Molecular cloning and functional expression of gicerin, a novel cell adhesion molecule that binds to neurite outgrowth factor

Neuron,

Vol. 12, 861-872,

April,

1994, Copyright

0 1994 by Cell Press

Molecular Cloning and Functional Expression of Gicerin, a Novel Cell Adhesion Molecule That Binds to Neurite Outgrowth Factor Eiichi Taira, Natsuki Takaha, Hideo Taniura, Cheol-Hee Kim, and Naomasa Miki Department of Pharmacology Osaka University School of Medicine 2-2 Yamadaoka Suita Osaka 565 Japan

Summary Gicerin is an integral membrane glycoprotein of about 82 kd that is transiently expressed in the developing CNS. Gicerin was first identified as a binding protein for neurite outgrowth factor (NOF), a member of the laminin family of extracellular matrix proteins. By isolating and sequencing a gicerin cDNA, we have found that this protein is a novel member of the immunoglobulin superfamily. The deduced protein (584 amino acids) consists of five immunoglobulin-like loop structures in an extracellular domain, a single transmembrane region, and a short cytoplasmic tail. Cells transfected stably with gicerin cDNA adhered to NOF and aggregated with each other, indicating that gicerin exhibits both heterophilic and homophilic adhesion activities. Introduction Growth of extensive neural processes is a distinctive feature of neurons critical for normal development of neuronal networks. Cell adhesion molecules, extracellular matrix (ECM) molecules, and their receptors govern this fundamental aspect of neural development (Edelman, 1988; Dodd and Jessell, 1988; Salzer and Colman, 1989; Reichardt and Tomaselli, 1991; Schachner, 1991). Cell adhesion molecules, such as N-CAM (Rutishauser et al., 1982) and cadherins (Takeichi, 19881, exhibit neurite-promoting activity by their homophilic binding properties, whereas ECM molecules, such as laminin and fibronectin, promote neurite extension by interacting with integrins (Evercooren et al., 1982; Bozyczko and Horwitz, 1986; Tomaselli et al., 1986). These adhesion molecules and their receptorsareexpressed temporallyand spatially to form neuronal networks. In the previous studies, we purified a neurite outgrowth factor (NOF) from chicken gizzard smooth muscle on the basis of its ability to promote neurite extension from ciliary ganglion neurons (Miki et al., 1981; Hayashi and Miki, 1985). NOF is a large (about 700 kd) ECM glycoprotein that also induces neurite extension from retinal ganglion, cerebellar, and dorsal root ganglion neurons and promotes granule cell migration in the developing cerebellum (Kato et al., 1992). The responsiveness of ciliary ganglion and retinal neurons to NOF changes markedly during devel-

opment. For example, ciliary ganglion neurons from embryonic day 6 (E6) to El0 embryos exhibit maximal neurite outgrowth in response to NlOF, and thereafter their responsiveness declines, even though the content of NOF in this tissue does not (Hayashi et al., 1987). To elucidate the molecular mechanisms regulating the sensitivity of neurons to NOF, we purified an NOFbinding protein from chicken gizzard smooth muscle membranes, which we refer to as gicerin, that migrates as a doublet of about 82 kd on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Taniura et al., 1991). We have also reported that gicerin (NOFbinding protein) is expressed in the developing retina and cerebellum and displays biochemical properties similar to those found in gizzard smooth muscle. In the retina and cerebellum of chick embryos, gicerin levels peak between E8 and El2 and then decrease markedly by E15-El8 (Kato et al., 1992). The temporal pattern of gicerin expression in both tissues coincides with the period of synapse formation, suggesting that NOF and gicerin mayplayacrucial role in this process. In this paper we describe the isolation of a cDNA encoding gicerin obtained by screening an expression library prepared from chicken gizzard with an antiserum against gicerin. The deduced amino acid sequence indicates that it is a novel member of the immunoglobulin (Ig) superfamily and has five immunoglobulin-like loops in an extracellular domain. Since Ig superfamily proteins generally exhibit cell adhesion activity by homophilic interactions between cells (Rutishauser et al., 1988) and in some cases by heterophilic binding of cells to the ECM (Cole and Glaser, 1986), we investigated the cell adhesion activity of gicerin by expressing it in mammalian cells. Our results indicate that gicerin exhibits both homophilic and heterophilic binding activities that mediate cellcell and cell-ECM interactions, respectively. Results Molecular Cloning of cDNAs Encoding Cicerin Rabbit antiserum against gicerin (NOF-binding protein) (Taniura et al., 1991), which had been preabsorbed with a lysate of the Lgtll host Escherichia coli Y1090, was used for screeningachicken gizzard cDNA library constructed in ahgtll expression vector (Clontech). Approximately 1 x 106 clones were screened, and 8 immunoreactive plaques were isolated. The inserts were subcloned into a phagemid cloning vector, Bluescript II SK(-), and sequenced. Three independent clones (LNOFB-A, -B, and -D) were obtained, and the longest one, LNOFB-B, contained a cDNA insert of 1.5 kb that spanned the 1.2 kb LNOFB-A cDNA insert. However, LNOFB-D (0.3 kb) did not overlap with the other 2 clones. The same library was rescreened by plaque hybridization using these cDNA

12 200.0

-

116.3

-

66.2 -

3

i.

