Monoclonal antibodies specific for human tumor-associated antigen 90K/Mac-2 binding protein: tools to examine protein conformation and function.
As part of our effort to identify glycoproteins that contribute to colon cancer progression, we have previously described a family of structurally related glycoproteins expressing beta1-6 branched asparagine(Asn)-linked oligosaccharides defined by monoclonal antibody (MAb 1H9), which are differentially expressed, processed, and glycosylated by human colon carcinoma cell lines (Laferté and Loh [1992]; Biochem J; 283:193-201). MAb 1H9 immunoprecipitates three glycoproteins having apparent sizes of 92-100, 66-70, and 25 kDa, the size heterogeneity attributable to cell-type specific glycosylation differences. We report on the basis of partial protein and cDNA sequence information, that the 100-kDa glycoprotein detected by MAb 1H9 is identical to the 90-kDa glycoprotein variably known as tumor-associated antigen 90K (TAA90K), Mac-2 binding protein, and cyclophilin C-associated protein. Using a PCR-based cloning strategy, the complete cDNA encoding TAA90K was cloned into the eukaryotic expression vector pCDNA-3 (pCD-TAA90K(wt)) and the protein expressed in COS-1 cells. A [(35)S]methionine-labeled 60-kDa polypeptide, processed to an endoglycosidase H-sensitive 74-kDa glycoprotein in the presence of dog pancreas microsomes, was detected in a coupled transcription/translation in vitro reaction. The in vitro-translated 60-kDa polypeptide and N-glycanase-treated TAA90K (60-kDa species) immunoprecipitated from HT29 cells were shown to be structurally identical by limited proteolytic peptide mapping. Using a new panel of 11 TAA90K-specific monoclonal antibodies, including five specific for human TAA90K and six cross-reactive with a 90-kDa species expressed by COS-1 cells, we have detected conformational differences between recombinant wild-type TAA90K, in vitro-synthesized TAA90K, and mutant forms of TAA90K containing point mutations at residues 189, 223, and 259. Furthermore, we have shown that these mutant forms of TAA90K, as well as a truncated form of TAA90K containing amino acid residues 1-383, are defective in secretion. These studies demonstrate the potential usefulness of TAA90K-specific monoclonal antibodies for examining the structure and function of TAA90K, and highlight the contribution of specific amino acid residues to its normal processing and secretion.
Laferté
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Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada. laferte@duke.usask.ca
Loh
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Keeler
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D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D014408:Biomarkers, Tumor; D015153:Blotting, Western; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D003110:Colonic Neoplasms / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D051379:Mice; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D016326:Extracellular Matrix Proteins
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D002352:Carrier Proteins; D018076:DNA, Complementary; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; C081856:LGALS3BP protein, human; D016326:Extracellular Matrix Proteins; C114782:TAA90K protein, human