Structural determinants for secretion of human salivary mucin MG1 (MUC5B): role of the cystine knot in dimer formation

Thesis (D.Sc)--Boston University, Henry M. Goldman School of Dental Medicine, 2003 (Department of Periodontology and Oral Biology).PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact open-help@bu.edu.Includes bibliography (leaves 153-193).ln order to investigate early steps in assembly of MUC5B mucin, CDNA Sequences encoding the C-terminal 151, 108 and 75 amino acids were cloned into the eukaryotic expression vector pSecTag2 to generate plasmids pM5B151, pM5B108 and pM5B75, respectively. Expression studies performed in COS-7 cells showed that pM5B151 and pM5B108, both containing a complete cystine knot motif, were Synthesized and secreted into the medium as disulfide-linked dimers. When electrophoresis was performed under reducing conditions, Western blot analysis revealed a series of four to five glycosylated monomers, consistent with the presence of three or four N-glycosylation sites in the C-terminal 108 or 151 amino acid residues of MUC5B. After denaturation and digestion with PNGase F alone, monomers derived from pM5B151 and pM5B108 exhibited a considerable shift in their apparent molecular weight. However, when monomers were digested with PNGase F in addition to Sialidase A and Endo-O-glycosidase, there was only a minor change in the electrophoretic mobility, suggesting that N-linked glycans are the predominant oligosaccharides on C-terminaI MUC5B monomers. COS-7 cells transfected with pM5B75, which does not contains the CK motif, did not synthesize or secrete detectable quantities of the encoded MUC5B domain. COS-7 cells transfected with pM5B151 and pM5B108 and treated with tunicamycin did not synthesize or secrete dimers but rather synthesized, but did not secrete, high molecular weight random disulfide linked multimers observed near the top of separating gels. In the presence of Brefeldin A, a inhibitor that deregulates Golgi function, COS-7 cells transfected with pM5B151 and pM5B108 were abIe to synthesize disulfide linked dimers although secretion was severely impaired. In pM5B108, mutation of Cys829 (the most N-terminal half-cystine participating in the cystine knot) to alanine had no effect on dimer formation. However, in pM5B108 mutation of Cys925 (the half-cystine homologous to the residue implicated in dimerization of von Willebrand factor and Transforming Growth Factor-[beta]) to alanine abolished dimer formation and secretion. These data showed that Cys925 plays an essential role in dimer formation of the C-terminal MUC5B domain containing the cysteine knot. Similar expression experiments were carried out with a gallbladder epithelial cell line, Which constitutively produces MUC5B. Results of transfection experiments with MUC5B constructs using this epithelial cell line were essentially identical to those obtained with COS-7 ce=s. Finally, these results suggested the suitability of COS-7 cells as a model system for investigating assembly of MUC5B and other human mucins