Journal of Neurochemislry Raven Press, Ltd., New York 0 I990 International Society for Neurochemistry
N-Cadherin Gene Maps to Human Chromosome 18 and Is Not Linked to the E-Cadherin Gene Frank S. Walsh, C. Howard Barton, Wendy Putt, Stephen E. Moore, *David Kelsell, *Nigel Spun, and *Peter N. Goodfellow Department of Experimental Pathology, UMDS, Guy’s Hospital, and *Imperial Cancer Research Fund, London, England
Abstract: cDNA clones encoding the human N-cadherin cell adhesion molecule have been isolated from an embryonic muscle library by screening with an oligonucleotide probe complementary to the chick brain sequence and chick brain cDNA probe XN2. Comparison of the predicted protein sequences revealed > 9 1% homology between chick brain, mouse brain, and human muscle N-cadherin cDNAs over the 748 amino acids of the mature, processed protein. A single polyadenylation site in the chick clone was also present and duplicated in the human muscle sequence. Immediately 3’ of the recognition site in chick a poly(A) tail ensued; however, in human an additional 800 bp of 3‘ untranslated sequence followed. Northern analysis identified a number of major Ncadherin mRNAs. These were of 5.2, 4.3, and 4.0 kb in C6
glioma, 4.3 and 4.0 kb in human foetal muscle cultures, and 4.3 kb in human embryonic brain and mouse brain with minor bands of 5.2 kb in human muscle and embryonic brain. Southern analysis of a panel of somatic cell hybrids allowed the human N-cadherin gene to be mapped to chromosome 18. This is distinct from the E-cadherin locus on chromosome 16. Therefore, it is likely that the cadherins have evolved from a common precursor gene that has undergone duplication and migration to other chromosomal locations. Key Words: Cell adhesion molecules-N-Cadherin-Gene mapping. Walsh F. S. et al. N-Cadherin gene maps to human chromosome 18 and is not linked to the E-cadherin gene. J. Neurochem. 55, 805-8 12 ( 1990).
The formation of selective adhesive interactions between cell surfaces in the nervous system is essential for the processes of cell recognition, sorting, and migration during development and in tissue regeneration (Edelman, 1986). The molecular basis of cell adhesion is being extensively investigated and these studies indicate that a number of adhesive interactions result from the expression of a relatively small number of cell adhesion molecules (CAMs). Two classes ofCAMs have been identified according to their requirement for function of extracellular Ca2+.Several Ca2+-independent CAMs such as N-CAM have been classified as members of the immunoglobulin (Ig) gene superfamily of recognition molecules based on sequence analysis (Edelman, 1987; Williams, 1987; Barton et al., 1988). Those studies suggested that the Ca2+-independent CAMs belonging to the Ig gene superfamily have evolved from a multifunctional primordial CAM, with tissue-specific CAMs arising much later in evolution (Edelman, 1987).
A putative gene family mediating Ca2+-dependent adhesion (the cadherins) has been identified (Takeichi, 1987), with the present three members of this family, called epithelial (E; Yoshida and Takeichi, 1982), neural (N; Hatta et al., 1985), and placental (P; Nose and Takeichi, 1986) cadherins, exhibiting precise patterns of cell and tissue expression. The cadherins have recently been cloned and the sequences of E-cadherin (Gallin et al., 1987; Nagafuchi et al., 1987; Ringwald et al., 1987; Sorkin et al., 1988), P-cadherin (Nose et al., 1987; Shimoyama et al., 1989), and N-cadherin (Hatta et al., 1988; Miyatani et al., 1989) reported. ECadherin appears to be identical to the protein called uvomorulin or L-CAM, which is a transmembrane cell surface glycoprotein of 125 kDa. N-cadherin is expressed in the brain and skeletal and cardiac muscle and is likely to be identical to the protein called ACAM or N-CAL-CAM (Volk and Geiger, 1986; Crittenden et al., 1988; Hatta et al., 1988). Extensive homology between the three main cadherin members in-
Received November 23, 1989; revised manuscript received January 6, 1990; accepted January 29, 1990.
Abbreviations used: CAM, cell adhesion molecule; E, N, and P, epithelial, neural, and placental; Ig, immunoglobulin; SDS, sodium dodecyl sulphate.
Address correspondence and reprint requests to Prof. F. S. Walsh at Department of Experimental Pathology, Guy’s Hospital, London Bridge, London SEI 9RT, U.K.
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dicates that they constitute a discrete gene family. Considerable evidence suggests that, like N-CAM and other members of the Ig superfamily, cadherins recognize and bind via a hornophilic mechanism (Takeichi, 1987). The region of greatest similarity within the cadherin gene family is found in the cytoplasmic domain, while extracellular regions have diverged quite considerably. This may explain the observations that each cadherin member binds only to itself and generally not to other cadherins in model systems. Indeed, the specificity is so restricted that it allows cells to sort out in vitro (Nose et al., 1988). The molecular structure of the cadherin genes is unclear at present and only E-cadherin has been studied in detail. The intron-exon structure of the E-cadherin gene in chicken (L-CAM) has been described (Sorkin et al., 1988), and E-cadherin has also been mapped to chromosome 8 in the mouse (Eistetter et al., 1988) and 16q in the human (Mansouri et al., 1988). However, little is known about the N- or P-cadherin genes and the molecular basis of the relationship between these genes is unclear. To approach these questions, cDNA clones encoding human N-cadherin have been isolated. Human N-cadherin is highly homologous to N-cadherin in chicken and mouse; in addition, we show that the human N-cadherin gene is located on chromosome 18. Thus, each cadherin member is likely to have arisen by gene duplication followed by dispersal to different chromosomal sites rather than alternative splicing or exon duplication of a single gene. MATERIALS AND METHODS Isolation of human N-cadherin cDNAs A cDNA library produced in Xgtl I from poly(A)+ RNA isolated from mixed cultures of human foetal muscle was screened by filter plaque hybridization ( I O5 recombinants) with a 43-base oligomer probe complementary to base pairs (bp) 2,369-2,412 of the chick N-cadherin sequence and encoding sequence within the cytoplasmic domain (Hatta et al., 1988). Hybridizations were performed overnight at 42°C in 3 X SSC (1 X SSC = I50 mM NaCI, 15 mM sodium citrate), 0.5%sodium dodecyl sulphate (SDS), 5 X Denhardt's, and 100 pg/ml denatured herring sperm DNA with 200 ng of end-labelled oligonucleotide probe. Filters were washed in 2 X SSC and 0.1% SDS at 37°C for 3 h and positive recombinants were purified by recloning. EcoRI inserts were prepared from positive plaques and were subcloned into the EcoRI site of M 13mp18 (Maniatis et al., 1982). Recombinant M I 3 subclones were isolated and DNA sequence analyses were performed by preparing a series of nested deletions (Henikoff, 1984), which were sequenced with Sequenase 2 and the dideoxy chain termination method. Sequence analysis in the reverse direction was performed by priming the o p posite orientation M 13 subclone with 17-baseoligonucleotide primers at 300-bp intervals generated to the sequence of the first strand or by use of deletion mutants prepared from the opposite orientation subclone. The sequence of clones isolated by this method encoded only the membrane proximal and cytoplasmic domains of human muscle N-cadherin. Additional 5' clones were isolated and analysed as described previously, by screening the same library with a chick brain
J. Neitrorliem , Val. 55. No. 3. 1990
cDNA probe,, XN2, bp 332-1,558. Hybridizations with cDNA probes (Dickson et al., 1986) were performed at 42°C in 50% formamide, 5 X Denhardt's, 5 X SSPE ( I X SSPE = 150 mMNaCI, 1 mMNaH2P04, 1 mM EDTA), 0. I % SDS, and 100 ng/ml denatured hemng sperm DNA. cDNA probes were labelled to a specific activity of lo9 cpm/pg using the multiprime rapid DNA labelling system (Amersham). Fillers were washed at 68°C for 60-90 min in 0.5 X SSC and 0.1% SDS.
