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

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Biochemistry and Molecular Biology

First Committee Member

K. L. Carraway, Committee Chair


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.)


Biology, Molecular

Link to Full Text


Link to Full Text