Characterization of the cysteine residues in MG2 and identification of heterotypic complexes involving MG2 and SIgA by peptide display
Soares, Rodrigo Villamarim
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1. CHARACTERIZATION OF THE CYSTEINE RESIDUES IN MG2: Human salivary mucin MG2 is a 180 kDa glycoprotein secreted by submandibular/sublingual and minor salivary glands. Secreted MG2 contains a domain with the only two cysteines (Cys45 and Cys50) in the polypeptide backbone and it has been shown that this domain in native and recombinant MG2 is involved in mucin binding to oral microbes. Since reduction and alkylation of MG2 was shown to abolish binding to oral microbes, the present study was undertaken to determine whether the cysteine residues exist in the dithiol or disulfide form. Electrophoretic analyses under reducing and non-reducing conditions showed that intermolecular disulfide bonds do not occur between MG2 molecules. The same incorporation of radiolabeled iodoacetamide into MG2 was obtained with or without prior reduction. When radiolabeled alkylated MG2 was digested with Endoproteinase LysC and the derived peptides were separated by reversed phase high performance liquid chromatography, radioactivity was found in two fractions. Mass spectral analyses of these fractions showed the presence of peptide 1 (Cys-Leu-His-Lys-) and peptide 2 (Arg-Cys-Arg-Pro-), both containing carboxymethylated cysteines. These results show that the cysteines in the structural motif associated with bacterial binding exist in the dithiol form and suggest the potential use of cysteine-containing peptides as agents to modify interactions of MG2 with microbes and oral surfaces. [TRUNCATED]
Thesis (D.Sc.D.)--Boston University, Henry M. Goldman School of Dental Medicine, 2002 (Periodontology and Oral Biology).Includes bibliographical references (leaves 127-152).
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