.'

45.0 Figure 1. lmmunoblot Analysis of Membrane Fractions from Chicken Gizzard Smooth Muscle with Antiserum and Epitope Selected Antibodies Antiserum against gicerin was epitope selected with the immunoreactive clone h-NOFB-B, which was treated with IPTG. As a control, the same procedure was also performed with this clone in the absence of IPTC. Twenty micrograms of the membrane fraction from chicken gizzard smooth muscle was subjected to 7.5% SDS-PAGE. The separated proteins were transferred to a PVDF membrane and reacted with the following antibodies: lane I, polyclonal antiserum against gicerin; lane 2, epitopeselected antibodies reacted with IPTG-induced immunoreactive clone LNOFB-B; lane 3, control antibodies prepared with noninduced immunoreactive clone LNOFB-B.

inserts as probes, since these clones did not contain the entire coding region of gicerin. Sixteen additional phage clones were isolated and subcloned into Bluescript II SK(-). Of these clones, we obtained an additional 7 independent clones. Comparison of the sequences obtained allowed us to identify the putative coding sequence for gicerin. Identification of Isolated cDNA Clones as a Cicerin cDNA The cDNA clones obtained were isolated on the basis oftheir abilityto encode protein recognized by a polyclonal antiserum to gicerin. To ensure that antibodies present in the antiserum which bind to the plaques also bind to authentic gicerin, we epitope selected the polyclonal antiserum using LNOFB-B. We exposed the antiserum to isopropyl-p-o-thiogalactopyranoside (IPTG) and then employed these affinity-purified antibodies in immunoblots of chicken gizzard extracts. As shown in Figure 1, the epitope-selected antibodies detected a doublet band identical to that observed with the crude anti-gicerin antiserum. However, when the epitope selection was performed with phage LNOFB-B in the absence of IPTG, staining was not observed. Similarly, antibodies that were epitope selected using IPTG-induced proteins produced by a nonimmunoreactive (negative) clone also did not detect gicerin (data not shown).

Nucleic Acid and Deduced Amino Acid Sequences of Cicerin The nucleotide and deduced amino acid sequences of gicerin are shown in Figure 2. Analysis of the nucleotide sequence revealed a long open reading frame consisting of 584 amino acids, but a polyadenylation signal and a poly(A) sequence were not found in the 3’ noncoding region. The consensus nucleotide sequence for an initiation codon was present in the vicinity of the first methionine codon, and several stop codons occurred just upstream of this codon. There were also several stop codons downstream of the stop codon. Based on the deduced amino acid sequence, a hydrophobic region is located near the N-terminus immediately after the initiation codon and presumably contains the signal sequence (Figure 3). We could not determine the N-terminal sequence of endogenous gicerin, since its N-terminal amino acid was blocked. The presence of a strongly hydrophobic region near the C-terminus indicates that gicerin may be composed of a large 539 amino acid extracellular domain followed by a transmembrane domain (24 amino acids, underlined in Figure 2) and a short cytoplasmic tail (21 amino acids). The putativeextracellular domain contained five Ig-like loop structures by periodic repeats of cysteine residue and eight potential N-linked glycosylation sites. The first two loops appear to belong to the V-set type, and the other three loops to the CZ-set type (Williams and Barclay, 1988; Santoni et al., 1988). A cysteine-rich region, which does not form an Ig-like loop, was found near the N-terminus of the first loop. The cytoplasmic region four had no tyrosine kinase domain, but contained possible phosphorylation sites for protein kinase A, protein kinase C, and calmodulin kinase II (residues 569, 573, 582, and 583). A computer search of the GenBank DNA data base did not detect any homologous sequences. Comparison of the predicted amino acid sequence of gicerin with PIR Genprot and Swissprot protein data bases revealed weak homology with some members of the Ig superfamily (Table 1) (Goad and Kaneshita, 1982), including SCl/DM-GRASP (Tanaka et al., 1991; Burns et al., 1991), N-CAM (Cunningham et al., 1987), V-CAM-l (Hession et al., 1992), myelin-associated glycoprotein (Sato et al., 1989), and carcinoembryonic antigen (Beauchemin et al., 1989). SCl/DM-GRASP, an adhesion molecule expressed in motor neurons of chick embryonic spinal cord, was the most homologous protein (Figure 4). Expression of Cicerin In Vivo Northern blot analysis was performed on total RNA extracted from a variety of chicken and chick embryonic tissues (Figure 5). Transcripts were detected in adult heart, skeletal muscle, and gizzard muscle, in the embryonic nervous system (retina and cerebellum),and in theembryonic kidney. Cicerin transcripts were barely detectable in the liver, retina, and brain,

Cloning 863

and Expression

of Gicerin

Figure 2. Nucleotide and Deduced Acid Sequences of Gicerin

Amino

The open reading frame begins at 196, and the first methionine is designated as amino acid +I. Cysteine residues are marked with asterisks, potential N-linked glycosylation sites are boxed, and the single putative transmembrane domain is underlined.

