Function of the CysD domain of the gel-forming MUC2 mucin

The colonic human MUC2 mucin forms a polymeric gel by covalent disulfide bonds in its N- and C-termini. The middle part of MUC2 is largely composed of two highly O-glycosylated mucin domains that are interrupted by a CysD domain of unknown function. We studied its function as recombinant proteins fused to a removable immunoglobulin Fc domain. Analysis of affinity-purified fusion proteins by native gel electrophoresis and gel filtration showed that they formed oligomeric complexes. Analysis of the individual isolated CysD parts showed that they formed dimers both when flanked by two MUC2 tandem repeats and without these. Cleavages of the two non-reduced CysD fusion proteins and analysis by MS revealed the localization of all five CysD disulfide bonds and that the predicted C-mannosylated site was not glycosylated. All disulfide bonds were within individual peptides showing that the domain was stabilized by intramolecular disulfide bonds and that CysD dimers were of non-covalent nature. These observations suggest that CysD domains act as non-covalent cross-links in the MUC2 gel, thereby determining the pore sizes of the mucus.     

The N terminus of the MUC2 mucin forms trimers that are held together within a trypsin-resistant core fragment

The N terminus of the human MUC2 mucin (amino acids 1-1397) has been expressed as a recombinant tagged protein in Chinese hamster ovary cells. The intracellular form was found to be an endoglycosidase H-sensitive monomer, whereas the secreted form was an oligomer that gave monomers upon disulfide bond reduction. The secreted MUC2 N terminus contained a trypsin-resistant core fragment. Edman sequencing and mass spectrometry of the peptides obtained localized this core fragment to the C-terminal end of the recombinant protein. This core retained its oligomeric nature with an apparent mass of approximately 240 kDa. Upon reduction, peptides of approximately 85 kDa were found, suggesting that the N terminus forms trimers. This interpretation was also supported by gel electrophoresis and gel filtration of the intact MUC2 N terminus. Electron microscopy revealed three globular domains each linked via an extended and flexible region to a central part in a trefoil-like manner. Immunostaining with gold-labeled antibodies localized the N-terminal end to the three globular structures, and the antibodies directed against the Myc and green fluorescent protein tags attached at the C terminus localized these to the stalk side of the central trefoil. The N terminus of the MUC2 mucin is thus assembled into trimers that contain proteolytically stable parts, suggesting that MUC2 can only be partly degraded by intestinal proteases and thus is able to maintain a mucin network protecting the intestine.     

Mucin complexes: characterization of the "link" component of submandibular mucus glycoprotein.

1. Analysis of the submandibular saliva revealed that the secretion consists of mucin complexed with 150 kDa fibronectin fragment and DNA. 2. The kallikreins, secreted by the submandibular gland, appear to be responsible for the fibronectin fragmentation, since an identical peptide was also generated when fibronectin was subjected to incubation with the submandibular saliva or the purified enzyme. 3. The results provide evidence that the 150 kDa glycopeptide so-called salivary mucin "link" component is neither an integral part of the mucin molecule, nor linked to mucin subunits by disulfide bonds, but is a fibronectin fragment which associates with mucin. 4. Using mucin monoclonal antibody (3G12), it was revealed that the nonglycosylated (naked) 8-12 kDa fragment of the mucin molecule is responsible for the interaction of mucin with other components of saliva. 5. Under physiological conditions, the interaction of mucin with fibronectin on the luminal surfaces may be relevant in building mucous barrier and protection of the delicate oral epithelium from damage.     

Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product

The MG1 population of mucins was isolated from human whole salivas by gel chromatography followed by isopycnic density gradient centrifugation. The reduced and alkylated MG1 mucins, separated by anion exchange chromatography, were of similar size (radius of gyration 55-64 nm) and molecular weight (2.5-2.9 x 10(6) Da). Two differently-charged populations of MG1 subunits were observed which showed different reactivity with monoclonal antibodies to glycan epitopes. Monosaccharide and amino acid compositional analyses indicated that the MG1 subunits had similar glycan structures on the same polypeptide. An antiserum recognizing the MUC5B mucin was reactive across the entire distribution, whereas antisera raised against the MUC2 and MUC5AC mucins showed no reactivity. Western blots of agarose gel electrophoresis of fractions across the anion exchange distribution indicated that the polypeptide underlying the mucins was the product of the MUC5B gene. Amino acid analysis and peptide mapping performed on the fragments produced by trypsin digestion of the two MG1 populations yielded data similar to that obtained for MUC5B mucin subunits prepared from respiratory mucus (Thornton et al., 1997) and confirmed that the MUC5B gene product was the predominant mucin polypeptide present. Isolation of the MG1 mucins from the secretions of the individual salivary glands (palatal, sublingual, and submandibular) indicate that the palatal gland is the source of the highly charged population of the MUC5B mucin.     

