Molecular characterization of a major high molecular weight mucin from human sublingual gland

Human submandibular/sublingual gland secretions containa multimeric high molecular weight mucin (MG1) and amonomeric low molecular weight mucin (MG2). MG2 isthe product of the MUC7 gene, whereas the gene for MG1has not been identified. Previously, we isolated a clone(pSM2-l) from a human sublingual gland cDNA expres-sion 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 whichpPM2-l was derived, Northern blots of human subman-dibular and sublingual gland RNA were hybridized with aseries of probes for tandem repeats found in the high mo-lecular weight secreted mucins MUC2, MUC3, MUC4,MUC5AC, MUC5B, and MUC6. The only known mucinexpressed at high levels in sublingual gland was MUC5B,and no known mucin was expressed at high levels in sub-mandibular gland. A series of overlapping clones was ob-tained by rescreening the sublingual gland cDNA libraryand by reverse transcriptase-polymerase chain reaction.The resulting clones connected pSM2-l to a series ofMUC5B tandem repeats at the 3' end of the repeat domainand provided the complete nudeotide and deduced aminosequence of the carboxyl terminal region of MG1. Thisregion is enriched with respect to cysteine (-10 mol %) andcontained a D domain and a carboxyl terminal domain thatcould be aligned with the corresponding domains in humanintestinal MUC2, human tracheobronchial MUC5AC, andhuman von Willebrand factor. The limited expression ofknown mucin genes, together with the considerable mucinsynthesizing capacity of submandibular gland, suggeststhat a novel (previously not described) mucin gene is ex-pressed in this gland and constitutes a portion of MG1 insalivary secretions.Key words: human sublingual gland/mucin/MGl/cDNA se-quence/MUC5BIntroductionSalivary mucins are important contributors to the lubricativeand rheological properties of whole saliva and thus play acritical role in physiological processes such as mastication,swallowing and speech (Cohen and Levine, 1989). Mucins areconstituents of the biofilm covering both hard and soft tissuesin the oral cavity providing protection from mechanical orchemical injury and from microbial assault (Mandel, 1987;Scannapieco and Levine, 1993; Tabak, 1995). Mucins are se-creted predominantly by a pair of submandibular glands, lo-cated medial to and partially under the mandible, and a pair ofsublingual glands, located anterior to the submandibular glandsunder the tongue and beneath the mucous membrane of thefloor of the mouth. The larger submandibular gland is a"mixed" gland containing both mucous and serous aciniwhereas the smaller sublingual gland is referred to as a "mu-cous" gland comprised almost entirely of mucous acini (TenCate, 1994). Both glands originate from budding of buccalepithelium in early development and are functionally classifiedas exocrine glands. Mucins are also produced to a much lesserextent by minor salivary glands (labial, buccal, and palatine)distributed throughout the submucosa in the oral cavity. Twodistinct mucins, named mucous glycoprotein 1 (MG1) and mu-cous glycoprotein 2 (MG2), have been isolated and partiallycharacterized with respect to their biochemical and functionalproperties (Levine et al., 1987; Loomis et al., 1987; Cohen andLevine, 1989; Scannapieco and Levine, 1993). These mucinswere isolated from secretion collected at the orifice of Whar-ton's duct located on the frenulum of the tongue. Since thissecretion is derived from both submandibular and sublingualglands, there is no way a priori to determine whether thesemucins are secreted by submandibular gland, sublingual gland,or both. MG1 is a large multimeric glycoprotein with an ap-parent molecular weight greater than 40 million, comprised ofsubunit monomers of approximately 3 x 10