including the adult chicken cerebellum. Northern blot analysis of these tissues revealed two prominent transcripts. The major band obtained in gizzard muscle migrated at -5.2 kb, whereas that found in heart was at -6.4 kb. Transcripts of both sizes, 5.2 kb and 6.4 kb, were observed in skeletal muscle and heart. Both embryonic retina and cerebellum displayed a major band that migrated at the same position as the band detected in gizzard muscle, but was slightly broader. We also carried out Southern blot analysis to determine the copy number of the gicerin gene. A single band of 15-20 kb was detected in each sample digested with Bglll, EcoRI, and BamHI, and in the case of Kpnl, about 15 kb and 3 kb bands were detected (data not shown). It is suggested that the gicerin gene is single. Since the transcription products change during development of the nervous system, we assessed the developmental profile of gicerin mRNA in the retina (Figure 6A). The amount of this mRNA (5.2 kb) was highest in E8 retina and thereafter decreased rapidly. After hatching, gicerin mRNA was barely detectable at the same position. Western blot analysis of the retina was also carried out to compare the developmental pattern of gicerin mRNA and protein expres-

sion (Figure 6B). The amount of gicerin protein was maximal in E8 retinaand decreased with development until this protein became almost wndetectable after hatching. These results indicate that the decrease in

Ammo

Figure Gicerin

3. Hydrophobicity

Aclds

Plot of the

Amino

Acid

Sequence

of

A putative signal sequence is indicated by an asterisk, and transmembrane domain is indicated by a bar. Hydrophobicity wasdetermined bythemethodof Kyteand Doolittle(l982),using a window of 17.

the

Neuron 864

Table

1. Proteins

Containing

Regions

Homologous

to Gicerin

Protein

Homologous Domain

% Identity

Score

Reference

SWDM-GRASP (chicken) SCIIDM-GRASP (chicken) N-CAM (chicken) N-CAM (chicken) V-CAM-l (mouse) MAC (human) CEA (human)

30-380 320-530 70-200 280-540 100-470 270-480 WI-520

30.6% 18.3% 21.2% 19.1% 21.9% 24.2% 18.5%

-405 -200 -133 -161 -285 -160 -270

Tanaka et al., 1991; Tanaka et al., 1991; Cunningham et al., Cunningham et al., Hession et al., 1992 Sat0 et al., 1989 Beauchemin et al.,

Identification the program

of proteins with regions homologous of Goad and Kaneshita (1982). MAC,

Transfection and Expression of Cicerin In Vitro A gicerin cDNA containing its entire coding region was subcloned into a mammalian expression vector, pcDNA1 (Invitrogen) to generate pcDNAl-NOFB. The construct was transfected into the mouse fibroblast cell line L929 (NCTC clone 929), which lacks endogenous gicerin, cadherin, and N-CAM and has been used for transfecting cell adhesion molecules, such as Ll (Miura et al., 1992; Mauro et al., 1992). The live cells transfected with pcDNAl-NOFB were examined by immunocytochemistrywith anti-gicerin antiserum. Cells transfected with pcDNAl-NOFB, but not the parental L929 cells, displayed prominent staining with anti-gicerin antiserum that appeared to reflect cell surface expression. The same experiment was also car-

was determined

according

to the

et al., 1991 et al., 1991

1989

to gicerin and calculation of % identity in these regions were myelin-associated glycoprotien; CEA, carcinoembryonic antigen.

gicerin protein parallels the decline in gicerin mRNA. These data are also consistent with the previous results of NOF ligand blot analysis in the developing retina (Taniura et al., 1991).

The alignment

Burns Burns 1987 1987

method

of Higgins

performed

using

ried out using stable transfectants of gicerin with comparable results (Figures 7A-7D). To investigate the function of gicerin, stable transfectants of L929 cells expressing gicerin were established by cotransfection of pcDNAl-NOFB with a neomycin resistance vector, pMAM-neo, and by selection with G418. The cell lines L-NOFB-1, -7, -8, and -10, which strongly expressed gicerin, were cloned and employed in further experiments. On immunoblot ana lysis, the membrane fraction of transfectant L-NOFB-1 cells exhibited a single band (90 kd) that corresponded to the upper band of a doublet band (82 and 90 kd) found in gizzard muscle (Figure 7E). The other cell lines also showed the same band. However, no bands were detectable in transfectant L-neo-1 cells, which expressed only a neomycin resistance gene. Cells Expressing Cicerin Adhere to NOF We examined the ability of cells expressing gicerin to bind to NOF. Purified NOF (Hayashi and Miki, 1985)

et al. (1992).

Identical

amino

acids

are indicated

by asterisks.

Cloning 865

and Expression

12

28s

-

18s

-

of Cicerin

3 4 5678

Figure 5. Northern Blot Analysis in Various Adult and Developing

910

1

2

3

4

zas18S-

of Chicken Tissues

Gicerin

Expression

Total RNA was isolated by the acid guanidinium thiocyanatephenol-chloroform method (Chomczynski et al., 1987). Twenty micrograms of total RNA from each tissue was loaded on a 1% agarose gel containing formaldehyde and transferred to a nylon membrane. The membrane was hybridized with a pZP]dCTPlabeled insert of 1-NOFB-B and washed with high stringency buffer. Lane 1, liver; lane 2, El4 kidney; lane 3, kidney; lane 4, E8 retina; lane 5, retina; lane 6, El2 cerebellum; lane 7, whole brain; lane 8, heart; lane 9, skeletal muscle; lane 10, gizzard. RNAwas obtained from adult tissues unless indicated otherwise. Positions of 28s and 18s ribosomal RNA markers are shown.