A high-density putative monomeric mucin is the major [35S]labelled macromolecular product of human colorectal mucins in organ culture

We have studied the biosynthesis of mucins in organ cultures of human colon using isopycnic density-gradient centrifugation following pulse labelling with [(35)S]sulphate and [(3)H]-D-glucosamine. A high-density [(35)S]sulphate labelled component, of larger size than MUC2 monomers, appeared in the tissue and also in the medium. It was not degraded by reduction, trypsin digestion, digestion with chondroitin ABC lyase or heparan sulphate III lyase, but was cleaved into smaller fragments following alkaline borohydride treatment and appears to be a monomeric, mucin-like molecule containing a protease-resistant domain with a larger hydrodynamic volume than MUC2 monomers. Although this macromolecule incorporated much more radiolabel than MUC2, it was not detected using chemical analysis and thus appears to be a component with a high metabolic turnover present in a very small amount. Most of the [(3)H]-D-glucosamine label was associated with low-density material that was well separated from MUC2, which was poorly labelled. Most of MUC2 was associated with the tissue as an 'insoluble' complex. The amount of MUC2 remained constant and its associated radiolabel increased only slightly with time. Analysis of the MUC2 subunits from the reduced 'insoluble' complex showed the typical reduction-insensitive oligomers and confirmed that the radiolabel was associated with this mucin. The large size of the [(35)S]-labelled putative monomeric mucin makes it difficult to separate it from reduced insoluble complex MUC2. As a result, many studies of intestinal mucin synthesis and secretion in the past have most likely been performed on 'mixtures' of this mucin and MUC2 and are thus not possible to interpret as the metabolic behaviour of oligomeric mucins.     

On the macromolecular composition of the zona pellucida from porcine oocytes

The chemical and immunological relation between the glycoprotein components of the pig oocyte zona pellucida resolved by two-dimensional polyacrylamide gel electrophoresis was investigated. After disulfide bond reduction, four microheterogeneous glycoprotein components with apparent molecular weights of 25K, 55K, 65K, and 90K were resolved. When disulfide bonds were left unreduced, two microheterogeneous glycoprotein components were resolved with apparent molecular weights of 55K and 90K. Actin was present, but as a contaminant of the zona pellucida rather than as a true component. The structural relation of these components was investigated using deglycosylation with trifluoromethane-sulfonic acid, limited proteolysis with Staphylococcus aureus V8 protease, amino acid and carbohydrate composition analyses, sequence analysis, and monoclonal antibodies. The 25K and 65K components comigrated with the 90K component when disulfide bonds were not reduced. When the intermolecular disulfide bonds crosslinking the two components were reduced, the 25K and 65K components behaved independently. The 25K and 65K components were derived from the 90K glycoprotein family by proteolysis. The 25K component originated from the C-terminal end, and the 65K component from the N-terminal end of the 90K glycoprotein. The 55K component was composed of two chemically and antigenically distinct glycoproteins, termed 55K alpha and 55K beta, that electrophoretically comigrated. The N-terminal amino acid of the 55K alpha family was blocked. The 55K beta family had an N-terminal amino acid sequence of Asp-Val-Pro-Thr-Ile-Gly-Leu-Ser-X-Ala-Pro-Thr. Thus, the two to four electrophoretic components of the zona pellucida observed on gel electrophoresis are derived from three glycoprotein families.     

MUC5AC,but not MUC2, is a prominent mucin in respiratory secretions

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 centrfugation 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.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate - CF cystic fibrosis - DFP diisopropylphosphofluoridate - DTT dithiothreitol - EDTA ethylenedinitrilotetraacetic acid - NEM N-ethylmaleimide - PAS periodic acid/Schiffs - PMSF phenylmethylsulphonyl fluoride - Tris Tris(hydroxymethyl)aminomethane - VNTR variable number of tandem repeats     

The structure of tracheobronchial mucins from cystic fibrosis and control patients.