-
Nucleic acid blot analysis Poly(A)+ RNA was isolated, fractionated by electrophoresis on I% glyoxal-agarose (Dickson et a]., 1986), and blotted onto Genescreen membranes as described by the manufacturer (NEN). Filters were probed with human muscle N-cadherin cDNA clone X I - 5 labelled to a specific activity of lo9 cpm/pg using the multiprime rapid labelling system (Amersham) in 50% formamide, 10%dextran sulphate, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, 0.2% Ficoll, 0.05 M Tris-HCI (pH 7 . 9 , 1 M NaCI, 0.1% sodium pyrophosphate, 1.0% SDS, and 100 pg/ml denatured hemng sperm DNA. Genomic DNA (10 pg) prepared from the somatic cell hybrids was digested to completion with EcoRI, fractionated on 0.7% agarose gels, blotted to nitrocellulose filters, and hybridized with 32P-labeled human muscle N-cadherin cDNA insert X I S as described (Walsh et al., 1986).
-
Somatic cell hybrids Interspecific somatic cell hybrids containing defined numbers of human chromosomes have been described previously and are listed in Table 1.
RESULTS Isolation and characterization of human N-cadherin cDNA clones The nucleotide and derived amino acid sequence for a full-length cDNA clone, XN2, encoding an 8 1.8kDa N-cadherin found in chick brain has recently been published (Hatta et al., 1988). A 43-base oligomer probe corresponding to 2,369-3,4 12 bp of the chick sequence was synthesized by phosphoramidate chemistry and used to screen a human muscle cDNA library in Xgt 1 1. Recombinants were identified by filter plaque hybridization and following plaque purification were prepared by plate lysis. Two clones, X1.5 and X4.10, from the nine isolates of the lo5 recombinants screened, with overlapping restriction endonuclease maps, contained sequence homologous to XN2. The largest clone, X4.10, failed to extend further 5' than nucleotide 1,794 in the chick sequence. The remaining seven clones exhibited differing restriction enzyme maps and were not studied further. Clones further 5' were isolated by screening the human muscle library with a Hind111 subfragment of AN2 (322-1,558 bp). One of the isolated recombinants, X14, contained two EcoRI fragments of -300 and 1,400 bp, both of which exhibited sequence homologous to chick XN2. Clones h1.5, X4.10, X14, and X 13 were fully characterized by DNA sequence analysis of M 13mp 18 subclones. Nested deletions of M 13 subclones were prepared by unidirectional deletion with exonuclease I11 (HenikofF, 1984) and appropriately
807
HUMAN N-CADHERIN TABLE 1. Sornutic cell hybrids usedfor mapping of hurnan N-cadherin gcne Human chromosome Cellline
I
2
3
4
5
6
7
8
9
10
12
II
13
14
15
16
17
18
19
20
21
22
X
N-Cadherin
+
CTP34B4 DT1.2.4 DT1.2 MOG34A4 FGI0 FST9/10 TWIN I9-Dl2 RJAZ RJDI DIS 20 Mogl3/10 HORP9.5 DUR4R3 SIF15 SIF4A3I 3W4CL5 CTP4 I A2
Reference i 7
3 4
5
+
6 7 8 9 10 11 12
13 14 15 16 17
Assignment of :he human N-cadherin gene to chromosome 18 was determined by Southern analysis of DNA from the 17 cell hybnds. Of the nine positive cell hybrids. the human chromosome 18 was the only common element. The table shows the human chromosomes contained in the hybrid cells (+ indicates presence, - indicates absence. t indicates not tested, and T indicates X/I 5 translocation). The presence or absence of the I .4-kb human N-cadherin gene fragment is indicated in the N-Cadherin column. The original descriptions of the cell hybrids used are as follows: ( I ) Jones et al.. 1976; ( 2 )Swallow et al., 1977; (3) Swallo\v el al., 1977; (4) Solomon el al., 1979; (5) Kielty et al.. 1982; (6) Kielty et al.. 1982; (7) Phillips et al., 1985: (8) Human Genetic Resources, ICRF; (9) Human Genetic Resources, I C R F (10) Heisterkamp et al.. 1982; (1 I ) Povey et al.. 1980 (12) Van Heynigen et al.. 1975; (13) Solomon et al.. 1976 (14) Edwards et al., 1985: (15) Edwards et al.. 1985; (16) Nabholz et al.. 1969: (17) Jones et al.. 1976.
sized selected subclones sequenced by using the chain termination methodology and sequence 11. The alignment of characterized human muscle Ncadherin cDNA clones is shown in Fig. 1 relative to
the full-length chick brain N-cadherin cDNA clone XN2. The combined sequences of clones X 14 and X4.10 are colinear with AN2 and encode a fully processed, mature N-cadherin protein; however, human clone A14
E
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FIG. 1. Alignment of human N-cadherin clones with the full-length clone XN2, showing the extent of the open reading frame. Restriction maps for human N-cadherin cDNA clones are aligned for sequence homology with a full-length chick clone. Restriction endonuclease cleavage sites are shown for 6arnHl (B), Hindlll (H), EcoRl (E), and Xmnl (X). The major open reading frame is depicted as a bar, signal peptide as a dotted bar, prepeptide as a hatched bar, and the hydrophobic membrane spanning domain as a filled bar; within the 3’ untranslated sequence (line), the positions of the consensus sequences for polyadenylation addition are indicated.
J A;etrrocheni , I’d 55. A’o 3, 1990
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FIG. 2. Above and opposite page: Comparison of nucleotide (a) and amino acid (b)sequence of chick and human N-cadherin. Nucleotide and derived amino acid sequences were compared over the length of the human N-cadherin mature protein and including 3' untranslated(a). The nucleotide similarity for chick and human is 77.8% over 2,696 bp and this increases to 91% over 747 amino acid residues not including conservativechanges. Consensus pdyadenylation addition sequences (ATTAAA) are underlined above and below the respective sequences as are EcoRl endonuclease cleavage sites. For the amino acid comparison, the hydrophobic membrane spanning domain is highlighted (underlined), showing the extensive homology within the cytoplasmic domain. Also indicated are the consensus sequences for N-linked glycosylation(r) above and below the respective sequences. Amino acid mismatches between the two sequences are indicated(*).
