Identification of Two Glycoforms of the MUC5B Mucin in Human Respiratory Mucus

Abstract

It has been demonstrated previously that respiratory secretions contain three oligomeric, gel-forming mucins; one of these was identified as the product of the MUC5AC gene (1). Here we demonstrate that the other two mucins are glycoforms of the MUC5B gene product. This was accomplished by trypsin treatment of the purified reduced mucin subunit populations and N-terminal sequencing of the liberated peptides. The products of trypsin digestion were separated by gel filtration into high molecular weight mucin glycopeptides and low molecular weight tryptic peptides. The latter were fractionated by reverse phase chromatography, and four of the major peptides were sequenced. Three of these peptides were identical to and contiguous within a 51-amino acid sequence deduced from a cDNA clone (JER57) encoding a portion of the MUC5B mucin. The other peptide is also present within this sequence but showed identity in only 9 of its 10 residues. A polyclonal antiserum raised against one of these peptides was reactive with the two putative MUC5B glycoforms. Analysis of the high molecular weight glycopeptides indicated that the MUC5B subunit contained different types and lengths of glycosylated domains; one domain of M r 7.3 ؋ 10 5 , two domains of M r 5.2 ؋ 10 5, and a third domain of M r 2.0 ؋ 10 5 . The amino acid composition of the larger two glycopeptides was similar in serine, threonine, and proline content but distinct from that of the smallest glycopeptide. Each of these domains in the mucin subunit is separated by a trypsin-sensitive region, and the relative abundance of the major peptides derived by proteolysis of these regions and their occurrence in a contiguous sequence suggest that they contain a common cysteine-rich motif.Respiratory tract mucus is the principal barrier in the lung against chemical and pathological insult. The physical properties of this gel-like secretion are due solely to high molecular weight O-linked glycoproteins termed mucins. Respiratory mucins are polydisperse in mass (M r 2-40 ϫ 10 6 ) and length (0.5-10 m) and can be fragmented into their constituent subunits (M r 2-3 ϫ 10 6

Stimulation of Airway Mucin Gene Expression by Interleukin (IL)-17 through IL-6 Paracrine/Autocrine Loop

“…Cytokines-We treated primary tracheobronchial epithelial cells grown in an air liquid interface with a panel of cytokines (IL-1␣, 1␤, 2, 3, 4, 5,6,7,8,9,10,11,12,13,15,16,17,18, and TNF␣) for 24 h. As shown in Fig. 1, our cytokine panel at 10 ng/ml showed that only IL-6 and IL-17 could induce a dramatic increase in expression of MUC5AC and MUC5B.…”

“…Cytokine, Antibody, and Inhibitor Treatment-Cytokines, IL-1␣, 1␤, 2, 3,4,5,6,7,8,9,10,11,12,13,15,16,17,18, and TNF␣ were purchased from R&D systems Inc. (Minneapolis, MN). They were dissolved in phosphate-based saline with 1% bovine serum albumin and added directly to the primary TBE cultures at concentrations of 10 and 50 ng/ml.…”

Interleukin-1β and Tumor Necrosis Factor-α Induce MUC5AC Overexpression through a Mechanism Involving ERK/p38 Mitogen-activated Protein Kinases-MSK1-CREB Activation in Human Airway Epithelial Cells

“…It has been reported that MUC5AC is the major mucin in human airways (22)(23)(24)44). The mechanism of the regulation of MUC5AC secretion by inflammatory cytokines in airway is very important, and the understanding of this mechanism may offer new therapeutic strategies for the inhibition of airway mucus hypersecretion.…”

“…Eighteen types of mucin genes have been discovered to date: MUC1 (5), MUC2 (6), MUC3 (7), MUC4 (8), MUC5AC (9), MUC5B (10), MUC6 (11), MUC7 (12), MUC8 (13), MUC9 (14), MUC10 (15), MUC11 (16), MUC12 (16), MUC13 (17), MUC15 (18), MUC16 (19), MUC17 (20), and MUC18 (21). Of these, MUC5AC and MUC5B are generally recognized to be the major airway mucins (22)(23)(24) because MUC5AC is highly expressed in the goblet cells of the human airway epithelium. Moreover, MUC5AC gene expression is known to be regulated by oxidative stress (25) and retinoic acid (26).…”

N-terminal Cleavage of the Salivary MUC5B Mucin

“…MUC5B has been identified in respiratory tract, endocervical secretions, and saliva (4,17) as well as in the colon (18) and gallbladder (18,19). MUC5B is the first mucin gene in which both the cDNA and genomic sequences are known (8).…”