Tracheobronchial mucin samples from control and cystic fibrosis patients were purified by gel filtration chromatography on Sephacryl S-1000 and by density gradient centrifugation. Normal secretions contained high molecular weight (approximately 10(7] mucins, whereas the cystic fibrosis secretions contained relatively small amounts of high molecular weight mucin together with larger quantities of lower molecular weight mucin fragments. These probably represent products of protease digestion. Reducing the disulfide bonds in either the control or cystic fibrosis high molecular weight mucin fractions released subunits of approximately 2000 kDa. Treating these subunits with trypsin released glycopeptides of 300 kDa. Trypsin treatment of unreduced mucin also released fragments of 2000 kDa that could be converted into 300-kDa glycopeptides upon disulfide bond reduction. Thus, protease-susceptible linkages within these mucins must be cross-linked by disulfide bonds so that the full effects of proteolytic degradation of mucins remain cryptic until disulfide bonds are reduced. Since various combinations of protease treatment and disulfide bond reduction release either 2000- or 300-kDa fragments, these fragments must represent important elements of mucin structure. The high molecular weight fractions of cystic fibrosis mucins appear to be indistinguishable from control mucins. Their amino acid compositions are the same, and various combinations of disulfide bond reduction and protease treatment release products of identical size and amino acid composition. Sulfate and carbohydrate compositions did vary considerably from sample to sample, but the limited number of samples tested did not demonstrate a cystic fibrosis-specific pattern. Thus, tracheobronchial mucins from cystic fibrosis and control patients are very similar, and both share the same generalized structure previously determined for salivary, cervical, and intestinal mucins.     

The sodium channel from rat brain. Separation and characterization of subunits

Procedures are described for separation of the alpha, beta 1, and beta 2 subunits of the voltage-sensitive sodium channel from rat brain by gel filtration in sodium dodecyl sulfate (SDS) before and after reduction of intersubunit disulfide bonds or by preparative SDS-gel electrophoresis. Partial proteolytic maps of the SDS-denatured subunits indicate that they are nonidentical polypeptides. They are all heavily glycosylated and contain complex carbohydrate chains that bind wheat germ agglutinin. The apparent molecular weights of the separated subunits were estimated by gradient SDS-gel electrophoresis, by Ferguson analysis of migration in SDS gels of fixed acrylamide concentration, or by gel filtration in SDS or guanidine hydrochloride. For the alpha subunit, SDS-gel electrophoresis under various conditions gives an average Mr of 260,000. Gel filtration methods give anomalously low values. Removal of carbohydrate by sequential treatment with neuraminidase and endoglycosidase F results in a sharp protein band with apparent Mr = 220,000, suggesting that 15% of the mass of the native alpha subunit is carbohydrate. Electrophoretic and gel filtration methods yield consistent molecular weight estimates for the beta subunits. The average values are: beta 1, Mr = 36,000, and beta 2, Mr = 33,000. Deglycosylation by treatment with endoglycosidase F, trifluoromethanesulfonic acid, or HF yields sharp protein bands with apparent Mr = 23,000 and 21,000 for the beta 1 and beta 2 subunits, respectively, suggesting that 36% of the mass of the native beta 1 and beta 2 subunits is carbohydrate.     

Isolation and characterisation of a sialic acid-binding lectin (carcinoscorpin) from Indian horseshoe crab Carcinoscorpius rotunda cauda

A sialic acid-binding lectin, carcinoscorpin, has been purified to apparent homogeneity in 40% yield from the Indian horseshoe carb, Carcinoscorpius rotunda cauda. This glycoprotein lectin of molecular weight 420,000 was composed of two non-identical subunits of molecular weights 27,000 and 28,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The hemagglutination activity of the lectin was susceptible to guanidine-HCl; modification of tyrosyl and tryptophanyl residues also inhibited the activity although alkylation of the -SH group, reduction of disulfide bonds or modification of amino and carboxyl groups were without any effect. The monomeric form of the lectin produced by succinylation of native protein was inactive in binding to sialoglycoconjugates.     

A novel protein derived from the MUC1 gene by alternative splicing and frameshifting

