(Received for publication, July 17, 1995; and in revised form, September 21, 1995)
From the
We have cloned a novel cDNA of gicerin, a cell adhesion molecule belonging to the immunoglobulin superfamily. Both gicerin isoforms share the same extracellular domain, which has five immunoglobulin-like loop structures and a transmembrane domain as s-gicerin, but differ in the cytoplasmic tail domain. As the newly identified form has a larger cytoplasmic domain than the previously reported form, we refer to them as l-gicerin and s-gicerin, respectively. l-gicerin is transcribed from a distinct mRNA containing an inserted sequence not found in s-gicerin mRNA which caused a frameshift for the coding region for a cytoplasmic domain. Previous studies demonstrated that gicerin showed a doublet band of 82 and 90 kDa in chicken gizzard smooth muscle. We report that the 82-kDa protein corresponds to s-gicerin and the 90-kDa protein to l-gicerin. We also found that the two gicerin isoforms are expressed differentially in the developing nervous system. Functional analysis of these gicerin isoforms in stable transfectants revealed that they had differ in their homophilic adhesion properties, as well as in heterophilic cell adhesion assayed with neurite outgrowth factor. In addition, these isoforms have neurite-promoting activity by their homophilic adhesion, but differ in their ability to promote neurite outgrowth.
Cell adhesion molecules are widely expressed in the nervous
system and play a crucial role in the organization of neural networks
during development(1, 2) . Members of the
immunoglobulin (Ig) superfamily are homophilic cell adhesion molecules,
expressed in the nervous systems of a variety of animal species (3, 4, 5) that participate in neurite
extension, fasciculation of nerve fibers, and neuronal migration (6, 7, 8, 9, 10) . Recent
studies provide evidence that they are also involved in synaptic
plasticity (11) and programmed cell death of
neurons(12) . These findings underscore the importance of Ig
superfamily molecules in cell-cell communication during nervous system
development. Cell adhesion molecules of the Ig superfamily are divided
into several subgroups. Members of one group have only Ig-like domains
in their extracellular domains; the others form a subgroup that have
fibronectin type III-related domains between Ig-like and transmembrane
domains (13) . Members of the first group include fasciclin
III(14) , neuromusculin(15) , and SC1/DM-GRASP/Ben (16, 17) that are mainly expressed in neurons, as well
as MAG ()(3, 18) and SMP (19) that
are found in glial cells. Ig superfamily molecules have also been
classified into two groups by their mode of membrane association, with
some containing a phosphatidylinositol anchor (20, 21) and others a transmembrane
domain(8, 22) . Interestingly, some Ig superfamily
members undergo alternative splicing to generate multiple forms that
differ in the type of membrane association
used(23, 24) . The alternatively spliced products are
differently expressed spatially and temporally and expected to play
different roles(4) . MAG, the molecule most homologous to
gicerin, is known to have two isoforms that differ in the length of
their cytoplasmic domains(3, 25) . These forms are
differentially regulated during development with only l-MAG found to be
associated with Fyn, a nonreceptor tyrosine kinase(26) .
Gicerin is a cell adhesion molecule with five Ig-like domains(27) . It was first identified as a binding protein for neurite outgrowth factor (NOF), a member of the laminin family(28, 29) . In the nervous system gicerin expression is transient, restricted to early stages of development when neurons extend neurites or undergo migration(30) , whereas constitutive expression is found in muscles and kidney. Introduction of gicerin into L929 fibroblast cells in vitro indicate that gicerin exhibits homophilic cell adhesion activity in addition the heterophilic adhesion to NOF(27) . In previous studies, we noted that Western blot analysis of endogenous gicerin in chicken gizzard reveals a doublet band, perhaps reflecting the presence of alternatively spliced products. In the present study, we identified and characterized a novel longer isoform of gicerin termed l-gicerin, which contains a different cytoplasmic tail than that found in the previously reported form of gicerin (s-gicerin (accession number D38559)). We have also investigated possible functional differences between these two gicerin isoforms and compared their expression during nervous system development.
Figure 1: DNA and deduced amino acid sequences of l-gicerin. Cysteine residues are marked with asterisks, and the single transmembrane domain is underlined. Sequences employed for RT-PCR primers and the SacI consensus sequence are underlined by thinner bars. The primer names are indicated above the sequences.
