RESUMEN
Heterotypic complexes between the high-molecular-weight mucin MG1 and other salivary proteins in human submandibular/sublingual secretion (HSMSL) could have a significant impact on the biological properties of these proteins in oral fluids in both health and disease. 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. Western blots of native MG1 probed with antibodies against 8 different salivary proteins showed that complexing occurs between MG1 and salivary amylase, proline-rich proteins (PRPs), statherins, and histatins but not MG1, sIgA, secretory component, or cystatins. When native MG1 was placed in 4 M guanidine hydrochloride and chromatographed on Sepharose CL-4B, ELISA measurement of column fractions showed that amylase, PRPs, statherins, and histatins were released. Interestingly, gel filtration resolved the material which eluted into 4 or 5 distinct peaks, suggesting that the released entities were heterotypic complexes. From these studies, the occurrence of at least three different types of complexes between MG1 and other salivary proteins has been identified. Type 1 complexes 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 during SDS-PAGE and by 4 M guanidine hydrochloride, but the released proteins appear to be complexes containing amylase, PRPs, statherins, and histatins. The possible functional role of heterotypic complexes between MG1 and other salivary proteins as a physiologic delivery system, a mechanism for protection against proteolysis, a repository for precursors of the acquired enamel pellicle, and a vehicle for modulation of the viscoelastic and rheological properties of saliva is discussed.
Asunto(s)
Amilasas/aislamiento & purificación , Mucinas/aislamiento & purificación , Prolina/aislamiento & purificación , Proteínas/aislamiento & purificación , Saliva/química , Proteínas y Péptidos Salivales/aislamiento & purificación , Aminoácidos/análisis , Amilasas/análisis , Western Blotting , Cromatografía Líquida de Alta Presión , Película Dental , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Peso Molecular , Mucinas/análisis , Prolina/análisis , Desnaturalización Proteica , Proteínas/análisis , Proteínas y Péptidos Salivales/análisisRESUMEN
MGI is a high-molecular-weight mucin secreted by mucous acinar cells in human submandibular and sublingual glands. We have recently shown that the tracheobronchial mucin MUC5B is a major component of MG1. MUC5B is organized into cysteine-rich N- and C-terminal regions that flank a central tandem-repeat region containing cysteine-rich subdomains and imperfect 29-residue tandem repeats. In earlier work, we have shown that this mucin selectively forms heterotypic complexes with amylase, proline-rich proteins, statherin, and histatins in salivary secretions, and the aim of this study was to identify specific binding domains within MUC5B using the yeast two-hybrid system. Interactions of cysteine-rich domains in the tandem-repeat region (Cys1-Cys4) and C-terminal region (Cys8a, Cys8b, Cys8c) of MUC5B with statherin and histatins were investigated. These studies indicated that histatin 1 selectively bound to Cysl and Cys2, whereas statherin and histatin 1, 3, and 5 selectively bound to Cys8a. Analysis of the primary sequences of the identified binding domains suggests that these domains most probably can fold into globular-like structures in the native mucin. A ProDom blast search revealed that sequences in Cys1, Cys2, and Cys8a exhibit similarity to domains in evolutionarily diverse extracellular proteins known to participate in a wide variety of protein-protein interactions.
Asunto(s)
Glicoproteínas/genética , Histidina/genética , Mucinas/genética , Proteínas/genética , Proteínas y Péptidos Salivales/genética , Amilasas/química , Western Blotting , Bronquios/metabolismo , Cisteína/química , Cisteína/genética , Electroforesis en Gel de Poliacrilamida , Glicoproteínas/química , Histidina/química , Humanos , Peso Molecular , Mucina 5B , Mucinas/química , Péptidos/química , Fosfoproteínas/química , Prolina/química , Dominios Proteicos Ricos en Prolina , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Proteínas/química , Proteínas y Péptidos Salivales/química , Glándula Sublingual/metabolismo , Glándula Submandibular/metabolismo , Secuencias Repetidas en Tándem/genética , Tráquea/metabolismo , Levaduras/genéticaRESUMEN
A human sublingual gland cDNA library was screened with a polyclonal antiserum against deglycosylated MG1 and a positive clone, pSM2-1, was isolated which codes for 196 amino acids in the carboxyl-terminal region of this mucin. This region is cysteine-rich and contains a C2-like domain upstream of the extreme carboxyl-terminal domain in which the arrangement of cysteines is nearly identical to that in human von Willebrand factor, human intestinal mucin MUC2, human tracheobronchial mucin MUC5 and porcine and bovine submaxillary gland mucins. Northern analyses with pSM2-1 showed MG1 transcripts are abundant in sublingual gland and barely detectable in submandibular gland. This study provides the first primary sequence data on human salivary mucin MG1 and the significance of the results is discussed with respect to the biosynthesis and differential expression of MG1 in human salivary glands.
Asunto(s)
Mucinas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Expresión Génica , Humanos , Datos de Secuencia Molecular , ARN Mensajero/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Glándula Sublingual/química , Factor de von Willebrand/genéticaRESUMEN
Human submandibular/sublingual gland secretions contain a multimeric high molecular weight mucin (MG1) and a monomeric low molecular weight mucin (MG2). MG2 is the product of the MUC7 gene, whereas the gene for MG1 has not been identified. Previously, we isolated a clone (pSM2-1) from a human sublingual gland cDNA expression library using an antibody against deglycosylated MG1 (Troxler et al., Biochem. Biophys. Res Commun., 217, 1112-1119, 1995). In order to identify the mucin gene from which pPM2-1 was derived, Northern blots of human submandibular and sublingual gland RNA were hybridized with a series of probes for tandem repeats found in the high molecular weight secreted mucins MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC6. The only known mucin expressed at high levels in sublingual gland was MUC5B, and no known mucin was expressed at high levels in submandibular gland. A series of overlapping clones was obtained by rescreening the sublingual gland cDNA library and by reverse transcriptase-polymerase chain reaction. The resulting clones connected pSM2-1 to a series of MUC5B tandem repeats at the 3' end of the repeat domain and provided the complete nucleotide and deduced amino sequence of the carboxyl terminal region of MG1. This region is enriched with respect to cysteine (approximately 10 mol %) and contained a D domain and a carboxyl terminal domain that could be aligned with the corresponding domains in human intestinal MUC2, human tracheobronchial MUC5AC, and human von Willebrand factor. The limited expression of known mucin genes, together with the considerable mucin synthesizing capacity of submandibular gland, suggests that a novel (previously not described) mucin gene is expressed in this gland and constitutes a portion of MG1 in salivary secretions.