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Identification of a new isoform of cell-cell adhesion molecule 105 (C-CAM), C-CAM4: a secretory protein with only one Ig domain.

ABSTRACT: A series of Southern blot hybridization experiments using probes derived from different regions of the rat liver cell-cell adhesion molecule 105 (C-CAM) cDNA revealed the presence of a 9.6 kb EcoRI genomic fragment that seemed to encode a unique C-CAM isoform. An RNase protection study showed that this c-CAM transcript was expressed in placenta, spleen, lung and large intestine. In contrast, the other C-CAM isoforms, C-CAM1 and C-CAM2, are expressed in liver and small intestine. This result also suggests that the new isoform, which we named C-CAM4, was indeed encoded by a new C-CAM gene. A rat placenta cDNA library was then screened and the full-length cDNA coding for C-CAM4 was isolated. The deduced protein contained 142 amino acids and had a calculated molecular mass of 15 kDa. C-CAM4 was composed of a leader sequence and the first V-like Ig domain typical of C-CAM-family proteins. However, C-CAM4 lacked the C-like Ig domains, the transmembrane domain, and the cytoplasmic domain found in other C-CAM isoforms. Thus, C-CAM4 is different from the other known C-CAMs in that it is a secreted protein. We have previously shown that the first Ig domain of C-CAM1 is crucial for its adhesion function. The V-like Ig domain of C-CAM4 had 92% and 89% sequence identity with the corresponding regions of C-CAM1 and C-cam2 respectively. Together these results suggest that C-CAM4 may play a role in regulating the function of other C-CAM family proteins.

SUBMITTER: Earley K

PROVIDER: S-EPMC1217277 | biostudies-other | 1996 May

REPOSITORIES: biostudies-other

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Project description:The neuron-glia cell adhesion molecule (Ng-CAM) mediates both neuron-neuron and neuron-glia adhesion; it is detected on SDS-PAGE as a predominant 135-kD glycoprotein, with minor components of 80, 190, and 210 kD. We have isolated cDNA clones encoding the entire sequence of chicken Ng-CAM. The predicted extracellular region includes six immunoglobulin-like domains followed by five fibronectin-type III repeats, structural features that are characteristic of several neural CAMs of the N-CAM superfamily. The amino acid sequence of chicken Ng-CAM is most similar to that of mouse L1 but the overall identity is only 40% and Ng-CAM contains a short fibronectin-like segment with an RGD sequence that has no counterpart in L1. These findings suggest that Ng-CAM and L1 may not be equivalent molecules in chicken and mouse. The amino-terminal sequences of the 210-, 190-, and 135-kD components of Ng-CAM are all the same as the predicted amino terminus of the molecule, whereas the 80-kD component begins within the third fibronectin repeat. The cDNA sequence is continuous across the junction between the 135- and 80-kD components, and a single 170-kD Ng-CAM polypeptide was isolated from tunicamycin-treated cells. In addition, all cDNA probes hybridized on Northern blots to a 6-kb RNA, and most hybridized to single bands on Southern blots. These results indicate that the Ng-CAM components are derived from a single polypeptide encoded by a single gene, and that the 135- and 80-kD components are generated from the 210/190-kD species by proteolytic cleavage. The 135-kD component contains most of the extracellular region including all of the immunoglobulin-like domains. It has no transmembrane segment, but it is tightly associated with the membrane. The 80-kD component contains two and a half type III repeats plus the RGD-containing segment, as well as the single transmembrane and cytoplasmic domains. These structural features of Ng-CAM provide a framework for understanding its multiple functions in neuron-neuron interactions, neurite fasciculation, and neuron-glia interactions.

Project description:Neuron-glia cell adhesion molecule (Ng-CAM) mediates cell adhesion between neurons homophilically and between neurons and glia heterophilically; it also promotes neurite outgrowth. In the chick brain, Ng-CAM is detected as glycoproteins of 190 and 210 kD (Ng-CAM200) with posttranslational cleavage products of 135 kD (F135, which contains most of the extracellular region) and 80 kD (F80, which includes the transmembrane and the cytoplasmic domains). To examine the functions of each of these components, we have expressed Ng-CAM200, F135, and F80 in murine L cells, and F135 and F80 as GST fusion proteins in the pGEX vector in bacteria. Appropriately transfected L cells expressed each of these proteins on their surfaces; F135 was also found in the media of cells transfected with Ng-CAM200 and F135. In addition to binding homophilically, cells transfected with Ng-CAM200 and F135 bound heterophilically to untransfected L cells, suggesting that there is a ligand for Ng-CAM on fibroblasts that may be related to the glial ligand. Detailed studies using the transfected cells and the fusion proteins indicated that both the homophilic and the heterophilic binding activities of Ng-CAM are localized in the F135 fragment of the molecule. The results also indicated that proteolytic cleavage of Ng-CAM200 is not required either for its expression on the cell surface or for cell adhesion and that there is an "anchor" for F135 on L cells (and presumably on neurons). In contrast to the cell binding results, the F80 but not the F135 fusion protein enhanced the outgrowth of neurites from dorsal root ganglion cells; this activity was associated with the FnIII repeats of F80. The observations that a protein corresponding to F135 contains the cell aggregation sites whereas one corresponding to the F80 has the ability to promote neurite outgrowth suggest that proteolytic cleavage may be an important event in regulating these Ng-CAM activities during embryonic development and neural regeneration.

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Identification of a new isoform of cell-cell adhesion molecule 105 (C-CAM), C-CAM4: a secretory protein with only one Ig domain.

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