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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.

Mucins were extracted from the epithelial surface and the submucosal tissue of human trachea in order to enrich glycoproteins from the goblet cells and the submucosal glands respectively. The macromolecules were purified using density-gradient centrifugation, and the presence of the MUC5AC mucin was investigated using an antiserum raised against a synthetic peptide based on the sequence of the MUC5AC apoprotein. Mucins from the surface epithelium showed a higher reactivity with the antiserum relative to carbohydrate than those from the submucosa, and ion-exchange HPLC of reduced subunits revealed the presence of two distinct mucin populations in the samples. The predominant species from the surface epithelium was more acidic than the major population from the submucosa and showed a strong reactivity with the anti-MUC5AC anti-serum. In contrast, the major portion of the submucosal mucins were less acidic and showed no MUC5AC reactivity, although a more acidic population did react with the antibody. Rate-zonal centrifugation showed that the MUC5AC mucin from the surface epithelium is smaller than the major submucosal mucin, and that both are composed of subunits. Immunolocalization confirmed that the MUC5AC mucin from human trachea originates from the goblet cells and that this glycoprotein is not a major product of the submucosal glands.

Airway mucus was collected from healthy and chronic bronchitic subjects. The chronic bronchitic sputum was separated into gel and sol phase by centrifugation and mucins were isolated using isopycnic density-gradient centrifugation in CsCl. The presence of the MUC5AC and MUC2 mucins was investigated with antisera raised against synthetic peptides with sequences from the respective apoproteins. The gel and sol phase of chronic bronchitic sputum as well as healthy respiratory secretions were shown to contain MUC5AC whereas the MUC2 mucin could not be detected. Rate-zonal centrifugation showed that the MUC5AC mucin was large, polydisperse in size and that reduction yielded subunits. Ion-exchange HPLC revealed the presence of two subunit populations in all secretions, the MUC5AC subunits always being the more acidic. MUC5AC is thus the first large, subunit-based, gel-forming respiratory mucin identified and this glycoprotein is biochemically distinct from at least one other population of large, gel-forming mucins also composed of subunits but lacking a genetic identity.

Mucous secretions were collected from tracheas of patients undergoing minor surgery under general anaesthesia with tracheal intubation, and mucus glycoproteins were isolated by using isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. 'Whole' mucins were excluded from a Sepharose CL-2B gel, whereas subunits obtained after reduction were included. Trypsin digestion of subunits afforded high-Mr glycopeptides (T-domains), which were further included in the gel. The latter fragments are heterogeneous and comprise two or three populations, as indicated by gel chromatography and ion-exchange h.p.l.c. Rate-zonal centrifugation showed that the 'whole' mucins are polydisperse in size, with a weight-average Mr of (14-16) x 10(6). The macromolecules were observed by electron microscopy, as linear and apparently flexible thread-like structures. Subunits and T-domains had weight-average contour lengths of 490 nm and 160 nm respectively. It is concluded that mucus glycoproteins are present in secretions from the healthy lower respiratory tract. The 'whole' tracheal mucins are assembled from subunits, which in turn can be fragmented into high-Mr glycopeptides corresponding to the oligosaccharide domains typically found in mucus glycoproteins. The size and macromolecular architecture of the tracheal mucins is thus similar to that observed for mucins from human cervical mucus, chronic bronchitic sputum and pig stomach, providing yet another example of this general design of these macromolecules, i.e. subunits assembled end-to-end into very large linear and flexible macromolecules.

Gastric MUC5AC and MUC6 mucins were studied using polyclonal antibodies. Immunohistochemistry showed MUC5AC to originate from the surface epithelium, whereas MUC6 was produced by the glands. Mucins from the surface epithelium or glands of corpus and antrum were purified using CsCl/4M guanidinium chloride density-gradient centrifugation. MUC5AC appeared as two distinct populations at 1.4 and 1.3g/ml, whereas MUC6, which was enriched in the gland tissue, appeared at 1.45g/ml. Reactivity with antibodies against the Leb structure (where Le represents the Lewis antigen) followed the MUC5AC distribution, whereas antibodies against the Ley structure and reactivity with the GlcNAc-selective Solanum tuberosum lectin coincided with MUC6, suggesting that the two mucins are glycosylated differently. Rate-zonal centrifugation of whole mucins and reduced subunits showed that both gastric MUC5AC and MUC6 are oligomeric glycoproteins composed of disulphide-bond linked subunits and that oligomeric MUC5AC was apparently smaller than MUC6. A heterogeneous population of ‘low-density’ MUC5AC mucins, which were smaller than the ‘high-density’ ones both before and after reduction, reacted with an antibody against a variable number tandem repeat sequence within MUC5AC, suggesting that they represent precursor forms of this mucin. Following ion-exchange HPLC, both MUC5AC and MUC6 appeared as several distinct populations, probably corresponding to ‘glycoforms’ of the mucins, the most highly charged of which were found in the gland tissue.