J Neurorhem., Vol 55, No. 3. 1990
HUMAN N-CADHERIN
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does not extend beyond or up to the initiation Met residue in chick XN2, but terminates at an Ala 16 residues 3’ of the start codon. In Fig. 2, comparisons are drawn between the full-length chick brain and derived human muscle DNA and protein sequences starting from the first codon/amino acid of the mature protein identified by direct protein sequencing (Hatta et al., 1988). At the DNA level, from the Asp codon, there is 77.8% similarity between chick and human sequences. However, clone X4.10 extends 77 1 bp farther 3’ and beyond the polyadenylation addition recognition sequence (ATTAAA) at 3,180 bp in XN2. Clone X4.10 does contain an analogous motif in a similar location, which is duplicated 7 bp farther 3’. The RNA from which X4.10 was derived clearly utilizes another more 3’ polyadenylation site and would consequently be derived from a different RNA size class. Comparisons between the derived amino acid sequence of the mature
748 912
human muscle N-cadherin revealed 9 I .O and 96.5% similarity for chick and mouse, respectively; this compares with a value of 9 I .8% between chick and mouse. The cytoplasmic region of the molecule shows a particularly high degree of conservation with only two and three mismatches in 160 residues between chick and mouse, respectively. Eight consensus sites for N-linked glycosylation were observed in the human sequence, including a consensus site within the cytoplasmic domain, and this compares with nine sites in chick and six sites in mouse. Northern analysis of N-cadherin DNA sequence analysis of human muscle clones has indicated that variability might exist for the N-cadherin mRNAs. To investigate this possibility, Northern blots of poly(A)+ RNA from C6 glioma, human foetal muscle, C2 myotube cultures, human embryonic brain, and J. iVeurochem , Vol. 55, No. 3, 1990
F. S. M'ALSH ET AL.
810 a
b
c
Chromosomal mapping of the human N-cadherin gene To determine the chromosomal location of the human N-cadherin gene, cDNA X 1.5 was used to probe genomic DNA digested with EcoRI from a panel of somatic cell hybrids. A 1.4-kb EcoRI fragment was diagnostic for the human N-cadherin gene and bands of 7.8, 8.0, and 8.4 kb for the mouse, hamster, and rat genes, respectively. A panel of 17 hybrids was then evaluated for the presence of the 1.4-kb EcoRI fragment indicative of the human N-cadherin (Fig. 4). Of the 17 hybrids analysed, positive 1.4-kb bands were identified for 9 cell hybrids. This unambiguously indicates that the human N-cadherin gene is located on chromosome 18 (Table 1).
e
d
5.2-
-28s
4.34.0-
-18s FIG. 3. Northern analysis of N-cadherin RNAs. Multiple RNA bands for N-cadherin transcripts were identified by Northern blot hybridization. Poly(A)+ RNA (8 pj) was probed with 32P-labeledcDNA X1.5. Exposures were selected to show heterogeneity of bands. Sizes of RNA in kilobases are shown on the left with the migration of 28s and 18s standards on the right. Lane a, adult mouse brain; lane b, embryonic human brain; lane c, C2 myotube; lane d, embryonic human muscle; lane e, C6 glioma cells.
DISCUSSION The Ca*'-dependent CAM N-cadherin has been found to be an important regulator of cell-cell interactions in the nervous system. Immunoperturbation studies have clearly shown that this homophilic CAM plays a dominant role in the process of neurite outgrowth over various cellular substrata such as astrocytes, Schwann cells, and myotubes (Bixby et al., 1987; Neugebauer et al., 1988; Tomaselli et al., 1988). Although multiple adhesive interactions may be operative in each of these model systems, N-cadherin appears to be important since antibody blockade leads to the greatest percentage decrease in neurite outgrowth. All studies on N-cadherin in the nervous system have shown that it is a single polypeptide chain of 125 kDa with no evidence to date of extensive microhet-
mouse brain were probed with cDNA from clone X1.5 (Fig. 3). As previously described for chick brain RNA, a single major band of 4.3 kb was identified in both embryonic human and adult mouse brain RNAs, whereas in C6 glioma additional RNA bands of 5.2 and 4.0 kb were identified. Consistent with an alternative choice of polyadenylation site selection indicated from analysis of muscle cDNAs, a minor 5.2-kb RNA band was present in samples from this source in addition to major bands of 4.0 and 4.3 kb. A similar minor band of 5.2 kb was also found in RNAs from embryonic human brain.
A
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-7.8
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FIG. 4. Chromosomal localization of the human N-cadherin gene. The chromosomal location of the N-cadherin gene was determined by probing a Southern blot of EcoRI-digested genomic DNA from the indicated cell hybrids. Characteristic bands of 1.4, 7.8, 8.0, and 8.4 kb were identified for human, mouse, hamster, and rat DNA, respectively. Hybrids positive for the human fragment (1.4 kb) are marked (+), while those that are negative are marked (-). Lane A, CTP34b4 (+); lane B, CTP41A2 (-); lane C, 3W4CL5 (-); lane D. SIF15 (-); lane E, SIF4A31 (-); lane F. DUR4R3 (-); lane G, SlR74ii (-); lane H, DTI .2.4 (+); lane I, DT1.2 (+); lane J, HORP9.5 (-); lane K, MOG34A4 (+); lane L, FGlO (+); lane M. FST9/10 (+); lane N, Twin 19-D12 (+); lane 0, human DNA; lane P, mouse DNA, lane Q, RJDl (+); lane R, RJA2 (+); lane S, FIR5 (+); lane T, CTP34B4 (+); lane U, MOG13/10 (-); lane V, DIS 20 (-); lane W, SIF4A31 (-).
