Role of the cystine knot in synthesis of human salivary mucin MG1 (MUC5B)
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Mucins are a family of large, heavily glycosylated proteins. Among different kinds of mucins, MUC5B is the most abundant mucin in human saliva. Structurally, mucins have a large central region formed of multiple tandem repeats, which is the site of glycosylation, and the amino- and carbooxyl-teminal regions which are rich in cysteines and are likely involved in establishing disulfade linkages within and among mucin monomers. The carboxyl-teminal region of MUC5B contains a cystine knot (CK) containing 11 half cystines, 6 of which are predicted to participate in three intrachain disulfide bonds. Therefore, the purpose of this investigation was to assess the role of these 11 cysteine residues in disulfide bond formation. To study the importance of the CK in MUC5B dimer formation, cDNA sequences encoding the carboxyl-terminal 108 amino acids were cIoned into the eukaryotic expression vector PSecTag2 to generate plasmid pM5B1O8. Meanwhile, each of the 11 cysteines in the carboxyl-terminal 108 residues of MUC5B was mutated to alanine by using two complimentary oligonucleotides containing the desired mutations, and mutant plasmids were estalblished. Expression studies performed in COS-7 ce11s showed that the cells transfected with pM5B1O8 secreted an immoreactive protein of -65 kDa which likely represents glycosylated disulfade linked dimers. Under reducing conditions, a series of immunoactive proteins ranging from -35 kDa to -50 kDa and a small quantity of a protein of -75 kDa were observed. The proteins in the -35 kDa to -50 kDa range likely represent variously glycosylated monomers with O, 1 , 2 or 3 glycans. COS-7 cells transfected with mutant plasmid C962A secreted -65 kDa dimers and higher molecular weight aggregates that likely represent randomly linked disulfide multimers. Mutants C879A, C893A, C902A, C906A, C926A, C940A, C956A and C958A did not secrete dimers but secreted relatively large quantities of higher molecular weight aggregates. Secretion was severely impaired in COS-7 ce11s transfected with C923A and C925A as evidenced by the absence of dimers and minimal secretion of higher molecular weight aggregates. Under reducing conditions, two groups of immunoreactive proteins were displayed, one corresponding to the typical -35 kDa to -50 kDa monomers and the other to immunoreactive proteins in the -75 kDa to -100 kDa. A notable exception was C923A where the only bands were found between -35 kDa to -50 kDa. These results showed that mutation of C879A and C926A, C902A and C956 and C906A and C958 of the predicted CK destroyed the ability to produce dimers. This suggests that all 3 of the disulfide bonds of the predicted CK are essential to orient one or more additional cysteines that participate in intermolecular disulfide linkages between the same or a different cysteine in a second monomer.
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 email@example.com.Thesis (MSD)--Boston University, Goldman School of Dental Medicine (Periodontology and Oral Biology).Includes bibliographical references: leaves 48-56.
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