NF200 Žpolyclonal, Sigma. or both, for 2 h at room temperature. A donkey anti-mouse fluorescein-conjugated andror a goat anti-rabbit rhodamine-conjugated ŽJackson Laboratories, both diluted 1:100 in PBS. were used and the coverslips mounted in DABCO ŽSigma.. Photography was performed on a Leica Diaplan microscope with an attached photomicro-
275
graphic system camera model MPS-52. Images were photographed under fluorescence illumination using Ektachrome 160 Dx tungsten film. For confocal microscopy, sections were dewaxed, blocked with 2% Žvrv. CWFSGr0.5% Žwrv. BSA and then incubated with the required antibody as described above. Tissue was prepared as previously described w23x. 2.6. CleaÕage of GPI anchors with the enzyme PI-PLC Neuronal culture coverslips were washed once with PBS and incubated with phosphatidylinositol-specific phospholipase C ŽPI-PLC; 0.5 Urcoverslip in 200 m l of PBS, Boehringer Mannheim. at 378C for 1 h. Control coverslips were incubated in an equal volume of PBS without PI-PLC. The coverslips were then fixed in 4% Žwrv. paraformaldehyde and treated as for immunofluorescence labeling of cultures. 2.7. Neuronal culture All cultures were maintained in Dulbecco’s modified Eagle’s medium ŽDME. supplemented with 5% Žvrv. fetal calf serum ŽFCS., 2.5% Žvrv. chick embryo extract ŽLife Technologies. and 25 mM KCl Žneuronal medium. w7x, in 7.5% CO 2 at 378C. For cerebellar cultures, cerebella were dissected from ED7 chick embryos as previously described w24x. For cortical cultures, forebrains were removed from ED8 and ‘‘to ED10’’ chicks, the cortex dissected away and treated as for cerebellar cultures.
3. Results 3.1. Protein sequencing, PCR and cloning Sequence from affinity purified AvGp50 was obtained either from the N-terminus or reduced and alkylated protein digested with Endo Lys-C or trypsin. Five peptide fragments Ž1–5. were produced and sequenced from the Endo Lys-C digests and two were produced and sequenced from the trypsin digests Ž6–7.. The sequences obtained from the peptides and N-terminal are shown in Table 1. The non-redundant search program BLAST was used to search the databases PDB, SwissProt, PIR, SPupdate, GenPept and GPupdate w2x. Peptide 3 was shown to have significant homology Ž72%., to the protein designated opioid binding protein cell adhesion molecule ŽOB-CAM. w26,34x. Subsequent alignment using the MacVector program to search the latest release of the Entrez database revealed the six remaining peptides can also be aligned within the protein sequence of OB-CAM. Oligonucleotide primers were designed using chick preferred codon usage w3x from all of the peptide fragments and the N-terminal sequence. A series of PCR reactions was performed based on the alignment of each of the
276
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
Table 1 Amino acid sequence of derived peptides from AvGp50 Fragment sequenced
Amino acid sequence
N-terminus Peptide 1 Peptide 2 Peptide 3 Peptide 4 Peptide 5 Peptide 6 Peptide 7
XDFTNGTDGITVNNGNXXPVFLSFVED CSLDPRVELEK RSPLNYSLRIQK VDFDEGTDNITVXQGDTAILXXFVEDDS SNEAATGRQALLRSEASA GFEGSEEYLWIL SEASQVPTPDFEV EFEGEEEYLEI
Five peptides Žpeptides 1–5. were produced and sequenced from Endo Lys-C aligests of AvGp50, whilst peptides 6 and 7 were produced and sequenced from trypsin digests. Amino acids are represented by the 1-letter amino acid code. The first amino acid of the N-terminus could not be determined and is designated by the symbol X. Peptide 3 and the N-terminus are likely to be the same region of sequence, as are peptides 5 and 7 Žsequenced independently..
peptides to OB-CAM. One of the reactions Žbetween peptide 3 in the 5X orientation and peptide 4 in the 3X orientation. gave a specific reaction product of approximately 600 base pairs. The fragment was subcloned into pGEM-T, with one positive clone obtained ŽpGM276r261-5.. The clone was originally sequenced using the internal pGEM-T SP6 site to verify its authenticity and later with the primers used to generate the original PCR clone. A subsequent sequencing reaction, using an internal primer, verified areas of sequence not fully covered in the initial sequencing. The 600 bp insert present in clone pGM276r261-5 aligned to the region from amino acid 36 to 257 of OB-CAM and is 60% identical at the amino acid level. The clone pGM276r26-5 was then used to screen an embryonic day 18 forebrain pUEX-1 library. Of the 200 000 colonies screened, two positive clones were identified with insert sizes of 1.58 and 1.4 kb. The two inserts are identical in their coding region and 3X-untranslated region ŽUTR. and differ only in their 5X-UTR Ždata not shown.. The 1.58 kb clone, designated pUEX762, covers 513 bp of 5X-UTR, the entire coding sequence of 1014 bp and 54 bp of 3X-UTR ŽFig. 1.. The 3X-UTR is unusually short and contains only a very short Poly A tail Ž11 As.. Also, there is no standard polyadenylation sequence, suggesting that the clone originated at an A-rich site and does not represent a full length clone for AvGp50. Interestingly, the bovine clone for OB-CAM originated at an A-rich site, although it contains slightly more 3X-UTR Ž795 bp. than the pUEX762 clone for AvGp50. Fig. 1 shows the entire
DNA sequence of pUEX762 and its deduced amino acid sequence. An open reading frame begins at bp 514 and extends through bp 1530. The single open reading frame encodes a 338 amino acid peptide containing a putative 31 amino acid signal peptide with a predicted cleavage site at Ser31 based on protein sequence from the N-terminal which designates valine at position 32 is the N-terminal amino acid of the mature protein. A 19 amino acid glycosyl phosphatidylinositol linkage sequence comprising hydrophobic amino acids is located at the C-terminal. The most likely cleavage site for GPI addition to occur is at Ser319 which is followed directly by two small amino acids, leucine and alanine. For GPI addition to occur, the sequence itself is not as crucial as the hydrophobicity and positioning of the sequence w6,13,15,17x. Hydrophobicity is largely achieved by a chain of between 15 and 30 amino acids rich in leucine at the C-terminal end of the peptide. As seen in Fig. 2, AvGp50 largely conforms to the requirements for GPI addition, although it differs significantly from the GPI addition sequence for either OB-CAM or neurotrimin. Further evidence that AvGp50 is GPI anchored is shown in experiments where cultured chick cortical or cerebellar neurons are treated with the phosphatidylinositol-specific phospholipase C Žsee Fig. 7.. 3.2. AÕGp50 forms a member of a specific subfamily of CAMs Subsequent database searches showed that AvGp50 was also significantly identical to two recently cloned proteins, neurotrimin w40x, and the limbic system-associated membrane protein, LAMP w29x. Alignment of the deduced amino acid sequence of AvGp50 to OB-CAM, neurotrimin and LAMP is shown in Fig. 2A. Alignment of AvGp50 to LAMP with which it shares most identity is shown in Fig. 2B. AvGp50 Ž338 amino acids. is marginally smaller than either OB-CAM Ž345 amino acids. or neurotrimin Ž344 amino acids., with gaps added in the coding sequence to maintain alignment. AvGp50 and LAMP are most similar, both sharing an identical length of 338 amino acids. The protein AvGp50 shares 50% identity to neurotrimin, 56% identity to OB-CAM and 90% identity to LAMP. OB-CAM contains 6 potential N-linked carbohydrate side chains, whereas AvGp50, neurotrimin and LAMP all contain 8 potential N-linked addition signals. Each of these proteins share 6 conserved cysteine residues which form the basis of the 3 Ig-like domains. Neurotrimin has an extra cysteine at postion 83 which is not conserved in either AvGp50,
X
X
Fig. 1. The insert in pUEX762 is 1586 bp in length and contains 513 bp of 5 -UTR and 54 bp of 3 -UTR. A single open reading frame contains the complete predicted coding sequence of AvGp50 with a methionine at bp 514 the putative start codon. Amino acids are represented by the 3-letter amino acid code below the nucleotide sequence. Potential N-linked carbohydrate side chain addition signals are underlined by dotted lines. The 6 cysteine residues postulated to be involved in C2 domain formation are designated by the symbol ‘v’. The theoretical GPI addition signal is underlined by the solid X bar. The first stop codon ŽTAA. lies at bp 1584–1586. The 3 -UTR is very ArT rich Ž93%. and consists mainly of TAA repeats, followed immediately by a string of 11 As.
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
277
278
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
279
Fig. 3. Three putative domains of AvGp50 Ždomain 1, domain 2 and domain 3. are aligned to representative C2 domains from N-CAM Žmouse., amalgam Ž Drosophila. and MAG Žmouse.. Conserved amino acids are in black boxes, conservative substitutions are in grey. Gaps created by the program to maintain alignment of the sequences are shown by the symbol ‘ Ø ’. The position of each of the 7 strands of a C2 domain are underlined. Alignment is performed by the latest release of the Pile-Up and Prettybox programs supplied by the Australian National Genomic Information Service ŽANGIS..
OB-CAM or LAMP. The first 2 domains of AvGp50 share the highest identity with the OB-CAM, neurotrimin and LAMP with identities of 61–63% between AvGp50, and each of OB-CAM and neurotrimin and 92–94% between AvGp50 and LAMP. Most of the variation between these molecules occurs in the third domain with identities of 42% between AvGp50 and each of OB-CAM and neurotrimin and 88% identity between AvGp50 and LAMP. Based on the high identities shared by these molecules we suggest AvGp50 to be a member of a new subfamily of the larger immunoglobulin superfamily, tentatively designated IgLONs. The high identity shared between AvGp50 and LAMP can be seen in Fig. 2B. The proteins share 90% identity although this figure can be as high as 95% if conservative substitutes are taken into account. LAMP and AvGp50 appear to have slightly different mature protein lengths, with LAMP Ž287 amino acids. one amino acid shorter than AvGp50. In particular, the cleavage site for the signal peptide would appear to be Pro28 in LAMP ŽFig. 2A. based on microsequencing data, whereas in AvGp50, the cleavage site is more likely to be at Ser31, based on protein sequence obtained from both the N-terminus and peptide fragments Žsee Table 1.. In addition, the predicted cleavage site for GPI addition in AvGp50 is Ser319, whereas in LAMP the predicted cleavage site is Asn315 w29x. In respect to GPI addition, however, neither site has been confirmed by alternative methods, making it difficult to predict which would be the more favored site.
