Molecular cloning, sequence, and specificity of expression of the gene encoding the low molecular weight human salivary mucin (MUC7)

Previous biochemical studies have determined that human saliva contains high and low molecular weight mucin glycoproteins (MG1 and MG2, respectively) that are structurally distinct. In this study, we describe the isolation and characterization of overlapping cDNA clones which code for the MG2 protein core. DNA sequencing revealed a translated region of 1131 nucleotides encoding a protein of 377 amino acid residues with a molecular mass of 39 kDa. The first 20 N-terminal residues were very hydrophobic and probably comprise the MG2 leader peptide. The region encoding the secreted protein can be divided into three distinct domains; unique 5'- and 3'-translated regions containing 4 and 1 potential N-glycosylation sites, respectively, and a central region of six almost perfect tandem repeats of 23 amino acid residues with a high number of Thr and Ser. No sequence homology with any other human or animal mucins, and no significant homology to any other proteins was found. MG2 mRNA is about 2.5 kilobases long, and its expression appears to be species-, tissue-, and cell-specific. We propose to name this gene MUC7 in accordance with the mucin genes cloned to date named MUC1-MUC6.     

Identification of a major human high molecular weight salivary mucin (MG1) as tracheobronchial mucin MUC5B

The strain G89HT of Clostridium argentinense obtained by culture selection of the prototype G89 strain producing high titers of type G botulinal toxin was studied. Its cultural, biochemical and toxigenic characteristics and the presence of plasmids were tested. Both strains showed similar physiological features and carried a 83 MDa plasmid. A 170 MDa plasmid was also recognized in the G89HT strain. Notably, this strain was better sporulating and showed a higher toxigenicity than the prototype G89 C. argentinense strain. These two characteristics might permit a long term storage and perhaps yield high antitoxin titres.     

MUC4 is a major component of salivary mucin MG1 secreted by the human submandibular gland

High molecular weight salivary mucin (MG1) is an important component of saliva, contributing to the lubricative and tissue-protective functions of this biological fluid. We have shown previously that the human mucin gene MUC5B is expressed at high levels in sublingual gland and is a significant constituent of MG1. Since many epithelia express multiple mucin genes, it seemed likely that MG1 in salivary secretions is also a heterogeneous mixture of mucin gene products. The aim of this study was to determine whether MUC4, a mucin shown in Northern blotting experiments to be expressed in salivary glands, was a significant protein component of MG1 in salivary secretions. Two cDNA clones containing MUC4 tandem repeats were isolated from a human submandibular gland cDNA library. In addition, recombinant MUC4 produced in a bacterial expression system cross-reacted with an antibody directed against deglycosylated MG1. This shows conclusively that human salivary mucin MG1 contains both MUC5B and MUC4 gene products suggesting that each mucin may perform distinct functions in the oral cavity.     

The MUC2 gene product: a human intestinal mucin

Sera from 82 cases of leptospirosis (confirmed by micro-agglutination tests or IFAT) and 108 patients with other diseases were investigated using the microcapsule agglutination test (MCAT) for leptospirosis. The overall sensitivity (90.2%), specificity (96.3%), positive predictive value (94.9%), negative predictive value (92.9%), and accuracy of the MCAT (93.7%) were encouraging. MCAT is simple, can be performed by unskilled personnel with minimum laboratory facilities, and produces results in 3 h. MCAT would be a reliable serodiagnostic test for rapidly screening individuals for leptospirosis, in various geographical areas of Thailand.     

Differential expression of human high-molecular-weight salivary mucin (MG1) and low-molecular-weight salivary mucin (MG2)

