Structure and biosynthesis of a sialomucin complex from 13762 rat mammary adenocarcinoma ascites cells

Cell surfaces of metastatic 13762 ascites rat mammary adenocarcinoma cells contain a sialomucin complex (SMC) which has been proposed to protect the cells from immunedestruction. This complex is composed of an approx. 600 kDa sialomucin (ASGP-1) and a 120 kDa transmembrane glycoprotein (ASGP-2).Interactions between ASGP-2 and the microfilament (MF) core were investigated utilizing proteolysis of microvilli at timed intervals followed by various extraction and detecting techniques to study surface, membrane-bound and MF-associated fragments. The earliest major proteolysis product was a 70 kDa membrane-bound fragment. At longer times a 60 kDa released fragment, 30-40 kDa membrane-bound fragment and 25-30 kDa membrane and MF-associated fragments were observed. Phalloidin shift analysis of MF-associated proteins indicated that the 25-30 kDa fragment was strongly associated with the MF core. We propose that ASGP-2 has a site for indirect association with the MF core near the membrane on a small segment.Early biosynthesis of SMC was investigated using pulse-chase labeling followed by biochemical analyses of peanut agglutinin (PNA) precipitates or immunoprecipitates. Under conditions which dissociate SMC, anti-ASGP-2 immunoprecipitated primarily components of 120 kDa (ASGP-2) and about 400 kDa (pSMC-1) from cell lysates. We propose that SMC complex is formed from common precursor pSMC-1 by proteolytic cleavage to yield 120 kDa ASGP-2 plus the precursor to ASGP-1 early in the transit pathway from the endoplasmic reticulum to the cell surface.Anti-ASGP-2 was used to screen a lambda gt11 expression library made from this cell line. A 1.3 cDNA clone which corresponds to about half of ASGP-2 polypeptide (60 kDa) was isolated. Northern blot analysis using this clone as probe revealed a transcript of about 9 kb in the 13762 cells, which is consistent with size of transcript for the polypeptide of SMC precursor. Analysis of the predicted protein sequence suggests that deduced peptide has a long extracellular domain (255 amino acids), followed by a short putative transmembrane segment (26 amino acids) and an intracellular carboxyl-terminal tail (20 amino acids). The sequence has 6 potential N-glycosylation sites and two internal repeats homologous to the EGF-like repeats found in many cell surface or secreted proteins. (Abstract shortened with permission of author.