We have previously demonstrated that AvGp50 has 5 N-linked carbohydrate side chains which when removed result in a protein of 34 kDa w23x. Presumably several of the potential glycosylation sequences are not functional ŽFig. 1.. The open reading frame gives a 288 amino acid mature protein with a predicted molecular weight of 32 kDa once the signal sequence and phosphatidylinositol linkage signals are removed. This is in agreement with our predicted molecular weight of 34 kDa Žas determined from SDS-PAGE. w23x. 3.3. Homology to Ig superfamily Like OB-CAM, neurotrimin and LAMP, AvGp50 shows significant homology to other members of the immunoglobulin superfamily including N-CAM w9x, MAG w4,33x and the Drosophila Ig superfamily member amalgam w37x. The alignment of the three Ig-like domains of AvGp50 to members of the Ig superfamily is shown in Fig. 3. The cysteine residues that form disulfide bonds of the Ig-like domains are completely conserved and the domains all have the classic features of C2 Ig-like domains w43x except the truncated domain 2 which is approximately 15 amino acids shorter than a classic C2 domain Žnormally around 100 amino acids.. This truncated second domain is shared between all members of the IgLON’s subfamily of CAMs. The folding pattern is typical of a C2 domain with one b-sheet formed from the C, F and G strands and the second from the D, E, B and A strands. The cysteine
Fig. 2. ŽA. The deduced amino acid sequence of insert from pUEX762 ŽAvGp50. is compared to the amino acid sequence for bovine OB-CAM, rat LAMP and neurotrimin. Identical amino acids are shown in black boxes Ž3 amino acids required for a consensus., conserved amino acids are boxed in grey. Gaps created by the program to maintain alignment of the sequences are shown by the symbol ‘v’. Overall AvGp50 is 56% identical to bovine OB-CAM, 50% identical to neurotrimin and 90% identical to LAMP. ŽB. Direct alignment of AvGp50 and LAMP. Amino acids are represented by the 1-letter code. Identical amino acids are indicated by the symbol ‘)’, conservative substitutions by the symbol ‘:’ and non-conservative substitutions by the symbol ‘ Ø ’, alignment was by the latest release of the MacVector program. Putative signal peptides are indicated in bold, putative GPI addition signals are underlined.
280
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
Fig. 4. Two mRNAs are detected by Northern analysis of 1-day-old chick tissue total RNA Ž20 m g per lane.. A 5.5 kb major message and 3.4 kb minor message is detected in forebrain Žlane 4. and cerebellum Žlane 5. but is not expressed in either kidney Žlane 1., liver Žlane 2. or heart Žlane 3..
residues in the B and F sheets allow for the formation of a disulfide bond and the stabilization of the two b-sheets. 3.4. Northern analysis Two mRNA species of approximately 5.5 kb Žmajor. and 3.4 kb Žminor. can be detected in total RNA from 1-day-old chick forebrain and cerebellum but not from the heart, liver or kidney ŽFig. 4.. Significantly, whilst OBCAM, neurotrimin and LAMP share significant homology, the transcripts from each of the genes are of different lengths ŽOB-CAM, 4.5 kb and 7.2 kb; neurotrimin, 3.2 and 4.0 kb; and LAMP, 1.6 kb and 8.0 kb.. Given that the library clone is 1.58 kb, it appears that the clone originated at an internal A-rich site and does not represent a full length cDNA. This is confirmed by the absence of a standard polyadenylation signal Žsee Fig. 1.. 3.5. In situ hybridization and confocal microscopy To characterize the localization of AvGp50 mRNA in the cerebellum Žwhere AvGp50 is highly expressed. an in situ hybridization was performed using the pGM276r261-5 600 bp insert. Fig. 5 shows a sagittal section of the
Fig. 5. In situ localization of AvGp50 mRNA in the 1-day-old chick cerebellum. Sense control Ža., anti-sense probes Žb and c.. By day 1 post hatch all neuronal cell types of the cerebellum express AvGp50 mRNA Žarrowheads. and cells present in the deep cerebellar nuclei also express AvGp50 mRNA Žc.. Magnification Ža and b.: =200; Žc. =1000.
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
cerebellum at post-hatch day 1. There is strong expression of the transcript in all neuronal cell types of the cerebellum including both the Purkinje and granule cells ŽFig. 5b, arrowheads. as well as the deep cerebellar nuclei ŽFig. 5c, arrowhead., with which the Purkinje cell axons make contact. The message expression in Purkinje cells suggested that AvGp50 is expressed in Purkinje cells despite no detectable levels of protein in either the cell bodies or dendrites w23x. Presumably the protein is located entirely in the axon. To test this hypothesis, sections of cerebellum were double labeled with an anti-neurofilament 200 antibody and the MabSA1.7. Fig. 6 shows a confocal image of a day 1 post-hatch cerebellum which has been double labeled with MabSA1.7 Žgreen. and a polyclonal anti-neurofilament 200 Žred. antibody. The arrowheads highlight a Purkinje cell axon as it leaves the cerebellum Ždouble label is represented by yellow.. The expression of AvGp50, therefore, appears to be predominantly axonal with strong expression on both the granule Žas shown by strong immunoreactivity of the molecular layer. and Purkinje cell axons w23x.
281
To determine the cellular localization of AvGp50, neuronal cultures were stained with either MabSA1.7 alone ŽFig. 7d and e, cerebellar. or double labeled with both MabSA1.7 and the polyclonal anti-NF200 antibody ŽFig. 7a–c, f and g; cortical.. Fig. 7a and b show a cortical neuronal culture after 3–4 days in vitro with punctate expression of AvGp50 on the surface of both the cell bodies and processes. This is in contrast to the intracellular expression of NF-200 ŽFig. 7c.. Likewise, cerebellar neuronal cultures maintained for 19 days in vitro ŽFig. 7d and e. show a similar pattern of expression of AvGp50, with strong punctate expression on the cell surface of cell bodies and processes. Expression of AvGp50 at the cell surface, in conjunction with the sequence data, suggests a potential role for the AvGp50 as a cell surface adhesion molecule. Treatment of forebrain cortical neuronal cultures with the enzyme phosphatidylinositol phospholipase C ŽFig. 7f and g. results in a complete loss of expression of AvGp50 at the cell surface ŽFig. 7f. but does not alter the expression of the intracellular protein neurofilament 200 ŽFig. 7g..
Fig. 6. The 1-day-old chick cerebellum was double labeled with the MabSA1.7 and a polyclonal anti-neurofilament 200 ŽNF200. antibody. A donkey anti-mouse fluorescein Žgreen. and goat anti-rabbit rhodamine Žred. labeled secondary antibodies were used to visualize reaction products. Where a double label occurs, the reaction product is visualized as yellow. A Purkinje cell axon Žarrowheads. is clearly visible as it leaves the base of the Purkinje cell Žunlabeled, arrow. and courses through the internal granular layer. Magnification =400. PCL, Purkinje cell layer; GCL, granule cell layer.
282
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
4. Discussion In this study we present the molecular characterization of AvGp50, the predicted structure as a phosphatidylinositol glycan linked cell surface molecule and a member of the immunoglobulin superfamily. In particular AvGp50 belongs to a specific subset of the immunoglobulin superfamily ŽIgLONs. comprising OB-CAM, neurotrimin and LAMP w29x. Purified protein was used to derive N-terminal and peptide fragments whose sequence were used in an intensive PCR screen. One of the PCR products gave a specific reaction product of 600 bp which, when sequenced, proved to contain all the other peptide fragments except peptide 6, which on final analysis lies outside the region of the PCR clone. The PCR fragment contained partial coding sequence which exhibited homology to both the bovine and rat OB-CAM. To further characterize AvGp50 the PCR fragment was used to screen an embryonic day 18 chicken forebrain library. Two positive clones were identified which differed only in their 5X-UTR and shared the entire coding sequence and 3X-UTR. The deduced amino acid sequence of AvGp50 gives a peptide of 338 amino acids Žmature protein of 288 amino acids. and a predicted molecular weight of 32 kDa, similar to our predicted molecular weight of 34 kDa as determined from SDS-PAGE w23x. Despite the protein containing 8 potential glycosylation sequences ŽAsn-X-SerrThr. defined by DNA sequencing, AvGp50 contains 5 N-linked carbohydrate side chains, as revealed by N-glycanase digestion w23x. This suggests that several of the potential sites are non-functional. The asparagine residue at amino acid 40 is a good candidate for glycosylation as it was absent from peptide 3 during protein sequencing. Evidence from bovine OB-CAM also points to this asparagine being glycosylated. In addition the amino acid 285 Žamino acid 279 AvGp50. is glycosylated on basis of the same criterion w34x. The protein contains six cysteine residues, all of which are conserved as in OB-CAM, neurotrimin and LAMP. The six cysteine residues divide the protein into 3 domains which share significant homology to the C2 domains characteristic of the immunoglobulin superfamily. Domains 1 and 3 each comprise approximately 100 amino acids, whilst domain 2 is slightly truncated, comprising 83 amino acids. Within the subfamily, domains 1 and 2 share the
283
highest identity with the most variability exhibited in the third domain. Evidence to suggest that the putative GPI addition signal is functional is derived from the observation that when neuronal cultures are treated with PI-PLC all immunoreactivity of the MabSA1.7 is lost, a treatment which does not alter the expression of the intracellular structural protein NF200. The structure of AvGp50 therefore consists of 3 C2 Ig-like domains anchored to the membrane by a GPI tail. The protein contains no transmembrane domains and no fibronectin type III repeats characteristic of other Ig superfamily members such as TAG-1 w16x. We propose that AvGp50 is a member of the subgroup of CAMs including OB-CAM, neurotrimin and LAMP. AvGp50 is most likely the avian homologue of LAMP, although there are significant differences between the expression patterns of AvGp50 and LAMP, suggesting that AvGp50 is a novel member in its own right. For example, AvGp50 is expressed at high levels in all regions of the chick brain with highest levels in the forebrain, cerebellum and tecta with slightly lower levels detected in the retina. AvGp50 is expressed in areas of high interneuronal contact, in the molecular layer and internal granule cell layer of the cerebellum and the inner and outer plexiform layers of the retina w23x. Furthermore AvGp50 is not detected on the cell bodies with expression restricted to neuronal processes of post-mitotic neurons w23x. Most significantly, however, AvGp50 is expressed relatively late in development with a sharp rise in protein expression at embryonic day 18 ŽED18. in the chick. Differences in the pattern of expression of the other members of the IgLON family exist. OB-CAM, by way of contrast, is expressed as early as ED18 in the rat ŽED9 in the chick. with maximum levels in the first postnatal weak and is not expressed in the cerebellum. Similarly, neurotrimin is expressed as early as ED15 in the rat Žequivalent to approximately ED4–5 in the chick., with expression peaking in the first postnatal week ŽED10–14 in the chick.. Likewise, LAMP is expressed as early as ED15 in the rat with expression limited to neurons of the limbic system w46,47x. Binding of LAMP is thought to be homophilic, as purified LAMP encourages binding and extension of LAMP positive, but not LAMP negative neurons in vitro w47x. Evidence from explant studies suggest that the binding of AvGp50 is heterophilic, rather than homophilic Ždata not shown.. In neuronal explant studies where AvGp50 was used as a substrate,
Fig. 7. Immunolocalization of AvGp50 in neuronal cultures. Purkinje cells, 19 days in vitro ŽDIV. Žd and e. were stained with MabSA1.7 alone and cortical neuronal cultures, 5 DIV Ža–c, f and g. were double labeled with the MabSA1.7 and a polyclonal antibody to the intracellular neurofilament protein NF200. A donkey anti-mouse flourescein labeled Žgreen. and goat anti-rabbit rhodamine labeled Žred. antibodies were used to visualize reaction products. AvGp50 is expressed on the cell surface of both neurites and cell bodies Ža, b, d, e: arrowheads.. This is in contrast to the intracellular expression of NF200 Žc and g: arrows.. AvGp50 is not expressed on the astrocytes which form the monolayer onto which neurons grow. Treatment of Purkinje cells with PI-PLC Žf and g. led to a complete loss of expression of AvGp50 Žf: curved arrow. from the cell surface but did not interfere with NF200 expression Žg: arrow.. Magnification =400.
