Co-localisation, heterophilic interactions and regulated expression of IgLON family proteins in the chick nervous system

Molecular Brain Research 82 (2000) 84–94 www.elsevier.com / locate / bres

Research report

Co-localisation, heterophilic interactions and regulated expression of IgLON family proteins in the chick nervous system Anthony P. Lodge, Mark R. Howard, Christine J. McNamee, Diana J. Moss* Department of Human Anatomy and Cell Biology, The University of Liverpool, New Medical School, Ashton St., Liverpool L69 3 GE, UK Accepted 25 July 2000

Abstract The chick glycoprotein GP55 has been shown to inhibit the growth and adhesion of DRG and forebrain neurons. GP55 consists of several members of the IgLON family, a group of glycoproteins including LAMP, OBCAM, CEPU-1 (chick) / neurotrimin (rat) and neurotractin (chick) / kilon (rat) thought to play a role in the guidance of growing axons. IgLONs belong to the Ig superfamily and have three C2 domains and a glycosyl phosphatidylinositol anchor which tethers them to the neuronal plasma membrane. We have now completed the deduced amino acid sequence for two isoforms of chicken OBCAM and used recombinant LAMP, OBCAM and CEPU-1 to raise antisera specific to these three IgLONs. LAMP and CEPU-1 are co-expressed on DRG and sympathetic neurons, while both overlapping and distinct expression patterns for LAMP, OBCAM and CEPU-1 are observed in retina. Analysis of IgLON mRNA expression reveals that alternatively spliced forms of LAMP and CEPU-1 are developmentally regulated. In an attempt to understand how the IgLONs function, we have begun to characterise their molecular interactions. LAMP and CEPU-1 have already been shown to interact homophilically. We now confirm that OBCAM will bind homophilically and also that LAMP, OBCAM and CEPU-1 will interact heterophilically with each other. We propose that IgLON activity will depend on the complement of IgLONs expressed by each neuron.  2000 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Axon guidance mechanisms and pathways Keywords: IgLON; Retina; GPI-anchor; Cell–cell recognition; Neurite outgrowth

1. Introduction During the development of the nervous system, neuronal axons are guided through the embryo by a variety of molecular cues which enable them to accurately form synapses with their appropriate targets [7,26]. The IgLON family proteins LAMP (limbic system-associated membrane protein), OBCAM (opioid-binding cell adhesion molecule), CEPU-1 / neurotrimin (NTM) (chick and rat orthologues, respectively) and neurotractin (chick) / kilon (rat) are thought to play a role in this process [8,19,21,23,25,28]. IgLONs are highly glycosylated members of the Ig superfamily with three C2-type Ig domains and are attached to the plasma membrane via a glycosyl *Corresponding author. Tel.: 144-151-794-5521; fax: 144-151-7945517. E-mail address: [email protected] (D.J. Moss).

phosphatidylinositol (GPI)-anchor. Each IgLON may be expressed as a variety of isoforms. For example, alternatively spliced mRNAs for chicken LAMP and CEPU-1 have been identified in which an optional exon encoding 11 (in CEPU-1) or 12 (in LAMP) amino acids (the ‘C-terminal insert’) is inserted just downstream of the exon for the third C2 domain. Chicken neurotractin (NTR) is expressed as two alternatively spliced forms, NTR-L and NTR-S, where the latter does not possess the third C2 domain of NTR-L [19]. In addition, two mRNAs for chicken LAMP, chicken CEPU-1 and rat OBCAM encode polypeptides which in each case are identical except for different (‘long’ or ‘short’) N-terminal signal peptides [4,17,25]. The reason why two distinct mRNAs are transcribed to produce IgLONs which, when fully processed, are structurally indistinguishable, is unclear. We have previously identified GP55, a GPI-linked glycoprotein from adult chicken brain, which inhibited the

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growth and adhesion of dorsal root ganglion (DRG) and forebrain neurons in vitro [5,29]. This activity was specific to GP55 and reversed by pertussis toxin, suggesting that GP55 interacts with a G protein-coupled transmembrane receptor [6]. Sequencing of GP55 cDNAs revealed that it consisted of at least three members of the IgLON family, namely LAMP, OBCAM (previously called GP55A) and CEPU-1 [29]. Other laboratories have begun to characterise the activities of the IgLONs individually. LAMP has been proposed to act as a homophilic cell recognition molecule [21,30]. It is expressed in the limbic cortex, the medial nucleus of the thalamus and may delineate organised neuronal circuits within the limbic system [20]. Support for this latter hypothesis is based on the ability of LAMP to enhance neurite extension from thalamic neurons which express LAMP, but to inhibit growth from thalamic neurons which do not express LAMP [18]. Nevertheless, LAMP is widely expressed beyond the limbic system. For example, it is present in both retina and cerebellum [11], but it is not known what role it performs in these areas. Chick CEPU-1 and its rat orthologue neurotrimin (NTM) have also been shown to interact homophilically [25,28]. Furthermore, NTM may act as a bifunctional agent on neurite outgrowth since it is reported to support neurite outgrowth from DRG neurons, and to a lesser extent hippocampal neurons, but fails to stimulate neurite outgrowth from sympathetic neurons [9]. Interestingly, DRG neurons continue to extend neurites on NTM even when neurons are treated with phosphatidylinositol-specific phospholipase C (PI-PLC) to remove GPI-linked glycoproteins, suggesting that NTM may act through a trans interaction with an as yet unidentified transmembrane receptor [9]. OBCAM was identified as a potential opioidbinding protein and there is evidence to suggest that it may be a component of an opiate receptor [3,10,14,15]. However, little is known about the effect of OBCAM on neurite outgrowth. The most recently identified member of the IgLON family is known as neurotractin (NTR) in chick or kilon in rat [8,19]. Interestingly, NTR-L stimulates neurite outgrowth from forebrain neurons while NTR-S does not. Considered together, the current data suggest that IgLONs may inhibit or enhance the growth of neurons and this may depend on the complement of IgLONs, and their receptors, expressed on the neuronal cell surface. Understanding the nature of IgLON-receptor interactions will be crucial for defining IgLON function. We have therefore begun to characterise the interactions of IgLONs with themselves and each other. To this end, we have cloned cDNAs encoding the complete ORFs of two OBCAM isoforms, thus enabling us to express recombinant OBCAM in addition to LAMP and CEPU-1. We have raised specific antisera to these three proteins and used them to demonstrate that different IgLONs may be coexpressed on some neurons but are differentially expressed on others. Analysis of mRNA expression has also identified two previously unknown isoforms of both LAMP

