Proteomic and functional anlaysis of salivary mucin (MUC5B) complexes
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The high molecular weight salivary mucin MG1, comprised primarily of the MUC5B gene product, is a major gel forming mucin in human submandibular/sublingual gland secretion (HSMSL). MG1 plays an important protective and lubricative function in the oral cavity. It protects hard and soft tissues from desiccation, mechanical abrasion and exogenous insults. MG1 forms complexes with other proteins and it has been suggested that these complexes may modulate the functional properties of both the mucin and the binding partners. The objectives of this study were: 1) to identify proteins which form complexes with MG1 using a proteomics approach and 2) to investigate the effect of complexing on the functional activities of some of these proteins in two assays: growth inhibition and yeast killing. In the first part, MG1 was isolated from HSMSL using size exclusion chromatography, complexes were dissociated with guanidine followed by rechromatography under denaturing conditions. Following electrophoresis on SOS gels, lanes containing dissociated complex partners were divided into equal slices, digested with trypsin in situ and subjected to MALDI TOF mass spectrometry. Later, the mass spectrometry results were confirmed by ELISA, Western blots and Far Western blots. In the second part of this study, complexes between MG1 and histatin and between MG1 and lactoferrin were reconstituted and their fungicidal and fungistatic functions were analyzed against Candida albicans. This study showed that rechromatography of dissociated mucin complexes yielded a void volume peak containing MG1 and a second large peak containing released mucin complex partners. Coomassie Blue stained gels of the latter peak revealed a smear of stained proteins as well as a few discrete bands. Analysis of tryptic peptides by MS/MS identified a variety of proteins including lysozyme, lactoferrin, myeloperoxidase, albumin, gp340 (salivary agglutinin), dermcidin, histatin, amylase, statherin, praline-rich proteins, prolactin-induced proteins, lipocalin, thioredoxin and calgranulins. The study also demonstrated that complexes containing MG1 and either histatin 5 or lactoferrin inhibited the growth of C. albicans by more than 50% and more than 45%, respectively. Interestingly, MG1 complexes with either lactoferrin or histatin 5 were not able to kill C. albicans cells. The identification of several proteins as MG 1 complex partners may localize these molecules in specific sites in the oral cavity, improve their resistance to proteolytic attack and prolong their residence time in the oral environment. To date this is the first study to identify MG1 complex partners by mass spectrometry and to analyze their fungicidal and fungistatic functions, suggesting that MG1 may have a novel protective role in the oral cavity as it could significantly modulate the functional properties of antimicrobial proteins through complex formation.
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 (D.Sc.D.)--Boston University, Henry M. Goldman School of Dental Medicine, 2007 (Dept. of Periodontology and Oral Biology).Includes bibliographical references: leaves 121-162.
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