J. Neirrochern , Val. 55, No. 3. 1990
HUMAN N-CADHERIN erogeneity due to specific glycosylation as has been found for other CAMs such as N-CAM. The limited molecular heterogeneity and relatively ubiquitous expression of N-cadherin on all classes of neurons have led to the suggestion that this protein may be involved in initial cell adhesion but may not have the ability to mediate specific pathway guidance (Dodd and Jessell, 1988). However, in the present study we have shown heterogeneity at the RNA level, which may reflect changes in the primary amino acid sequence. Further studies will be required to resolve this issue. The recent cloning of chicken N-cadherin and its subsequent expression in L-cells and neuroblastoma cells have shown that neurons from the optic nerve can recognize and use this molecule as a guidance cue (Matsunaga et al., 1988). In this study cDNA clones for the full coding sequence of the mature, fully processed human muscle N-cadherin have been isolated. Two approaches were utilized to obtain this information: First, a synthetic oligonucleotide to a region likely to be best conserved across species identified cytoplasmic and membrane proximal domain sequences, whereas a cDNA subfragment of AN2 yielded the remaining sequence. We have no direct evidence to date, in the form of overlapping clones, indicating that human clones X4.10 and h I4 are derived from the same RNA species, although it is highly likely. The sequence spanning the junction of clones X4.10 and XI4 is colinear with the chick sequence, although direct proof will require additional RNA analyses. The human sequence is likely to utilize a similar initiating Met codon as the chick and mouse genes, since the human sequence shows strong similarity for the first 30 residues particularly with the mouse. It is of interest to note that the 5amino acid deletion in the mouse compared to the chick sequence is also present in the human. The conservation of amino acid sequence within the cytoplasmic domain is particularly high with two (99%) and three (98%) mismatches in I60 residues for chick and mouse, respectively, compared to the human sequence. The maintenance of this amino acid sequence suggests that it has an important and conserved role for N-cadherin function in all species. Evidence for alternative polyadenylation site selection has been obtained through study of human N-cadherin clones since the 3' end of the human cDNA clone X4.10 extends beyond the 3' end of the used polyadenylation site in chick, but contains a duplicated site in an analogous position to that of the chick transcript. It is not known whether the usage of additional polyadenylation sites represents an alternative splicing choice to utilize separate exons or usage of two sites in a single exon. In the N-CAM gene both situations occur, with the closest parallel being the 2.9- and 5.2-kb mRNAs, which have in common three coding sequences. To date, only one of the three cadherin members has been mapped to its respective chromosome. E-cadherin has been mapped to chromosome 8 in the mouse
811
(Eistetter et al., 1988) and chromosome 16q in the human (Mansouri et al., 1988). To understand more fully the evolution of this gene family, it is important to extend this analysis to other cadherin members. In the present study, the human N-cadherin gene was shown to be present on chromosome 18 and is therefore clearly not a spliced derivative of E-cadherin. Thus, the functional specificity of the N- and E-cadherin gene products arose via evolution following duplication of a single gene. There appear to be some parallels with the Ig superfamily (Edelman, 1987; Williams, 1987), except that the cadherins have not evolved beyond tissue specific adhesion while the Ig gene superfamily contains a much greater diversity of function. Chromosome I8 has not been found to encode any protein that is highly expressed in the nervous system except myelin basic protein (Sparkes et al., 1987). It will be of interest to determine the subchromosomal location of the N-cadherin gene since there are a number of tumours associated with breakpoints of this chromosome and in particular a region on 18q that is likely involved in tumour suppression in colorectal cancers (Fearon et al., 1990). Acknowledgment: This work was supported by grants from the Muscular Dystrophy Group of Great Britain and the Wellcome Trust.
REFERENCES Barton C. H., Dickson G., Cower H. J., Rowett L. H., Putt W.. Elsom V., Moore S. E.. Goridis C.. and Walsh F. S. (1988) Complete sequence and in v i m expression of a tissue specific phosphatidylinositol-linked N-CAM isoform from skeletal muscle. Development 104, 165-173. Bixby J. L., Pratt R. S., Lilien J.. and Reichardt L. F. (1987) Neurite outgrowth on muscle cell surfaces involves extracellular matrix receptors as well as Ca2+-dependentand -independent cell adhesion molecules. Proc. Nu//. Acad. Sci C'SA 84, 2555-2559. Crittenden S. L., Rutishauser U.. and Lilien J. (1988) Identification of two structural types of calciumdependent adhesion molecules in the chicken embryo. Proc. Nut/. .4cad. Sci. CTS.4 85, 34643468. Dickson J. G., Prentice H. M.. Kenimer J. G.. and Walsh F. S. (1986) Identification and characterisation of neuron-specific and developmentally regulated gene transcripts in the chick embryo spinal cord. J. Neirrocliein. 46, 787-793. Dodd J. and Jessell T. M. (1989) Axon guidance and the patterning of neuronal projections in vertebrates. Science 242,692-699. Edelman G. M. ( 1 986) Cell adhesion molecules in the regulation of animal form and tissue pattern. ,4nnit. Rev. Ce// Biol 2, 81116. Edelman G. M. (1987) CAMs and Igs: cell adhesion and the evolutionary origins of immunity. Iin~nzrnd.Rev. 100, 11-45. Edwards Y. H., Parkar M., Povey S., West L. F.. Fanington F. M.. and Solomon E. (1985) Human myosin heavy chain genes assigned to chromosome I 7 using a human cDNA clone as probe. Ann. Hitm. Gene/. 49, 101-109. Eistetter H. R., Adolph S.. Ringwald M., Simon-Chazottes D., Schuh R., Guenet J. L., and Kemler R. (1988) Chromosomal mapping of the gene coding for the mouse cell adhesion molecule uvomorulin. Proc. Nut/. k u d . Sci. C?S.485, 3489-3493. Fearon E. R.. Cho K. R., Nigro J . M., Kern S. E., Simons J. W., Ruppert J. M., Hamilton S. R.. Presinger A. C., Thomas G., Kinzler K. W.. and Vogelstein B. (1990) Identification ofa chro-