284
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
AvGp50 did not significantly increase or decrease the length of neurites from ED10 forebrain explants over control levels, suggesting that AvGp50 does not act to stimulate or inhibit neurite outgrowth per se. However, as a receptor for AvGp50 has not yet been characterized, it is possible that the binding of AvGp50 may be heterophilic, with the receptor not expressed at this time in vitro. Significant differences are also seen in the molecular weights of each protein with AvGp50 the smallest Ž50 kDa., followed by OB-CAM Ž58 kDa. and then neurotrimin Ž65 kDa. and LAMP Ž68 kDa.. The differential expression seen between each member of the IgLON’s subfamily lends no clues as to the potential function of this group, although LAMP has been shown to specifically enhance neurite outgrowth in the limbic system, suggesting that this member may function in the targeting of neurites in selected neuronal circuits. Struyk et al. w40x suggest a similar role for neurotrimin in targeting of specific sets of neurites during development. Whether this is true for for AvGp50 will require further experimentation. The 1-day-old chick cerebellum makes an excellent model system in which to study protein expression because of the relatively few well defined cell types which are segregated into highly structured layers. In situ hybridization reveals that by day 1 post hatch, high levels of RNA transcripts are detected in both the Purkinje and granule cells as well as the deep cerebellar nuclei. AvGp50 is expressed predominantly on axons in the cerebellum, although expression on dendrites, particularly granule cell dendrites, has not been ruled out. There appears to be no expression on Purkinje dendrites with staining in the molecular layer most likely expressed on granule cell axons w23x. Earlier in development, at ED18, staining on migrating granule cell axons is more easily seen w23x. In double labeling experiments, it is possible to follow the expression of AvGp50 on an identified Purkinje cell axon as it leaves the base of a Purkinje cell body and courses its way through the internal granule cell layer ŽFig. 6.. The deduced structure of AvGp50 as a member of the immunoglobulin superfamily member and potentially as a cell adhesion molecule is confirmed by the cell surface expression of AvGp50 in neuronal cultures. Interestingly, in neuronal culture, the protein loses its restricted expression pattern and is localized on the cell surface of both processes and cell bodies, presumably due to the lack of environmental cues. Recently a 55 kDa glycoprotein isolated from chick by Clarke and Moss w8x, thought to be GPI linked, was shown to inhibit the outgrowth of both forebrain and dorsal root ganglion neurons. As the protein is expressed relatively late in development, unlike LAMP, OB-CAM and neurotrimin, which are all expressed relatively early, it is interesting to speculate on its function. Interesting observations regarding AvGp50’s possible function have been derived from preliminary experiments which show AvGp50 to be enriched in detergent resistant membrane ŽDRM. complexes iso-
lated from avian brain ŽR.C. Henke, K.A. Hancox and P.L. Jeffrey, J. Neurosci. Res., 1996, in press.. DRM complexes are thought to operate as a medium whereby GPIlinked proteins can interact with members of the kinase signaling pathway such as fyn and yes w19,41x. Despite having been clearly demonstrated to be a member of the immunoglobulin superfamily and more specifically a member of the IgLON’s subfamily of CAMs, both by cellular biology and molecular cloning experiments, AvGp50 does not appear to directly influence neurite outgrowth in ED10 explant cultures. Further studies, however, will be required to determine AvGp50’s exact function in the nervous system and the significance of AvGp50’s enrichment in DRM complexes. Acknowledgements We acknowledge the technical assistance of Mrs. J. Meaney in tissue culture experiments and Mrs. C. Smythe with the confocal microscope. References w1x Afar, D.E.H., Marius, R.M., Salzer, J.L., Stanners, C.P., Braun, P.E. and Bell, J.C., Cell adhesion properties of myelin-associated glycoprotein in L cell fibroblasts, J. Neurosci. Res., 29 Ž1991. 429–436. w2x Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J., Basic local alignment search tool, J. Mol. Biol., 215 Ž1990. 403–410. w3x Aota, S., Gojobori, T., Ishabashi, F., Maruyama, T. and Ikemura, T., Codon usage tabulated from the GenBank genetic sequence data, Nucleic Acids Res., 16 Ž1988. Suppl. 315–402. w4x Arquint, M., Roder, J., Chai, L.-S., Down, J., Wilkinson, D., Bayley, H., Braun, P. and Dunn, R., Molecular cloning and primary structure of myelin associated glycoprotein, Proc. Natl. Acad. Sci. (USA), 84 Ž1987. 600–604. w5x Barthels, D., Santoni, M.-J., Wille, W., Ruppert, C., Chaix, J.-C., Hirsch, M.-R., Fontecilla-Camps, J.C. and Goridis, C., Isolation and nucleotide sequence of mouse NCAM cDNA that codes for a Mr 79000 polypeptide without a membrane spanning region, EMBO J., 6 Ž1987. 907–914. w6x Caras, I.W., Weddell, G.N. and Williams, S.R., Analysis of the signal for attachment of a glycophospholipid membrane anchor, J. Cell Biol., 108 Ž1989. 1387–1396. w7x Chen, E.W. and Chiu, A.Y., Early stages in the development of spinal motor neurons, J. Comp. Neurol., 320 Ž1992. 291–303. w8x Clarke, G.A. and Moss, D.J., Identification of a novel protein from adult chicken brain that inhibits neurite outgrowth, J. Cell Sci., 107 Ž1994. 3393–3402. w9x Cunningham, B.A., Hemperley, J.J., Murray, B.A., Prediger, E.A., Brackenbury, R. and Edelman, G.M., Neural cell adhesion molecule: structure, immunoglubulin-like domains, cell surface modulation and alternative RNA splicing, Science, 236 Ž1987. 799–806. w10x DeBernado, A.P. and Chang, S., Native and recombinant DMGRASP selectively supports neurite extension from neurons that express GRASP, DeÕ. Biol., 169 Ž1995. 65–75. w11x Doherty, P., Williams, E. and Walsh, F.S., A soluble chimeric form of the L1 glycoprotein stimulates neurite outgrowth, Neuron, 14 Ž1995. 57–66. w12x Dowsing, B.J., Gooley, A.A., Gunning, P., Cunningham, A. and Jeffrey, P.L., Molecular cloning and primary structure of the avian Thy-1 glycoprotein, Mol. Brain Res., 14 Ž1992. 250–260.
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285 w13x Englund, P.T., The structure and biosynthesis of glycosyl phospatidylinositol protein anchors, Annu. ReÕ. Biochem., 62 Ž1993. 121–138. w14x Fahring, T., Landa, C., Pesheva, P., Kuhn, K. and Schachner, M., Characterisation of binding properties of the myelin-associated glycoprotein to extracellular matrix constituents, EMBO J., 6 Ž1987. 2875–2883. w15x Ferguson, M.A.J. and Williams, A.F., Cell-surface anchoring of proteins via glycosylphosphatidylinositol structures, Annu. ReÕ. Biochem., 57 Ž1988. 285–320. w16x Furley, A.J., Morton, S.B., Manalo, D., Karagogeos, D., Dodd, J. and Jessel, T.M., The axonal glycoprotein TAG-1 is an immunoglobulin superfamily member with neurite outgrowth-promoting activity, Cell, 61 Ž1990. 157–170. w17x Gerber, L.D., Kodukula, K. and Udenfriend, S., Phosphatidylinositol glycan ŽPI-G. anchored membrane proteins: amino acid requirements adjacent to the site of cleavage and PI-G attachment in the COOH-terminal signal peptide, J. Biol. Chem., 267 Ž1992. 12168– 12173. w18x Goodman, C.S., Bastiani, M.J., Doe, C.Q., Du Lac, S., Helfand, S.L., Kuwada, J.Y. and Thomas, J.B., Cell recognition during neuronal development, Science, 225 Ž1984. 1271–1279. w19x Gorodinsky, A. and Harris, D.A., Glycolipid-anchored proteins in neuroblastoma cells form detergent-resistant complexes without caveolin, J. Cell Biol., 129 Ž1995. 619–627. w20x Grumet, M., Hoffman, S., Chuong, C.-M. and Edelman, G.M., Polypeptide components and binding functions of neuron-glia cell adhesion molecules, Proc. Natl. Acad. Sci. USA, 81 Ž1984. 7989– 7993. w21x Grumet, M., Mauro, V., Burgoon, M.P., Edelman, G.M. and Cunningham, B.A., Structure of a new nervous system glycoprotein, Nr-CAM and its relationship to subgroups of neural cell adhesion molecules, J. Cell Biol., 113 Ž1991. 1399–1412. w22x Grunstein, M. and Hogness, D., Colony hybridisation: a method for the isolation of cloned cDNAs that contain a specific gene, Proc. Natl. Acad. Sci. USA, 72 Ž1975. 3961–3965. w23x Hancox, K.A., Sheppard, A.M. and Jeffrey, P.L., Characterisation of a novel glycoprotein ŽAVGP50. in the avian nervous system, with a monoclonal antibody, DeÕ. Brain Res., 70 Ž1992. 25–37. w24x Jeffrey, P.L., Meaney, J., Tolhurst, O. and Weinberger, R.P., Epigenetic factors controlling the development of avian Purkinje neurons, J. Neurosci. Methods, Ž1996. in press. w25x Lindner, J., Rathjen, F.G. and Schachner, M., L1 mono- and polyclonal antibodies modify cell migration in early postnatal mouse cerebellum, Nature, 305 Ž1983. 427–430. w26x Lippman, D.A., Lee, N.M. and Loh, H.H., Opioid-binding cell adhesion molecule ŽOBCAM. – related clones from a rat brain cDNA library, Gene, 117 Ž1992. 249–254. w27x Mukhopadhyay, G., Doherty, P., Walsh, F.S., Crocker, P.R. and Filbin, M.T., A novel role for myelin-associated glycoprotein as an inhibitor of axonal regeneration, Neuron, 13 Ž1994. 757–767. w28x Patel, N.H., Snow, P.M. and Goodman, C.S., Characterisation and cloning of Fasciclin III: a glycoprotein expressed on a subset of neurons and axon pathways in Drosophila, Cell, 48 Ž1987. 975–988. w29x Pimenta, A.F., Zhukareva, V., Barbe, M.F., Reinoso, B.S., Grimley, C., Henzel, W., Fischer, I. and Levitt, P., The limbic system-associated membrane protein is an Ig superfamily member that mediates selective neuronal growth and axon targeting, Neuron, 15 Ž1995. 287–297. w30x Pollerberg, G.E. and Beck-Sickinger, A., A functional role for the middle extracellular region of the neural cell adhesion molecule N-CAM in axonal fasciculation and orientation, DeÕ. Biol., 156 Ž1993. 324–340.