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and CEPU-1, and the expression pattern of all four isoforms of these two IgLONs is regulated during development. Finally, we show that LAMP, OBCAM and CEPU-1 not only interact homophilically with themselves but also heterophilically with each other. We therefore propose that IgLON-receptor interactions will be dependent on the identity and developmental status of the neurons on which they are expressed.

2. Materials and methods

2.1. PCR amplification of OBCAM cDNAs Template cDNA was synthesised from 5 mg of E18 chick brain total RNA (purified using Trizol Reagent (Life Technologies)) in a 20-ml reaction containing 20 mM Tris–HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl 2 , 10 mM DTT, 500 mM each of dATP, dCTP, dGTP, dTTP, 0.5 mg oligo(dT) and 200 U Superscript II (Life Technologies). The a1 OBCAM cDNA (see results for terminology) was amplified with the sense primer 59-GTT GTG GCT GTC GAG A / GAT GG-39 (based on the Kozak sequence of rat, bovine and human OBCAM sequences, Genbank M88710, M88711, L34774 and X12672), and the antisense primer 59-CAC TCC CTT ATC AAA AGT CGA GGA-39 (based on the E14S sequence [29]). The a2 OBCAM cDNA was amplified with the sense primer 59-ACC ACA GCC TCG AGA TGT ACC-39, identical to bases 624–644 of the rat OBCAM sequence (Genbank M88709) and the antisense primer described above. PCRs were performed in a reaction mix of 50 ml containing 20 mM Tris–HCl, pH 8.4, 50 mM KCl, 1 mM MgCl 2 , 200 mM each of dATP, dCTP, dGTP, dTTP, 0.2 mM each primer, 1 ml template cDNA and 2.5 U Taq DNA polymerase (Life Technologies) and were analysed on 1% MacroABgarose gels (Abgene). The PCR-amplified cDNAs were purified by agarose gel electrophoresis and subcloned into pCR2.1TOPO (Invitrogen). Two clones for both a1 and a2 were sequenced via dideoxy chain termination on an Applied Biosystems automated DNA sequencer and the sequences were identical.

2.2. Expression and purification of recombinant Fcchimeric IgLONs A CEPU-1 cDNA fragment encoding the full open reading frame (bases 37–1183, Genbank Z72497) was amplified from E18 chick brain cDNA using the primers 59-TGG CAG CAG GCA GTG AGT GGG-39 (sense) and 59-CTT TCT GCT GTG GTG GTC GTG GC-39 (antisense) and subcloned into pGEM-TEasy (Promega). LAMP [29], CEPU-1 and a2 OBCAM (this paper) cDNAs were then amplified by PCR with a sense primer designed to introduce a HindIII site at the 59 end, and an antisense primer which eliminated the sequence of the open-reading

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frame encoding the C-terminal hydrophobic domain specifying the addition of the GPI anchor and which added a BamHI site to the 39 end of each cDNA. The primers used were: 59-GGA AGC TTG CAG TGA GTG GGG GAA GG-39 (sense) and 59-GGA GGA TCC ACT TAC CTG TTC GCC GCC ACG CAC CGC TGT TGC-39 (antisense) for CEPU-1; 59-CAG CGA AGC TTA CCG AGA CCA GCC A-39 (sense) and 59-CCA AGC TCA GGA TCC ACT TAC CTG TTC TCC CCG TGC C-39 (antisense) for LAMP; and 59-GAA GCT TAC AGC CTC GAG ATG TAC C-39 (sense) and 59-GGA TCC ACT TAC CTG TGG AGG CTG CAT TGC CAC TGC-39 (antisense) for OBCAM. The amplified cDNAs were ligated into pGEM-TEasy (Promega), excised with HindIII and BamHI, and subcloned into pIG-1 (a kind gift from Professor F. Walsh) in fusion with a gene segment encoding the Fc region of human IgG [24]. The modified cDNAs were finally excised from recombinant pIG-1 and ligated into pcDNA3 (Invitrogen) at the HindIII and NotI sites to improve the efficiency of recombinant plasmid purification. For protein expression, the recombinant plasmids were used to transfect, via calcium phosphate precipitation, 15-cm dishes (50 mg plasmid per dish) seeded with approximately 160310 4 COS-7 cells in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U / ml penicillin and 100 mg / ml streptomycin sulphate (all from Life Technologies). Cells were exposed to the plasmid for 16 h before replacing the medium with DMEM containing 1% FBS, 2 mM L-glutamine, 100 U / ml penicillin and 100 mg / ml streptomycin sulphate. After 5–6 days, recombinant Fc-chimeric proteins were purified from the medium on protein A-agarose (Repligen) according to standard techniques [12].

IGEPAL, 100 mU / ml PI-PLC (Sigma), incubated at 378C for 2 h and centrifuged at 50 0003g for 1 h at 48C to recover proteins released by PI-PLC into the supernatant. Twenty ml of each supernatant was then run on replicate SDS–polyacrylamide gels under both reducing and nonreducing conditions, transferred to nitrocellulose (Pall Gelman Sciences) and probed with each antiserum. Bound antibody was detected with HRP-conjugated goat anti-rat immunoglobulins (Sigma) and Super Signal West Pico Chemiluminescent Substrate (Pierce). Specificity of the antisera was also tested by indirect immunofluorescence using COS-7 cells transiently transfected with LAMP, OBCAM or CEPU-1 cDNAs in pcDNA3 such that they were expressed as GPI-anchored proteins on the cell surface. Bound antibody was detected with goat anti-rat biotin-conjugated immunoglobulins (Pierce) followed by streptavidin–Texas Red (AP Biotech). For some experiments, anti-Fc antibodies were removed from the antisera on human IgG (Sigma) coupled to Sepharose CL-4B (AP Biotech).

