Biochemical and molecular characterization of human high molecular weight salivary mucin MGI and its protein complexes
Iontcheva, Iveta I.
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Mucins are high molecular weight glycoproteins which are secreted by epithelial cells throughout the gastrointestinal, respiratory and urogenital tracts in human body and play a major role in the non-immune defense of mucous membranes from various environmental insults. Human mucins are encoded in at least eight distinct genes(ie., MUC1 to MUC8). MG1 a high molecular weight mucin in human submandibular/sublingual secretions (Loomis et al., 1987). In this studies partial sequences of carboxyl as well as amino-terminal regions of the MG1 gene are shown. The nucleotide and deduced amino acid sequences of the carboxyl-terminal region of MG1 were reported (Troxler et al., 1995) and recently it has been shown that MG1 from sublingual gland is identical to MUC5B gene product (Troxler et al., 1997). Thus MUC5B appears to be the principal mucin, expressed in human sublingual gland. The publication and biochemical studies of mucins are greatly impaired by the high content of oligosaccharides and the high molecular weight of these glycoproteins. Most of the mucins have polymeric structure and have been shown to be involved in interactions with another proteins (Snyder et al., 1982; Biesbrock et al., 1991). Although little is known about the functionai significance of heterotypic complexing in saliva, this process has the potential to modify the bioIogical activity of salivary proteins and to provide protection from proteolytic enzymes in the oral environment. In this investigation we describe a mild procedure for isolation and purification of native MG1 by gel filtration chromatography on Sepharose CL-2B which does not involve dialysis, lyophilization, use of denaturing agents or covalent modification. We have shown that amylase, PRPs, statherin and histatins selectively from heterotypic complexes with high molecular weight salivary mucin MG1 (Iontcheva et al., 1997). In these studies the occurrence of at least three different types of complexes between MG1 and other salivary proteins have been identified. Type 1 complexes occur between MG1and other salivary proteins are dissociated by SDS-PAGE and in 4 M guanidine hydrochloride. Type II complexes are not dissociated under these conditions. Type III complexes are dissociated under the conditions of SDS-PAGE and in 4 M guanidine hydrochloride but the released proteins themselves appear to be complexes between amylase, PRPs, statherins and histatins. Based on this biochemical data we explored interactions between MUC5B C-terminal domains with statherin and histatin in vivo, using the yeast two-hybrid system. The yeast-two hybrid system demonstrated differential binding of statherin and histatin binding domain of GAL4 chimeras to mucin activation domain of GAL4 chimeras. Complexing was observed only between statherin or histatin baits and the 5’domain, but not with the middle or 3’domains of the C-terminal region of MUC5B. We also explored interactions between statherin and another salivary proteins by screening a submandibular cDNA library with statherin as a bait in the yeast two-hybrid system. This method has also been successfully used to identify in vivo complexes between statherin and statherin and histatin. Heterotypic complexes among salivary proteins could have a significant impact on the biological properties of salivary proteins in oral fluids in both health and disease.
Thesis (D.Sc.D.)--Boston University, Henry M. Goldman School of Dental Medicine, 1997.Includes bibliographical references: (leaves 163-183).
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