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mosome 18q gene that is altered in colorectal cancers. Science 247,49-56. Gallin W. J., Sorkin B. C., Edelman G. M., and Cunningham B. A. (1987) Sequence analysis of a cDNA clone encoding the liver cell adhesion molecule (L-CAM). Proc. Null. Acad. Sci. USA 84,2808-28 12. Hatta K., Okada T. S., and Takeichi M. (1985) A monoclonal antibody disrupting calcium-dependent cell-cell adhesion of brain tissues: possible role of its target antigen in animal pattern formation. Proc. Nail. rlcad. Sci. USA 82, 2789-2793. Hatta K., Nose A,, Nagafuchi A,, and Takeichi M. (1988) Cloning and expression of cDNA encoding a neural calciumdependent cell adhesion molecule: its identity in the cadherin gene family. J. Cell Biol. 106, 873-88 1. Heisterkamp N., Groffen J., Stephenson J. R., Spurr N. K., Goodfellow P. N., Solomon E.. Camtt B., and Bodmer W. F. (1982) Chromosomal localization of human cellular homologues of two viral oncogenes. Nature 299, 747-749. Henikoff S. (1984) Unidirectional digestion with exonuclease 111creates targeted breakpoints for DNA sequencing. Gene 28, 351359. Jones E. A,. Goodfellow P. N.. Kennett R. H., and Bodmer W. F. (1976)The independent expression of HLA and BZmicroglobulin on human-mouse hybrids. Somatic Cell Genet. 2,483-496. Kielty C. M., Povey S.. and Hopkinson D. A. (1982) Regulation of expression of liver-specific enzymes 111. Further analysis of a series of rat hepatoma X human somatic cell hybrids. Ann. Hum. Genet. 46, 307-363. Maniatis T.. Fritsh, E. F., and Sambrook J. (1982)Molecular Cloning: A Lahoratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Mansouri M., Spurr N., Goodfellow P. N., and Kemler R. (1988) Characterisation and chromosomal localization of the gene encoding the human cell adhesion molecule uvomorulin. DiJerentiation 38, 67-7 I . Matsunaga M.. Hatta K.. Nagafuchi A,, and Takeichi M. (1988) Guidance of optic nerve fibres by N-cadherin adhesion molecules. Nature 334, 62-64. Miyatani S., Shimamura K., Hatta M., Nagafuchi A., Nose A., Matsunaga M., Hatta K., and Takeichi M. (1989) Neural cadherin: role in selective cell-cell adhesion. Science 245, 63 1-635. Nabhloz M., Miggiano V., and Bodmer W. (1969) Genetic analysis with human-mouse somatic cell hybrids. Nafure 223, 358-363. Nagafuchi A,, Shirayoshi Y., Okazaki K., Yasuda K., and Takeichi M. (1987) Transfection of cell adhesion properties by exogenously introduced E-cadherin cDNA. Nature 329, 341-343. Neugebauer K. M.. Tomaselli K. J., Lilien J.. and Reichardt L. F. (1988) N-Cadherin, N-CAM, and integrins promote retinal neurite outgrowth on astrocytes in vilro. J. Cell Biof. 107, 1 1771187. Nose A. and Takeichi M. (1986) A novel cadherin cell adhesion molecule: its expression patterns associated with implantation and organogenesis of mouse embryos. J . Cell Biol. 103, 26492658. Nose A., Nagafuchi A,, and Takeichi M. (1987)Isolation of placental cadherin cDNA: identification of a novel gene family of cellcell adhesion molecules. EMBO J. 6, 3655-366 I . Nose A., Nagafuchi A,, and Takeichi M. (1988) Expressed recombinant cadherins mediate cell sorting in model systems. Cell 54, 993- 1001. Phillips J. R., Shephard E. A,, Povey S., Davis M. B., Kelsey G.,
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Monteiro M., West L. F., and Cowell J. (1985) A cytocrome P450 gene family mapped to human chromosome 19. Ann. Hum. Genet. 49,267-274. Povey S., Jeremiah S. J., Barker R. F., Hopkinson D. A., Robson E. B., Cook P. J. L., Solomon E., Bobrow M., Camtt B., and Buckton K. E. (1 980) Assignment of human locus determining phosphoglycolate phosphatase (PGP) to chromosome 16. Ann. Hum. Genet. 43, 241-248. Ringwald M., Schuh R., Vestweber D., Eistetter H., Lottspeich F., Engel J., Dolz R., Jahnig F., Epplen J., Mayer S., Muller C., and Kemler R. (1987) The structure of cell adhesion molecule uvomorulin. Insights into the molecular mechanism of Ca++-dependentcell adhesion. EMBO J. 6,3647-3653. Shimoyama Y., Yoshida T., Terada M., Shimosato Y., Abe O., and Hirohashi S. (1989) Molecular cloning of a human Ca2+dependent ceU-ceU adhesion molecule homologous to mouse placental cadherin: its low expression in human placental tissues. J. Cell Bi01. 109, 1787-1794. Solomon E., Bobrow M., Goodfellow P. N., Bodmer W. F., Swallow D. M., Povey S., and Noel B. (1976) Human gene mapping using an X/autosome translocation. Somaiic Cell Genet. 2, 125140. Solomon E., Swallow D., Burgess S., and Evans L. (1979) Assignment of the human glucosidase gene (GLU) to chromosome I7 using somatic cell hybrids. Ann. Hum. Genet. 42, 273-28 1 . Sorkin B. C., Hemperly J. J., Edelman G. M., and Cunningham B. A. (1988) Structure of the gene for the liver cell adhesion molecule, L-CAM. Proc. Nail. Acad. Sci. USA. 85, 76 17-762 1. Sparkes R. S., Mohandas T., Heinzmann C., Roth H. J., Klisak I., and Campagnoni A. T. (1987) Assignment of the myelin basic protein gene to human chromosome I8q22qter. Hum. Genet. 75, 147-150. Swallow D. M., Solomon E., and Pajunen L. (1977) Immunochemical analysis of the N-acetyl hexosaminidases in human-mouse hybrids made using a double selective system. C~mgenet.Cell Genef. 18, 136-148. Takeichi M. (1987) Cadherins: a molecular family essential for selective cell-cell adhesion and animal morphogenesis. Trends Genet. 3, 213-217. Tomaselli K. J., Neugebauer K. M., Bixby J. L., Lilien J., and Reichardt L. F. (1988) N-Cadherins and integrins: two receptor systems that mediate neuronal process outgrowth on astrocyte surfaces. Neuron 1 , 33-43. Van Heyningen V., Bobrow M., Bodmer W. F., Gardiner S. E., Povey S., and Hopkinson D. A. (1975) Chromosome assignment of some human loci: mitochondria1 malate dehydrogenase to 7, mannose phosphate isomerase and pyruvate kinase to 15 and probably esterase D to 13. Ann. Hum. Genei. 38, 295-303. Volk T. and Geiger B. (1986) A-CAM: a 135Kd receptor of intercellular adherens junctions I. Immunodetection, microscopic localization and biochemical studies. J. Cell Biol. 103, 14411450. Walsh F. S., Putt W., Dickson J . G., Quinn C. A,, Cox R. D., Webb M., Spurr N., and Goodfellow P. N. (1986) Human N-CAM gene; mapping to chromosome I 1 by analysis of somatic cell hybrids with mouse and human cDNA probes. Mol. Brain Res. 1, 197-200. Williams A. F. (1987) A year in the life of the immunoglobulin superfamily. Immunol. Today 8,298-303. Yoshida C. and Takeichi M. (1982) Teratocarcinoma cell adhesion: identification of a cell-surface protein involved in calciumdependent cell aggregation. Cell 28, 2 17-224.
N-Cadherin Gene Maps to Human Chromosome 18 and Is Not Linked to the E-Cadherin Gene Frank S. Walsh, C. Howard Barton, Wendy Putt, Stephen E. Moore, *David Kelsell, *Nigel Spun, and *Peter N. Goodfellow Department of Experimental Pathology, UMDS, Guy’s Hospital, and *Imperial Cancer Research Fund, London, England
Abstract: cDNA clones encoding the human N-cadherin cell adhesion molecule have been isolated from an embryonic muscle library by screening with an oligonucleotide probe complementary to the chick brain sequence and chick brain cDNA probe XN2. Comparison of the predicted protein sequences revealed > 9 1% homology between chick brain, mouse brain, and human muscle N-cadherin cDNAs over the 748 amino acids of the mature, processed protein. A single polyadenylation site in the chick clone was also present and duplicated in the human muscle sequence. Immediately 3’ of the recognition site in chick a poly(A) tail ensued; however, in human an additional 800 bp of 3‘ untranslated sequence followed. Northern analysis identified a number of major Ncadherin mRNAs. These were of 5.2, 4.3, and 4.0 kb in C6
glioma, 4.3 and 4.0 kb in human foetal muscle cultures, and 4.3 kb in human embryonic brain and mouse brain with minor bands of 5.2 kb in human muscle and embryonic brain. Southern analysis of a panel of somatic cell hybrids allowed the human N-cadherin gene to be mapped to chromosome 18. This is distinct from the E-cadherin locus on chromosome 16. Therefore, it is likely that the cadherins have evolved from a common precursor gene that has undergone duplication and migration to other chromosomal locations. Key Words: Cell adhesion molecules-N-Cadherin-Gene mapping. Walsh F. S. et al. N-Cadherin gene maps to human chromosome 18 and is not linked to the E-cadherin gene. J. Neurochem. 55, 805-8 12 ( 1990).