285
w31x Rathjen, F.G. and Schachner, M., Immunocytological and biochemical characterization of a new neuronal cell surface component ŽL1 antigen. which involved in cell adhesion, EMBO J., 3 Ž1984. 1–10. w32x Rathjen, F.G., Wolff, J.M., Frank, R., Bonhoeffer, F. and Rutishauser, U., Membrane glycoproteins involved in neurite fasciculation, J. Cell Biol., 104 Ž1987. 343–353. w33x Salzer, J.L., Holmes, W.P. and Colman, D.R., The amino acid sequence of the myelin-associated glycoproteins: homology to the immunoglobulin gene superfamily, J. Cell Biol., 104 Ž1987. 957– 965. w34x Schofield, P.R., McFarland, K.C., Hayflick, J.S., Wilcox, J.N., Cho, T.M., Roy, S., Lee, N.M., Loh, H.H. and Seeburg, P.H., Molecular characterization of a new immunoglobulin superfamily protein with potential roles in opioid binding and cell contact, EMBO J., 8 Ž1989. 489–495. w35x Schwanzel-Fukuda, M., Abraham, S., Crossin, K.L., Edelman, G.M. and Pfaff, D.W., Immunocytochemical demonstration of neural cell adhesion molecule ŽN-CAM. along the migration route of luteinizing hormone-releasing hormone ŽLHRH. neurons in mice, J. Comp. Neurol., 321 Ž1992. 1–18. w36x Schwanzel-Fukuda, M.S., Reinhard, G.R., Abraham, S., Crossin, K.L., Edelman, G.M. and Pfaff, D.W., Antibody to neural cell adhesion molecule can disrupt the migration of luteinizing hormone releasing hormone neurons into the mouse brain, J. Comp. Neurol., 342 Ž1994. 174–185. w37x Seeger, M.A., Haffley, L. and Kaufman, T.C., Characterisation of amalgam: a member of the immunoglobulin superfamily from Drosophila, Cell, 55 Ž1988. 589–600. w38x Southern, E.M., Detection of specific sequences among DNA fragments separated by gel electrophoresis, J. Mol. Biol., 98 Ž1975. 503–517. w39x Stallcup, W.B., Beasley, L.L. and Levine, J.M., Antibody against nerve growth factor-inducible large external ŽNILE. glycoprotein labels nerve fibre tracts in the developing rat nervous system, J. Neurosci., 5 Ž1985. 1090–1101. w40x Struyk, A.F., Canoll, P.D., Wolfgang, M.J., Rosen, C.L., D’Eustachio, P. and Salzer, J.L., Cloning of neurotrimin defines a new subfamily of differentially expressed neural cell adhesion molecules, J. Neurosci., 15 Ž1995. 2141–2156. w41x Thomas, P.M. and Samelson, L.E., The glycosylphosphatidylanchored Thy-1 molecule interacts with the p60fyn protein tyrosine kinase in T cells, J. Biol. Chem., 267 Ž1992. 12317–12322. w42x Wade-Evans, A. and Jenkins, J.R., Precise epitope mapping of the murine transformation-associated protein p53, EMBO J., 4 Ž1985. 699–706. w43x Williams, A.F. and Barclay, A.N., The immunoglobulin superfamily: domains for cell surface recognition, Annu. ReÕ. Immunol., 6 Ž1988. 381–405. w44x Williams, A.F. and Gagnon, J., Neuronal cell Thy-1 glycoprotein: homology with immunoglobulins, Science, 216 Ž1982. 696–703. w45x Yoshihara, Y., Kawasaki, M., Tani, A., Tamada, A., Nagata, S., Kagamiyama, H. and Mori, K., BIG-1: a new TAG-1rF3-related member of the immunoglobulin superfamily with neurite outgrowth-promoting activity, Neuron, 13 Ž1994. 415–426. w46x Zacco, A., Cooper, V., Chantler, P.D., Fisher-Hyland, S., Horton, H.L. and Levitt, P., Isolation, biochemical characterization and ultrastructural analysis of the limbic system-associated membrane protein ŽLAMP., a protein expressed by neurons comprising functional neural circuits, J. Neurosci., 10 Ž1990. 73–90. w47x Zhukareva, V. and Levitt, P., The limbic system-associated membrane protein ŽLAMP. selectively mediates interactions with specific central neuron populations, DeÕelopment, 121 Ž1995. 1161–1172.
Research report
AvGp50, a predominantly axonally expressed glycoprotein, is a member of the IgLON’s subfamily of cell adhesion molecules ž CAMs/ K.A. Hancox a , A.A. Gooley b, P.L. Jeffrey b
a,)
a DeÕelopmental Neurobiology, Children’s Medical Research Institute, Westmead, NSW, Australia Macquarie UniÕersity Center for Analytical Biotechnology, Macquarie UniÕersity, North Ryde, NSW, Australia
Accepted 6 August 1996
Abstract We have previously reported a 50 kDa glycoprotein ŽAvGp50. expressed specifically in the chick nervous system wHancox, K.A., Sheppard, A.M. and Jeffrey, P.L., Characterisation of a novel glycoprotein ŽAVGP50. in the avian nervous system, with a monoclonal antibody, DeÕ. Brain Res., 70 Ž1992. 25–37x, and we present its molecular characterization. A PCR fragment was generated following sequencing of peptide and N-terminal fragments derived from purified AvGp50. A 1.58 kb clone ŽpUEX762. containing the 5X-UTR, the entire coding sequence and a short 3X-UTR was then isolated from a chick embryonic day 18 forebrain library. The deduced amino acid sequence encodes a 338 amino acid peptide containing a 31 amino acid signal peptide at the N-terminal and a 19 amino acid phosphatidylinositol glycan linkage sequence at the C-terminal. The mature protein contains three C2-immunoglobulin-like domains and a glycosyl phosphatidylinositol anchor and shares significant homology to other members of the immunoglobulin superfamily, including neural cell adhesion molecule ŽN-CAM., myelin-associated glycoprotein ŽMAG. and the Drosophila protein Amalgam. AvGp50 exhibits highest sequence identity to a recently classified subgroup of the immunoglobulin superfamily ŽIgLONs – immunoglobulin LAMP, OBCAM and neurotrimin – classified by Pimenta et al. wPimenta, A.F., Zhukareva, V., Barbe, M.F., Reinoso, B.S., Grimley, C., Henzel, W., Fischer, I. and Levitt, P., The limbic system-associated membrane protein is an Ig superfamily member that mediates selective neuronal growth and axon targeting, Neuron, 15 Ž1995. 287–297x, comprising the opioid binding cell adhesion molecule ŽOBCAM., neurotrimin and the limbic system-associated membrane protein ŽLAMP. suggesting that AvGp50 is a member of this subgroup. AvGp50 is expressed predominantly on the cell surface of axons, in particular Purkinje cell and granule cell axons in the cerebellum. In cerebellar and forebrain neuronal cultures, protein expression is exclusively located at the cell surface. Despite its cell surface localization, AvGp50 does not directly influence the outgrowth of neurons from explant cultures from ED8 to ED10 chick forebrain, prompting the suggestion that AvGp50 may act in later maturational events. Keywords: AvGp50; Axonally expressed; Immunoglobulin superfamily; Opioid binding cell adhesion molecule ŽOB-CAM.; Limbic system-associated membrane protein ŽLAMP.; Neurotrimin
1. Introduction The formation of the nervous system depends on the correct expression of a specific subset of the large number of cell surface proteins and extracellular matrix molecules, which interact to control neuronal migration, axonal extension, fasciculation and synapse formation. Numerous cell adhesion molecules ŽCAMs., including members of the immunoglobulin superfamily, have been shown to be involved in many aspects of nervous system development ) Corresponding author. Children’s Medical Research Institute, Locked Bag 23, Wentworthville, NSW 2145, Australia. Fax: q61 Ž2. 687-2120; E-mail: [email protected]
including cell-cell and cell-substrate adhesion w1,10,11,14,16x, axonal outgrowth and target recognition w18,27,28x, fasciculation w20,21,30–32,39x, synaptogenesis and migration w21,25,35,36x. The majority of immunoglobulin superfamily members are attached to the membrane via a single transmembrane spanning domain which is hydrophobic, however, an increasing number, initially represented by Thy-1, have been shown to be linked to the membrane via a phosphatidylinositol glycan moiety w5,16,28,29,40,44,45x. Previously a monoclonal antibody MabSA1.7 had been used to characterize the avian neuronal specific glycoprotein, AvGp50 w23x. AvGp50 is expressed at high levels in the chick forebrain, cerebellum and tecta, with slightly
0169-328Xr97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 6 . 0 0 2 2 8 - 8
274
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
lower levels detected in the retina and spinal cord. The protein is expressed at low levels until embryonic day 18 when expresssion is upregulated in all neuronal tissues. Immunohistochemically, the protein has been shown to localize to areas of interneuronal contact, for example, the molecular layer of the cerebellum and both the inner and outer plexiform layers of the retina. It is absent from cell bodies and is only expressed on neuronal processes. By Western blot analysis, AvGp50 was shown to be enriched in both plasma membranes and synaptic plasma membranes. The pattern of expression and cellular localization makes AvGp50 a potential candidate for functional involvement in the development of the chick nervous system. In this present study we have determined the molecular characteristics of AvGp50. A specific PCR clone was generated between peptide fragments derived from AvGp50. This was then used to obtain a near full length cDNA clone for AvGp50. Its deduced amino acid sequence revealed that AvGP50 is significantly homologous Ž21–25%. to members of the immunoglobulin superfamily including N-CAM, MAG and the Drosophila cell adhesion molecule amalgam. In particular AvGp50 is 56% identical to the opiate binding cell adhesion molecule OB-CAM w26,34x and 50% identical to the protein neurotrimin w40x. Together with the limbic system-associated membrane protein ŽLAMP. w29x, with which AvGp50 shares it highest sequence identity Ž90%., AvGp50 forms a specific subset of the immunoglobulin superfamily known as the IgLONs Žimmunoglobulins LAMP, OB-CAM and neurotrimin w29x..
