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.

T h e mucosal surfaces represent an impressive barrier between the 'inside' of the body and the 'outside' world. This 'front line' is under constant threat from micro-organisms, degradative enzymes and other noxious agents and is, therefore, as a first line of defence, covered by a gellike secretionmucus. T h e polymer matrix of this biofilm is provided by high-M, glycoproteins referred to as mucus glycoproteins or mucins. The mucin superfamilyMucins constitute a family of glycoproteins traditionally regarded as secretory products from epithelial surfaces. T h e apoprotein is substituted with a large number of oligosaccharides attached via an 0-glycosidic linkage between serine and/or threonine and GalNAc. In a typical mucin, the 0-linked glycansoften referred to as mucintype oligosaccharidesare enriched within serine/threonine-rich domains. These often contain proline, which is believed to give the protein core an extended conformation, in particular after substitution with the link GalNAc [ l ] , and it is therefore likely that one major function of the serinelthreonine-rich regions is to provide a matrix for the presentation of carbohydrate structures. Similar sequences have been identified in, for example, some membrane-bound glycoproteins, and a 'mucin-like stretch' has become an expression used to denote such domains with multiple sites for substitution with $To whom correspondence should be addressed. 0-linked oligosaccharides in any protein. For example, glycoproteins carrying receptor structures for selectins have been referred to as endo-

Little is known about whether the properties of respiratory mucins are altered as a result of airway irritation, but histochemical studies of respiratory tract secretory cells show a more 'acidic' staining pattern after exposure to tobacco smoke. Furthermore it has been suggested that proteoglycans are the major glycoconjugates in 'normal' respiratory secretions, whereas mucins predominate in sputum. To investigate these observations further, mucins from secretions collected from the tracheal surface of healthy non-smoking 'normal' subjects and sputum from patients with chronic bronchitis were compared. All samples contained one major mucin population after density-gradient centrifugation, and a small amount of 'denser' mucin was present in some chronic bronchitic and one of the 'normal' samples. Proteoglycans were not a major component of 'normal' secretions. The major mucin population from chronic bronchitic samples had molecular masses between 10 and 30 MDa and behaved as random coils in solution. Whole mucins from 'normal' individuals and chronic bronchitic patients were excluded from Sepharose CL-2B, whereas reduced subunits were included. Proteolysis of subunits yielded two populations of high-molecular-mass glycopeptides differing in size, suggesting the presence of two different tandem repeat regions in the mucins. Finally, mucins from patients with chronic bronchitis are less, rather than more, acidic than those from 'normal' individuals. Mucins from bronchitic sputum and 'normal' secretions are thus similar in their macromolecular properties, but differ slightly in charge density.

Bovine trachea in organ culture secretes mucus containing a 'high-density' (1.46 g/ml) and a 'low-density' (1.37 g/ml) mucin similar to those identified previously in bovine respiratory secretions [Hovenberg, Carlstedt and Davies (1997) Biochem. J. 321, 117-123]. After pulse-labelling, autoradiography showed uptake of [35S]sulphate by both epithelial goblet cells and submucosal glands, while [3H]proline was mainly incorporated into the ciliated surface epithelial cells. After 24 h of radiolabelling, neither the high- nor the low-density mucin in the secreted mucus gel was heavily radiolabelled with the precursors. In contrast, a population of molecules banding at 1.50 g/ml was heavily radiolabelled with [35S]sulphate. This component was smaller than the high-density mucin from the mucus gel and was insensitive to reduction or digestion with chondroitin ABC lyase or heparan sulphate lyase. The molecules yielded two populations of high-Mr glycopeptides upon trypsin digestion, were sensitive to keratanase and endo-beta-galactosidase digestion and contained O-linked glycans. Extracts of the surface epithelium and submucosal tissue after radiolabelling showed that the high- and low-density mucins in the tissue were also poorly radiolabelled. Thus, under these conditions, the radiolabelled precursors were not effectively incorporated into the large oligomeric mucins but into a high-Mr monomeric species. This study suggests that data obtained in investigations where mucins are radiolabelled and studied without further separation into distinct components may rather reflect the turnover of this 'novel' monomeric species than the large oligomeric mucins.