Two distinct mucin components of saliva, MG1 and MG2, have been identified based on chemical composition and molecular weights (high and low, respectively) in saliva. With the aim of characterizing the expression pattern of salivary mucins, we have prepared monoclonal antibodies (MAbs) directed against the peptide core of MG1 and against a synthetic peptide derived from the MG2 (MUC7) sequence. MAb PANH2 raised against partially deglycosylated MG1 stained a high-molecular-weight smear in Western blots of partially purified MG1. PANH2 binding was increased by deglycosylation with trifluoromethanesulfonic acid as well as with subsequent periodate treatment, and was eliminated by pronase treatment, strongly suggesting that MAb PANH2 was directed to a peptide epitope of MG1. MAb PANH3 raised against a synthetic peptide derived from the MG2 (MUC7) sequence reacted with the native molecule and stained a narrow smear of ca. 200,000 to 210,000 in Western blots of concentrated saliva and a lower-molecular-weight smear of trifluoromethanesulfonic-acid-treated MG2. Immunohistology on frozen sections of human salivary glands showed that MAb PANH2 selectively labeled mucous cells, whereas MAb PANH3 labeled subpopulations of serous cells. Double-direct immunofluorescence staining with PANH2 and PANH3 demonstrated that the staining patterns were non-overlapping. The development of these antibody probes will facilitate studies of mucin expression in diseases of salivary glands.     

Association of ulcerative colitis with rare VNTR alleles of the human intestinal mucin gene, MUC3

Ulcerative colitis (UC), a common form of inflammatory bowel disease, is a multifactorial disorder with significant genetic influence. Recently, evidence of linkage on chromosome 7q near the intestinal mucin gene MUC3 was reported by an affected sib-pair analysis. Previous reports indicate a possible mucin abnormality in UC patients, but whether genetic differences in a specific mucin gene are associated with UC is unknown. Here we analysed polymorphisms of variable number of tandem repeats (VNTRs) within this gene using DNAs obtained from 243 Japanese (75 patients with UC and 168 controls), and to confirm the result we undertook a two-stage examination using 328 Caucasian samples (72 and 85 with UC in the first and second stages, respectively, and 171 controls). When the frequency of patients carrying one or two rare VNTR alleles was compared with that of controls, a significant increase was found first in Japanese patients (odds ratio 2.72, 95% CI 1.17-6.32, P = 0. 0308). In Caucasians, the odds ratio was 2.80 (95% CI 1.36-5.75, P = 0.0079) in the first stage, 2.43 (95% CI 1.20-4.92, P = 0.0196) in the second stage and 2.60 (95% CI 1.41-4.80, P = 0.0024) in total. The overall odds ratio was 2.64 (95% CI 1.60-4.33, P = 0.0001). This result suggests that rare alleles of the MUC3 gene may confer genetic predisposition to UC.     

Significance of MUC1 and MUC2 mucin expression in colorectal cancer

AIMS: To compare the lineage specific distribution of MUC1 and MUC2 mucins in normal colorectal mucosa and adenocarcinoma and to identify pathological correlations. METHODS: Paraffin wax sections from 51 colorectal cancers were examined for the expression of MUC1 and MUC2, non-O-acetyl sialic acid and the carbohydrate epitopes Lex, Ley, sialosyl-Lex, sialosyl-Tn, and Tn using standard histochemical methods. RESULTS: MUC1, Lex and Ley co-localised with columnar cell secretions, whereas MUC2, mild periodic acid Schiff and sialosyl-Tn co-localised with goblet cell mucin in both normal and malignant tissues. Sialosyl-Lex and Tn were associated with both lineages. In normal tissues MUC1, Lex and Ley showed only trace expression by crypt base columnar cells. Cancers could be classified into four phenotypes (MUC2+/MUC1-, MUC2+/MUC1+, MUC2-/MUC1+, MUC2-/MUC1-). Particular phenotypes showed significant correlations with cancer type, lymph node spread and peritumoral lymphocytic infiltration and trends falling short of significance in relation to grade of differentiation and contiguous adenoma. CONCLUSIONS: Classification of colorectal cancer by means of lineage specific function may be relevant to both pathogenesis and prognosis.     

Effects of glycosylation on fragments of tumour associated human epithelial mucin MUC1

The glycodecapeptide AcPAPGS(alpha GalNAc)T(alpha GalNAc)APPA and the C-terminal glycohexapeptide AcS(alpha GalNAc)T(alpha GalNAc)APPA have been synthesized by applying the N-terminal Fmoc group in combination with the heptyl ester cleavable by lipase-catalyzed hydrolysis at pH 7. The solution conformation of these MUC1-related synthetic glycopeptides and the control, non-glycosylated decapeptide AcPAPGSTAPPA have been investigated using NMR spectroscopy. The structural studies indicate that the glycohexapeptide has a folded structure in solution. For this molecule, unrestrained molecular dynamics has been used to confirm the presence of the observed solution through-space connections. The results indicate that the non-globular nature of MUC1 is due to both protein core sequence and the effect of carbohydrate.     