Figure 3: Expression of s-gicerin and l-gicerin RNA transcripts in chicken gizzard smooth muscle. A, cDNA structures of each gicerin and the location of primers. An open box indicates the open reading frame, a black box the transmembrane domain, and the striped box the l-gicerin-specific insert. Locations of each primer are indicated. B, gel electrophoresis of RT-PCR products. Total RNA was amplified by RT-PCR with both gicerin and l-gicerin-specific primers. An aliquot of the products was digested by restriction endonuclease SacI, and the products were electrophoresed in a 2% agarose gel and stained with ethidium bromide. Lane M, size marker; lane 1, G-5`/G-3` primer set which amplify both l- and s-gicerin (2083 and 1963 bp each); lane 2, G-5`/I-3` primer set which amplify only l-gicerin (1837 bp); lane 3, I-5`/G-3` primer set which amplify only l-gicerin (288 bp); lane 4, products in lane 1 were digested by SacI (1629, 1509, and 454 bp); lane 5, product in lane 2 was digested by SacI (1383 and 454 bp).
Figure 2: Comparison of cytoplasmic sequences of l-gicerin and s-gicerin. Amino acid sequences of cytoplasmic domains of both s-gicerin and l-gicerin are indicated with cDNA sequences. The numbers shown correspond to the amino acids indicated in Fig. 1. Amino acid residues employed for constructing multiple antigen peptides for generating l-gicerin-specific antiserum are shown in bold and underlined. The oligopeptide names are indicated above the sequences.
Figure 4: Immunoprecipitation analysis of chicken gizzard gicerin. The Nonidet P-40-extracted fraction of chicken gizzard and the immunoprecipitated samples were separated by 7.5% SDS-PAGE under reducing condition. Western blot analysis was performed with anti-gicerin antiserum (lanes 1-3) or with anti-l-gicerin-specific antiserum (lane 4-6) followed by horseradish peroxidase-conjugated secondary antibodies. Lane 1, Nonidet P-40-extracted fraction of gizzard; lane 2, immunoprecipitated with protein A-Sepharose loaded with anti-gicerin antibodies; lane 3, immunoprecipitated with protein A-Sepharose loaded with anti-l-gicerin-specific antibodies; lane 4, Nonidet P-40-extracted fraction of gizzard; lane 5, immunoprecipitated with protein A-Sepharose loaded with anti-gicerin antibodies; lane 6, immunoprecipitated with protein A-Sepharose loaded with anti-l-gicerin-specific antibodies.
Figure 5: Deglycosylation analysis of gizzard gicerin. Nonidet P-40-extracted fraction and the N-glycosidase F-digested samples were separated by 7.5% SDS-PAGE under reducing conditions. Western blot analysis was performed with anti-gicerin antiserum (lanes 1 and 2) or with anti-l-gicerin-specific antiserum (lanes 3 and 4) followed by horseradish peroxidase-conjugated secondary antibodies. Lane 1, Nonidet P-40-extracted fraction; lane 2, Nonidet P-40-extracted fraction digested by N-glycosidase F; lane 3, Nonidet P-40-extracted fraction; lane 4, Nonidet P-40-extracted fraction digested by N-glycosidase F.
Figure 6: Expression of gicerin in the nervous system. 20 µg of Nonidet P-40-extracted fraction from each tissue were separated by 7.5% SDS-PAGE under reducing conditions. Western blot analysis was performed with anti-gicerin antiserum (A) or with anti-l-gicerin-specific antiserum (B) followed by horseradish peroxidase-conjugated secondary antibodies. Lanes 1, gizzard; lanes 2, embryonic day 8 retina; lanes 3, embryonic day 18 retina; lanes 4, embryonic day 6 optic tectum; lanes 5, embryonic day 18 optic tectum; lanes 6, embryonic day 11 cerebellum; lane 7, embryonic day 18 cerebellum. The big band of about 53 kDa (B) is a degenerated product of that gicerin that was sometimes observed in repeatedly frozen samples.
Figure 7: Comparison of gicerin isoforms expressed in cell lines. Nonidet P-40-extracted fraction (A) and the N-glycosidase F-digested samples (B) were separated by 7.5% SDS-PAGE under reducing conditions. Western blot analysis was performed with anti-gicerin antiserum (lanes 1-3) or with anti-l-gicerin-specific antiserum (lanes 4-6) followed by horseradish peroxidase-conjugated secondary antibodies. A, Nonidet P-40 extracts; B, N-glycosidase F-digested samples; lanes 1 and 4, gizzard; lanes 2 and 5, s-gicerin transfectants; lanes 3 and 6, l-gicerin transfectants (l-lG-1 and l-lG-3, respectively).