2.3. Preparation and characterisation of polyclonal antisera

2.5. Frozen sections

Recombinant LAMP-Fc, OBCAM-Fc and CEPU-1-Fc were used to raise individual polyclonal antisera in Wistar strain rats as previously described [29], using approximately 50 mg of chimeric protein for immunisations and boosts. The specificity of each antiserum was determined by Western blotting against recombinant IgLONs isolated by PI-PLC treatment of stably transfected CHO cells expressing LAMP, OBCAM or CEPU-1 individually. Wild-type CHO cells were used as a control. PI-PLC treatment was performed as follows. The cells were grown to confluency in a 15-cm dish before harvesting by scraping into 1 ml of PBS and centrifuging at 10003g for 5 min. The cell pellet was then extracted with 500 ml of 50 mM MOPS, pH 7.4, 5 mM EGTA, 0.5 mM DTT, 5 mM ZnSO 4 , 0.16% IGEPAL containing COMPLETEE protease inhibitors (Roche Molecular Biochemicals) and centrifuged at 50 0003g for 30 min at 48C. The pellet was resuspended in 200 ml of 50 mM MOPS, pH 7.4, 2 mM EDTA, 0.16%

2.4. Primary neuronal cultures Embryonic day 10–15 DRG and sympathetic neurons were grown as described previously [1], except that 13-mm coverslips coated for 2 h with 20 mg / ml laminin (Sigma) were used. Immunofluorescent staining was performed on cultured cells by incubating them sequentially with antiIgLON rat antiserum (1:50 or 1:100) in PBS containing 5% bovine serum albumin (BSA) for 30 min at room temperature, followed by goat anti-rat biotin (1:100) and streptavidin–Texas Red (1:50). Cells were then fixed in 4% formaldehyde and mounted in Airvol as described previously [1].

Retinae were dissected, fixed and sectioned as described previously [29]. Tissue sections were blocked with 0.1 M glycine, pH 7.4, containing 1% BSA for 30 min, then stained sequentially with anti-IgLON antisera and goat anti-rat Texas Red, both at 1:50 in 0.12 M sodium phosphate, pH 7.4, containing 1% BSA.

2.6. RT-PCR analysis of IgLON isoform expression To prepare template cDNA, total RNA was first prepared using Trizol Reagent (Life Technologies) from retina (E5, E7, E10, E12, E14, E17, E20), DRGs (E7, E14), sympathetic ganglia (E10) and sciatic nerve (E10). Firststrand cDNA synthesis was performed as described earlier. PCRs were then performed to amplify cDNA fragments of LAMP, OBCAM, CEPU-1 and GAPDH. The primers used were: LAMP, 59-CAT CCA TCC GTC CGT CCA CAT A-39 (sense) and 59-AAC ACT TGC TGA GTA GGC AGA GCA-39 (antisense); OBCAM, 59-GTC GGA GAA

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GGA CTA TGG CAA CT-39 (sense) and 59-CAC TCC CTT ATC AAA AGT CGA GGA-39 (antisense); CEPU-1, 59-GAC GAC AAG CGG CTG GCT GAA-39 (sense) and 59-CTT TCT GCT GTG GTG GTC GTG GC-39 (antisense); GAPDH, 59-TCC AAG TGG TGG CCA TCA ATG ATC-39 (sense) and 59-TTC TGG GCA GCA CCT GTG TCA TC-39 (antisense). All PCRs were performed in a reaction mix of 50 ml containing 20 mM Tris–HCl, pH 8.4, 50 mM KCl, 1 mM MgCl 2 , 200 mM each of dATP, dCTP, dGTP, dTTP, 0.2 mM each primer, 1 ml template cDNA and 2.5 U Taq DNA polymerase (Life Technologies). PCRs were analysed on 4% MicroABgarose (Abgene) gels. For Southern blot analysis, gels were transferred to a nylon membrane (Schleicher and Schuell), baked at 1208C for 30 min to fix the DNA fragments, blocked in digoxygenin (DIG) EasyHyb solution (Roche Molecular Biochemicals) and probed with DIG-labelled cDNA fragments synthesised by random priming using DIG High Prime (Roche Molecular Biochemicals). Following hybridisation for 6 h at 428C, membranes were washed at 688C with 0.13 SSC, 0.1% SDS for three times of 20 min each. Bound probe was then detected with alkaline phosphatase-conjugated anti-DIG Fab fragments and CSPD chemiluminescent substrate (both from Roche Molecular Biochemicals).

2.7. Binding assays using recombinant IgLONs COS-7 cells were plated on 13-mm coverslips in a 24-well plate at a concentration of 1310 4 cells per well and transfected via calcium phosphate precipitation with LAMP, OBCAM or CEPU-1 cDNAs in pcDNA3. Fortyeight hours post-transfection, cells were incubated for 30 min in 50 ml of 0.12 M sodium phosphate, pH 7.4, 1% BSA containing 25 mg / ml of one of LAMP-Fc, OBCAMFc or CEPU-1-Fc. Human IgG or OX40L-Fc [2] were used as negative controls. Cells were then incubated with an Fc domain-specific biotinylated rabbit anti-human antibody (Sigma) at 1:350, followed by streptavidin–Texas Red (AP Biotech) at 1:100. After fixation in 4% paraformaldehyde, coverslips were mounted in Airvol as previously described [1].