The formation of selective adhesive interactions between cell surfaces in the nervous system is essential for the processes of cell recognition, sorting, and migration during development and in tissue regeneration (Edelman, 1986). The molecular basis of cell adhesion is being extensively investigated and these studies indicate that a number of adhesive interactions result from the expression of a relatively small number of cell adhesion molecules (CAMs). Two classes ofCAMs have been identified according to their requirement for function of extracellular Ca2+.Several Ca2+-independent CAMs such as N-CAM have been classified as members of the immunoglobulin (Ig) gene superfamily of recognition molecules based on sequence analysis (Edelman, 1987; Williams, 1987; Barton et al., 1988). Those studies suggested that the Ca2+-independent CAMs belonging to the Ig gene superfamily have evolved from a multifunctional primordial CAM, with tissue-specific CAMs arising much later in evolution (Edelman, 1987).
A putative gene family mediating Ca2+-dependent adhesion (the cadherins) has been identified (Takeichi, 1987), with the present three members of this family, called epithelial (E; Yoshida and Takeichi, 1982), neural (N; Hatta et al., 1985), and placental (P; Nose and Takeichi, 1986) cadherins, exhibiting precise patterns of cell and tissue expression. The cadherins have recently been cloned and the sequences of E-cadherin (Gallin et al., 1987; Nagafuchi et al., 1987; Ringwald et al., 1987; Sorkin et al., 1988), P-cadherin (Nose et al., 1987; Shimoyama et al., 1989), and N-cadherin (Hatta et al., 1988; Miyatani et al., 1989) reported. ECadherin appears to be identical to the protein called uvomorulin or L-CAM, which is a transmembrane cell surface glycoprotein of 125 kDa. N-cadherin is expressed in the brain and skeletal and cardiac muscle and is likely to be identical to the protein called ACAM or N-CAL-CAM (Volk and Geiger, 1986; Crittenden et al., 1988; Hatta et al., 1988). Extensive homology between the three main cadherin members in-
Received November 23, 1989; revised manuscript received January 6, 1990; accepted January 29, 1990.
Abbreviations used: CAM, cell adhesion molecule; E, N, and P, epithelial, neural, and placental; Ig, immunoglobulin; SDS, sodium dodecyl sulphate.
Address correspondence and reprint requests to Prof. F. S. Walsh at Department of Experimental Pathology, Guy’s Hospital, London Bridge, London SEI 9RT, U.K.
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dicates that they constitute a discrete gene family. Considerable evidence suggests that, like N-CAM and other members of the Ig superfamily, cadherins recognize and bind via a hornophilic mechanism (Takeichi, 1987). The region of greatest similarity within the cadherin gene family is found in the cytoplasmic domain, while extracellular regions have diverged quite considerably. This may explain the observations that each cadherin member binds only to itself and generally not to other cadherins in model systems. Indeed, the specificity is so restricted that it allows cells to sort out in vitro (Nose et al., 1988). The molecular structure of the cadherin genes is unclear at present and only E-cadherin has been studied in detail. The intron-exon structure of the E-cadherin gene in chicken (L-CAM) has been described (Sorkin et al., 1988), and E-cadherin has also been mapped to chromosome 8 in the mouse (Eistetter et al., 1988) and 16q in the human (Mansouri et al., 1988). However, little is known about the N- or P-cadherin genes and the molecular basis of the relationship between these genes is unclear. To approach these questions, cDNA clones encoding human N-cadherin have been isolated. Human N-cadherin is highly homologous to N-cadherin in chicken and mouse; in addition, we show that the human N-cadherin gene is located on chromosome 18. Thus, each cadherin member is likely to have arisen by gene duplication followed by dispersal to different chromosomal sites rather than alternative splicing or exon duplication of a single gene. MATERIALS AND METHODS Isolation of human N-cadherin cDNAs A cDNA library produced in Xgtl I from poly(A)+ RNA isolated from mixed cultures of human foetal muscle was screened by filter plaque hybridization ( I O5 recombinants) with a 43-base oligomer probe complementary to base pairs (bp) 2,369-2,412 of the chick N-cadherin sequence and encoding sequence within the cytoplasmic domain (Hatta et al., 1988). Hybridizations were performed overnight at 42°C in 3 X SSC (1 X SSC = I50 mM NaCI, 15 mM sodium citrate), 0.5%sodium dodecyl sulphate (SDS), 5 X Denhardt's, and 100 pg/ml denatured herring sperm DNA with 200 ng of end-labelled oligonucleotide probe. Filters were washed in 2 X SSC and 0.1% SDS at 37°C for 3 h and positive recombinants were purified by recloning. EcoRI inserts were prepared from positive plaques and were subcloned into the EcoRI site of M 13mp18 (Maniatis et al., 1982). Recombinant M I 3 subclones were isolated and DNA sequence analyses were performed by preparing a series of nested deletions (Henikoff, 1984), which were sequenced with Sequenase 2 and the dideoxy chain termination method. Sequence analysis in the reverse direction was performed by priming the o p posite orientation M 13 subclone with 17-baseoligonucleotide primers at 300-bp intervals generated to the sequence of the first strand or by use of deletion mutants prepared from the opposite orientation subclone. The sequence of clones isolated by this method encoded only the membrane proximal and cytoplasmic domains of human muscle N-cadherin. Additional 5' clones were isolated and analysed as described previously, by screening the same library with a chick brain
J. Neitrorliem , Val. 55. No. 3. 1990
cDNA probe,, XN2, bp 332-1,558. Hybridizations with cDNA probes (Dickson et al., 1986) were performed at 42°C in 50% formamide, 5 X Denhardt's, 5 X SSPE ( I X SSPE = 150 mMNaCI, 1 mMNaH2P04, 1 mM EDTA), 0. I % SDS, and 100 ng/ml denatured hemng sperm DNA. cDNA probes were labelled to a specific activity of lo9 cpm/pg using the multiprime rapid DNA labelling system (Amersham). Fillers were washed at 68°C for 60-90 min in 0.5 X SSC and 0.1% SDS.
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Nucleic acid blot analysis Poly(A)+ RNA was isolated, fractionated by electrophoresis on I% glyoxal-agarose (Dickson et a]., 1986), and blotted onto Genescreen membranes as described by the manufacturer (NEN). Filters were probed with human muscle N-cadherin cDNA clone X I - 5 labelled to a specific activity of lo9 cpm/pg using the multiprime rapid labelling system (Amersham) in 50% formamide, 10%dextran sulphate, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, 0.2% Ficoll, 0.05 M Tris-HCI (pH 7 . 9 , 1 M NaCI, 0.1% sodium pyrophosphate, 1.0% SDS, and 100 pg/ml denatured hemng sperm DNA. Genomic DNA (10 pg) prepared from the somatic cell hybrids was digested to completion with EcoRI, fractionated on 0.7% agarose gels, blotted to nitrocellulose filters, and hybridized with 32P-labeled human muscle N-cadherin cDNA insert X I S as described (Walsh et al., 1986).
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Somatic cell hybrids Interspecific somatic cell hybrids containing defined numbers of human chromosomes have been described previously and are listed in Table 1.