2. Materials and methods 2.1. Protein purificationr protein sequencing Immunoaffinity purified AvGp50 was prepared as previously described w23x. Briefly, a deoxycholate ŽDOC. extract of 1-day-old chick brains was passed over an MabSA1.7 monoclonal antibody immunoaffinity column. Purified protein was eluted in 500 mM diethylaminer0.5% Žwrv. deoxycholate pH 11.5, immediately neutralized with solid glycine and then dialysed against Tris-buffered saline ŽTBS. pH 7.4 for 48 h at 48C. Protein sequencing was carried out by two independent laboratories. Macromolecular Resources, Colorado, USA: 1–2 nmol AvGp50 was resuspended in 1% Žwrv. sodium dodecyl sulfate ŽSDS. and digested with 10% Žwrw. trypsin. The resulting pellet was resuspended in 70% Žvrv. trifluoroacetic acid, incubated for 1 h and then diluted dropwise with water until fully resuspended. The peptide fragments were separated on a reverse phase-HPLC column and sequenced on a Applied Biosystems 473A protein sequencer using standard Edman chemistry. Macquarie UniÕersity Center for Analytical Biotechnol-
ogy, Macquarie UniÕersity, Australia: For peptide fragments, AvGp50 was reduced and alkylated with vinylpyridene, digested with 2.5% Žwrw. Endoproteinase Lys-C ŽBoehringer Mannheim. for 2 = 2 h at 378C in 25 mM Tris-HClr0.1 mM ethylenediaminetetraacetic acid ŽEDTA.r0.025% Žwrv. SDS pH 8.6. The peptide fragments were separated on a C18 reversed-phase HPLC column and sequenced on an Applied Biosystems 470A protein sequencer using standard Edman chemistry. For N-terminal sequence, AvGp50 was electrophoresed on SDS-PAGE, transferred to PVDF ŽBio-Rad. nylon membrane and directly sequenced using a MilliGen 6600 Prosequencer using standard Edman chemistry. 2.2. PCR and library screening Oligonucleotides were designed from protein sequence using chick preferred codon usage w3x. PCR was performed using a Hybaid OmniGene thermocycler in 500 mM KClr100 mM Tris-HClr4.5 mM magnesium chloride pH 8.4 and 1 mgrml gelatin. Oligonucleotide primers were added to a final concentration of 50 m M. Single stranded cDNA was reverse transcribed from total adult forebrain RNA using 200 U Moloney murine leukemia virus ŽMMLV. reverse transcriptase w42x, 50–100 ng of the resulting cDNA was added per PCR reaction. The reaction mix was denatured for 5 min at 948C, Taq ŽBoehringer Mannheim. added and PCR products amplified by 30 cycles of 948C, 1 min Ždenature.; 608C, 1 min Žanneal.; 728C, 1 min Žextend. with a final extension of 5 min at 728C. PCR products of interest were purified using QIAEX resin ŽBresatec. according to the manufacturer’s specifications and subcloned into pGEM-T ŽPromega. for sequencing. Sequencing was performed on an Applied Biosystems 373A automated DNA sequencer using the Applied Biosystems dyedeoxy terminator reactions kit. A 600 bp fragment ŽpGM276r261-5. generated between two of the peptide fragments was used to screen approximately 200 000 colonies from an embryonic day 18 forebrain oligo dT primed pUEX-1 library constructed as previously described w12x. Colony screening was performed as described by Grunstein and Hogness w22x. Lifts were prehybridized in a buffer containing 6 = SSC Ž20 = is 3 M NaClr300 mM sodium citrate pH 7.0.r50 mM sodium di-hydrogen phosphater5 = Denharts Ž1% Žwrv . Ficollr1% Žwrv. polyvinylpyrrolidoner1% Žwrv. BSA. and 10% Žwrv. dextran sulfate at 608C for 6 h and hybridized overnight in fresh buffer containing pGM276r261-5 labeled with w a- 32 PxdCTP at approximately 1.5 = 10 6 countsrml of hybridization solution. The lifts were washed twice with 0.5 = SSCr0.1% Žwrv. SDS for 1 h and twice with 0.1 = SSCr0.1% Žwrv. SDS for 45 min at 608C before being autoradiographed on Kodak X-OMAT X-ray film. Positive colonies were further purified by a second round of screening.
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
2.3. Northern analysis Total mRNA was purified from 1-day-old chick cerebellum and forebrain using TRIzol total RNA isolation reagent ŽLife Technologies. and resuspended in 0.1% Žwrv. SDS. Poly Aq RNA was purified from total RNA using PolyA Tract mRNA Isolation Systems kit ŽPharmacia.. For analysis, samples were electrophoresed on a 1% agaroserformaldehyde gel and blotted onto nylon filters overnight in 20 = SSC by the method of Southern w38x. Hybridization was carried out as described for library screening except that the temperature was increased to 658C and washes were 2 = 0.5 = SSCr0.1% Žwrv. SDS for 30 min and 1 = 0.1 = SSCr0.1% Žwrv. SDS for 30 min. 2.4. In situ hybridization Sense and anti-sense digoxygenin RNA probes were transcribed from linearized pGM276r261-5 using SP6rT7 Digoxigenin ŽDIG . Transcription kit ŽBoehringer Mannheim.. Tissue for in situ hybridization was fixed in 4% Žwrv. paraformaldehyde in PBS, pH 7.4 overnight at 48C washed and dehydrated, cleared in 3 successive changes of xylene and then infiltrated with paraffin wax at 658C. Sections Ž5 m m. were collected onto HClrethanol cleaned and baked slides subbed with 1 mgrml poly-Llysine ŽSigma.. Sections were dewaxed with histolene, hydrated in decreasing concentrations of ethanol, washed in 2 = SSC and prehybridized for 2 h at room temperature in a buffer containing 50% Žvrv. deionized formamider4 = SSCr1= Denhardt’srsonicated herring sperm DNA Ž0.5 mgrml.ryeast tRNA Ž0.25 mgrml. and 10% Žwrv. dextran sulfate. DIG-labeled probe was added at a concentration of 2 mgrml in fresh buffer and incubation was overnight at 558C in a humid chamber. Slides were blocked and then incubated with anti-DIG alkaline phosphatase conjugated secondary antibody ŽBoehringer Mannheim. diluted 1:500. Color was developed using a BCIPrNBT detection kit ŽLife Technologies. in 100 mM Tris-HClr100 mM NaClr50 mM MgCl, pH 9.5. Slides were dehydrated, cleared in histolene and mounted in Entellan. 2.5. Immunofluorescence Cultures were fixed in 4% Žwrv. paraformaldehyde in PBS for 30 min and then blocked with 2% Žvrv. cold water fish skin gelatin ŽCWFSG.r0.5% Žwrv. bovine serum albumin ŽBSA.. Cells were incubated with either MabSA1.7 or anti-neurofilament NF200 Žpolyclonal, Sigma. or both, for 2 h at room temperature. A donkey anti-mouse fluorescein-conjugated andror a goat anti-rabbit rhodamine-conjugated ŽJackson Laboratories, both diluted 1:100 in PBS. were used and the coverslips mounted in DABCO ŽSigma.. Photography was performed on a Leica Diaplan microscope with an attached photomicro-
275
graphic system camera model MPS-52. Images were photographed under fluorescence illumination using Ektachrome 160 Dx tungsten film. For confocal microscopy, sections were dewaxed, blocked with 2% Žvrv. CWFSGr0.5% Žwrv. BSA and then incubated with the required antibody as described above. Tissue was prepared as previously described w23x. 2.6. CleaÕage of GPI anchors with the enzyme PI-PLC Neuronal culture coverslips were washed once with PBS and incubated with phosphatidylinositol-specific phospholipase C ŽPI-PLC; 0.5 Urcoverslip in 200 m l of PBS, Boehringer Mannheim. at 378C for 1 h. Control coverslips were incubated in an equal volume of PBS without PI-PLC. The coverslips were then fixed in 4% Žwrv. paraformaldehyde and treated as for immunofluorescence labeling of cultures. 2.7. Neuronal culture All cultures were maintained in Dulbecco’s modified Eagle’s medium ŽDME. supplemented with 5% Žvrv. fetal calf serum ŽFCS., 2.5% Žvrv. chick embryo extract ŽLife Technologies. and 25 mM KCl Žneuronal medium. w7x, in 7.5% CO 2 at 378C. For cerebellar cultures, cerebella were dissected from ED7 chick embryos as previously described w24x. For cortical cultures, forebrains were removed from ED8 and ‘‘to ED10’’ chicks, the cortex dissected away and treated as for cerebellar cultures.
3. Results 3.1. Protein sequencing, PCR and cloning Sequence from affinity purified AvGp50 was obtained either from the N-terminus or reduced and alkylated protein digested with Endo Lys-C or trypsin. Five peptide fragments Ž1–5. were produced and sequenced from the Endo Lys-C digests and two were produced and sequenced from the trypsin digests Ž6–7.. The sequences obtained from the peptides and N-terminal are shown in Table 1. The non-redundant search program BLAST was used to search the databases PDB, SwissProt, PIR, SPupdate, GenPept and GPupdate w2x. Peptide 3 was shown to have significant homology Ž72%., to the protein designated opioid binding protein cell adhesion molecule ŽOB-CAM. w26,34x. Subsequent alignment using the MacVector program to search the latest release of the Entrez database revealed the six remaining peptides can also be aligned within the protein sequence of OB-CAM. Oligonucleotide primers were designed using chick preferred codon usage w3x from all of the peptide fragments and the N-terminal sequence. A series of PCR reactions was performed based on the alignment of each of the
276
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
Table 1 Amino acid sequence of derived peptides from AvGp50 Fragment sequenced
Amino acid sequence
N-terminus Peptide 1 Peptide 2 Peptide 3 Peptide 4 Peptide 5 Peptide 6 Peptide 7
XDFTNGTDGITVNNGNXXPVFLSFVED CSLDPRVELEK RSPLNYSLRIQK VDFDEGTDNITVXQGDTAILXXFVEDDS SNEAATGRQALLRSEASA GFEGSEEYLWIL SEASQVPTPDFEV EFEGEEEYLEI
Five peptides Žpeptides 1–5. were produced and sequenced from Endo Lys-C aligests of AvGp50, whilst peptides 6 and 7 were produced and sequenced from trypsin digests. Amino acids are represented by the 1-letter amino acid code. The first amino acid of the N-terminus could not be determined and is designated by the symbol X. Peptide 3 and the N-terminus are likely to be the same region of sequence, as are peptides 5 and 7 Žsequenced independently..