2.8. Miscellaneous techniques SDS–PAGE and Western blotting were performed as previously described [29]. Proteins were separated under reducing condition (with 2-mercaptoethanol) or under nonreducing conditions (in the absence of 2-mercaptoethanol). Protein was assayed by the modified Bradford assay of Stoscheck [27], using human IgG as the standard. General nucleic acid manipulations were performed according to standard techniques [22]. GPI-linked proteins were prepared from retina (E7, E10, E12, E14, E20) by digestion with PI-PLC, as described for transfected CHO cells.

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3. Results

3.1. Cloning of chicken OBCAM cDNAs We previously cloned a cDNA (termed E14S) encoding a polypeptide (termed GP55A) which had closest homology to OBCAM but was incomplete at the 59 end of the ORF. It proved difficult to obtain a complete GP55A cDNA from libraries and so we adopted a PCR-based strategy to complete this sequence. We used two different sense primers, based on published mammalian cDNAs encoding OBCAM isoforms with both long and short N-terminal signal peptides, and an antisense primer, complementary to part of the E14S sequence, to amplify two different cDNAs with complete ORFs (see Section 2). Both cDNAs encoded the same mature protein but differed in the sequence of their N-terminal signal peptide (Fig. 1). At the amino acid level, the sequence of the mature GP55A protein (i.e., minus the N-terminal signal peptide) was 89% identical to rat, bovine and human OBCAM, consistent with it being the chicken orthologue of these proteins (data not shown). We have therefore adopted the name OBCAM, rather than GP55A, for the chicken protein(s). Furthemore, we have given the prefix ‘a1’ to the cDNA encoding chicken OBCAM with the long Nterminal signal peptide, and the prefix ‘a2’ to the cDNA encoding chicken OBCAM with the short N-terminal signal peptide. The sequences of the two cDNAs are identical except for the region which encodes the signal peptides, suggesting that both mRNAs arise from an alternatively spliced transcript of one gene. However, we have not obtained any evidence to suggest that the OBCAM gene possesses an exon which would encode a C-terminal insert, as is seen for LAMP and CEPU-1 (see later).

3.2. Production of antisera specific to LAMP, OBCAM and CEPU-1 Recombinant LAMP, OBCAM and CEPU-1 Fc-fusion proteins were used to raise individual antisera (see Section 2). To determine the specificity of the antisera, each one was tested against recombinant LAMP, OBCAM and CEPU-1 isolated from transfected CHO cell lines by digestion with PI-PLC. Proteins separated under reducing (data not shown) and non-reduceing conditions were specifically detected on Western blots (Fig. 2a). Each antiserum also specifically detected its native antigen on transiently transfected COS-7 cells (Fig. 2b) but did not stain either non-transfected cells or transfected cells expressing a different IgLON antigen. Two antisera were raised against each molecule and one OBCAM antiserum, one CEPU-1 antiserum and both LAMP antisera were specific for their respective antigens. The antisera have not been tested against the recently identified neurotractin. However, neurotractin has lower homology with LAMP,

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Fig. 1. Comparison of chicken OBCAM with rat IgLON sequences. Two complete cDNAs were amplified by PCR using sense primers based on mammalian OBCAM sequences and an antisense primer based on the partial GP55A cDNA, E14S [29]. The alignment shows that the GP55A isoforms are indeed the chicken orthologues of rat OBCAM, and they have been re-named thus. Note that the two mature proteins encoded by the a1 (long N-terminal signal peptide) and a2 (short signal peptide) OBCAM cDNAs are identical following cleavage of the signal sequence. The cleaved N-terminal sequences are shown in italics, as predicted by amino acid microsequencing of N-terminal peptides derived from rat LAMP [21]. The C-terminal sequences in italics are predicted to be removed prior to GPI-anchor addition. The amino acid homologies shared by the mature chicken OBCAM and rat IgLON sequences (i.e., not including N- and C-terminal signal sequences are: chicken OBCAM versus rat OBCAM, 89%; chicken OBCAM versus rat LAMP, 55%; chicken OBCAM versus rat neurotrimin, 73%; chicken OBCAM versus rat kilon, 47%. The alignment was created using the GCG program PILEUP and edited using Boxshade (http: / / www.ch.embnet.org / software / BOX form.html). The Genbank accession numbers for the chicken a1 and a2 OBCAM sequences ] are, respectively, Y08170 and AF292934. The accession numbers for the rat sequences used in this alignment are: OBCAM (long signal peptide isoform), M88710 and M88711; OBCAM (short signal peptide isoform), M88709; LAMP, U31554; neurotrimin, U16845; kilon, AB017139.

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Fig. 2. Antisera raised against recombinant IgLON proteins are specific for their respective antigens. (A) To test the specificity of IgLON antisera on Western blots, GPI-linked glycoproteins from wild-type CHO cells and CHO cells transfected with LAMP, OBCAM and CEPU-1 were prepared as described in Section 2. Samples were separated on non-reducing SDS–polyacrylamide gels, transferred to nitrocellulose and probed with LAMP, OBCAM or CEPU-1 antisera at 1:1000 followed by goat anti-rat HRP at 1:2000. Each antiserum was highly specific for its antigen. Only slight background staining was observed in the absence of the correct antigen and this was also seen in non-transfected cells. (B) To test the ability of the IgLON antisera to recognise native antigens, COS-7 cells were transiently transfected with LAMP (a), OBCAM (b), CEPU-1 (c) cDNA or mock transfected (d) and stained with the matching antiserum at 1:100 followed by goat anti-rat biotin at 1:100 and streptavidin–Texas Red at 1:50. Negligable staining was observed with any antiserum on mock transfected cells, CEPU-1 is shown as an example (d). Approximately 30% of the cells stained corresponding to the expected transfection efficiency. Where a non-matching antiserum was used, no staining was observed with either rat LAMP antisera although one OBCAM antiserum detected CEPU-1 transfected cells at 1:50, and one CEPU-1 antiserum detected OBCAM transfected cells at 1:50 (not shown).

