Molecular Cloning and Characterization of a Novel Human Classic Cadherin Homologous with Mouse Muscle Cadherin

Abstract

We used a novel cDNA cloning method based on the cadherin-␤-catenin protein interaction and identified a new human classic-type cadherin, which we named cadherin-15, from adult brain and skeletal muscle cDNA libraries. Sequence analysis revealed that this cadherin was closely related to mouse muscle cadherin and seemed to be its human counterpart. However, its deduced amino acid sequence differed from that of mouse muscle cadherin in that it had an extra 31-amino acid sequence at its C terminus that has been found neither in mouse muscle cadherin nor in any other known classic cadherin. Analysis of cadherin-15 protein expressed in L fibroblasts showed that it was cleaved proteolytically, expressed on the cell surfaces as a mature form of about 124-kDa, and functioned as a cell-cell adhesion molecule in a homophilic and specific manner, but Ca 2؉ did not protect it against degradation by trypsin. Our findings also suggest that cadherin-15 mediates cell-cell adhesion with a binding strength comparable to that of E-cadherin.It is generally accepted that various molecules that have the extracellular subdomain (EC) 1 structure of the classic cadherins in common constitute a large gene family, the cadherin superfamily. This superfamily includes the classic cadherins (1), truncated-type cadherins, which lack the characteristic cytoplasmic domain of the classic cadherins (2-4), desmosomal cadherins, which are localized in desmosomes (5-7), protocadherins, which have more than five extracellular subdomains (8 -9), and molecules showing high similarities to rat LI-cadherin (10 -12).Each classic cadherin comprises a signal sequence and a precursor region, which are both cleaved by intracellular proteolytic processing, five cadherin extracellular subdomain repeats, a transmembrane domain and a characteristic cytoplasmic domain, which is highly conserved among the subclasses and is indispensable for association with catenins, the ensuing linkage to the cytoskeleton and full functioning as a Ca 2ϩ -dependent cell-cell adhesion molecule (13-15). It is now understood that these cadherins play essential roles in various morphogenetic events in multicellular organisms (1). The first classic cadherins to be identified were E-, N-, and P-cadherins, as a result of the establishment of their respective blocking antibodies (16 -23), and then V-cadherin was identified using a blocking monoclonal antibody (24). Thereafter, several cadherins were identified (25-28) by determining their cross-reactivities with antibodies raised against conserved peptide sequences or cross-hybridization with cDNA fragments of known cadherins. Over the past few years, the existence of more classic cadherin molecules has been demonstrated by PCR-based cDNA cloning methods (3, 29 -34).Full cDNA cloning of nine independent human classic cadherin molecules has been reported, i.e. E-, N-, and P-cadherins and cadherin-4, -5, -6, -8, -11 (OB-cadherin), and -12 (3, 29, 30, 31, 35-37). We are interested in how many classic cadherin molecules actually exist in humans...

Biochemical Characterization and Functional Analysis of Two Type II Classic Cadherins, Cadherin-6 and -14, and Comparison with E-cadherin

“…A cadherin-6 cDNA fragment corresponding to EC5, a region with low homology to the other classic cadherins (12)(13)(14), and containing KpnI and HindIII sites at the 5Ј-and 3Ј-ends, respectively, was amplified by polymerase chain reaction, digested with KpnI and HindIII, and ligated to a prokaryotic expression vector, pR-SET B (Invitrogen Corp.) that had been cleaved with the same enzymes. The resultant plasmid was introduced into an Escherichia coli strain, BL21(DE3)pLysS (Novagen), and expression of the fusion protein was induced by 1 mM isopropyl-␤-D-thiogalactoside.…”

“…Other Biochemical Procedures-Immunoprecipitation, N-terminal amino acid sequencing, and determination of the trypsin sensitivity was performed as described previously (14). Exposure of cadherin molecules on the surface of the transfected L cells was also examined as described previously (14).…”

“…Exposure of cadherin molecules on the surface of the transfected L cells was also examined as described previously (14).…”

“…The human classic cadherins E-, N-, and P-cadherin and cadherin-4 have been classified as type I, and cadherin-6, -8, -11, -12, and -14 are classified as type II. Although cadherin-5 and -15 (Mcadherin) have been proposed to be type II and I, respectively (2), we think that they do not clearly belong to either subgroup (14).…”

“…We recently reported that the susceptibility of cadherin-15 to trypsin digestion in the presence of Ca ϩ causes a low aggregation rate in the short term assay (14). We therefore examined the trypsin sensitivities of cadherin-6 and -14 (Fig.…”