Genes that have been designated the name "MUC" code for proteins comprising mucin domains. These proteins may be involved in barrier and protective functions. The first such gene to be characterized and sequenced is the MUC1 gene. Here we report a novel small protein derived from the MUC1 gene by alternative splicing that does not contain the hallmark of mucin proteins, the mucin domain. This protein termed MUC1/ZD retains the same N-terminal MUC1 sequences as all of the other known MUC1 protein isoforms. The common N-terminal sequences comprise the signal peptide and a subsequent stretch of 30 amino acids. In contrast, the MUC1/ZD C-terminal 43 amino acids are novel and result from a reading frameshift engendered by a splicing event that forms MUC1/ZD. The expression of MUC1/ZD at the protein level in human tissues is demonstrated by Western blotting, immunohistochemistry, immunoprecipitation, and an ELISA. Utilization was made of affinity-purified MUC1/ZD-specific polyclonal antibodies as well as two different monoclonal antibodies that are monospecific for the MUC1/ZD protein. The MUC1/ZD protein is expressed in tissues as an oligomeric complex composed of monomers linked by disulfide bonds contributed by MUC1/ZD cysteine residues. MUC1/ZD protein expression did not parallel that of the tandem-repeat array-containing MUC1 protein. Results presented here demonstrate for the first time the expression of a novel MUC1 protein isoform MUC1/ZD, which is generated by an alternative splicing event that both deletes the tandem-repeat array and leads to a C-terminal reading frameshift.     

Identification of the glucagon receptor by covalent labeling with a radiolabeled photoreactive glucagon analogue

The photoreactive 125I-labeled glucagon-NAPS [125I-labeled 2-[2-nitro-4-azidophenyl)sulfenyl]-Trp25-glucagon] was used to label the glucagon receptor sites in rat liver plasma membranes. The proteins labeled were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without reduction with dithiothreitol. The photoaffinity peptide specifically labeled a number of bands with apparent molecular weights greater than 200000 and probably at least two protein bands in the molecular weight range 52000-70000. The relative amounts of radioactivity associated with these bands and their relative mobilities differed in samples from reduced and unreduced membranes. Their relative mobilities also differed with percent acrylamide cross-linking, suggesting a glycoprotein nature and the presence of intramolecular disulfide bonds. A nonspecifically labeled band with an apparent molecular weight of 27000-28000 also displayed a similar behavior. Photolabeling in the presence of 0.1 mM guanosine 5'-triphosphate (GTP) decreased the amount of radiolabeling of these bands, suggesting their involvement in the glucagon stimulation of adenylate cyclase. The photolabeled receptor in the membranes, solubilized with Lubrol-PX and fractionated on an Ultrogel AcA22 column, eluted with an apparent molecular weight of 200000-250000. Addition of GTP to the solubilized glucagon receptor of nonirradiated membranes caused complete dissociation of the complex. Gel electrophoresis of the partially purified radiolabeled receptor identified the same protein components observed in photolabeled membranes. These results indicate that the glucagon receptor is an oligomer probably composed of at least two different subunits that are linked together or greatly stabilized by disulfide bonds. They also show that 125I-labeled glucagon-NAPS can be used effectively to covalently label the putative glucagon receptor and thus aid in its further characterization.     

Structure of canine tracheobronchial mucin glycoprotein

Canine tracheal mucin glycoprotein was isolated from beagle dogs fitted with tracheal pouches. Following exclusion chromatography on Sepharose CL-4B, noncovalently associated proteins were further resolved by dissociative density gradient centrifugation in CsBr-guanidinium chloride, and the mucin was then extracted with chloroform-methanol. The delipidated high-density product obtained had a nominal molecular weight of about 10(6) and an overall composition characteristic for a mucin glycoprotein, viz., a high content of serine and threonine, about 80% carbohydrate by weight, the absence of mannose or uronic acid, measurable ester sulfate, and a Pronase-resistant domain of molecular weight (1.75-3.0) X 10(5) which contains essentially all of the saccharide residues. Noncovalently bound lipid amounted to 6-10% by weight and was primarily cholesterol and cholesteryl esters. Cleavage of disulfide bonds by performic acid oxidation resulted in the release of a protein (Mr 65,000) not otherwise resolved by sodium dodecyl sulfate gel electrophoresis or the purification scheme.     

On the molecular weight of mitochondrially synthesized subunits of rat liver cytochrome oxidase.

The subunit composition of cytochrome c oxidase from rat liver mitochondria was studied by dodecylsulfate polyacrylamide gel electrophoresis. The apparent molecular weight of the seven subunits are in reasonable agreement with published data on cytochrome c oxidase subunits from other sources. Two additional subunits were found if the electrophoresis was performed with 8m urea, due to splitting of the smallest subunit. Performic acid oxidation of the isolated subunits I and II increased the apparent molecular weights from 38000 to 48000 and from 24500 to 29000, respectively, accompained by a normalization of the anomalous behaviour of subunit I in the Ferguson plot. It is suggested that performic acid, by splitting extremely inaccessible disulfide bridges, mediates full complexing of the subunits by dodecylsulfate, thus permitting the determination of the real molecular weights by dodecylsulfate polyacrylamide gel electrophoresis.     