was applied in spots onto nitrocellulose-coated test dishes, and bovine serum albumin (BSA) and laminin were used as controls. L-NOFB-1 cells, stable gicerin transfectants, and parental L929 cells were dissociated by incubating with a mild trypsin-EDTA solution and pipetting gently to keep gicerin intact on the cell surface. Their ability to adhere to the dishes was examined using the method of Lemmon et al. (1989). Only L-NOFB-1 cells, and not parental L929 cells, adhered to the NOF-coated spots after 30 min (Figures 8A and 8C), and adhesion was suppressed by preincubating cells with anti-gicerin antibodies (Figure 8E). Neither the transfectants nor the parental cells adhered to the control spots coated with BSA (Figures 88 and 8D). Furthermore, cells expressing gicerin did not adhere to spots of laminin (Figure 8F). Similar results were obtained by using other cell lines expressing gicerin (L-NOFBJ, -8, and -10). These data suggest that gicerin has the ability to adhere to NOF. Aggregation of Cells Expressing Gicerin Cell adhesion molecules in the Ig superfamily generally adhere to each other and exhibit cell adhesion activity(homophilicadhesion).Sincegicerin isamember of this Ig superfamily, we suspected that it may also display homophilic adhesion properties. We investigated this possibility using several stable transfectants expressing gicerin, L-NOFB-1, -7, -8, and -10.

12

205.0

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116.5

-

80.0

-

49.5

-

Figure 6. Northern sion during Retinal

3

4

5

/

and immunoblot Development

Analyses

of Gicerin

Expres-

(A) Northern blot analysis of chicken retina gicerin expression during development. Each lane was loaded with 20 ug of total RNA. Lane 1, E8; lane 2, E14; lane 3, EM; lane 4, adult. (B) Immunoblot analysis of chicken retina gicerin expression during development. Each lanewas loaded with 20 ug of gizzard and retinal membrane fractions. Lane 1, gizzard; lane 2, E8 retina; lane 3, El4 retina; lane 4, El8 retina; lane 5, adult retina.

The stable transfectants and control parental L929 cells were gently dissociated as in the adhesion experiment described above. The dissociated cells were placed in a COz incubator at 37%, and aliquots were removed every IS min for estimating the degree of aggregation. Marked differences in aggregation between transfectants and parental L929 cells were apparent, as transfected cells formed large aggregates whereas parent L929 cells did not (Figure 9A). This cell aggregation was blocked with antigicerin antibodies, but not with control antibodies (Figwre 9B). The aggregation patterns of parental L929 cells, L-NOFB-1 cells, and L-NOFB-1 cells treated with anti-gicerin antibodies are shown in Figures IOA-IOC. In addition to this,

Neuron 866

the mixing experiment gated with each other transfected cells (Figure that gicerin also mediates tween cells. in

gicerin

transfectants

aggre-

and did not bind to unIOD). These results suggest homophilic interactions be-

Discussion

205.0

-

116.5

-

49.5

-

Figure 7. lmmunocytochemistry and lmmunoblot Analyses of Gicerin Transfectants (A-D) lmmunostaining of the stable gicerin transfectant L-NOFB-1. LB29 cells were stably transfected with pcDNAl-NOFB and the neomycin gene, pMAM-neo (L-NOFB-I), or with pMAM-neo only (L-neo-I). Live cell immunocytochemistry was carried out with anti-gicerin antiserum. L-NOFBI cells displayed intense staining that appeared to reflect localization on the cytoplasmic membrane, but L-neol cells were unstained. (A and B) L-NOFB-1; (C and D) L-neo-1; (A and C) live cell immunocytochemistry; (B and D) phase-contrast microscopy. Comparable results were ob tained with all the other cell lines expressing gicerin (L-NOFB-7, -B, and -10). Bar, 40 urn. (E) lmmunoblot analysis of chicken gizzard and stable transfectants L-NOFB-1 and L-neo-1. L929 cells were stably transfected with pcDNAl-NOFB (L-NOFB-1) or the neomycin gene, pMAM-neo (L-neol). Each lane was loaded with 20 ug of extracts. Lane 1, membrane fraction of chicken gizzard reacted with antiserum against gicerin; lane 2, membrane frac-

