Dissertation

Molecular analysis of a low molecular weight mucin glycoprotein from rat submandibular glands

Albone, Earl Francis

1995

Overview

Mucins are secreted glycoproteins which aid to protect and hydrate the exposed epithelial surfaces of mucosal systems. Until recently, detailed sequence and structural analysis of the protein moiety was limited by their resistance to standard biochemical and analytical analysis. In order to understand better the structure of apomucins and how their synthesis and expression is regulated, a low molecular weight mucin from rat submandibular glands (RSMG) was purified using a combination of anion exchange, gel filtration, and high-performance liquid chromatography. The mucin was chemically deglycosylated and partial amino acid sequence was obtained by Edman degradation. The sequence information was used to amplify mucin-like sequences by polymerase chain reaction, which were then used to isolate full length cDNA clones. The conceptually translated apoprotein consists of a signal peptide, followed by a secreted protein consisting of three separate domains--a non-glycosylated N-terminal domain, a central repeat domain consisting of eleven tandem repeats of thirteen amino acids, and a non-repetitive C-terminal domain. The last two domains are presumably heavily O-glycosylated due to their large content of hydroxyamino acids. Their differing ratios of hydroxyamino acid to proline suggest that they may represent two distinctly different domains of O-glycosylation. Transcripts corresponding to the cDNAs were found to be limited in their expression to the rat submandibular gland. Southern blot analysis indicated that the gene showed limited allelic polymorphism in the number of tandem repeats, a common feature of mucin genes. A cDNA clone was used to isolate overlapping lambda phage genomic clones, which showed a gene structure of three exons of 106 bp, 69 bp, and 991 bp, respectively, separated by introns of 0.9 kb and 12.5 kb. Sequence analysis of 3.8 kb of 5 $\\sp\\prime$flanking DNA revealed no homology with any other mucin or salivary genes cloned to date. Analysis of expression of the gene by Northern blot analysis from rats chronically exposed to the$\\beta$ -andrenergic agonist isoproterenol revealed no differences between agonist-exposed rats and saline controls. This gene represents only the third mucin gene, and the first secreted mucin, cloned in its entirety. Accordingly, the gene is referred to as RSM-1.

• Publisher: ProQuest Dissertations & Theses
• Subject: Biochemistry / Molecular biology
• ISBN: 9798641388427