peptides to OB-CAM. One of the reactions Žbetween peptide 3 in the 5X orientation and peptide 4 in the 3X orientation. gave a specific reaction product of approximately 600 base pairs. The fragment was subcloned into pGEM-T, with one positive clone obtained ŽpGM276r261-5.. The clone was originally sequenced using the internal pGEM-T SP6 site to verify its authenticity and later with the primers used to generate the original PCR clone. A subsequent sequencing reaction, using an internal primer, verified areas of sequence not fully covered in the initial sequencing. The 600 bp insert present in clone pGM276r261-5 aligned to the region from amino acid 36 to 257 of OB-CAM and is 60% identical at the amino acid level. The clone pGM276r26-5 was then used to screen an embryonic day 18 forebrain pUEX-1 library. Of the 200 000 colonies screened, two positive clones were identified with insert sizes of 1.58 and 1.4 kb. The two inserts are identical in their coding region and 3X-untranslated region ŽUTR. and differ only in their 5X-UTR Ždata not shown.. The 1.58 kb clone, designated pUEX762, covers 513 bp of 5X-UTR, the entire coding sequence of 1014 bp and 54 bp of 3X-UTR ŽFig. 1.. The 3X-UTR is unusually short and contains only a very short Poly A tail Ž11 As.. Also, there is no standard polyadenylation sequence, suggesting that the clone originated at an A-rich site and does not represent a full length clone for AvGp50. Interestingly, the bovine clone for OB-CAM originated at an A-rich site, although it contains slightly more 3X-UTR Ž795 bp. than the pUEX762 clone for AvGp50. Fig. 1 shows the entire
DNA sequence of pUEX762 and its deduced amino acid sequence. An open reading frame begins at bp 514 and extends through bp 1530. The single open reading frame encodes a 338 amino acid peptide containing a putative 31 amino acid signal peptide with a predicted cleavage site at Ser31 based on protein sequence from the N-terminal which designates valine at position 32 is the N-terminal amino acid of the mature protein. A 19 amino acid glycosyl phosphatidylinositol linkage sequence comprising hydrophobic amino acids is located at the C-terminal. The most likely cleavage site for GPI addition to occur is at Ser319 which is followed directly by two small amino acids, leucine and alanine. For GPI addition to occur, the sequence itself is not as crucial as the hydrophobicity and positioning of the sequence w6,13,15,17x. Hydrophobicity is largely achieved by a chain of between 15 and 30 amino acids rich in leucine at the C-terminal end of the peptide. As seen in Fig. 2, AvGp50 largely conforms to the requirements for GPI addition, although it differs significantly from the GPI addition sequence for either OB-CAM or neurotrimin. Further evidence that AvGp50 is GPI anchored is shown in experiments where cultured chick cortical or cerebellar neurons are treated with the phosphatidylinositol-specific phospholipase C Žsee Fig. 7.. 3.2. AÕGp50 forms a member of a specific subfamily of CAMs Subsequent database searches showed that AvGp50 was also significantly identical to two recently cloned proteins, neurotrimin w40x, and the limbic system-associated membrane protein, LAMP w29x. Alignment of the deduced amino acid sequence of AvGp50 to OB-CAM, neurotrimin and LAMP is shown in Fig. 2A. Alignment of AvGp50 to LAMP with which it shares most identity is shown in Fig. 2B. AvGp50 Ž338 amino acids. is marginally smaller than either OB-CAM Ž345 amino acids. or neurotrimin Ž344 amino acids., with gaps added in the coding sequence to maintain alignment. AvGp50 and LAMP are most similar, both sharing an identical length of 338 amino acids. The protein AvGp50 shares 50% identity to neurotrimin, 56% identity to OB-CAM and 90% identity to LAMP. OB-CAM contains 6 potential N-linked carbohydrate side chains, whereas AvGp50, neurotrimin and LAMP all contain 8 potential N-linked addition signals. Each of these proteins share 6 conserved cysteine residues which form the basis of the 3 Ig-like domains. Neurotrimin has an extra cysteine at postion 83 which is not conserved in either AvGp50,
X
X
Fig. 1. The insert in pUEX762 is 1586 bp in length and contains 513 bp of 5 -UTR and 54 bp of 3 -UTR. A single open reading frame contains the complete predicted coding sequence of AvGp50 with a methionine at bp 514 the putative start codon. Amino acids are represented by the 3-letter amino acid code below the nucleotide sequence. Potential N-linked carbohydrate side chain addition signals are underlined by dotted lines. The 6 cysteine residues postulated to be involved in C2 domain formation are designated by the symbol ‘v’. The theoretical GPI addition signal is underlined by the solid X bar. The first stop codon ŽTAA. lies at bp 1584–1586. The 3 -UTR is very ArT rich Ž93%. and consists mainly of TAA repeats, followed immediately by a string of 11 As.
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
277
278
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
279
Fig. 3. Three putative domains of AvGp50 Ždomain 1, domain 2 and domain 3. are aligned to representative C2 domains from N-CAM Žmouse., amalgam Ž Drosophila. and MAG Žmouse.. Conserved amino acids are in black boxes, conservative substitutions are in grey. Gaps created by the program to maintain alignment of the sequences are shown by the symbol ‘ Ø ’. The position of each of the 7 strands of a C2 domain are underlined. Alignment is performed by the latest release of the Pile-Up and Prettybox programs supplied by the Australian National Genomic Information Service ŽANGIS..
OB-CAM or LAMP. The first 2 domains of AvGp50 share the highest identity with the OB-CAM, neurotrimin and LAMP with identities of 61–63% between AvGp50, and each of OB-CAM and neurotrimin and 92–94% between AvGp50 and LAMP. Most of the variation between these molecules occurs in the third domain with identities of 42% between AvGp50 and each of OB-CAM and neurotrimin and 88% identity between AvGp50 and LAMP. Based on the high identities shared by these molecules we suggest AvGp50 to be a member of a new subfamily of the larger immunoglobulin superfamily, tentatively designated IgLONs. The high identity shared between AvGp50 and LAMP can be seen in Fig. 2B. The proteins share 90% identity although this figure can be as high as 95% if conservative substitutes are taken into account. LAMP and AvGp50 appear to have slightly different mature protein lengths, with LAMP Ž287 amino acids. one amino acid shorter than AvGp50. In particular, the cleavage site for the signal peptide would appear to be Pro28 in LAMP ŽFig. 2A. based on microsequencing data, whereas in AvGp50, the cleavage site is more likely to be at Ser31, based on protein sequence obtained from both the N-terminus and peptide fragments Žsee Table 1.. In addition, the predicted cleavage site for GPI addition in AvGp50 is Ser319, whereas in LAMP the predicted cleavage site is Asn315 w29x. In respect to GPI addition, however, neither site has been confirmed by alternative methods, making it difficult to predict which would be the more favored site.
We have previously demonstrated that AvGp50 has 5 N-linked carbohydrate side chains which when removed result in a protein of 34 kDa w23x. Presumably several of the potential glycosylation sequences are not functional ŽFig. 1.. The open reading frame gives a 288 amino acid mature protein with a predicted molecular weight of 32 kDa once the signal sequence and phosphatidylinositol linkage signals are removed. This is in agreement with our predicted molecular weight of 34 kDa Žas determined from SDS-PAGE. w23x. 3.3. Homology to Ig superfamily Like OB-CAM, neurotrimin and LAMP, AvGp50 shows significant homology to other members of the immunoglobulin superfamily including N-CAM w9x, MAG w4,33x and the Drosophila Ig superfamily member amalgam w37x. The alignment of the three Ig-like domains of AvGp50 to members of the Ig superfamily is shown in Fig. 3. The cysteine residues that form disulfide bonds of the Ig-like domains are completely conserved and the domains all have the classic features of C2 Ig-like domains w43x except the truncated domain 2 which is approximately 15 amino acids shorter than a classic C2 domain Žnormally around 100 amino acids.. This truncated second domain is shared between all members of the IgLON’s subfamily of CAMs. The folding pattern is typical of a C2 domain with one b-sheet formed from the C, F and G strands and the second from the D, E, B and A strands. The cysteine
Fig. 2. ŽA. The deduced amino acid sequence of insert from pUEX762 ŽAvGp50. is compared to the amino acid sequence for bovine OB-CAM, rat LAMP and neurotrimin. Identical amino acids are shown in black boxes Ž3 amino acids required for a consensus., conserved amino acids are boxed in grey. Gaps created by the program to maintain alignment of the sequences are shown by the symbol ‘v’. Overall AvGp50 is 56% identical to bovine OB-CAM, 50% identical to neurotrimin and 90% identical to LAMP. ŽB. Direct alignment of AvGp50 and LAMP. Amino acids are represented by the 1-letter code. Identical amino acids are indicated by the symbol ‘)’, conservative substitutions by the symbol ‘:’ and non-conservative substitutions by the symbol ‘ Ø ’, alignment was by the latest release of the MacVector program. Putative signal peptides are indicated in bold, putative GPI addition signals are underlined.
280
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
Fig. 4. Two mRNAs are detected by Northern analysis of 1-day-old chick tissue total RNA Ž20 m g per lane.. A 5.5 kb major message and 3.4 kb minor message is detected in forebrain Žlane 4. and cerebellum Žlane 5. but is not expressed in either kidney Žlane 1., liver Žlane 2. or heart Žlane 3..
residues in the B and F sheets allow for the formation of a disulfide bond and the stabilization of the two b-sheets. 3.4. Northern analysis Two mRNA species of approximately 5.5 kb Žmajor. and 3.4 kb Žminor. can be detected in total RNA from 1-day-old chick forebrain and cerebellum but not from the heart, liver or kidney ŽFig. 4.. Significantly, whilst OBCAM, neurotrimin and LAMP share significant homology, the transcripts from each of the genes are of different lengths ŽOB-CAM, 4.5 kb and 7.2 kb; neurotrimin, 3.2 and 4.0 kb; and LAMP, 1.6 kb and 8.0 kb.. Given that the library clone is 1.58 kb, it appears that the clone originated at an internal A-rich site and does not represent a full length cDNA. This is confirmed by the absence of a standard polyadenylation signal Žsee Fig. 1.. 3.5. In situ hybridization and confocal microscopy To characterize the localization of AvGp50 mRNA in the cerebellum Žwhere AvGp50 is highly expressed. an in situ hybridization was performed using the pGM276r261-5 600 bp insert. Fig. 5 shows a sagittal section of the
Fig. 5. In situ localization of AvGp50 mRNA in the 1-day-old chick cerebellum. Sense control Ža., anti-sense probes Žb and c.. By day 1 post hatch all neuronal cell types of the cerebellum express AvGp50 mRNA Žarrowheads. and cells present in the deep cerebellar nuclei also express AvGp50 mRNA Žc.. Magnification Ža and b.: =200; Žc. =1000.
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
cerebellum at post-hatch day 1. There is strong expression of the transcript in all neuronal cell types of the cerebellum including both the Purkinje and granule cells ŽFig. 5b, arrowheads. as well as the deep cerebellar nuclei ŽFig. 5c, arrowhead., with which the Purkinje cell axons make contact. The message expression in Purkinje cells suggested that AvGp50 is expressed in Purkinje cells despite no detectable levels of protein in either the cell bodies or dendrites w23x. Presumably the protein is located entirely in the axon. To test this hypothesis, sections of cerebellum were double labeled with an anti-neurofilament 200 antibody and the MabSA1.7. Fig. 6 shows a confocal image of a day 1 post-hatch cerebellum which has been double labeled with MabSA1.7 Žgreen. and a polyclonal anti-neurofilament 200 Žred. antibody. The arrowheads highlight a Purkinje cell axon as it leaves the cerebellum Ždouble label is represented by yellow.. The expression of AvGp50, therefore, appears to be predominantly axonal with strong expression on both the granule Žas shown by strong immunoreactivity of the molecular layer. and Purkinje cell axons w23x.