OBCAM and CEPU-1 than CEPU-1 has with OBCAM (data not shown). Of all the IgLONs, OBCAM and CEPU1 share highest homology and so the fact that our OBCAM antisera did not cross-react with CEPU-1, and vice versa, suggests that our antisera are unlikely to cross-react with neurotractin. The availability of specific antisera allowed us to confirm that GP55 from adult chick brain [5] contained LAMP, OBCAM and CEPU-1 (data not shown). It is likely it also contains neurotractin.

3.3. Overlapping and distinct expression patterns of IgLONs on cultured neurons and in the retina We have previously shown that neurite outgrowth from DRG neurons is inhibited by GP55 [5,6] and so we were interested to determine which IgLONs these neurons express. LAMP and CEPU-1 were co-expressed on DRG neurons from E10 to E15 (Fig. 3a–d), in each case most if not all neurons were stained. IgLONs are also expressed on cultured sympathetic neurons. Again, LAMP and CEPU-1 were clearly stained on most if not all neurons. (Fig. 3e,f). OBCAM expression on both DRG and sympathetic neurons was hard to detect convincingly although whether this

is due to variation between the anti-IgLON antisera or variation in IgLON expression is unclear. However the three antisera work at the same maximum tire on blots and on transfected COS and CHO cells. We had previously observed expression of IgLONs (GP55) in the developing retina [29]. Western blots using the three specific antisera revealed trace expression only early in development (E6 and E7) but this showed a marked increase by E20 for all three IgLONs (Fig. 4a). Immunolocalisation on frozen sections revealed that LAMP and CEPU-1 were first detectable at E15 and staining was restricted to the outer plexiform layer (OPL). Little staining was observed for OBCAM at this age. At E18, CEPU-1 is most abundant in the OPL but there is also expression on amacrine and bipolar neuronal soma (Fig. 4b). Little or no expression of CEPU-1 was observed on the inner plexiform layer (IPL) or retinal ganglion cells (RGC). LAMP, however, was strongly expressed on the IPL and optic fibre layer (OFL) as well as the OPL. OBCAM also showed a distinctive distribution at E18. It was absent from the OPL and stains layers within the IPL. Thus, CEPU-1 and LAMP are co-expressed in the OPL, LAMP and OBCAM are co-expressed in the IPL and all

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three are co-expressed on amacrine cells and to a lesser degree on bipolar cell soma.

3.4. Developmental regulation of IgLON isoform expression

Fig. 3. LAMP and CEPU-1 are co-expressed on DRG and sympathetic neurons. Neurons were grown for 24 h in culture and stained with LAMP or CEPU-1 antisera at 1:50 followed by goat anti-rat biotin at 1:100, then streptavidin–Texas Red at 1:50. (A) E10 DRG neurons and (B) E15 DRG neurons, both stained with LAMP antiserum; (C) E10 DRG neurons and (D) E15 DRG neurons, both stained with CEPU-1 antiserum; (E) E10 sympathetic neurons stained with LAMP serum; (F) E10 sympathetic neurons stained with CEPU-1 antiserum; (G) E10 DRG neurons and (H) sympathetic neurons both stained with pre-immune serum. Scale bar, 20 mm.

The immunofluorescent staining and Western blotting experiments described above did not provide us with any information regarding which isoforms of LAMP, OBCAM and CEPU-1 were being expressed in retina or cultured neurons. In the chick, both LAMP and CEPU-1 possess an optional C-terminal insert (12 amino acids in LAMP and 11 amino acids in CEPU-1). We were particularly interested in seeing if there was any difference in the timing and ratio of expression of the mRNAs encoding LAMP and CEPU-1 with and without the exon for the C-terminal insert. (For simplicity we will refer to the proteins without the C terminal insert as aLAMP and aCEPU-1 whilst bLAMP and bCEPU-1 refer to the protein with the C terminal insert). We therefore designed primer pairs to amplify LAMP and CEPU-1 cDNA fragments which included the site of splicing for the exon encoding the C-terminal insert. We also designed primers which would indicate whether the OBCAM gene contains an exon for a C-terminal insert, and GAPDH was amplified as a control. We have analysed LAMP and CEPU-1 mRNA expression in retina, dorsal root and sympathetic ganglia (Fig. 5a,b). As well as observing bands corresponding to fragments with and without the characterised alternative exon (a and b isoforms), we observed two additional bands for LAMP and CEPU-1 of higher molecular weight than the

Fig. 4. LAMP, CEPU-1 and OBCAM are co-expressed in the developing chick retina. (A) Retinae were dissected from chick embryos of various ages and an enriched fraction of GPI-linked glycoproteins was prepared from each as described for transfected CHO cells (Section 2) and separated on non-reducing SDS–polyacrylamide gels. In all cases, trace expression was observed at E7 and the level of each increased considerably by E20. (B) E18 retina were stained with LAMP, OBCAM or CEPU-1 antisera followed by goat anti-rat Texas Red. CEPU-1 was detected principally in the outer plexiform layer (OPL) while LAMP was expressed in the OPL, inner plexiform layer (IPL) and optic fibre layer (OFL). OBCAM was absent from the OPL but was expressed in the IPL and OFL. Scale bar, 50 mm.

