MUC5B is a major gel-forming, oligomeric mucin from human salivary gland, respiratory tract and endocervix: identification of glycoforms and C-terminal cleavage

Abstract

Mucins from human whole saliva, as well as from respiratory- and cervical-tract secretions, were subjected to density-gradient centrifugation in CsCl/0.5 M guanidinium chloride. A polydisperse population of MUC5B mucins was demonstrated in all samples using anti-peptide antisera (LUM5B-2, LUM5B-3 and LUM5B-4) raised against sequences within the MUC5B mucin. The sequences recognized by the LUM5B-2 and LUM5B-3 antisera are located within the domains flanking the highly glycosylated regions of MUC5B, and reduction increased the reactivity with these antibodies, suggesting that the epitopes are partially shielded and that these regions are folded and stabilized by disulphide bonds. Rate-zonal centrifugation before and after reduction showed MUC5B to be a large oligomeric mucin composed of disulphide-linked subunits. In saliva and respiratory-tract secretions, populations of MUC5B mucins with different charge densities were identified by ion-exchange HPLC, suggesting the presence of MUC5B 'glycoforms'. In trachea, the F2 monoclonal antibody against the sulpho-Lewis C structure reacted preferentially with the later-to-be-eluted populations. An antibody (LUM5B-4) recognizing a sequence in the C-terminal domain of MUC5B identified, after reduction, the mucin subunits as well as smaller fragments, suggesting that some of the MUC5B mucins are cleaved within the C-terminal domain. Immunohistochemistry revealed that MUC5B is produced by cells dispersed throughout the human submandibular and sublingual glands, in the airway submucosal glands as well as the goblet cells, and in the epithelium and glands of the endocervix. The F2 antibody stained a subpopulation of the MUC5B-producing cells in the airway submucosal glands, suggesting that different cells may produce different glycoforms of MUC5B in this tissue.

Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus

“…4). In particular, the mucin glycoform MUC5B has been identified as the major secreted form of mucin in the mucosal layers protecting the CV tract (45,46), lungs (46,47), nose (48), and eye (49). The mucin content, Ϸ1-3% by weight, is also similar among cervical, nasal, and lung mucus (50)(51)(52)(53)(54).…”

MUC16 is produced in tracheal surface epithelium and submucosal glands and is present in secretions from normal human airway and cultured bronchial epithelial cells

“…Amsterdam, The Netherlands. The LUM2-3, LUM5B-2 and LUM5-1 antisera, recognizing MUC2, MUC5B and MUC5AC respectively, have been described previously (Hovenberg, Davies, & Carlstedt, 1996;Wickström, Davies, Eriksen, Veermann, & Carlstedt, 1998). The sequences NLQYEEDMRHPG (in the repeat domain of MUC16 upstream of the binding sites for the OC 125 and M11 classes of antibodies) and RRRKKEGEYNVQQQC (in the MUC16 cytoplasmic tail) were used to raise LUM16-2 and LUM16-4 polyclonal antisera ( Figure 1a).…”

“…The plates were blocked for 1 h with 0.15M NaCl, All samples studied with the LUM2-3 and LUM5B-2 antisera were subjected to reduction and alkylation before analysis as described previously cells (Hovenberg, Davies, & Carlstedt, 1996;Wickström, Davies, Eriksen, Veermann, & Carlstedt, 1998). Reactivity was detected using an alkaline phosphatase-conjugated swine anti-rabbit serum diluted 1:2000 in blocking solution with nitrophenyl phosphate (2mg/ml in 5mM…”

“…However, MUC16 has also been detected in cervical mucus (Nustad et al, 2002) and corneal epithelial cells (Argueso, Spurr-Michaud, Russo, Tisdale, & Gipson, 2003), suggesting that it may be a component of normal mucosal secretions at some sites. In the -Human airway MUC16 -5/14/2007 4 airway, large oligomeric mucins: MUC5AC derived mainly from the goblet cells (Hovenberg, Davies, & Carlstedt, 1996) and MUC5B originating largely from the submucosal gland mucous cells (Wickström, Davies, Eriksen, Veermann, & Carlstedt, 1998) have been identified as the major components of the mucus gel network. However in our studies, where airway tissue was metabolically labelled with 35 S-sulphate or 3 H-monosaccharides, radiolabelled "mucin-like" molecules were also present in secretions (Somerville et al, 1992;Svitacheva, Hovenberg, & Davies 1998;Svitacheva, & Davies, 2001).…”

Activity of lactoperoxidase when adsorbed on protein layers

“…Human salivary high molecular weight mucin (MUC5B) was purified from collected saliva according to Wickstrom et al [10] and dialyzed using a Spectra/Por ® Dialysis Membrane, MWCO: 6-8000 from Spectrum Europe (Breda, The Netherlands) according to Lindh et al [11]. All protein solutions were prepared freshly before each experiment.…”