Figure 8:
Aggregation patterns of parental L929
cells and stable s- or l-gicerin transfectants. Cells were dissociated
by mild trypsinization and resuspended in the culture medium. They were
incubated in a CO incubator at 37 °C and photographed
in suspension after 1 h. In some experiments, 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; B, l-sG-1, stable transfectants of s-gicerin; C,
l-lG-1, stable transfectants of l-gicerin; D, l-lG-1
pretreated with gicerin antibodies. Bar, 120
mm.
Figure 9:
Aggregation pattern of gicerin
transfectants. A, time course of aggregation of
l-gicerin-stable transfectants, s-gicerin transfectants, and parental
L929 cells. Cells (cell lines l-lG-1, -3, and -5) were mildly
trypsinized and incubated at 37 °C. 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 N/N
(see
``Experimental Procedures''). Each point represents an
average of four independent experiments. In the experiments conducted
in the presence of anti-gicerin antibodies, cells were preincubated
with them 30 min before starting the aggregation time course. Results
from parental L929 cells and s-gicerin transfectants are reported as
the mean ± S.D. of the values measured for each of several cell
lines (27) .
Figure 10: Neurite extension from embryonic CG neurons on a feeder layer of gicerin transfectants. Dissociated CG neurons from embryonic day 8 chick retina were cultured for 24 h on confluent monolayers of control L929 (A), l-sG-1 (B), and l-lG-1 (C) cells. The cultures were fixed and immunostained for MAP2. Bar, 25 mm.
Figure 11: Comparison of neurite extension. Dissociated CG neurons from embryonic day 8 chick retina were cultured for 24 h on confluent monolayers of control L929 or gicerin transfectants (l-sG-1 and -2 and l-lG-1 and -3). To asses inhibition of neurite extension by anti-gicerin antibodies, both the CG neurons and feeder cells were preincubated with antibodies 30 min before mixing the cultures. The cultures were fixed and immunostained with anti-MAP2 antibodies. Neurons with neurites longer than the cell body were scored as neurite positive. More than 1000 neurons were examined microscopically for each condition shown.
Figure 12: NOF adhesion activity. Purified NOF or control laminin was spotted on nitrocellulose-coated dishes. Only the right half of each field is coated with NOF or laminin. Adhesion of each gicerin transfectant to the dish was examined by phase-contrast microscopy. Adhesion of l-sG-1 (A) and l-lG-1 (C) was blocked by preincubating with anti-gicerin antibodies: l-sG-1 (B) and l-lG-1 (D). No adhesion of l-lG-1 to laminin (E) or parental L929 to NOF (F) was observed. The same results were obtained with other transfectants. Bar, 120 mm.
In the present paper, we isolated a novel isoform of gicerin (l-gicerin) that differs from the previously reported isoform s-gicerin in the cytoplasmic tail domain. Comparison of the functional activity of these isoforms revealed that l-gicerin was not as effective as s-gicerin in assays of both homophilic and heterophilic adhesion. These findings indicate that the cytoplasmic domain is not merely a passive structural feature but exerts an important influence on the adhesive properties of the extracellular domain.
Gicerin was originally purified from chicken gizzard as a doublet with molecular sizes of 82 and 90 kDa(29) . Recently, we have reported the molecular cloning and functional analysis of gicerin, which we renamed s-gicerin here(27) . However, these studies did not clarify the origin of the doublet band. Since gicerin mRNA from gizzard exhibited a broad single band of about 5.2 kilobases, we considered two likely possibilities: 1) the two bands on SDS-PAGE reflect different post-transcriptional modification such as glycosylation or 2) they were translated from different mRNA species with similar size.
Following digestion of gizzard membrane extracts with N-glycosidase F, two bands migrating as 70 and 64 kDa proteins were detected by Western blot analysis. The predicted molecular mass of s-gicerin was 63.9 kDa, matching the size of the smaller digested product. The presence of the 70-kDa gicerin immunoreactive band favored the existence of two different gicerin proteins. Of note, precedent for this alternative is provided by MAG, a homologous molecule that has two isoforms detected as a doublet band on Western blot analysis(3, 18) . To pursue this possibility, we screened for a novel gicerin isoform and isolated a cDNA clone that encoded a larger isoform of gicerin (l-gicerin) which differed from s-gicerin only in the cytoplasmic coding region. The predicted molecular mass was calculated to be 69.0 kDa, corresponding to the larger band (70 kDa) seen following N-glycosidase F digestion. We also confirmed the existence of two different mRNAs by RT-PCR analysis. The expected size products were obtained and confirmed by restriction analysis (Fig. 3B) and sequencing both ends of the products. Southern blot analysis indicated the presence of only a single gicerin gene in the genome (data not shown). We, therefore, concluded that these two mRNA species were generated by alternative splicing.