Aberrant expression of MUC5AC and MUC6 gastric mucin genes in colorectal polyps

Altered mucin glycosylation and the de novo appearance of gastric mucin antigens have been described in colonic adenomas. The purpose of our study was to determine if expression of the gastric mucin genes MUC5AC and MUC6 occurs in colorectal adenomas and whether this correlates with histopathologic criteria of malignant potential. Immunohistochemical staining using antibodies against MUC5AC and MUC6 tandem repeat synthetic peptides was performed on specimens of normal colon mucosa (n = 26), hyperplastic polyps (n = 9) and adenomatous polyps (n = 111). Mucin mRNA levels were determined using RNase protection assays using riboprobes corresponding to unique non-repetitive sequences. MUC5AC and MUC6 staining were rarely detected and of low intensity in normal colon and hyperplastic polyps. The number of immunoreactive polyps and intensity of MUC5AC and MUC6 staining were greatest in larger adenomas of moderate villous histology and dysplasia. MUC5AC and MUC6 staining tended to decrease in highly villous polyps with severe dysplasia. Increased MUC5AC mRNA levels were found in 26/45 of adenomas tested compared with 0/9 normal colon specimens. MUC6 mRNA levels were found in 20/45 of adenomas compared with 1/9 normal colon specimens. MUC5AC and MUC6 mRNA were present more frequently and at higher levels in polyps with intermediate stages of size, villous histology and dysplasia. We conclude that aberrant expression of MUC5AC and MUC6 mucin genes is likely responsible for an expanded repertoire of mucin antigen expression in colorectal neoplasia.     

Biophysical approaches to salivary mucin structure, conformation and dynamics

Our understanding of the origins of the physical and biochemical properties of mucous glycoproteins is incomplete and not with out controversy. Recent molecular biological and biophysical studies revealing the architecture and solution structure and dynamics of a series of salivary mucins, invaluable toward resolving many of these questions, are discussed. Mucins are very large, structurally heterogeneous, and highly expanded molecules with the carbohydrate playing a key role in maintaining the extended mucin conformation.     

Analysis of Candida albicans adhesion to salivary mucin

Clearance of Candida albicans from the oral cavity is thought to be mediated via specific receptor-ligand interactions between salivary constituents and the fungus. Since surfaces in the oral cavity are normally coated with a saliva-derived pellicle, specific interactions between salivary constituents and C. albicans may also contribute to adhesion of C. albicans to the oral mucosa and dental prostheses. Therefore, the purpose of this study was to identify salivary constituents to which C. albicans is capable of binding. A solid-phase overlay assay was used in which electrophoretically separated rat and human salivary constituents bound to membrane filters were incubated with radiolabelled C. albicans cells. C. albicans adhered to a single salivary component from each host. Correlation of cell-binding activity with specific monoclonal antibody (MAb)-binding activity indicated that the constituent bound by C. albicans in human saliva was low-molecular-weight mucin (MG2) and that in rat saliva was rat submandibular gland (RSMG) mucin. Further studies showed an identical cell hybridization signal and MAb colocalization by using RSMG ductal saliva and an aqueous RSMG extract in the solid-phase overlay assay. Analysis of cell binding to the aqueous extract of RSMG fractionated by anion-exchange chromatography demonstrated that C. albicans binding was restricted to an acidic subfraction of the RSMG extract, which also bound the RSMG mucin-specific MAb. The Candida-binding fraction contained predominantly RSMG mucin glycoprotein and also a noncovalently associated, chloroform-extractable material. Furthermore, we identified two strains of C. albicans which differed severalfold in the ability to bind RSMG mucin in the overlay assay. These results suggest that C. albicans binds to only a specific subfraction of RSMG mucin and that the two C. albicans strains tested differ in the ability to bind RSMG mucin subfractions.     