281
To determine the cellular localization of AvGp50, neuronal cultures were stained with either MabSA1.7 alone ŽFig. 7d and e, cerebellar. or double labeled with both MabSA1.7 and the polyclonal anti-NF200 antibody ŽFig. 7a–c, f and g; cortical.. Fig. 7a and b show a cortical neuronal culture after 3–4 days in vitro with punctate expression of AvGp50 on the surface of both the cell bodies and processes. This is in contrast to the intracellular expression of NF-200 ŽFig. 7c.. Likewise, cerebellar neuronal cultures maintained for 19 days in vitro ŽFig. 7d and e. show a similar pattern of expression of AvGp50, with strong punctate expression on the cell surface of cell bodies and processes. Expression of AvGp50 at the cell surface, in conjunction with the sequence data, suggests a potential role for the AvGp50 as a cell surface adhesion molecule. Treatment of forebrain cortical neuronal cultures with the enzyme phosphatidylinositol phospholipase C ŽFig. 7f and g. results in a complete loss of expression of AvGp50 at the cell surface ŽFig. 7f. but does not alter the expression of the intracellular protein neurofilament 200 ŽFig. 7g..
Fig. 6. The 1-day-old chick cerebellum was double labeled with the MabSA1.7 and a polyclonal anti-neurofilament 200 ŽNF200. antibody. A donkey anti-mouse fluorescein Žgreen. and goat anti-rabbit rhodamine Žred. labeled secondary antibodies were used to visualize reaction products. Where a double label occurs, the reaction product is visualized as yellow. A Purkinje cell axon Žarrowheads. is clearly visible as it leaves the base of the Purkinje cell Žunlabeled, arrow. and courses through the internal granular layer. Magnification =400. PCL, Purkinje cell layer; GCL, granule cell layer.
282
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
4. Discussion In this study we present the molecular characterization of AvGp50, the predicted structure as a phosphatidylinositol glycan linked cell surface molecule and a member of the immunoglobulin superfamily. In particular AvGp50 belongs to a specific subset of the immunoglobulin superfamily ŽIgLONs. comprising OB-CAM, neurotrimin and LAMP w29x. Purified protein was used to derive N-terminal and peptide fragments whose sequence were used in an intensive PCR screen. One of the PCR products gave a specific reaction product of 600 bp which, when sequenced, proved to contain all the other peptide fragments except peptide 6, which on final analysis lies outside the region of the PCR clone. The PCR fragment contained partial coding sequence which exhibited homology to both the bovine and rat OB-CAM. To further characterize AvGp50 the PCR fragment was used to screen an embryonic day 18 chicken forebrain library. Two positive clones were identified which differed only in their 5X-UTR and shared the entire coding sequence and 3X-UTR. The deduced amino acid sequence of AvGp50 gives a peptide of 338 amino acids Žmature protein of 288 amino acids. and a predicted molecular weight of 32 kDa, similar to our predicted molecular weight of 34 kDa as determined from SDS-PAGE w23x. Despite the protein containing 8 potential glycosylation sequences ŽAsn-X-SerrThr. defined by DNA sequencing, AvGp50 contains 5 N-linked carbohydrate side chains, as revealed by N-glycanase digestion w23x. This suggests that several of the potential sites are non-functional. The asparagine residue at amino acid 40 is a good candidate for glycosylation as it was absent from peptide 3 during protein sequencing. Evidence from bovine OB-CAM also points to this asparagine being glycosylated. In addition the amino acid 285 Žamino acid 279 AvGp50. is glycosylated on basis of the same criterion w34x. The protein contains six cysteine residues, all of which are conserved as in OB-CAM, neurotrimin and LAMP. The six cysteine residues divide the protein into 3 domains which share significant homology to the C2 domains characteristic of the immunoglobulin superfamily. Domains 1 and 3 each comprise approximately 100 amino acids, whilst domain 2 is slightly truncated, comprising 83 amino acids. Within the subfamily, domains 1 and 2 share the
283
highest identity with the most variability exhibited in the third domain. Evidence to suggest that the putative GPI addition signal is functional is derived from the observation that when neuronal cultures are treated with PI-PLC all immunoreactivity of the MabSA1.7 is lost, a treatment which does not alter the expression of the intracellular structural protein NF200. The structure of AvGp50 therefore consists of 3 C2 Ig-like domains anchored to the membrane by a GPI tail. The protein contains no transmembrane domains and no fibronectin type III repeats characteristic of other Ig superfamily members such as TAG-1 w16x. We propose that AvGp50 is a member of the subgroup of CAMs including OB-CAM, neurotrimin and LAMP. AvGp50 is most likely the avian homologue of LAMP, although there are significant differences between the expression patterns of AvGp50 and LAMP, suggesting that AvGp50 is a novel member in its own right. For example, AvGp50 is expressed at high levels in all regions of the chick brain with highest levels in the forebrain, cerebellum and tecta with slightly lower levels detected in the retina. AvGp50 is expressed in areas of high interneuronal contact, in the molecular layer and internal granule cell layer of the cerebellum and the inner and outer plexiform layers of the retina w23x. Furthermore AvGp50 is not detected on the cell bodies with expression restricted to neuronal processes of post-mitotic neurons w23x. Most significantly, however, AvGp50 is expressed relatively late in development with a sharp rise in protein expression at embryonic day 18 ŽED18. in the chick. Differences in the pattern of expression of the other members of the IgLON family exist. OB-CAM, by way of contrast, is expressed as early as ED18 in the rat ŽED9 in the chick. with maximum levels in the first postnatal weak and is not expressed in the cerebellum. Similarly, neurotrimin is expressed as early as ED15 in the rat Žequivalent to approximately ED4–5 in the chick., with expression peaking in the first postnatal week ŽED10–14 in the chick.. Likewise, LAMP is expressed as early as ED15 in the rat with expression limited to neurons of the limbic system w46,47x. Binding of LAMP is thought to be homophilic, as purified LAMP encourages binding and extension of LAMP positive, but not LAMP negative neurons in vitro w47x. Evidence from explant studies suggest that the binding of AvGp50 is heterophilic, rather than homophilic Ždata not shown.. In neuronal explant studies where AvGp50 was used as a substrate,
Fig. 7. Immunolocalization of AvGp50 in neuronal cultures. Purkinje cells, 19 days in vitro ŽDIV. Žd and e. were stained with MabSA1.7 alone and cortical neuronal cultures, 5 DIV Ža–c, f and g. were double labeled with the MabSA1.7 and a polyclonal antibody to the intracellular neurofilament protein NF200. A donkey anti-mouse flourescein labeled Žgreen. and goat anti-rabbit rhodamine labeled Žred. antibodies were used to visualize reaction products. AvGp50 is expressed on the cell surface of both neurites and cell bodies Ža, b, d, e: arrowheads.. This is in contrast to the intracellular expression of NF200 Žc and g: arrows.. AvGp50 is not expressed on the astrocytes which form the monolayer onto which neurons grow. Treatment of Purkinje cells with PI-PLC Žf and g. led to a complete loss of expression of AvGp50 Žf: curved arrow. from the cell surface but did not interfere with NF200 expression Žg: arrow.. Magnification =400.
284
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285
AvGp50 did not significantly increase or decrease the length of neurites from ED10 forebrain explants over control levels, suggesting that AvGp50 does not act to stimulate or inhibit neurite outgrowth per se. However, as a receptor for AvGp50 has not yet been characterized, it is possible that the binding of AvGp50 may be heterophilic, with the receptor not expressed at this time in vitro. Significant differences are also seen in the molecular weights of each protein with AvGp50 the smallest Ž50 kDa., followed by OB-CAM Ž58 kDa. and then neurotrimin Ž65 kDa. and LAMP Ž68 kDa.. The differential expression seen between each member of the IgLON’s subfamily lends no clues as to the potential function of this group, although LAMP has been shown to specifically enhance neurite outgrowth in the limbic system, suggesting that this member may function in the targeting of neurites in selected neuronal circuits. Struyk et al. w40x suggest a similar role for neurotrimin in targeting of specific sets of neurites during development. Whether this is true for for AvGp50 will require further experimentation. The 1-day-old chick cerebellum makes an excellent model system in which to study protein expression because of the relatively few well defined cell types which are segregated into highly structured layers. In situ hybridization reveals that by day 1 post hatch, high levels of RNA transcripts are detected in both the Purkinje and granule cells as well as the deep cerebellar nuclei. AvGp50 is expressed predominantly on axons in the cerebellum, although expression on dendrites, particularly granule cell dendrites, has not been ruled out. There appears to be no expression on Purkinje dendrites with staining in the molecular layer most likely expressed on granule cell axons w23x. Earlier in development, at ED18, staining on migrating granule cell axons is more easily seen w23x. In double labeling experiments, it is possible to follow the expression of AvGp50 on an identified Purkinje cell axon as it leaves the base of a Purkinje cell body and courses its way through the internal granule cell layer ŽFig. 6.. The deduced structure of AvGp50 as a member of the immunoglobulin superfamily member and potentially as a cell adhesion molecule is confirmed by the cell surface expression of AvGp50 in neuronal cultures. Interestingly, in neuronal culture, the protein loses its restricted expression pattern and is localized on the cell surface of both processes and cell bodies, presumably due to the lack of environmental cues. Recently a 55 kDa glycoprotein isolated from chick by Clarke and Moss w8x, thought to be GPI linked, was shown to inhibit the outgrowth of both forebrain and dorsal root ganglion neurons. As the protein is expressed relatively late in development, unlike LAMP, OB-CAM and neurotrimin, which are all expressed relatively early, it is interesting to speculate on its function. Interesting observations regarding AvGp50’s possible function have been derived from preliminary experiments which show AvGp50 to be enriched in detergent resistant membrane ŽDRM. complexes iso-
lated from avian brain ŽR.C. Henke, K.A. Hancox and P.L. Jeffrey, J. Neurosci. Res., 1996, in press.. DRM complexes are thought to operate as a medium whereby GPIlinked proteins can interact with members of the kinase signaling pathway such as fyn and yes w19,41x. Despite having been clearly demonstrated to be a member of the immunoglobulin superfamily and more specifically a member of the IgLON’s subfamily of CAMs, both by cellular biology and molecular cloning experiments, AvGp50 does not appear to directly influence neurite outgrowth in ED10 explant cultures. Further studies, however, will be required to determine AvGp50’s exact function in the nervous system and the significance of AvGp50’s enrichment in DRM complexes. Acknowledgements We acknowledge the technical assistance of Mrs. J. Meaney in tissue culture experiments and Mrs. C. Smythe with the confocal microscope. References w1x Afar, D.E.H., Marius, R.M., Salzer, J.L., Stanners, C.P., Braun, P.E. and Bell, J.C., Cell adhesion properties of myelin-associated glycoprotein in L cell fibroblasts, J. Neurosci. Res., 29 Ž1991. 429–436. w2x Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J., Basic local alignment search tool, J. Mol. Biol., 215 Ž1990. 403–410. w3x Aota, S., Gojobori, T., Ishabashi, F., Maruyama, T. and Ikemura, T., Codon usage tabulated from the GenBank genetic sequence data, Nucleic Acids Res., 16 Ž1988. Suppl. 315–402. w4x Arquint, M., Roder, J., Chai, L.-S., Down, J., Wilkinson, D., Bayley, H., Braun, P. and Dunn, R., Molecular cloning and primary structure of myelin associated glycoprotein, Proc. Natl. Acad. Sci. (USA), 84 Ž1987. 600–604. w5x Barthels, D., Santoni, M.-J., Wille, W., Ruppert, C., Chaix, J.-C., Hirsch, M.-R., Fontecilla-Camps, J.C. and Goridis, C., Isolation and nucleotide sequence of mouse NCAM cDNA that codes for a Mr 79000 polypeptide without a membrane spanning region, EMBO J., 6 Ž1987. 907–914. w6x Caras, I.W., Weddell, G.N. and Williams, S.R., Analysis of the signal for attachment of a glycophospholipid membrane anchor, J. Cell Biol., 108 Ž1989. 1387–1396. w7x Chen, E.W. and Chiu, A.Y., Early stages in the development of spinal motor neurons, J. Comp. Neurol., 320 Ž1992. 291–303. w8x Clarke, G.A. and Moss, D.J., Identification of a novel protein from adult chicken brain that inhibits neurite outgrowth, J. Cell Sci., 107 Ž1994. 3393–3402. w9x Cunningham, B.A., Hemperley, J.J., Murray, B.A., Prediger, E.A., Brackenbury, R. and Edelman, G.M., Neural cell adhesion molecule: structure, immunoglubulin-like domains, cell surface modulation and alternative RNA splicing, Science, 236 Ž1987. 799–806. w10x DeBernado, A.P. and Chang, S., Native and recombinant DMGRASP selectively supports neurite extension from neurons that express GRASP, DeÕ. Biol., 169 Ž1995. 65–75. w11x Doherty, P., Williams, E. and Walsh, F.S., A soluble chimeric form of the L1 glycoprotein stimulates neurite outgrowth, Neuron, 14 Ž1995. 57–66. w12x Dowsing, B.J., Gooley, A.A., Gunning, P., Cunningham, A. and Jeffrey, P.L., Molecular cloning and primary structure of the avian Thy-1 glycoprotein, Mol. Brain Res., 14 Ž1992. 250–260.