RESULTS Isolation and characterization of human N-cadherin cDNA clones The nucleotide and derived amino acid sequence for a full-length cDNA clone, XN2, encoding an 8 1.8kDa N-cadherin found in chick brain has recently been published (Hatta et al., 1988). A 43-base oligomer probe corresponding to 2,369-3,4 12 bp of the chick sequence was synthesized by phosphoramidate chemistry and used to screen a human muscle cDNA library in Xgt 1 1. Recombinants were identified by filter plaque hybridization and following plaque purification were prepared by plate lysis. Two clones, X1.5 and X4.10, from the nine isolates of the lo5 recombinants screened, with overlapping restriction endonuclease maps, contained sequence homologous to XN2. The largest clone, X4.10, failed to extend further 5' than nucleotide 1,794 in the chick sequence. The remaining seven clones exhibited differing restriction enzyme maps and were not studied further. Clones further 5' were isolated by screening the human muscle library with a Hind111 subfragment of AN2 (322-1,558 bp). One of the isolated recombinants, X14, contained two EcoRI fragments of -300 and 1,400 bp, both of which exhibited sequence homologous to chick XN2. Clones h1.5, X4.10, X14, and X 13 were fully characterized by DNA sequence analysis of M 13mp 18 subclones. Nested deletions of M 13 subclones were prepared by unidirectional deletion with exonuclease I11 (HenikofF, 1984) and appropriately
807
HUMAN N-CADHERIN TABLE 1. Sornutic cell hybrids usedfor mapping of hurnan N-cadherin gcne Human chromosome Cellline
I
2
3
4
5
6
7
8
9
10
12
II
13
14
15
16
17
18
19
20
21
22
X
N-Cadherin
+
CTP34B4 DT1.2.4 DT1.2 MOG34A4 FGI0 FST9/10 TWIN I9-Dl2 RJAZ RJDI DIS 20 Mogl3/10 HORP9.5 DUR4R3 SIF15 SIF4A3I 3W4CL5 CTP4 I A2
Reference i 7
3 4
5
+
6 7 8 9 10 11 12
13 14 15 16 17
Assignment of :he human N-cadherin gene to chromosome 18 was determined by Southern analysis of DNA from the 17 cell hybnds. Of the nine positive cell hybrids. the human chromosome 18 was the only common element. The table shows the human chromosomes contained in the hybrid cells (+ indicates presence, - indicates absence. t indicates not tested, and T indicates X/I 5 translocation). The presence or absence of the I .4-kb human N-cadherin gene fragment is indicated in the N-Cadherin column. The original descriptions of the cell hybrids used are as follows: ( I ) Jones et al.. 1976; ( 2 )Swallow et al., 1977; (3) Swallo\v el al., 1977; (4) Solomon el al., 1979; (5) Kielty et al.. 1982; (6) Kielty et al.. 1982; (7) Phillips et al., 1985: (8) Human Genetic Resources, ICRF; (9) Human Genetic Resources, I C R F (10) Heisterkamp et al.. 1982; (1 I ) Povey et al.. 1980 (12) Van Heynigen et al.. 1975; (13) Solomon et al.. 1976 (14) Edwards et al., 1985: (15) Edwards et al.. 1985; (16) Nabholz et al.. 1969: (17) Jones et al.. 1976.
sized selected subclones sequenced by using the chain termination methodology and sequence 11. The alignment of characterized human muscle Ncadherin cDNA clones is shown in Fig. 1 relative to
the full-length chick brain N-cadherin cDNA clone XN2. The combined sequences of clones X 14 and X4.10 are colinear with AN2 and encode a fully processed, mature N-cadherin protein; however, human clone A14
E
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n 1
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E X
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I
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FIG. 1. Alignment of human N-cadherin clones with the full-length clone XN2, showing the extent of the open reading frame. Restriction maps for human N-cadherin cDNA clones are aligned for sequence homology with a full-length chick clone. Restriction endonuclease cleavage sites are shown for 6arnHl (B), Hindlll (H), EcoRl (E), and Xmnl (X). The major open reading frame is depicted as a bar, signal peptide as a dotted bar, prepeptide as a hatched bar, and the hydrophobic membrane spanning domain as a filled bar; within the 3’ untranslated sequence (line), the positions of the consensus sequences for polyadenylation addition are indicated.
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FIG. 2. Above and opposite page: Comparison of nucleotide (a) and amino acid (b)sequence of chick and human N-cadherin. Nucleotide and derived amino acid sequences were compared over the length of the human N-cadherin mature protein and including 3' untranslated(a). The nucleotide similarity for chick and human is 77.8% over 2,696 bp and this increases to 91% over 747 amino acid residues not including conservativechanges. Consensus pdyadenylation addition sequences (ATTAAA) are underlined above and below the respective sequences as are EcoRl endonuclease cleavage sites. For the amino acid comparison, the hydrophobic membrane spanning domain is highlighted (underlined), showing the extensive homology within the cytoplasmic domain. Also indicated are the consensus sequences for N-linked glycosylation(r) above and below the respective sequences. Amino acid mismatches between the two sequences are indicated(*).
J Neurorhem., Vol 55, No. 3. 1990
HUMAN N-CADHERIN
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does not extend beyond or up to the initiation Met residue in chick XN2, but terminates at an Ala 16 residues 3’ of the start codon. In Fig. 2, comparisons are drawn between the full-length chick brain and derived human muscle DNA and protein sequences starting from the first codon/amino acid of the mature protein identified by direct protein sequencing (Hatta et al., 1988). At the DNA level, from the Asp codon, there is 77.8% similarity between chick and human sequences. However, clone X4.10 extends 77 1 bp farther 3’ and beyond the polyadenylation addition recognition sequence (ATTAAA) at 3,180 bp in XN2. Clone X4.10 does contain an analogous motif in a similar location, which is duplicated 7 bp farther 3’. The RNA from which X4.10 was derived clearly utilizes another more 3’ polyadenylation site and would consequently be derived from a different RNA size class. Comparisons between the derived amino acid sequence of the mature
748 912
human muscle N-cadherin revealed 9 I .O and 96.5% similarity for chick and mouse, respectively; this compares with a value of 9 I .8% between chick and mouse. The cytoplasmic region of the molecule shows a particularly high degree of conservation with only two and three mismatches in 160 residues between chick and mouse, respectively. Eight consensus sites for N-linked glycosylation were observed in the human sequence, including a consensus site within the cytoplasmic domain, and this compares with nine sites in chick and six sites in mouse. Northern analysis of N-cadherin DNA sequence analysis of human muscle clones has indicated that variability might exist for the N-cadherin mRNAs. To investigate this possibility, Northern blots of poly(A)+ RNA from C6 glioma, human foetal muscle, C2 myotube cultures, human embryonic brain, and J. iVeurochem , Vol. 55, No. 3, 1990
F. S. M'ALSH ET AL.
810 a
b
c
Chromosomal mapping of the human N-cadherin gene To determine the chromosomal location of the human N-cadherin gene, cDNA X 1.5 was used to probe genomic DNA digested with EcoRI from a panel of somatic cell hybrids. A 1.4-kb EcoRI fragment was diagnostic for the human N-cadherin gene and bands of 7.8, 8.0, and 8.4 kb for the mouse, hamster, and rat genes, respectively. A panel of 17 hybrids was then evaluated for the presence of the 1.4-kb EcoRI fragment indicative of the human N-cadherin (Fig. 4). Of the 17 hybrids analysed, positive 1.4-kb bands were identified for 9 cell hybrids. This unambiguously indicates that the human N-cadherin gene is located on chromosome 18 (Table 1).
e
d
5.2-
-28s
4.34.0-
-18s FIG. 3. Northern analysis of N-cadherin RNAs. Multiple RNA bands for N-cadherin transcripts were identified by Northern blot hybridization. Poly(A)+ RNA (8 pj) was probed with 32P-labeledcDNA X1.5. Exposures were selected to show heterogeneity of bands. Sizes of RNA in kilobases are shown on the left with the migration of 28s and 18s standards on the right. Lane a, adult mouse brain; lane b, embryonic human brain; lane c, C2 myotube; lane d, embryonic human muscle; lane e, C6 glioma cells.
