Immunochemical characterization of two isoforms of rat liver ecto-ATPase that show an immunological and structural identity with a glycoprotein cell-adhesion molecule with Mr 105,000

• Published in: Biochemical journal 1991-08, Vol.278 ( Pt 1) (Pt 1), p.155-161
• Main Authors: Lin, S H, Culic, O, Flanagan, D, Hixson, D C
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - analysis; Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - genetics; Amino Acid Sequence; Animals; Antigens, CD; Blotting, Western; Cell Adhesion Molecules - analysis; Cell Adhesion Molecules - chemistry; Cell Line; DNA - genetics; Electrophoresis, Polyacrylamide Gel; Immunosorbent Techniques; Liver - enzymology; Molecular Sequence Data; Molecular Weight; Peptide Fragments - chemistry; Peptide Mapping; Rats; Transfection
• ISSN: 0264-6021; 1470-8728

One of the cell-adhesion molecules (CAMs) responsible for rat hepatocyte aggregation has been described as a glycoprotein having an Mr of 105,000 (cell-CAM105). The Mr and localization of cell-CAM105 in liver membranes are very similar to those of liver ecto-ATPase, an ATPase with its nucleotide-hydrolysing site localized on the outside of the cell membrane. The protein sequence of the ecto-ATPase has been deduced from cDNA cloning. Structural analysis of the sequence indicates that the ecto-ATPase has immunoglobulin-like domains and is a member of the immunoglobulin superfamily. Since a group of proteins in the immunoglobulin superfamily has been shown to have functions related to cell adhesion, the structural characteristics of the ecto-ATPase further led to the possibility that the ecto-ATPase may have functions related to cell adhesion. In this paper, using the cDNA for the ecto-ATPase, the anti-peptide antibodies produced against peptides derived from the ecto-ATPase cDNA sequence and monoclonal antibodies against the cell-CAM105, we present evidence of identity between cell-CAM105 and ecto-ATPase. First, in Western immunoblots, two anti-cell-CAM105 monoclonal antibodies cross-reacted with the purified ecto-ATPase. Secondly, in immunodepletion experiments, antibodies against the ecto-ATPase depleted the same protein recognized by the anti-cell-CAM105 antibodies. Thirdly, in two-dimensional gel-electrophoretic analysis, anti-peptide antibodies generated against an extracellular N-terminal peptide and the intracellular C-terminal peptides of the ecto-ATPase immunoprecipitated proteins of similar isoelectric points and Mr values to those of the cell-CAM105. Fourthly, proteins immunoprecipitated by anti-ecto-ATPase antibodies and anti-cell-CAM105 antibodies have similar V8-proteinase-digest peptide maps. Finally, monoclonal antibodies against the cell-CAM105 specifically recognized the protein expressed in COS cells transfected with the ecto-ATPase cDNA. These results indicate that the ecto-ATPase cDNA codes for a protein that is identical with the cell-CAM105. Since the ecto-ATPase has structural features of immunoglobulin domains, the identity of cell-CAM105 with ecto-ATPase leads to the conclusion that this liver CAM, similarly to neuronal CAM, is also a member of the immunoglobulin supergene family. Furthermore, immunological studies indicate that the cell-CAM105/ecto-ATPase is composed of two isoforms of different C-terminal sequences. The associatio