The "insoluble" glycoprotein complex was isolated from human colonic tissue and mucin subunits were prepared following reduction. Antibodies raised against peptide sequences within MUC2 revealed that virtually all of this mucin occurs in the insoluble glycoprotein complex. In addition, reduction released a 120-kDa Cterminal MUC2 fragment, showing that proteolytic cleavage in this domain may occur and leave the fragment attached to the complex via disulfide bonds. The variable number tandem repeat region and the irregular repeat domain were isolated after trypsin digestion and shown to have molecular weights of 930,000 and 180,000, respectively, suggesting a molecular weight for the entire MUC2 monomer of approximately 1.5 million. Gel chromatography and agarose gel electrophoresis revealed several populations of MUC2 subunits, and analytical ultracentrifugation showed that these have molecular weights on the order of 2 million, 4 million, and 5 million, corresponding to monomers, dimers, and trimers, respectively. Agarose gel electrophoresis of subunits from individuals expressing both a "long" and a "short" MUC2 allele revealed a larger number of populations, consistent with the presence of short and long monomers and oligomers arising from permutations of the two types of monomers. In addition to disulfide bonds, MUC2 monomers are apparently joined by a "novel," reduction-insensitive bond.The mucosal surface of the gastrointestinal tract is protected by a visco-elastic mucus gel formed by high molecular mass (0.5-25 ϫ 10 6 ) glycoproteins referred to as mucins. The protein backbones of mucins are heavily substituted with O-linked oligosaccharides attached to serine and/or threonine residues, and these amino acids are, together with proline, typically enriched within so-called mucin domains. Several mucin domains may occur in a single mucin subunit and these regions are flanked by less heavily glycosylated segments of the protein core. Large secreted mucins from the respiratory tract, stomach, and endocervix have been shown to be linear oligomers formed by subunits joined via disulfide bonds (1, 2). However, less is known about the macromolecular architecture of intestinal mucins.The mucin genes MUC2, MUC3, MUC4, MUC5B, and MUC6 are expressed in normal human colon (3-7). MUC2 is believed to be the dominating mucus-forming species in this tissue and is, so far, the only "large" mucin of which the cDNA has been fully sequenced (8). The apoprotein contains two mucin domains, which differ in length and are separated by a cysteinecontaining region (Fig. 1A). The longer domain, which is referred to as the variable number tandem repeat (VNTR) 1 region is composed largely of tandemly repeated 23 amino acid peptide units, which vary in number between alleles and are rich in threonine and proline (9, 10). The shorter mucin domain comprises a 347-amino acid-long irregular repeat rich in threonine, serine, and proline. The regions flanking the mucin domains each show a high degree of similarity to the N-and C-terminal cyst...

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by an impaired epidermal barrier, dysregulation of innate and adaptive immunity, and a high susceptibility to bacterial colonization and infection. In the present study, bacterial biofilm was visualized by electron microscopy at the surface of AD skin. Correspondingly, Staphylococcus aureus (S. aureus) isolates from lesional skin of patients with AD, produced a substantial amount of biofilm in vitro. S. aureus biofilms showed less susceptibility to killing by the antimicrobial peptide LL-37 when compared with results obtained using planktonic cells. Confocal microscopy analysis showed that LL-37 binds to the S. aureus biofilms. Immuno-gold staining of S. aureus biofilm of AD skin detected the S. aureus derived protease staphopain adjacent to the bacteria. In vitro, staphopain B degraded LL-37 into shorter peptide fragments. Further, LL-37 significantly inhibited S. aureus biofilm formation, but no such effects were observed for the degradation products. The data presented here provide novel information on staphopains present in S. aureus biofilms in vivo, and illustrate the complex interplay between biofilm and LL-37 in skin of AD patients, possibly leading to a disturbed host defense, which facilitates bacterial persistence.