Biochemical analysis of a human epithelial surface antigen: differential cell expression and processing.
Epithelial surface antigen (ESA) is a glycoprotein with a distribution in vivo that is largely confined to human epithelial cells. Previous studies using a mouse monoclonal antibody (MH99) detecting ESA had shown that the antigen immunoprecipitated from most epithelial cancer cell lines has two chains (38,000 and 32,000 Da) when separated under reducing conditions and only one (38,000 Da) under nonreducing conditions. We now show that the 38-kDa band observed under nonreducing conditions consists of two species, one a 38-kDa single chain protein and the other a disulfide-linked dimer consisting of the 32-kDa chain bonded to a previously unrecognized 6-kDa chain. Pulse-chase studies have shown that ESA is synthesized as a 34-kDa protein which is glycosylated to a 38-kDa glycoprotein containing both high mannose and complex carbohydrate chains. With longer chase periods, a 32-kDa species also appears. Peptide mapping, together with the pulse-chase data, suggests that the 32- and 6-kDa species are formed from the 38-kDa protein, probably by limited proteolysis. Epithelial cell lines differ in their ratios of 38/32-kDa species, some cell lines having only the 38-kDa form. Incubation of radiolabeled extracts of cells having only the 38-kDa protein with unlabeled extracts of the other cell types resulted in progressive conversion of the 38-kDa species to the 32- and 6-kDa forms. Only cell lines expressing both forms of ESA are able to carry out this cleavage of the 38-kDa protein. This is a novel mechanism for generating cell-type related differences in cell surface glycoprotein expression. Finally, sequential immunoprecipitation experiments showed that the antigen detected by Ab MH99 is closely related or identical to that detected by Ab 17-1A, a previously described colon cancer antigen.
Thampoe
I J
IJ
Memorial Sloan-Kettering Institute, New York, New York 10021.
Ng
J S
JS
Lloyd
K O
KO
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D002277:Carcinoma / Q000276:immunology; D002460:Cell Line; D006026:Glycoside Hydrolases; D006801:Humans; D006868:Hydrolysis; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D018396:Mucin-1; D010084:Oxidation-Reduction; D010449:Peptide Mapping
D000911:Antibodies, Monoclonal; D008562:Membrane Glycoproteins; D018396:Mucin-1; D006026:Glycoside Hydrolases