We then examined whether the doublet band detected with SDS-PAGE originates from these two mRNA species, as expected. An antiserum specific for l-gicerin was produced and reacted with only the upper 90-kDa band of the doublet seen in gizzard extracts and the upper 70-kDa band following N-glycosidase F treatment. These two molecules did not co-precipitated when immunoprecipitated with l-gicerin-specific antiserum, even under mild condition in the presence of 0.5% Nonidet P-40, suggesting that they do not form a tight dimer.
The distribution of both subtypes was examined in the developing nervous system by Western blot analysis. In the retina, only s-gicerin was detected. In contrast, only l-gicerin was found in the optic tectum. Both gicerins were expressed in the cerebellum. Differential expression among cell types may help account for different responses to NOF and gicerin seen in these regions. We have reported that neurons in cultured retinal explants that express s-gicerin selectively extend neurites in response to NOF(29) , while the cerebellar explants cultured on NOF display migration of neurons (probably granular cells) in addition to neurite extension(30) .
To examine functional differences between these two gicerin isoforms, we established cell lines expressing each alone. Although the degree of l-gicerin expression differed a little among three cell lines tested, similar cell aggregation activities were observed among the l-gicerin transfectants. Presumably, the amount of gicerin expressed was over a threshold amount needed to achieve a maximal cell adhesion activity. A similar result was obtained in previous experiments examining s-gicerin transfectants(27) . Experiments investigating NCAM and cadherin activity have revealed that the NCAM displays a threshold value of expression for neurite extension, while cadherin shows a graded concentration-dependent activity(41, 42, 43) . Experiments on MAG also support the idea that cell adhesion activity reaches a plateau once above a threshold concentration(44) . These reports indicate that this type of threshold or plateau profile may be a general feature shared by a wide variety of cell adhesion molecules in the Ig superfamily.
We compared the aggregation activity of these
gicerin transfectants and found that l-gicerin transfectants were less
effective than s-gicerin transfectants. In addition to the degree of
aggregation indicated by the score, N/N
, l-gicerin transfectants
did not form as large aggregates as s-gicerin transfectants did (Fig. 8). Experiments assaying neurite extension from ciliary
ganglion neurons cultured on a feeder layer of transfectants also
showed that s-gicerin transfectants were more effective than l-gicerin
transfectants. We could not find any difference in the morphological
features of CG neurites such as the number or length of neurites or
neurite branches induced by s- or l-gicerin transfectants. These
results suggest that a structural change in the cytoplasmic tail of
gicerin modulates cell adhesion activity. Of note, neurite extension
from rat cerebellar neurons grown on a feeder layer of NCAM 140
transfectants is stronger than that induced by NCAM 180 transfectants
that have a larger cytoplasmic tail(45) . Furthermore, our
observation that the adhesive activity of l-gicerin toward NOF was less
than s-gicerin's supports the idea that the extracellular domain
itself possesses cell adhesion activity which is modified by the
structure of the cyctoplasmic domain.
Introduction of a frameshift and an insert alters the sequence of the l-gicerin cytoplasmic domain and also leads to deletion of the C-terminal portion of s-gicerin. The protein structure of l-gicerin predicted by the Chou-Fasman method contains foldings which may hide some residues in the cytoplasmic domain. We speculate that one of the following mechanisms underlies the decreased activity of l-gicerin: 1) the l-gicerin-specific C terminus of the cytoplasmic domain blocks a crucial domain for cell adhesion located in the common N terminus half of the cytoplasmic domain found in both gicerins, 2) the tail of l-gicerin lacks a domain present in s-gicerin needed for maximal adhesion activity, or 3) the long tail of l-gicerin modifies the structural conformation of the extracellular domain directly responsible for adhesion. In addition to these intrinsic structural consideration, it is also important to consider potential difference between s- and l-gicerin in interaction with other molecules in the cytoplasm(46, 47) . The large isoform of the most homologous molecule MAG (l-MAG) has a phosphotyrosine in its C terminus thought to interact with Fyn kinase(26) . The deduced amino acid sequences of the l-gicerin cytoplasmic domain shows a weak homology with l-MAG with the tyrosine phosphorylation site conserved. Preliminary data indicate that the tyrosine residue of l-gicerin in gizzard is also phosphorylated. Further studies directed at examining the structural conformation of the tail and its interaction with other proteins may provide important clues to elucidating the mechanisms of adherence and signal transduction mediating gicerin.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D49849 [GenBank](l-gicerin).