Characterization of pepsin-resistant collagen-like tail subunit fragments of 18S and 14S acetylcholinesterase from Electrophorus electricus

Digestion of 18S and 14S acetylcholinesterase from eel electric organ with pepsin at 15 degrees C for 6 h results in extensive degradation of the catalytic subunits, but a major portion of the collagen-like tail structure associated with these enzyme forms resists degradation. The pepsin-resistant structures partially aggregate and can be isolated by gel exclusion chromatography on Sepharose CL-6B in buffered 1 M sodium chloride. The largest structure, denoted F3, has a molecular weight of 72 000 according to gel electrophoresis in sodium dodecyl sulfate and is composed of three 24 000 molecular weight polypeptides linked by intersubunit disulfide bonds. This structure is largely, but not completely, a collagen-like triple helix as indicated by a circular dichroism spectrum typical of triple-helical collagen and an amino acid composition characterized by 27% glycine, 5% hydroxyproline, and 5% hydroxylysine. Continued pepsin action results in degradation of the disulfide linkage region such that disulfide-linked dimers F2 and finally F1 monomers become the predominant forms in sodium dodecyl sulfate. Digested samples in which either F3 or F2 predominate have virtually identical circular dichroic spectra and amino acid compositions and generate similar diffuse 24 000 molecular weight polypeptides following disulfide reduction. Thus the intersubunit disulfide linkages in F3 must occur close to the end(s) of the fragment polypeptide chains. Pepsin conversion of F3 to F2 is particularly accelerated between 25 and 30 degrees C, suggesting that the triple-helical structure in the disulfide linkage region undergoes thermal destabilization in this temperature range. Digestion at 40 degrees C yields presumably triple-helical F1 structures devoid of disulfide linkages, although their degradation to small fragments can be detected at this temperature. The question of whether the three tail subunits that give rise to F1 polypeptides are identical remains open.     

N-terminal cleavage of the salivary MUC5B mucin. Analogy with the Van Willebrand propolypeptide?

Sequence similarities between the oligomeric mucins (MUC2, MUC5AC, MUC5B) and the von Willebrand factor suggest that they may be assembled in a similar way. After oligomerization, a fragment corresponding to the D1 and D2 domains is released from the von Willebrand factor. This cleavage does not appear to occur in pig submaxillary mucin, the only mammalian mucin in which this cleavage has been examined thus far, but whether other oligomeric mucins undergo N terminus proteolysis is not known. Antibodies recognizing the D1, D2, D3, and the first Cys domains in MUC5B were established and used to investigate to what extent proteolytic cleavage occurs within the N-terminal part of salivary MUC5B. The antibodies against the D1 and D2 domains identified a polypeptide corresponding in size to a MUC5B fragment generated by cleavage within the D' domain analogously with the von Willebrand factor propolypeptide. The antibodies did not recognize the main mucin population, suggesting that the major part of salivary MUC5B is subjected to this cleavage. An antibody recognizing the D3 domain was used to reveal a second cleavage site in the "soluble" but not in the "insoluble" MUC5B fraction: the first structural difference observed between soluble and insoluble salivary MUC5B. The identification of these cleavage events shows that the N-terminal sites for MUC5B oligomerization are present in the D3 domain and/or in domains located C-terminal to this part of the molecule.     

Isolation and characterization of the major androgen-dependent glycoprotein of canine prostatic fluid

Canine prostatic fluid, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions, is characterized by the presence of a single diffuse band (Mr approximately 30,000) which accounts for over 90% of the total protein. The biosynthesis of this protein is under androgen control. Castration results in the disappearance of this protein, whereas its presence in the prostate can be maintained in the castrated animal with exogenous androgen. Analysis of the native protein by isoelectric focusing revealed the presence of 10-13 charged variants with pI values in the range of 6.5 to 8.4. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed that each isoform is constructed of two dissimilar polypeptide subunits covalently linked through disulfide bonds. One subunit has a molecular weight of 15,000 (H chain); the second subunit (L chain) has a variable molecular weight in the 12,000-14,000 range. The H and L subunits have been purified by preparative isoelectric focusing and chemically characterized. Based on tryptic peptide mapping, NH2-terminal analysis, amino acid and carbohydrate composition, the H and L subunits are structurally unrelated and consequently appear to be unique gene products. Furthermore, the L subunit is glycosylated which potentially accounts for its size heterogeneity. Quantitative NH2-terminal analysis indicated that the H and L subunits are present in the native molecule in a ratio of 2:1, suggesting that the native molecule is a trimer with an apparent molecular weight of 43,000. Based on electrophoretic data, the glycoprotein also constitutes the major fraction of the soluble protein in canine prostatic tissue; its presence is organ specific. This glycoprotein should prove useful as a marker for prostatic function under varying hormonal and environmental conditions.     

