Biosynthesis of the cell surface sialomucin complex of ascites 13762 rat mammary adenocarcinoma cells from a high molecular weight precursor.
Cell surfaces of metastatic 13762 ascites rat mammary adenocarcinoma cells are covered with a sialomucin complex composed of the high Mr sialomucin ASGP-1 (approximately 600,000) and a concanavalin A-binding, integral membrane glycoprotein ASGP-2 (120,000). Antibodies prepared against ASGP-2 and deglycosylated ASGP-1 react on immunoblots of ascites cells or their isolated microvilli with the Mr = 120,000 species and the high Mr sialomucin, respectively. No cross-reactivity was observed. Under complex dissociating conditions, anti-ASGP-2 immunoprecipitated primarily components of Mr = 120,000 and about 400,000 from lysates of cells labeled for 1 h with mannose, glucosamine, and threonine. Under similar conditions, anti-ASGP-1 immunoprecipitated the Mr = 400,000 component and a second major labeled component of about 330,000. Pulse-chase labeling with 35S-labeled amino acids followed by immunoprecipitation with anti-ASGP-2 indicated a precursor-product relationship for the Mr = 400,000 component, designated pSMC-1 (precursor, sialomucin complex), and ASGP-2. Similar pulse-chase analyses of threonine-labeled cells using anti-ASGP-1 showed equivalent amounts of immunoprecipitated pSMC-1 and pSMC-2, both of which disappeared with kinetics similar to those observed for pSMC-1 immunoprecipitated with anti-ASGP-2. A precursor-product relationship of both pSMC-1 and pSMC-2 to ASGP-1 was suggested by combined precipitations with anti-ASGP-1 and peanut agglutinin, which precipitates ASGP-1 specifically. Immunoblot and lectin blot analyses indicated that pSMC-1 and pSMC-2 from the immunoprecipitates bind anti-ASGP-2, anti-ASGP-1, and concanavalin A. Moreover, these three components can also be labeled with mannose; the mannose was removed from 30-min pulse-labeled anti-ASGP-2 immunoprecipitates by incubation with endo-beta-N-acetylglucosaminidase H, indicating the presence of only high mannose N-linked oligosaccharides in pSMC-1. One-dimensional peptide maps of 35S-labeled pSMC-1 and Mr = 120,000 ASGP-2 showed several corresponding bands. These results indicate that both ASGP-1 and ASGP-2 can be synthesized from a common high Mr precursor. We propose that complex is formed from pSMC-1 by proteolytic cleavage to yield Mr = 120,000 ASGP-2 plus the precursor to ASGP-1 early in the transit pathway from the endoplasmic reticulum to the cell surface.
Sheng
Z Q
ZQ
Department of Cell Biology, University of Miami School of Medicine, Florida 33101.
Hull
S R
SR
Carraway
K L
KL
D000230:Adenocarcinoma / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005944:Glucosamine / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism; D008970:Molecular Weight; D009077:Mucins / Q000096:biosynthesis* / Q000302:isolation & purification; D011487:Protein Conformation; D051381:Rats; D052116:Sialomucins; D013912:Threonine / Q000378:metabolism
D009077:Mucins; D052116:Sialomucins; D013912:Threonine; D005944:Glucosamine; D008358:Mannose