Cicerin is an integral membrane glycoprotein expressed transiently in the developing nervous system and constitutively in muscle. Gicerin is maximally expressed in retinal and cerebellar neurons during the period of synapse formation and cellular migration and thereafter decreases as development progresses, whereas NOF levels remain constant (Hayashi et al,, 1987; Taniura et al., 1991; Kato et al., 1992). To investigate the function of gicerin in developing neurons, we have used anti-gicerin antibodies to isolate cDNAs encoding gicerin. Several lines of evidence indicate that clones isolated by the antibody screening strategy encode gicerin: -Epitope-selected antibodies, which had bound to the IPTG-induced products of L-NOFB-B, recognized a doublet band in extracts of gizzard muscle identical to that detected with the antiserum. -Northern blot analysis showed that the developmental profile of gicerin mRNA expression in the retina paralleled that of gicerin protein expression, as determined by Western blot analysis. -Cells transfected with pcDNAl-NOFB, a mammalian expression vector containing a gicerin cDNA and processed for live cell immunocytochemistry with the anti-gicerin antiserum displayed prominent cell surface staining. Western blot analysis of these transfectants with the anti-gicerin antiserum detected a single band identical to one of the doublet bands observed in gizzard muscle. -The transfectants exhibited an ability to bind to NOF. Northern blot analysis detected two major transcripts of 6.4 kb and 5.2 kb. This may reflect alternative splicing, as Southern blot analysis indicated the presence of a single gene. Western blot analysis of the developmental profile of gicerin in the retina detected a single band of 82 kd, which displayed maximal expression in E8-El0 retina and then decreased during development, as assayed by NOF ligand blot analysis (Taniura et al., 1991). The appearance of a single band is discussed later. Northern blot analysis demonstrated that gicerin mRNA showed a similar profile, indicating that regulation of gicerin expression reflects changes in transcription. The main transcript of - 5.2 kb decreased dramatically after peak expression in E8-El0 retina. In adult retina,

tion of L-NOFB-1 reacted with antiserum membrane fraction of L-neo-1 reacted gicerin.

against gicerin; with antiserum

lane 3, against

Cloning 867

and Expression

of Gicerin

Figure 8. Adhesion of Stable Gicerin fectants to NOF-Coated Dish

Trans-

Purified NOF (A, C, atid E), ESA (B and D), and laminin (F) were spotted on nitrocellulosecoated dishes. Oply the right half of each field is coated wi h NOF, BSA, or laminin. Adhesion of L-N4 FB-1 (C, D, E, and F) and the parental L929 tells (A and B) to the dishes was examined by phase-contrast microscopy. Adhesiori NOF (C) was block4 cells with anti-gicerin 120 pm.

A

d & U & &

0.6 Nt/No 0.4

parental L929 L-NOFB-1 L-NOFB-7 L-NOFB-8 L-NOFE-10

40

20

time

60

(mm)

B

o.oi--..-

. .._

-

20

0

40

60

time (mm) Figure

9. Time

Course

of Cell Aggregation

(A) Time course of aggregation gicerin transfectants. Parental

of parental L929 cells and stable L929 cells and stable transfectant

of L-NOFB-1 cells to by preincubating antibodies (E). Bar,

trace amounts of the 6.4 kb transcript were detected. These may be derived from blood vessels of the choroid, since gicerin is also expressed in blood vessels (data not shown) and choroid contamination is inevitable in dissecting adult retina. The assignment of the initiation methionine is consistent with the consensus sequence of Kozak (1989): the purine at position -3 and the guanine at position +4, which are important for transcription, are conserved, and several stop codons occur just upstream of this methionine. The products of pcDNAl-NOFB expressed in mammalian cells were detected on the cell surface by immunocytochemistty of live cells and were also observed in the membrane fraction by immunoblot analysis. These results indicate that the putative signal sequence functions in the transfected cells and gicerin was targeted properly to the cytoplasmic membrane. The extracellular domain of gicerin contains eight potential N-linked glycosylation sites and many serines and threonines that are poten-

cells (cell line L-NOFB-1, -7, 8, and -10) were mildly trypsinized and placed in a CO? incubator. An aliquot of each sample was withdrawn every 15 min after gentle mixing, and the particle number was counted in a hemocytometer. The degree of cell aggregation was estimated by the index Nt/NO (see Experimental Procedures). Each point represents an average of four independent experiments. (B) Time course of aggregation of parental L929 cells and stable gicerin transfectants in the presence of anti-gicerin antibodies. Cells stably transfected with gicerin (L-NOFB-1) were treated with anti-gicerin antibodies or with control preimmune antibodies at a final conaentration of 0.2 mgl ml and kepton icefor3Omin beforestartingthe incubation. Each point represents an average of four independent experiments.

Figure

IO. Patterns

of Cell Aggregation

(A-C) Aggregation patterns of parental L929 cells, stable gicerin transfectants, and stable gicerin transfectants treated with antigicerin antibodies. Cells weredissociated by mild trypsinization and resuspended in the culture medium. They were incubated in a CO* incubator at 37OC and photographed in suspension after 1 hr. In some experiments, anti-gicerin antibodies were added at a final concentration of 0.2 mg/ml and kept on ice for 30 min before starting the incubation. (A) Parental L929 cells; (6) L-NOFB-1, stable transfectants of gicerin; (C) L-NOFB-1 treated with anti-gicerin antibodies. Bar, 120 Wm. (D-E) Aggregation pattern of the mixing experiment of transfectants with parental L929 cells. Cells were dissociated by mild trypsinization and resuspended in the culture medium. Parental L929 cells were prelabeled with Dil and mixed with transfectants at 1:l. The mixture was incubated in a CO? incubator at 37OC and photographed in suspension after 1 hr.(D) Phase-contrast microscopy;(E) Dil was visualized by fluorescence. Note theaggregation of transfectants excluding untransfected cells. Bar, 40 pm.

