Pro-inflammatory cytokines up-regulate MUC1 gene expression in oral epithelial cells.

The membrane-bound mucin MUC1 is expressed ubiquitously on epithelial surfaces and is thought to provide protection from bacterial and chemical injury. The present study was undertaken to determine whether MUC1 was expressed in cultured oral epithelial cells and whether expression is modulated by pro-inflammatory mediators released as part of the host response to infection by oral pathogens. Northern and Western blotting experiments showed that KB cells express MUC1 mRNA and protein. When cells were treated with interleukins (IL-1beta, IL-6), tumor necrosis factor-alpha (TNF-alpha), or interferon-gamma (IFN-gamma), or combinations of these, real-time PCR demonstrated that MUC1 mRNA increased 1.4- to 3.2-fold. Interestingly, a significant increase in levels of MUC1 protein was also observed. While no effect was observed when KB cells were incubated with LPS from Porphyromonas gingivalis, infection of KB monolayers with this oral pathogen caused a 2.85-fold increase in MUC1 transcript levels. These results suggest that increased MUC1 synthesis may be a key element in the host response to infection with oral pathogens.

MG2 and lactoferrin form a heterotypic complex in salivary secretions.

Protein-protein interactions are necessary for homeostasis to be maintained and for biological systems to be integrated. Heterotypic complexes occur in saliva, and a complex between MG2 and SIgA has been suggested to promote microbial clearance from the oral cavity. In this study, we used a peptide display library to investigate previously unrecognized heterotypic complexes involving MG2 and other proteins. The library was panned with MG2 12 times, and analyses of clones identified the sequence Ala-Leu-Leu-Cys-, which occurs in salivary lactoferrin. Blotting experiments confirmed that MG2 and lactoferrin form a heterotypic complex in vitro and in vivo. Periodate treatment of MG2 did not affect the interaction. A synthetic lactoferrin peptide containing the motif Ala-Leu-Leu-Cys-blocked the interaction between MG2 and lactoferrin, confirming the specificity of the interaction identified by panning. This complex may enhance the properties of these salivary components in the oral environment.

The effects of duration and intensity of stimulation on total protein and mucin concentrations in resting and stimulated whole saliva.

The present investigation has characterized the influence of the duration and intensity of stimulation on the secretion pattern of total protein and salivary mucins MG1 and MG2 in whole saliva. Resting and stimulated whole saliva was collected from six healthy subjects on 2 consecutive days. Whole saliva was collected for 2 five-minute intervals under resting conditions followed by collection under masticatory stimulation induced by the chewing of parafilm (1 g) at 10 or 60 strokes/min for 15 min. Flow rates were different under the 2 levels of stimulation. The concentration of total protein was different in resting and stimulated whole saliva but was not affected by the duration or intensity of stimulation. Analysis of mucin concentrations determined by capture ELISAs revealed that the pattern of MG1 secretion was similar to that of total protein. The pattern of MG2 secretion was unique in that no differences were observed in the concentration of this mucin under resting and stimulated conditions. This study shows that the pattern of protein secretion in whole saliva does not reflect the combined pattern observed for protein secretion in parotid and submandibular/sublingual glands, and that the secretion patterns of MG1 and MG2 in whole saliva are quite different from one another.

The Amount of MUC5B mucin in cervical mucus peaks at midcycle.

The physical character and amount of mucus secreted by the endocervix changes dramatically during the menstrual cycle to facilitate sperm migration at the time of midcycle ovulation. Mucins are highly glycosylated, high-molecular-weight proteins, which are the major structural components of the protective mucus gel covering all wet-surfaced epithelia, including that of the endocervix. We have previously demonstrated that the endocervical epithelium expresses messenger RNA (mRNA) of three of the large gel-forming mucins, designated MUC5AC, MUC5B, and MUC6, with mRNA of MUC5B predominating. Because mucin protein levels may be regulated posttranscriptionally, measurement of MUC5B protein levels with cycle are needed for correlation to mRNA levels. Measurement of specific mucin gene products within mucus secretions has been limited by availability of specific, well-characterized antibodies and by volume requirements of the isolation protocols for mucins, which include CsCl density centrifugation and fraction isolation. To measure MUC5B protein within the cervical mucus through the hormone cycle, we developed a polyclonal antibody specific to the mucin. The antibody, designated no. 799, is to a synthetic peptide mimicking a 19-amino-acid segment of an intercysteine-rich region within the D4 domain in the 3' region of the MUC5B protein. It recognizes native as well as denatured MUC5B on immunoblot, is preadsorbable with its peptide, and binds to apical secretory vesicles of epithelia expressing MUC5B. We used the MUC5B antibody along with a cervical mucin standard cervical mucin isolate in enzyme-linked immunosorbent assay to determine the relative amount of MUC5B mucin in samples of human cervical mucus taken through the menstrual cycle. We demonstrate a peak of MUC5B mucin in human cervical mucus collected at midcycle, compared with mucus from early or late in the cycle. This peak in MUC5B content coincides with the change in mucus character that occurs at midcycle, suggesting that this large mucin species may be important to sperm transit to the uterus.

