Molecular cloning of a major human gall bladder mucin: complete C-terminal sequence and genomic organization of MUC5B

Abstract

Gall bladder mucin has been shown to play a central role in the pathogenesis of cholesterol gallstone disease. While cloning and sequencing studies have provided a wealth of information on the structure of other gastrointestinal and respiratory mucins, nothing is known about the primary structure of human gall bladder mucin. In this study, we show that the tracheobronchial mucin MUC5B is a major mucin gene product expressed in the gall bladder. Antibodies directed against deglycosylated human gall bladder mucin were used to screen a gall bladder cDNA expression library, and most of the isolated clones contained repetitive sequences nearly identical with those in the tandem repeat region of MUC5B. An additional clone (hGBM2-3) contained an open reading frame coding for a 389 residue cysteine-rich sequence. The arrangement of cysteine residues in this sequence was very similar to that in the C-terminal regions of MUC2, MUC5AC and human von Willebrand factor. This cysteine-rich sequence was connected to a series of degenerate MUC5B tandem repeats in a 7.5 kb HincII genomic DNA fragment. This fragment, with ten exons and nine introns, contained MUC5B repeats in exon 1 and a 469 residue cysteine-rich sequence in exons 2–10 that provided a 152 nucleotide overlap with cDNA clone hGBM2-3. Interestingly, the exon–intron junctions in the MUC5B genomic fragment occurred at positions equivalent to those in the D4 domain of human von Willebrand factor, suggesting that these proteins evolved from a common evolutionary ancestor through addition or deletion of exons encoding functional domains.

Human MUC5AC mucin dimerizes in the rough endoplasmic reticulum, similarly to the MUC2 mucin

“…In the far C-terminal end of many growth factors there is a cysteine motif named the ' cystine knot ', known to be directly involved in dimerization [29]. This ' cystine knot ' motif is also found in vWF and the mucins pig submaxillary mucin, MUC5AC, MUC2, MUC5B [30,31] and MUC6 [32]. Despite the similarity in the position of the cysteine residues involved in the ' cystine knot ' motif and the biosynthetic similarities of the MUC2 and MUC5AC mucins shown here, they do not heterodimerize when formed in the same cell, as shown here and also recently by van Klinken et al [33].…”

Expression of Mucins in Mucoid Otitis Media

“…At both ends of the molecule, MUC5B contains abundant cysteine residues (Keates et al 1997) which are potential residues for formation of disulfide bonds inter-or intramolecularly and serve as a biochemical basis for dimerization or polymerization. Dimerization of mucin monomers is known to occur at the endoplasmic reticulum (Asker et al 1998;Dekker and Strous 1990); however, it is not clear where dimerized mucins polymerize.…”

Small gallstones, preserved gallbladder motility, and fast crystallization are associated with pancreatitis†‡

“…7,39 Several MUC genes (MUC1, MUC3, MUC4, MUC5B, MUC6) are expressed in human gallbladder mucosa. 40,41 Upregulation of these genes could lead to increased gallbladder mucin concentrations. Although mucin secretion has been suggested to be stimulated by cholesterol supersaturation, 22 hydrophobic bile salts, 42 or gallbladder wall inflammation, 43 we found no differences between pancreatitis and symptomatic gallstone patients in this respect.…”