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3.5. Homophilic and heterophilic interactions between LAMP, OBCAM and CEPU-1

Fig. 5. Developmental expression of IgLON isoforms. (A) RT-PCR was carried out on cDNA prepared from retina between E7 and E20. GAPDH primers showed that approximately equal quantities of cDNA template were added to each PCR reaction. aCEPU-1 (lowest band), all LAMP isoforms and OBCAM were expressed across the age range. Expression of bCEPU-1 (second lowest band) and two higher molecular weight isoforms (top two bands) was observed later in development. Only one isoform (a) was observed for OBCAM. (B) RT-PCR was carried out on cDNA prepared from E7 and E14 DRG and E10 sympathetic ganglia (SYMP). GAPDH primers showed consistent levels of cDNA template in all samples. LAMP, OBCAM and CEPU-1 were expressed in DRG and sympathetic neurons. Additional isoforms of CEPU-1 only were seen in these cultured neurons. All primers were tested against cDNA for LAMP, OBCAM and CEPU-1 and were specific for their respective sequences (not shown).

two expected fragments. The upper two bands frequently ran as a single band and have so far proved intractable to sequencing. Nevertheless, Southern blotting and sequencing of the cDNA surrounding the exon confirm the bands are isoforms of LAMP and CEPU-1 (data not shown). It was interesting to observe the expression of all four LAMP and CEPU-1 mRNAs during development. In the retina, all four forms of LAMP were present throughout development (Fig. 5a). aCEPU-1 was expressed between E7 and E20. In contrast, bCEPU-1 and the higher molecular weight isoforms appeared more abundant later in development although these experiments were not quantitative. Expression of only a single isoform of OBCAM was detected and its level remained constant during this developmental period (Fig. 5a). DRGs express all four forms of CEPU-1 (Fig. 5b), whereas only the major form of LAMP is detectable and a single band is observed again for OBCAM. In sympathetic neurons all three IgLONs are again expressed but only the major isoform is observed. It is interesting to note that OBCAM mRNA is easily detectable in both DRG and sympathetic ganglia despite the difficulties in observing OBCAM expression by immunofluorescence.

It has previously been shown that LAMP and CEPU-1 bind homophilically although neurotractin does not. We therefore wished to test whether the remaining member of the IgLON family, OBCAM, will act as a homophilic CAM. As shown in Fig. 6, LAMP-Fc, OBCAM-Fc and CEPU-1-Fc will bind to transiently transfected COS-7 cells which express, respectively, LAMP, OBCAM or CEPU-1 on their cell surface. The chimeric Fc-fusion proteins did not bind to non-transfected COS-7 cells and a control molecule, OX40L-Fc, did not interact with any of the transfected cells [2]. Therefore, LAMP, OBCAM and CEPU-1 are all capable of interacting homophilically with themselves. In all cases, 25 mg / ml of chimeric protein was used, irrespective of whether transiently transfected COS cells (Fig. 6) or stably transfected CHO cell lines were stained (data not shown).

Fig. 6. LAMP, OBCAM and CEPU-1 bind themselves homophilically. COS-7 cells were transfected with OBCAM (A,D), LAMP (B) or CEPU1 (C) and then stained with OBCAM-Fc (A) LAMP-Fc (B) CEPU-1-Fc (C) or OX40L-Fc (D) at 25 mg / ml. Bound chimeric proteins were detected with goat anti-human biotin and streptavidin–Texas Red. Transfected cells could frequently be distinguished from non-transfected cells by morphology; they either extended one or more neurite-like processes (see A) or large veils of cytoplasm similar to lamellipodia (see bottom cell in B). Transfected cells were clearly stained with the appropriate chimeric protein while non-transfected cells showed no staining. An unrelated protein, OX40L-Fc, also showed no staining either on transfected or non-transfected cells. Scale bar, 25 mm.

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Fig. 7. LAMP, OBCAM and CEPU-1 bind heterophilically to each other.. COS-7 cells transfected with LAMP cDNA were stained with CEPU-1-Fc (A) or OBCAM-Fc (D); cells transfected with OBCAM cDNA were stained with CEPU-1-Fc (B) or LAMP-FC (E) and cells transfected with CEPU-1 cDNA were stained with OBCAM-Fc (C) or LAMP-Fc (F), showing that LAMP, OBCAM and CEPU-1 can all interact with each other as well as themselves. A non-transfected cell is depicted by the arrowhead in (B). Scale bar 25 mm.

Since two or more IgLONs may be co-expressed on the same neurons at the same time (see above), it was interesting to determine whether LAMP, OBCAM and CEPU-1 were capable of interacting with each other. Surprisingly, LAMP-Fc bound to COS-7 cells transfected with OBCAM or CEPU-1 as well as to COS-7 cells transfected with LAMP. The same was true for OBCAMFc binding to COS-7 cells transfected with LAMP or CEPU-1, and CEPU-1-Fc binding to COS-7 cells transfected with LAMP or OBCAM (Fig. 7). In each case, 25 mg / ml of chimeric protein were used. Proteins such as OX40L-Fc or human IgG, which are unrelated to IgLONs but have the same Fc domain as the chimeric ligands we have used in these experiments, gave no indication of binding. Thus, it appears that each IgLON may have a choice of binding partners.

4. Discussion The IgLON family now consists of the four proteins LAMP, OBCAM, CEPU-1 / NTM and NTR / kilon which are expressed as a variety of isoforms due to alternative mRNA splicing. In this paper, we have used specific antisera and RT-PCR to examine the expression of LAMP, OBCAM and CEPU-1 on chick DRG and sympathetic neurons and in the developing retina. These results demonstrate, for the first time, localisation of two or more IgLON