tial O-linked glycosylation sites. The molecular weight of gicerin from the cDNA was calculated to be 64.6 kd, and the molecular size expressed in the fibroblast transfectants was about 90 kd. However, digestion of the gicerin doublet band (82 and 90 kd) found in gizzard muscle with N-glycosidase F and 0-glycanase reduced the molecular size to about 64 kd (data not shown), which corresponds closely to a molecular weight calculated from the gicerin cDNA. At present, it is unclear whether the doublet gicerin protein bands reflect alternatively spliced mRNAs that have different numbers of glycosylation sites, or whether the doublet bands stem from different tissue-specific and cell type-dependent patterns of glycosylation of a single mRNA species. Further analysis of the structure of the gicerin gene and its transcripts, currently underway, may help clarify this point. A structural model of the gicerin molecule is proposed in Figure 11. There are several cysteine residues about every 50 amino acid residues, which are assumed to form five Ig-like loop structures. The first two loops located in the N-terminus appear to be of the V-set type, and the other three loops are of the CZset type, although V- and C2-set types are structural designations (Williams and Barclay, 1988). The

function of 6 cysteine residues in the N-terminal portion, including the signal sequence, is unknown. The short cytoplasmic tail is composed of 21 amino acids without homology with known functional domains, such as tyrosine kinase domains, but with four putative phosphorylation sites, which may form the basis for its interaction with other signal transduction molecules. Gicerin is a novel member of the Ig superfamily and has weak homology with SCl/DM-GRASP, N-CAM, V-CAM-l, myelin-associated glycoprotein, and carcinoembryonic antigen, all of which have Ig-like loop structures in their extracellular domain (Table 1). SCl/ DM-GRASP is the most similar, with a homology of about 30% (Figure 4), whereas the others have less than 25% homology. We emphasize that the poor homology with these Ig superfamily members indicates that gicerin cannot be classified as closely related to any of these proteins (Goad and Kaneshita, 1982). Cicerin

was

originally

isolated

as a membrane

pro-

tein that binds to NOF, a laminin family member (Hayashi and Miki, 1985). Since adhesion molecules in the Ig superfamily such as N-CAM generally interact with each other (homophilic adhesion) in the nervous system (Rutishauseret al., 1982), the heterophilic binding

Cloning 869

and Expression

of Cicerin

Figure

NH? V

V

c2

c2

c2

SiteS in the cy’toplasmic tail. Extracellular domain, amino acids l-539; transmembrane Potential N-linked Rlvcosvlation sites are indicated bv asterisks. Predicted serine by a circled P. ’

property of gicerin is unexpected. In the vascular system, ICAMs, which are cell adhesion molecules of the Ig superfamily expressed in endothelial cells of blood vessels, interact heterophilically in leukocytes with LFA-1, an integrin family member (Makgoba et al., 1988; Marlin and Springer, 1987). N-CAM shows heterophilic adhesion with an ECM protein, heparin (Cole and Glaser, 1986). Recently, it has been reported that contactinlFl1 interacts with tenascin (Zisch et al., 1992), Fll with restrictin (Brummendorf et al., 1993), and F3 with Jl-160/180 (Pesheva et al., 1993). All these reports indicate that some cell adhesion molecules belonging to the lg superfamily interact with ECM proteins heterophilically. We carried out cell adhesion assays to ensure that gicerin interacts with NOF. Stable transfectants of gicerin bound only to an NOFcoated spot, and not to control, BSA, or laminin spots, whereas the parent L929 cells did not bind to NOF. Antibodies against gicerin completely blocked binding to NOF. The NOF used in the assay was over 95% purity (Hayashi and Miki, 1985) and did not include gicerin or other ECM molecules, such as laminin, fibronectin, and vitronectin (data not shown). Similarly, anti-gicerin antibodies inhibit both the neurite-promoting activity of ciliary ganglion neurons (Taniura et al., 1991) and the migration of cerebellar granular cells by NOF (Kato et al., 1992). From these data, it is evident that gicerin binds to NOF and participates in its neurite-promoting activity. We further examined whether gicerin also has homophilic binding activity, as do other Ig superfamily molecules. Stable transfectants of gicerin adhered to each other and formed large aggregates in the culture medium, whereas the control parental cells aggregated poorly (Figures IOA-IOC). Furthermore, gicerin transfectants did not bind to untransfected cells when they were incubated after mixing (Figures IOD and IOE). These results strongly indicate that gicerin also has homophilic adhesion activity. In addition to gicerin’s role in neurite extension and cell migration, it may also participate in neurite fasciculation. Ig superfamily molecules are thought to contribute to neurite fasciculation by homophilic adhesion (Rathjen et al., 1987; Chang et al., 1987). Conceivably, the homophilic adhesion activity of gicerin

11. Predicted

Structure

of Gicerin

Cicerin is depicted as an integral membrane glycoprotein that has a large extracellular domain, a single transmembrane domain, and a short cytoplasmic tail. Cysteine disulfide linkages in the extracellular domain are thought to form five Ig-like loops. The first two loops are of the V-set type, and the other three loops of the C2set type. There are eight potential N-linked glycosylation sites in the extracellular do main and four predicted phosphorylation domain, 540-565; cytoplasmicdomain, 564-584. and threonine phospho@ion sites are indicated

could also promote formation of neurite fascicles. Besides its transient expression in the nervous system, gicerin is also expressed constitutively in muscles. Although the function of gicerin in muscle is unknown, it could be involved in the adhesion of muscle bundles or the maintenance of neuromuscular junctions. Introduction of mutant gicerin genes in vivo and studies of denervation may help in elucidating the role of gicerin in the nervous system and in muscle. Experimental