K.A. Hancox et al.r Molecular Brain Research 44 (1997) 273–285 w13x Englund, P.T., The structure and biosynthesis of glycosyl phospatidylinositol protein anchors, Annu. ReÕ. Biochem., 62 Ž1993. 121–138. w14x Fahring, T., Landa, C., Pesheva, P., Kuhn, K. and Schachner, M., Characterisation of binding properties of the myelin-associated glycoprotein to extracellular matrix constituents, EMBO J., 6 Ž1987. 2875–2883. w15x Ferguson, M.A.J. and Williams, A.F., Cell-surface anchoring of proteins via glycosylphosphatidylinositol structures, Annu. ReÕ. Biochem., 57 Ž1988. 285–320. w16x Furley, A.J., Morton, S.B., Manalo, D., Karagogeos, D., Dodd, J. and Jessel, T.M., The axonal glycoprotein TAG-1 is an immunoglobulin superfamily member with neurite outgrowth-promoting activity, Cell, 61 Ž1990. 157–170. w17x Gerber, L.D., Kodukula, K. and Udenfriend, S., Phosphatidylinositol glycan ŽPI-G. anchored membrane proteins: amino acid requirements adjacent to the site of cleavage and PI-G attachment in the COOH-terminal signal peptide, J. Biol. Chem., 267 Ž1992. 12168– 12173. w18x Goodman, C.S., Bastiani, M.J., Doe, C.Q., Du Lac, S., Helfand, S.L., Kuwada, J.Y. and Thomas, J.B., Cell recognition during neuronal development, Science, 225 Ž1984. 1271–1279. w19x Gorodinsky, A. and Harris, D.A., Glycolipid-anchored proteins in neuroblastoma cells form detergent-resistant complexes without caveolin, J. Cell Biol., 129 Ž1995. 619–627. w20x Grumet, M., Hoffman, S., Chuong, C.-M. and Edelman, G.M., Polypeptide components and binding functions of neuron-glia cell adhesion molecules, Proc. Natl. Acad. Sci. USA, 81 Ž1984. 7989– 7993. w21x Grumet, M., Mauro, V., Burgoon, M.P., Edelman, G.M. and Cunningham, B.A., Structure of a new nervous system glycoprotein, Nr-CAM and its relationship to subgroups of neural cell adhesion molecules, J. Cell Biol., 113 Ž1991. 1399–1412. w22x Grunstein, M. and Hogness, D., Colony hybridisation: a method for the isolation of cloned cDNAs that contain a specific gene, Proc. Natl. Acad. Sci. USA, 72 Ž1975. 3961–3965. w23x Hancox, K.A., Sheppard, A.M. and Jeffrey, P.L., Characterisation of a novel glycoprotein ŽAVGP50. in the avian nervous system, with a monoclonal antibody, DeÕ. Brain Res., 70 Ž1992. 25–37. w24x Jeffrey, P.L., Meaney, J., Tolhurst, O. and Weinberger, R.P., Epigenetic factors controlling the development of avian Purkinje neurons, J. Neurosci. Methods, Ž1996. in press. w25x Lindner, J., Rathjen, F.G. and Schachner, M., L1 mono- and polyclonal antibodies modify cell migration in early postnatal mouse cerebellum, Nature, 305 Ž1983. 427–430. w26x Lippman, D.A., Lee, N.M. and Loh, H.H., Opioid-binding cell adhesion molecule ŽOBCAM. – related clones from a rat brain cDNA library, Gene, 117 Ž1992. 249–254. w27x Mukhopadhyay, G., Doherty, P., Walsh, F.S., Crocker, P.R. and Filbin, M.T., A novel role for myelin-associated glycoprotein as an inhibitor of axonal regeneration, Neuron, 13 Ž1994. 757–767. w28x Patel, N.H., Snow, P.M. and Goodman, C.S., Characterisation and cloning of Fasciclin III: a glycoprotein expressed on a subset of neurons and axon pathways in Drosophila, Cell, 48 Ž1987. 975–988. w29x Pimenta, A.F., Zhukareva, V., Barbe, M.F., Reinoso, B.S., Grimley, C., Henzel, W., Fischer, I. and Levitt, P., The limbic system-associated membrane protein is an Ig superfamily member that mediates selective neuronal growth and axon targeting, Neuron, 15 Ž1995. 287–297. w30x Pollerberg, G.E. and Beck-Sickinger, A., A functional role for the middle extracellular region of the neural cell adhesion molecule N-CAM in axonal fasciculation and orientation, DeÕ. Biol., 156 Ž1993. 324–340.
285
w31x Rathjen, F.G. and Schachner, M., Immunocytological and biochemical characterization of a new neuronal cell surface component ŽL1 antigen. which involved in cell adhesion, EMBO J., 3 Ž1984. 1–10. w32x Rathjen, F.G., Wolff, J.M., Frank, R., Bonhoeffer, F. and Rutishauser, U., Membrane glycoproteins involved in neurite fasciculation, J. Cell Biol., 104 Ž1987. 343–353. w33x Salzer, J.L., Holmes, W.P. and Colman, D.R., The amino acid sequence of the myelin-associated glycoproteins: homology to the immunoglobulin gene superfamily, J. Cell Biol., 104 Ž1987. 957– 965. w34x Schofield, P.R., McFarland, K.C., Hayflick, J.S., Wilcox, J.N., Cho, T.M., Roy, S., Lee, N.M., Loh, H.H. and Seeburg, P.H., Molecular characterization of a new immunoglobulin superfamily protein with potential roles in opioid binding and cell contact, EMBO J., 8 Ž1989. 489–495. w35x Schwanzel-Fukuda, M., Abraham, S., Crossin, K.L., Edelman, G.M. and Pfaff, D.W., Immunocytochemical demonstration of neural cell adhesion molecule ŽN-CAM. along the migration route of luteinizing hormone-releasing hormone ŽLHRH. neurons in mice, J. Comp. Neurol., 321 Ž1992. 1–18. w36x Schwanzel-Fukuda, M.S., Reinhard, G.R., Abraham, S., Crossin, K.L., Edelman, G.M. and Pfaff, D.W., Antibody to neural cell adhesion molecule can disrupt the migration of luteinizing hormone releasing hormone neurons into the mouse brain, J. Comp. Neurol., 342 Ž1994. 174–185. w37x Seeger, M.A., Haffley, L. and Kaufman, T.C., Characterisation of amalgam: a member of the immunoglobulin superfamily from Drosophila, Cell, 55 Ž1988. 589–600. w38x Southern, E.M., Detection of specific sequences among DNA fragments separated by gel electrophoresis, J. Mol. Biol., 98 Ž1975. 503–517. w39x Stallcup, W.B., Beasley, L.L. and Levine, J.M., Antibody against nerve growth factor-inducible large external ŽNILE. glycoprotein labels nerve fibre tracts in the developing rat nervous system, J. Neurosci., 5 Ž1985. 1090–1101. w40x Struyk, A.F., Canoll, P.D., Wolfgang, M.J., Rosen, C.L., D’Eustachio, P. and Salzer, J.L., Cloning of neurotrimin defines a new subfamily of differentially expressed neural cell adhesion molecules, J. Neurosci., 15 Ž1995. 2141–2156. w41x Thomas, P.M. and Samelson, L.E., The glycosylphosphatidylanchored Thy-1 molecule interacts with the p60fyn protein tyrosine kinase in T cells, J. Biol. Chem., 267 Ž1992. 12317–12322. w42x Wade-Evans, A. and Jenkins, J.R., Precise epitope mapping of the murine transformation-associated protein p53, EMBO J., 4 Ž1985. 699–706. w43x Williams, A.F. and Barclay, A.N., The immunoglobulin superfamily: domains for cell surface recognition, Annu. ReÕ. Immunol., 6 Ž1988. 381–405. w44x Williams, A.F. and Gagnon, J., Neuronal cell Thy-1 glycoprotein: homology with immunoglobulins, Science, 216 Ž1982. 696–703. w45x Yoshihara, Y., Kawasaki, M., Tani, A., Tamada, A., Nagata, S., Kagamiyama, H. and Mori, K., BIG-1: a new TAG-1rF3-related member of the immunoglobulin superfamily with neurite outgrowth-promoting activity, Neuron, 13 Ž1994. 415–426. w46x Zacco, A., Cooper, V., Chantler, P.D., Fisher-Hyland, S., Horton, H.L. and Levitt, P., Isolation, biochemical characterization and ultrastructural analysis of the limbic system-associated membrane protein ŽLAMP., a protein expressed by neurons comprising functional neural circuits, J. Neurosci., 10 Ž1990. 73–90. w47x Zhukareva, V. and Levitt, P., The limbic system-associated membrane protein ŽLAMP. selectively mediates interactions with specific central neuron populations, DeÕelopment, 121 Ž1995. 1161–1172.