proteins on the same neurons. This prompted us to study the ability of these three IgLON family proteins to interact with themselves and each other. Both DRG and sympathetic neurons co-express LAMP and CEPU-1 and RTPCR results also demonstrate that OBCAM is expressed on DRG and sympathetic neurons. This confirms previous reports that IgLONs are expressed on DRG neurons [28] and, in contrast to previous reports, provides clear evidence that they are also expressed on sympathetic neurons. LAMP, OBCAM and CEPU-1 are co-expressed in the retina at E18 although the expression pattern is, in each case, distinct. LAMP and CEPU-1 are both expressed in the OPL, suggesting co-expression on horizontal cells and a possible role in the formation of synaptic interactions between these cells and rods, cones and bipolar cells. LAMP and OBCAM are co-expressed in the IPL and both have a striated appearance. Western blotting revealed that trace amounts of all three proteins were present from E7 in the retina but became more abundant later in development. No specific immunofluorescence was detected on frozen sections of retina before E15 with any of the three antibodies, in contrast to a previous report in which LAMP was shown to be expressed on RGC at E7 [4]. However, our expression pattern for LAMP was consistent with that seen for chicken AvGp50 / LAMP [11]. It was surprising to discover that RT-PCR experiments suggested that LAMP, OBCAM and CEPU-1 expression was not developmentally regulated between E7 and E20. Previous experiments have indicated that GPI-linked glycoproteins have a long half life compared to most transmembrane and cytoplasmic proteins, suggesting they have low rates of degradation [16]. Our results may therefore demonstrate that, while IgLON mRNAs may be synthesised from early in development, the proteins only begin to accumulate much later. The proteins also remain abundant in the adult [11,29]. Since alternatively spliced mRNAs for chicken LAMP and CEPU-1 have previously been characterised, we designed RT-PCR experiments to enable us to compare their expression. These experiments also revealed the expression of two other isoforms of both LAMP and CEPU-1 of higher molecular weight than the previously described cDNAs. Based on the design of our RT-PCR experiments, we predict that the higher molecular weight isoforms of LAMP and CEPU-1 identified in this paper will have an additional or different alternatively spliced exon to the b isoforms. For LAMP, the b and higher molecular weight isoform mRNAs were expressed at the highest levels early in retinal development while the reverse was true for CEPU-1. Indeed, bCEPU-1 and the high molecular weight isoforms may be adult forms of CEPU-1 since they appear late in development in DRG neurons as well as retina. We have cloned two isoforms of chicken OBCAM termed a1 (long signal peptide) and a2 (short signal peptide). Since the a1 and a2 mRNAs will have different 59 non-coding sequences, their transcription and translation could be differentially regulated. For

A.P. Lodge et al. / Molecular Brain Research 82 (2000) 84 – 94

chicken LAMP, the a1 isoform (or g19 isoform) is expressed early in development and the a2 isoform (or g9 isoform) is not expressed until later [4]. This may reflect a mechanism which regulates the transcription of the b isoform since, so far, the expression of this isoform has only been observed in conjunction with the a1 (long) signal peptide. a1 and a2 isoforms of CEPU-1 have also been identified and this may similarly provide an explanation for the regulation of bCEPU-1 expression [13]. The role of the different IgLON protein isoforms is at present unclear. Known interactions of IgLON family proteins include homophilic and heterophilic interactions which will occur between IgLONs on opposing cell membranes, i.e., trans interactions. LAMP [30], OBCAM (Fig. 6) and CEPU-1 / NTM [9] all undergo homophilic interactions but neurotractin does not [19]. In addition, LAMP, OBCAM and CEPU-1 all interact heterophilically with each other (Fig. 7), and LAMP and CEPU-1 interact with NTR [19]. The ability of OBCAM to interact with NTR has not yet been characterised. In addition to these trans interactions, neurotrimin has been reported to form homo dimers and homo trimers in the plane of the membrane, i.e., cis interactions [9]. The IgLONs may also interact in cis with a transmembrane G protein-coupled receptor since pertussis toxin will inhibit the activity of IgLONs (GP55) on DRG neurons [6]. Any or all of these interactions could be modulated by the expression of the different alternatively spliced isoforms which, for LAMP and CEPU-1 at least, changes dynamically during development. Certainly, the absence of the third C2 domain from the short form of neurotractin (NTR-S) alters its activity compared to NTR-L in which the third C2 domain is present [19], although the relative expression of NTR-L and NTR-S mRNAs has not yet been studied. IgLON interactions may be further modified by the expression of another alternatively spliced isoform of CEPU-1 which is secreted rather than GPI-anchored to the neuronal plasma membrane (Lodge et al. in preparation). We have shown here that IgLONs are capable of heterophilic interactions and are frequently co-expressed on neurons, suggesting that caution will be required in interpreting the activity of any one IgLON considered in isolation. However, it is worth considering that the binding experiments we have carried out have used IgLONs expressed on the surface of COS-7 cells which express only one IgLON and may not express a cis-interacting transmembrane IgLON receptor. Thus, the interactions between IgLONs on the surface of neurons may be more stringently regulated. Furthermore, it is worthy of note that NTM-Fc bound to the surface of neurons from which GPI-linked glycoproteins had been removed, suggesting IgLONs can also form heterophilic trans interactions with transmembrane glycoproteins [9]. To understand how IgLON family proteins function it is now important to characterise both the cis- and trans-interacting proteins with which their various isoforms interact in vivo.