Procedures

Protein Extraction and Western Blot Analysis Cultured cells were harvested with a cell scraper after rinsing twice with Ca*+- and Mgz’-free Hanks’ solution. Cultured cells or freshly dissected chicken tissues were homogenized in phosphate-buffered saline (PBS) with a polytron homogenizer. The homogenate was centrifuged at 1,000 x g for 10 min, and the supernatant was centrifuged at 18,000 x g for 30 min. The pellet was solubilized by incubating it in IO mM Tris-acetate buffer (pH 8.0), 1 mM EDTA, 0.5% Nonidet P-40 at 4OC for 1 hr on a rotating shaker and then centrifuged at 40,080 x g for 90 min. The resultant supernatant was used for Western blot analysis CTaniura et al., 1991). Protein concentrations were measured as As2 with the BCA protein assay reagent (Pierce) and compared with BSA standards. Equal amounts of protein (20 ug) from each sample were run on 7.5% SDS-PAGE and electrotransferred to a polyvinyl difluoride (PVDF) membrane (Bio+Rad). The blots were blocked with 2% skim milk in TS (150 mM NaCl and 10 mM TrisHCI buffer [pH 8.01) and then incubated with priman/ antiserum or antibodies diluted in TS containing 2% $kim milk (1:lOOO) for 1 hr at room temperature. After washing with TST (TS containing 0.05% Tween 20) six times, the PVDF membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Cappel) in TS containing 2% skim milk (1:iCKtO) for 1 hr at room temperature. Then, after washing with TST four times and washing with TS twice, the blots were developed with diaminobenzidine and photographed. Screening A lgtll cDNA library prepared from chicken gizzard (Clontech) was used for cDNA screening. The cDNA library containing 10,000 recombinants was plated with E. coli YlO90 on 92 mm dishes. The plates were covered with nitrocellulose filters (Schleicher & Schuell) soaked in 10 mM IPTC and screened by using rabbit antiserum against gicerin (Taniura et al., 1991) that had been preabsorbed with Y1090 lysates (Huynh et al., 1985). For this purpose, a 50 ml culture of Y10!30 was resuspended in 2 ml of distilled water, boiled for 10 min, and then incubated with anti-gicerin antiserum at 4OC for 2 hr. The supernatant was centrifuged at 10,000 x g for 15 min and then used for screening (1:5000). Horseradish peroxidase-conjugated goat IgC (Cap-

pel) was used as a secondary antibody. Using this procedure, we isolated 8 immunoreactive clones from this library and 3 independent clones, kNOFB-A, -B, and -D, were obtained. The same library was then rescreened with DNA probes derived from clones k-NOFB-B and -D. The DNA probes were prepared by the random primer method with p2P]dCTP (Amersham), and screening was carried out with standard procedures (Maniatis et al., 1982). Epitope Selection Epitope selection was performed according to the method of Weinberger et al. (1985). Positive phage clone bNOFB-B was plated with E. coli Y1090 onto a dish, incubated for 4 hr, and covered with nitrocellulose filters (Schleicher 81 Schuell) soaked in 10 mM IPTG or distilled water. These filters were washed with TS buffer (pH 8.0) with 0.02% NaN, and 0.5% skim milk for I hr at room temperature. The filters were incubated with rabbit antiserum diluted in TS containing 0.02% NaNl and 0.5% skim milk (1:500) overnight at 4OC. These filters were washed three times with TS containing 0.02% NaNl and 0.5% Triton X-100 for IO min and then rinsed with distilled water. The epitope-selected antibodies were collected with elution buffer (5 mM glycineHCI buffer [pH 2.3],150 mM NaCI, 0.1 mglml BSA). Buffers were immediately neutralized with 1 M Tris to pH 7.8. These antibody preparations were used for Western blot analysis. Sequencing and Analysis of Cicerin cDNA Purified cDNA inserts from positive phage were subcloned into a Bluescript II SK(-) phagemid vector (Stratagene). The nucleic acid sequences were determined by the dideoxy chain termination method of Sanger et al. (1977) using double-stranded DNA astemplatefor Sequenase(USB). Initially, bothendsofthecDNA inserts were sequenced, and then internal fragments were obtained by digestion with restriction enzymes. In addition, se quential deletion clones were generated using exonuclease III (Takara), and both strands were sequenced. Northern Blot Analysis Northern blot analysis was carried out according to the method of Maniatis et al. (1982). Total RNA was isolated from several tissues by the acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987). The amount.of RNA was measured at OD,. Twenty micrograms of the RNA samples was run on 1% agarose gels containing formaldehyde and transferred toa Hybond N membrane(Amersham).The blots were hybridized with a cDNA probe derived from I.-NOFB-B and washed under high stringency conditions (0.1 x SSPE, 0.1% SDS, 65OC, 10 min). Expression of Gicerin cDNA Transfected into Mammalian Cells Mouse fibroblast 1929 cells (NCTC 929) (1 x 105 per well) were plated on a two chamber glass slide. After incubating them overnight in a COz incubator at 37OC, they were transfected with 1 pg of pcDNAl-NOFB, the mammalian expression vector pcDNA1 (Invitrogen) containing a full-length gicerin cDNA insert, by using Lipofectin (BRL). Cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics-antimycotics (100 U/ml penicillin, 100 mglml streptomycin, and 250 rig/ml amphotericin B). After 2 days of transfection, the cells were processed for immunocytochemistry. To obtain stable transfectants of L929 cells expressing gicerin, the expression vector pcDNAl-NOFB was cotransfected with a neomycin-resistant vector, pMAM-neo (Clontech), by using Lipofectin (1O:l). AS a control, only pMAM-neo was transfected, to generate L-neo-1. TWO days after transfection, G418 (Geneticin, CIBCO)wasadded (0.6mglmlfinal concentration).After2weeks, G415resistant cloneswere isolated and examined by immunocytochemistry with anti-gicerin antiserum. Four strongly positive clones (L-NDFB-I, -7, 8, and -10) were used in further experiments.