Purification and characterization of Tora-bean (Phaseolus vulgaris) lectin

A lectin purified from the Tora-bean (Phaseolus vulgaris) by affinity chromatography with Con-A Sepharose was shown to be a glycoprotein containing 7.8% neutral sugars (D-mannose, N-acetyl-D-glucosamine, L-fucose, and D-xylose, in a molar ratio of 9.6 : 2.0 : 0.6 : 0.7). Its molecular weight was 130,000, as estimated by exclusion gel chromatography, and SDS gel electrophoresis showed that it consists of four subunits of molecular weight 32,000. The lectin reacts with various glycoproteins, i.e., blood group substances, human parotid salivary glycoprotein, fetuin, and bovine submaxillary mucin. Divalent cations, such as Ca2+, Mn2+, and Mg2+, appear to stimulate its reactivity. Inhibition tests using the glycopeptide fragment from fetuin and some oligosaccharides, as well as the binding test with 14C-N-acetyl-lactosamine suggest that the sequence of D-galactose, N-acetyl-D-glucosamine, and D-mannose residues in the carbohydrate chain of fetuin is essential for binding.     

Assembly of the Respiratory Mucin MUC5B: A NEW MODEL FOR A GEL-FORMING MUCIN*

Mucins are essential components in mucus gels that form protective barriers at all epithelial surfaces, but much remains unknown about their assembly, intragranular organization, and post-secretion unfurling to form mucus. MUC5B is a major polymeric mucin expressed by respiratory epithelia, and we investigated the molecular mechanisms involved during its assembly. Studies of intact polymeric MUC5B revealed a single high affinity calcium-binding site, distinct from multiple low affinity sites on each MUC5B monomer. Self-diffusion studies with intact MUC5B showed that calcium binding at the protein site catalyzed reversible cross-links between MUC5B chains to form networks. The site of cross-linking was identified in the MUC5B D3-domain as it was specifically blocked by D3 peptide antibodies. Biophysical analysis and single particle EM of recombinant MUC5B N terminus (D1D2D′D3; NT5B) and subdomains (D1, D1-D2, D2-D′-D3, and D3) generated structural models of monomers and disulfide-linked dimers and suggested that MUC5B multimerizes by disulfide linkage between D3-domains to form linear polymer chains. Moreover, these analyses revealed reversible homotypic interactions of NT5B at low pH and in high calcium, between disulfide-linked NT5B dimers, but not monomers. These results enable a model of MUC5B to be derived, which predicts mechanisms of mucin intracellular assembly and storage, which may be common to the other major gel-forming polymeric mucins.     

Subunit structure of porcine submaxillary mucin

The structure of a high molecular weight fraction of porcine submaxillary mucin was studied by using degradative techniques. Reduction of disulfide linkages released mucin subunits together with an associated protein(s) of approximately 140 kDa. The molecular weights of the subunits ranged from approximately 0.5 x 10(6) to 2.5 x 10(6). Trypsinization of subunits generated glycosylated domains and small, poorly glycosylated or nonglycosylated tryptic peptides. The glycosylated domains, which have an average molecular weight of approximately 270K, possess an unusual amino acid composition containing only nine different amino acids. The minor amino acids which are absent from the glycosylated domains but which are consistently present in both the mucin and the mucin subunits were recovered in the tryptic peptides. Pronase digestion of the glycosylated domains generated smaller fragments of approximately 17 kDa. Comparing these results to the partial cDNA sequence for porcine submaxillary mucin reported by Timpte et al. [(1988) J. Biol. Chem. 263, 1081-1088] suggests that the glycosylated domains consist of variable numbers of the 81 amino acid tandem repeat observed in the cDNA sequence. Further, the fact that porcine submaxillary mucin contains subunits, link proteins, and glycosylated domains suggests that its structure is similar to that described for cervical and intestinal mucins. Intact mucin, mucin "subunits", and the glycosylated domains are all polydisperse with respect to molecular weight, indicating that mucin polydispersity is due to variability in the number of units linked together as well as to variability in the size of the units.