Immunoquantification of human salivary mucins MG1 and MG2 in stimulated whole saliva: factors influencing mucin levels.

While more and more is known about the structure and function of human salivary mucins, there is relatively little information on quantification of these glycoconjugates in whole saliva and on factors influencing their secretion. The goal of the present work was to develop capture ELISAs that would allow for rapid, inexpensive, and reliable measurement of the salivary mucins MG1 and MG2, and to use these immunological procedures to investigate the significance of age, gender, flow rate, and protein concentration on mucin levels in whole saliva. Previously, we described a rabbit polyclonal antibody against MG1 (Troxler et al., 1995) and a rabbit polyclonal peptide antibody against an epitope in the N-terminal region of MG2 (Liu et al., 1999) which were used to develop the capture ELISAs. We verified the accuracy and specificity of these assays by showing correct measurement of known quantities of purified MG1 or MG2 added to whole saliva and lack of cross-reactivity between mucins and heterologous antisera on Western blots or in ELISAs. Whole saliva was collected from 60 subjects under conditions of masticatory stimulation, flow rates were recorded, and mucin concentrations were determined. The results showed that the mean concentration of MG1 and MG2 was 23.3 +/- 14.6 mg% and 13.3 +/- 11.6 mg%, respectively, and that mucins constitute approximately 16% of the total protein in whole saliva. No significant correlations were found between mucin levels and age or flow rate; however, a significant correlation was found between MG2 levels and total protein concentration. Furthermore, there were statistically significant gender differences in flow rate and MG1 levels, but not in MG2 levels. The availability of these immunoassays for quantification of MG1 and MG2 will help to elucidate the role of mucin in oral health and disease.

Salivary mucin: a factor in the lower prevalence of gastroesophageal reflux disease in African-Americans?

OBJECTIVES: Organic and inorganic constituents of saliva have been implicated as protective components in the esophagus, and deficiencies in one or more of these factors in different races may be an important element in the prevalence of gastroesophageal reflux disease (GERD). To determine whether there are differences in the concentration of salivary mucins between different racial groups, we measured the concentration of mucous glycoprotein MG1 and mucous glycoprotein MG2 in whole saliva of African-Americans and Caucasians. METHODS: Whole saliva was collected from 19 African-American (four male, 15 female; mean age 34 yr, range 19-53 yr) and 25 Caucasian (11 male, 14 female; mean age 31 yr, range 20-51 yr) volunteers under masticatory stimulation (1 g Parafilm, 60 strokes/min) between 11:00 AM and 12:00 noon. Total salivary carbohydrate was measured with a periodic acid-Schiff assay and total protein by absorbance at 215 nm. Immunological reagents were employed to quantify MG2 in a combined enzyme-linked immunosorbent assay/enzyme linked lectin assay (ELISA/ELLA) and to quantify MG1 in a capture ELISA. RESULTS: The total carbohydrate, protein, MG1 and MG2 values were 24.4 +/- 11.9, 243.5 +/- 62.7, 21.8 +/- 13.4, and 11.6 +/- 9.5 mg% for African-Americans, and the corresponding values were 23.3 +/- 9.3, 221.7 +/- 39.7, 25.7 +/- 16.2, and 10.9 +/- 8.7 mg% for Caucasians. There was no statistical difference for any of the parameters measured between the two groups. Furthermore, it was shown that no correlation existed between salivary flow rate and the concentration of carbohydrate, protein, or salivary mucins in African-Americans and in Caucasians. These results show that flow rate did not influence the measured values for salivary parameters in the two groups. CONCLUSIONS: No differences were found in the concentration of salivary mucins MG1 and MG2 in whole saliva of African-Americans and Caucasians, and it seems unlikely that variations in mucin levels influence the prevalence of GERD in these groups.

No significant difference in antigenicity or tissue transglutaminase substrate specificity of Irish and US wheat gliadins.

The prevalence of clinical celiac disease has been shown to vary both across time and between genetically similar populations. Differences in wheat antigenicity and transglutaminase substrate properties are a possible explanation for these differences. This study assessed the antigenicity and transglutaminase substrate specificities of gliadins from regions of high and low celiac disease prevalence. Gliadin was extracted from three commercial US wheat sources and two Irish sources. SDS-PAGE and western blotting revealed minor, but significant variations in the gliadin extracts. However, ELISA showed no difference in the antigenicity of these gliadins. Transglutaminase pretreatment of gliadin resulted in no significant change in gliadin antigenicity and kinetic studies showed that the Kms of the various gliadins were very similar. Purified IgA and IgG had no effect on transglutaminase activity. In summary, minor variations in wheat gliadins are unlikely to explain the observed differences in disease expression across genetically similar populations.

