Monoclonal antibodies specific for human tumor-associated antigen 90K/Mac-2 binding protein: Tools to examine protein conformation and function
✍ Scribed by Suzanne Laferté; Lambert C. Loh; Vicki Keeler
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 312 KB
- Volume
- 77
- Category
- Article
- ISSN
- 0730-2312
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✦ Synopsis
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 1-6 branched asparagine(Asn)-linked oligosaccharides defined by monoclonal antibody (MAb 1H9), which are differentially expressed, processed, and glycosylated by human colon carcinoma cell lines (Laferte ´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.