Cell Adhesion Assays Cell adhesion experiments were carried out according to the method of Lemmon et al. (1989). A 20 cm2 nitrocellulose filter strip (Schleicher & Schuell) was dissolved in 48 ml of methanol. Aliquots (0.25 ml) of this solution were rapidly spread over 35 mm test dishes and allowed to dry. Aliquots (4 ul) of each test sample containing either 10 rig/ml purified NOF, or 100 rig/ml BSAor laminin were spotted on the dried surface and incubated for 1 hr in a humidified CO2 incubator at 37OC. The dishes were washed with DMEM containing 10% FCS three times and preincubated with the medium containing 10% FCS for 1 hr to block nonspecific binding. Subconfluent monolayers of gicerin transfectants (L-NOFB-1, -7, -8, and -10) or parental L 929 cells were washed with Ca2+- and Mg2+-free Hanks’solution twice and then treated with a solution containing 0.001% trypsin and 0.01 mM EDTA for 10 min at 37°C. The cells were collected, dissociated by gentle pipetting, and suspended in DMEM containing 10% FCS at 2 x 106 cells per ml (Urushihara et al., 1979; Tanaka et al., 1991). Under these treatment conditions, gicerin remained intact. The cells were then kept on ice to inhibit aggregation. Aliquots (2 ml) of the cell suspension were plated on the test dishes. To assess inhibition of adhesion by antibodies against gicerin, the cells were preincubated with the antibodies at a final concentration of 0.2 mglml on ice for 30 min before plating on the test dishes. The dishes were kept in CO1 at 37°C for 30 min, washed twice with Hanks’ solution containing Caz+ and Mg*‘, and fixed with Zamboni’s solution. Adhesion to the dishes was then assessed by phase-contrast microscopy. Cell Aggregation Assays Subconfluent monolayers of permanent gicerin transfectants (L-NOFB-1, -7, -8, and -10) or parent L929 cells were washed with Ca2+- and Mg*+-free Hanks’ solution twice and then treated with 0.001% trypsin, 0.01 mM EDTA for 10 min at 37’C. The cells were collected, dissociated by gentle pipetting, and suspended in DMEM containing 10% FCS at 2 x 106 cells per ml (Urushihara et al., 1979; Tanaka et al., 1991). The cells were kept on ice to inhibit aggregation. In some experiments, antibodies were added at a final concentration of 0.2 mg/ml and kept on ice for 30 min before starting the aggregation assay. To quantitate aggregation, a2 ml aliquot of thecell suspension was transferred into a 50 ml polypropylene tube that had been precoated with FCS for 1 hr. The assay was carried out by incubating the cell suspension at 37OC in a CO* incubator without agitation. A small aliquot was withdrawn every 15 min after gentle mixing, and the particles were counted in a hemocytometer. The degree of cell aggregation was estimated by the index Nt/NO according to conventional methods CTakeichi, 197 Tanaka et al., 1991). The index NO was the total particle number at the initial incubation time (0 min), and the index Nt was the total particle number at the incubation time t. Experiments were performed four times, and the scores were averaged. Photographs were taken of cells in suspension after a 1 hr incubation. In the experiment mixing transfectants with parental L929 cells, parental L929 cells were labeled by incubation with 15 ng/ ml Dil for 2 hr (Elkins et al., 1990; Miura et al., 1992) and then mixed at a I:1 ratio with transfectants; total cell number was 2 x 106 cells per ml. The aggregation assay described above was used. tmmunocytochemistry and Microscopic Examination of live Cells Subconfluent monolayers of cells were collected in the same way as for adhesion and aggregation assays. They were incubated with a primary antibody against gicerin in Hanks’solution (1:800) for 2 hr at room temperature. After washing with Hanks’ solution twice, cells were incubated with an FITC-conjugated goat secondary antibody against rabbit IgG (Cappel) (1:lOOO) for 1 hr and washed twice. Live cells were then examined under a fluorescence microscope and a phase-contrast microscope (Nikon).

Cloning 871

and Expression

of Cicerin

Acknowledgments

them.

We thank Dr. J. Baraban for critical reading of the manuscript and discussions. This work was supported by the Mitsubishi Foundation and the Uehara Memorial Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

November

12, 1993; revised

February

15,1994.

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