DISCUSSION The Ca*'-dependent CAM N-cadherin has been found to be an important regulator of cell-cell interactions in the nervous system. Immunoperturbation studies have clearly shown that this homophilic CAM plays a dominant role in the process of neurite outgrowth over various cellular substrata such as astrocytes, Schwann cells, and myotubes (Bixby et al., 1987; Neugebauer et al., 1988; Tomaselli et al., 1988). Although multiple adhesive interactions may be operative in each of these model systems, N-cadherin appears to be important since antibody blockade leads to the greatest percentage decrease in neurite outgrowth. All studies on N-cadherin in the nervous system have shown that it is a single polypeptide chain of 125 kDa with no evidence to date of extensive microhet-
mouse brain were probed with cDNA from clone X1.5 (Fig. 3). As previously described for chick brain RNA, a single major band of 4.3 kb was identified in both embryonic human and adult mouse brain RNAs, whereas in C6 glioma additional RNA bands of 5.2 and 4.0 kb were identified. Consistent with an alternative choice of polyadenylation site selection indicated from analysis of muscle cDNAs, a minor 5.2-kb RNA band was present in samples from this source in addition to major bands of 4.0 and 4.3 kb. A similar minor band of 5.2 kb was also found in RNAs from embryonic human brain.
A
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FIG. 4. Chromosomal localization of the human N-cadherin gene. The chromosomal location of the N-cadherin gene was determined by probing a Southern blot of EcoRI-digested genomic DNA from the indicated cell hybrids. Characteristic bands of 1.4, 7.8, 8.0, and 8.4 kb were identified for human, mouse, hamster, and rat DNA, respectively. Hybrids positive for the human fragment (1.4 kb) are marked (+), while those that are negative are marked (-). Lane A, CTP34b4 (+); lane B, CTP41A2 (-); lane C, 3W4CL5 (-); lane D. SIF15 (-); lane E, SIF4A31 (-); lane F. DUR4R3 (-); lane G, SlR74ii (-); lane H, DTI .2.4 (+); lane I, DT1.2 (+); lane J, HORP9.5 (-); lane K, MOG34A4 (+); lane L, FGlO (+); lane M. FST9/10 (+); lane N, Twin 19-D12 (+); lane 0, human DNA; lane P, mouse DNA, lane Q, RJDl (+); lane R, RJA2 (+); lane S, FIR5 (+); lane T, CTP34B4 (+); lane U, MOG13/10 (-); lane V, DIS 20 (-); lane W, SIF4A31 (-).
J. Neirrochern , Val. 55, No. 3. 1990
HUMAN N-CADHERIN erogeneity due to specific glycosylation as has been found for other CAMs such as N-CAM. The limited molecular heterogeneity and relatively ubiquitous expression of N-cadherin on all classes of neurons have led to the suggestion that this protein may be involved in initial cell adhesion but may not have the ability to mediate specific pathway guidance (Dodd and Jessell, 1988). However, in the present study we have shown heterogeneity at the RNA level, which may reflect changes in the primary amino acid sequence. Further studies will be required to resolve this issue. The recent cloning of chicken N-cadherin and its subsequent expression in L-cells and neuroblastoma cells have shown that neurons from the optic nerve can recognize and use this molecule as a guidance cue (Matsunaga et al., 1988). In this study cDNA clones for the full coding sequence of the mature, fully processed human muscle N-cadherin have been isolated. Two approaches were utilized to obtain this information: First, a synthetic oligonucleotide to a region likely to be best conserved across species identified cytoplasmic and membrane proximal domain sequences, whereas a cDNA subfragment of AN2 yielded the remaining sequence. We have no direct evidence to date, in the form of overlapping clones, indicating that human clones X4.10 and h I4 are derived from the same RNA species, although it is highly likely. The sequence spanning the junction of clones X4.10 and XI4 is colinear with the chick sequence, although direct proof will require additional RNA analyses. The human sequence is likely to utilize a similar initiating Met codon as the chick and mouse genes, since the human sequence shows strong similarity for the first 30 residues particularly with the mouse. It is of interest to note that the 5amino acid deletion in the mouse compared to the chick sequence is also present in the human. The conservation of amino acid sequence within the cytoplasmic domain is particularly high with two (99%) and three (98%) mismatches in I60 residues for chick and mouse, respectively, compared to the human sequence. The maintenance of this amino acid sequence suggests that it has an important and conserved role for N-cadherin function in all species. Evidence for alternative polyadenylation site selection has been obtained through study of human N-cadherin clones since the 3' end of the human cDNA clone X4.10 extends beyond the 3' end of the used polyadenylation site in chick, but contains a duplicated site in an analogous position to that of the chick transcript. It is not known whether the usage of additional polyadenylation sites represents an alternative splicing choice to utilize separate exons or usage of two sites in a single exon. In the N-CAM gene both situations occur, with the closest parallel being the 2.9- and 5.2-kb mRNAs, which have in common three coding sequences. To date, only one of the three cadherin members has been mapped to its respective chromosome. E-cadherin has been mapped to chromosome 8 in the mouse
811
(Eistetter et al., 1988) and chromosome 16q in the human (Mansouri et al., 1988). To understand more fully the evolution of this gene family, it is important to extend this analysis to other cadherin members. In the present study, the human N-cadherin gene was shown to be present on chromosome 18 and is therefore clearly not a spliced derivative of E-cadherin. Thus, the functional specificity of the N- and E-cadherin gene products arose via evolution following duplication of a single gene. There appear to be some parallels with the Ig superfamily (Edelman, 1987; Williams, 1987), except that the cadherins have not evolved beyond tissue specific adhesion while the Ig gene superfamily contains a much greater diversity of function. Chromosome I8 has not been found to encode any protein that is highly expressed in the nervous system except myelin basic protein (Sparkes et al., 1987). It will be of interest to determine the subchromosomal location of the N-cadherin gene since there are a number of tumours associated with breakpoints of this chromosome and in particular a region on 18q that is likely involved in tumour suppression in colorectal cancers (Fearon et al., 1990). Acknowledgment: This work was supported by grants from the Muscular Dystrophy Group of Great Britain and the Wellcome Trust.
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