Chromosomal localization of a human mucin gene (MUC8) and cloning of the cDNA corresponding to the carboxy terminus

The objective of this study was to determine the predictability of endosseous implants placed in a maxillary sinus grafted with a mixture of bovine porous bone mineral and demineralized freeze-dried bone. Sixty implants were placed in 20 patients representing 28 sinuses using either a one- or two-stage technique. After an implant loading period of more than 2 years, the survival rate (eg, a clinically functioning implant without signs of mobility or infection) varied from 90% to 96%. No infections or other complications were encountered. The data suggest that this treatment regimen can result in a high rate of survival.     

Molecular cloning of human gastric mucin MUC5AC reveals conserved cysteine-rich D-domains and a putative leucine zipper motif

The central limitation hypothesis postulates that energy budgets are limited by the capacity for energy assimilation. Contradictory evidence from in vitro studies of nutrient uptake capacity of intestinal tissues indicates a margin, however, that could allow a higher rate of energy acquisition than actually measured. As a more direct test, I measured nutrient uptake capacity in vivo. This is possible in animals such as the neotropical nectar-feeding bats (Glossophaginae: Phyllostomidae) that have both rapid gut transit times and high daily metabolic needs. Here, during the steady-state period of feeding, the rate of food intake is equal to the rate of food processing and food egestion. Therefore, the rate of food absorption can be determined directly from the rate of food ingestion. Maximal feeding rates were elicited by limiting the time period available for feeding during the 24-hr day through manipulation of the light/dark (LD) cycle. During 4-hr nights (LD 20:4), sugar intake averaged 488 J/g/hr and was 73% higher than during 12-hr nights (LD12:12). A 16.4-g bat would assimilate 96 kJ/day if feeding at this maximal rate during a regular 12-hr foraging period. This would be sufficient for maintaining a positive energy balance even during extreme physical activity under the natural conditions of its tropical environment. Nutrient assimilation capacity could thus not be identified as the single central factor setting the metabolic ceiling.     

The high-molecular-weight human mucin is the primary salivary carrier of ABH, Le(a), and Le(b) blood group antigens

Because many bacteria interact with the carbohydrate portions of receptor molecules, factors controlling glycosylation probably influence the ability of salivary components to mediate bacterial adherence/clearance. Important sources of diversity in glycosylation are the ABO, secretor, and Lewis genes, which code for glycosyltransferases that add specific sugar sequences to the termini of carbohydrate chains of glycolipids and glycoproteins. We identified, by Western blotting, salivary glycoproteins carrying the ABH and Le(a) or Le(b) antigens. Samples of whole, unstimulated saliva were obtained from 19 subjects whose blood group was determined by agglutination of red blood cells with specific antisera. After centrifugation, the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose. Glycoproteins carrying blood group antigens were identified by staining the blot with monoclonal antisera specific for the A, B, H, Le(a), or Le(b) antigens. The most intensely staining component from all the samples migrated at the same position as the high-molecular-weight mucin. Saliva samples from the nonsecretors contained only the Le(a) antigen. Samples from the secretors contained one or more of the ABH antigens and, variably, the Le(b) antigen. In all cases, the salivary blood group antigens corresponded to those found on the red blood cells of the same subject. The functional consequences of the expression of blood group antigens on the high-molecular-weight mucin are not known, but their presence could modulate the adherence of certain oral microorganisms that interact preferentially with this molecule.     

Dimerization of the human MUC2 mucin in the endoplasmic reticulum is followed by a N-glycosylation-dependent transfer of the mono- and dimers to the Golgi apparatus

Pulse-chase experiments in the colon cell line LS 174T combined with subcellular fractionation by sucrose density gradient centrifugation showed that the initial dimerization of the MUC2 apomucin started directly after translocation of the apomucin into the rough endoplasmic reticulum as detected by calnexin reactivity. As the mono- and dimers were chased, O-glycosylated MUC2 mono- and dimers were precipitated using an O-glycosylation-insensitive antiserum against the N-terminal domain of the MUC2 mucin. These O-glycosylated species were precipitated from the fractions that comigrated with the galactosyltransferase activity during the subcellular fractionation, indicating that not only MUC2 dimers but also a significant amount of monomers are transferred into the Golgi apparatus. Inhibition of N-glycosylation with tunicamycin treatment slowed down the rate of dimerization and introduced further oligomerization of the MUC2 apomucin in the endoplasmic reticulum. Results of two-dimensional gel electrophoresis demonstrated that these oligomers (putative tri- and tetramers) were stabilized by disulfide bonds. The non-N-glycosylated species of the MUC2 mucin were retained in the endoplasmic reticulum because no O-glycosylated species were precipitated after inhibition by tunicamycin. This suggests that N-glycans of MUC2 are necessary for the correct folding and dimerization of the MUC2 mucin.     