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References [1] T.E. Allsopp, D.J. Moss, A developmentally regulated chicken neuronal protein associated with the cortical cytoskeleton, J. Neurosci. 9 (1989) 13–24. [2] A. al-Shamkhani, M.L. Birkeland, M. Puklavec, M.H. Brown, W. James, A.N. Barclay, OX40 is differentially expressed on activated rat and mouse T cells and is the sole receptor for the OX40 ligand, Eur. J. Immunol. 26 (1996) 1695–1699. [3] D.K. Ann, J. Hasegawa, J.L. Ko, S.T. Chen, N.M. Lee, H.H. Loh, Specific reduction of delta-opioid receptor-binding in transfected Ng108-15 cells, J. Biol. Chem. 267 (1992) 7921–7926. [4] T. Brummendorf, F. Spaltmann, U. Treubert, Cloning and characterization of a neural cell recognition molecule on axons of the retinotectal system and spinal cord, Eur. J. Neurosci. 9 (1997) 1105–1116. [5] G.A. Clarke, D.J. Moss, Identification of a novel protein from adult chicken brain that inhibits neurite outgrowth, J. Cell Sci. 107 (1994) 3393–3402. [6] G.A. Clarke, D.J. Moss, GP55 inhibits both cell adhesion and growth of neurons, but not non-neuronal cells via a G-proteincoupled receptor, Eur. J. Neurosci. 9 (1997) 334–341. [7] G. Cook, D. Tannahill, R. Keynes, Axon guidance to and from choice points, Curr. Opin. Neurobiol. 8 (1998) 64–72. [8] N. Funatsu, S. Miyata, H. Kumanogoh, M. Shigeta, K. Hamada, Y. Endo, Y. Sokawa, S. Maekawa, Characterization of a novel rat brain glycosylphosphatidylinositol-anchored protein (Kilon), a member of the IgLON cell adhesion molecule family, J. Biol. Chem. 274 (1999) 8224–8230. [9] O.D. Gil, G. Zanazzi, A.F. Struyk, J.L. Salzer, Neurotrimin mediates bifunctional effects on neurite outgrowth via hemophilic and heterophilic interactions, J. Neurosci. 18 (1998) 9312–9325. [10] P. Govitrapong, X.H. Zhang, H.H. Loh, N.M. Lee, Transfection of Ng108-15 cells with antisense opioid-binding cell-adhesion molecule cDNA alters opioid receptor-G-protein interaction, J. Biol. Chem. 268 (1993) 18280–18285. [11] K.A. Hancox, A.M. Sheppard, P.L. Jeffrey, Characterisation of a novel glycoprotein (AvGp50) in the avian nervous system, with a monoclonal antibody, Dev. Brain Res. 70 (1992) 25–37. [12] E. Harlow, D. Lane, in: Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988. [13] Y. Kimura, K. Shirabe, M. Fukushima, M. Takeshita, H. Tanaka, CEPU-1, an immunoglobulin superfamily molecule, has cell adhesion activity and shows dynamic expression patterns in chick embryonic spinal cord, Neurosci. Res. 34 (1999) 245–255. [14] C.M. Lane, R. Elde, H.H. Loh, N.M. Lee, Regulation of an opioidbinding protein in Ng108-15 cells parallels regulation of deltaopioid receptors, Proc. Natl. Acad. Sci. USA 89 (1992) 11234– 11238. [15] C.M. Lane, C.R.N. Jones, T. Reisine, L. Yu, N.M. Lee, Alteration of obcam conformation as a result of opioid receptor expression and opioid ligand treatment, Brain Res. 698 (1995) 15–22. [16] P. Lemansky, S.H. Fatemi, B. Gorican, S. Meyale, R. Rossero, A.M. Tartakoff, Dynamics and longevity of the glycolipid-anchored membrane-protein, Thy-1, J. Cell Biol. 110 (1990) 1525–1531. [17] D.A. Lippman, N.M. Lee, H.H. Loh, Opioid-binding cell-adhesion molecule (obcam)-related clones from a rat-brain cDNA library, Gene 117 (1992) 249–254. [18] F. Mann, V. Zhukareva, A. Pimenta, P. Levitt, J. Bolz, Membraneassociated molecules guide limbic and nonlimbic thalamocortical projections, J. Neurosci. 18 (1998) 9409–9419. [19] A. Marg, P. Sirim, F. Spaltmann, A. Plagge, G. Kauselmann, F. Buck, F.G. Rathjen, T. Brummendorf, Neurotractin, a novel neurite outgrowth-promoting Ig-like protein that interacts with CEPU-1 and LAMP, J. Cell Biol. 145 (1999) 865–876. [20] A.F. Pimenta, B.S. Reinoso, P. Levitt, Expression of the mRNAs

94

[21]

[22]

[23]

[24]

[25]

A.P. Lodge et al. / Molecular Brain Research 82 (2000) 84 – 94 encoding the limbic system-associated membrane protein (LAMP). 2. Fetal rat brain, J. Comparative Neurol. 375 (1996) 289–302. A.F. Pimenta, V. Zhukareva, M.F. Barbe, B.S. Reinoso, C. Grimley, W. Henzel, I. Fischer, P. Levitt, The limbic system-associated membrane-protein is an Ig superfamily member that mediates selective neuronal growth and axon targeting, Neuron 15 (1995) 287–297. J. Sambrook, E.F. Fritsch, T. Maniatis, in: Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989. P.R. Schofield, K.C. McFarland, J.S. Hayflick, J.N. Wilcox, T.M. Cho, S. Roy, N.M. Lee, H.H. Loh, P.H. Seeburg, Molecular characterization of a new immunoglobulin superfamily protein with potential roles in opioid binding and cell contact, EMBO J. 8 (1989) 489–495. D.L. Simmons, Cloning cell surface molecules by transient expression in mammalian cells, in: D. Hartley (Ed.), Cellular Interactions in Development: A Practical Approach, Oxford University Press, Oxford, 1993, pp. 95–126. F. Spaltmann, T. Brummendorf, CEPU-1, a novel immunoglobulin

[26] [27]

[28]

[29]

[30]

superfamily molecule, is expressed by developing cerebellar Purkinje cells, J. Neurosci. 16 (1996) 1770–1779. E.T. Stoeckli, L.T. Landmesser, Axon guidance at choice points, Curr. Opin. Neurobiol. 8 (1998) 73–79. C.M. Stoscheck, Increased uniformity in the response of the coomassie blue G protein assay to different proteins, Anal. Biochem. 184 (1990) 111–116. A.F. Struyk, P.D. Canoll, M.J. Wolfgang, C.L. Rosen, P. Deustachio, J.L. Salzer, Cloning of neurotrimin defines a new subfamily of differentially expressed neural cell-adhesion molecules, J. Neurosci. 15 (1995) 2141–2156. D.J.A. Wilson, D.S. Kim, G.A. Clarke, S. MarshallClarke, D.J. Moss, A family of glycoproteins (GP55), which inhibit neurite outgrowth, are members of the Ig superfamily and are related to OBCAM, neurotrimin, LAMP and CEPU-1, J. Cell Sci. 109 (1996) 3129–3138. V. Zhukareva, P. Levitt, The limbic system-associated membraneprotein (Lamp) selectively mediates interactions with specific central neuron populations, Development 121 (1995) 1161–1172.