Effect of O-glycosylated mucin on invasion and metastasis of HM7 human colon cancer cells

Mucinous colorectal cancers have a poorer prognosis than colorectal cancers which produce a low amount of mucin, but the exact mechanism is not well understood. The present study was undertaken to elucidate the possible mechanisms of invasion and metastasis of colon cancer cells producing high levels of mucin using mucin glycosylation inhibitor, benzyl-alpha-N-acetylgalactosamine. The binding activity of treated HM7 cells to endothelial leukocyte adhesion molecule (ELAM-1) was significantly decreased and fixed cell binding of MoAb SNH-3 and 19-9 (specific for sialyl Le(x) and sialyl Le(a), respectively) was also significantly decreased. Metalloproteinase activity in conditioned medium and invasion of matrigel-coated porous filters by treated HM7 cells were decreased. However, there was no difference between control and treated HM7 cells in terms of matrix protein binding. These results suggest that O-glycosylated mucin is important in the invasive and metastatic properties of HM7 human colon cancer cells.     

Structural basis for the recognition of carbohydrates by human galectin-7

Knowledge about carbohydrate recognition domains of galectins, formerly known as S-type animal lectins, is important in understanding their role(s) in cell-cell interactions. Here we report the crystal structure of human galectin-7 (hGal-7), in free form and in the presence of galactose, galactosamine, lactose, and N-acetyl-lactosamine at high resolution. This is the first structure of a galectin determined in both free and carbohydrate-bound forms. The structure shows a fold similar to that of the prototype galectins -1 and -2, but has greater similarity to a related galectin molecule, Gal-10. Even though the carbohydrate-binding residues are conserved, there are significant changes in this pocket due to shortening of a loop structure. The monomeric hGal-7 molecule exists as a dimer in the crystals, but adopts a packing arrangement considerably different from that of Gal-1 and Gal-2, which has implications for carbohydrate recognition.     

Human low-molecular-weight salivary mucin expresses the sialyl lewisx determinant and has L-selectin ligand activity

Previously we showed that the low-molecular-weight mucin (MG2, encoded by MUC7), a major component of human submandibular/sublingual saliva, is a bacterial receptor that coats the tooth surface. Here we tested the hypothesis that the structure of its carbohydrate residues contains important information about its function. Purified MG2 (Mr 120 000) was digested with trypsin, and the resulting Mr 90 000 fragment, which carried primarily O-linked oligosaccharides, was subjected to reductive beta-elimination. The released oligosaccharides were characterized by using nuclear magnetic resonance spectroscopy and mass spectrometry. Of the 41 different structures we detected, the most prominent included NeuAcalpha2-->3Galbeta1-->3GalNAc-ol (sialyl-T antigen), Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->6(Galbeta1 -->3)GalNAc-ol [type 2 core with Lewisx (Lex) determinant], and NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6(Galbeta1--> 3) GalNAc-ol [type 2 core with sialyl Lex (sLex) determinant]. We also detected di-, tri-, and pentasaccharides with one sulfate group. Lex, sLex, and related sulfated structures are ligands for selectins, adhesion molecules that mediate leukocyte trafficking. Therefore, we investigated whether MG2 was a selectin ligand. In an enzyme-linked immunosorbent assay, L-selectin chimeras interacted with immobilized MG2 in a Ca2+-dependent manner. L-Selectin chimeras also bound to MG2 immobilized on nitrocellulose. Together, these results suggest that the saccharides that MG2 carries could specify some of its important functions, which may include mediating leukocyte interactions in the oral cavity.