Molecular mapping of statherin- and histatin-binding domains in human salivary mucin MG1 (MUC5B) by the yeast two-hybrid system.

MGI is a high-molecular-weight mucin secreted by mucous acinar cells in human submandibular and sublingual glands. We have recently shown that the tracheobronchial mucin MUC5B is a major component of MG1. MUC5B is organized into cysteine-rich N- and C-terminal regions that flank a central tandem-repeat region containing cysteine-rich subdomains and imperfect 29-residue tandem repeats. In earlier work, we have shown that this mucin selectively forms heterotypic complexes with amylase, proline-rich proteins, statherin, and histatins in salivary secretions, and the aim of this study was to identify specific binding domains within MUC5B using the yeast two-hybrid system. Interactions of cysteine-rich domains in the tandem-repeat region (Cys1-Cys4) and C-terminal region (Cys8a, Cys8b, Cys8c) of MUC5B with statherin and histatins were investigated. These studies indicated that histatin 1 selectively bound to Cysl and Cys2, whereas statherin and histatin 1, 3, and 5 selectively bound to Cys8a. Analysis of the primary sequences of the identified binding domains suggests that these domains most probably can fold into globular-like structures in the native mucin. A ProDom blast search revealed that sequences in Cys1, Cys2, and Cys8a exhibit similarity to domains in evolutionarily diverse extracellular proteins known to participate in a wide variety of protein-protein interactions.

The recombinant N-terminal region of human salivary mucin MG2 (MUC7) contains a binding domain for oral Streptococci and exhibits candidacidal activity.

MG2 (the MUC7 gene product) is a low-molecular-mass mucin found in human submandibular/sublingual secretions. This mucin is believed to agglutinate a variety of microbes and thus is considered an important component of the non-immune host defence system in the oral cavity. We have shown that MUC7 can bind to cariogenic strains of Streptococcus mutans and that this binding requires a structural determinant in the N-terminal region. In the present study an expression construct, pNMuc7, encoding the N-terminal 144 amino acids of MUC7 was generated, and the recombinant protein rNMUC7 was expressed in Escherichia coli. Purified rNMUC7 was characterized and the binding of this protein to oral bacteria was investigated in an established assay. The results showed that the recombinant protein bound to S. mutans ATCC 25175 and ATCC 33402, and that alkylation of the two cysteine residues (Cys(45) and Cys(50)) resulted in the complete loss of bacterial binding. This suggests that binding of MUC7 to S. mutans occurs between the N-terminal region of the mucin molecule and the bacterial surface, and that this interaction is dependent on a cysteine-containing domain within this region of MUC7. In addition, the killing activity of rNMUC7 was compared with that of the candidacidal salivary protein histatin 5 in an established Candida albicans (ATCC 44505) blastoconidia killing assay. It was found that the LD(50) values of rNMUC7 and histatin 5 were comparable, and that the recombinant protein displayed significant killing activity at the physiological concentration range of MUC7 in whole saliva. This study is the first to show that the N-terminal region of MUC7 contains a structural determinant for bacterial binding and that this region exhibits candidacidal activity.

Heterogeneity of high-molecular-weight human salivary mucins.

The existence of high-molecular-weight glycoproteins in saliva and salivary secretions has been recognized for nearly 30 years. These proteins, called mucins, are essential for oral health and perform many diverse functions in the oral cavity. Mucins have been intensively studied, and much has been learned about their biochemical properties and their interactions with oral micro-organisms and other salivary proteins. In the past several years, the major high-molecular-weight mucin in salivary secretions has been identified as MUC5B, one of a family of 11 human mucin gene products expressed in tissue-specific patterns in the gastrointestinal, respiratory, and reproductive tracts. MUC5B is one of four gel-forming mucins which exist as multimeric proteins with molecular weights greater than 20-40 million daltons. The heavily glycosylated mucin multimers form viscous layers which protect underlying epithelial surfaces from microbial, mechanical, and chemical assault. Another class of mucin molecules, the membrane-bound mucins, is structurally and functionally distinct from the gel-forming mucins. These proteins do not form multimers and can exist as both secreted and membrane-bound forms, with the latter anchored to epithelial cell membranes through a short membrane-spanning domain. In the present work, we show that two of the membrane-bound mucins, MUC1 and MUC4, are expressed in all major human salivary glands as well as in buccal epithelial cells. While the functions of these mucins in the oral environment are not understood, it is possible that they form a structural framework on the cell surface which not only is cytoprotective, but also may serve as a scaffold upon which MUC5B, and possibly other salivary proteins, assemble.

A recombinant bovine gallbladder mucin polypeptide binds biliary lipids and accelerates cholesterol crystal appearance time.

BACKGROUND & AIMS: Mucin has a central role in the pathogenesis of cholesterol gallstones, in part because of its ability to bind biliary lipids and accelerate cholesterol crystal appearance time. Previous studies have localized these properties to nonglycosylated mucin domains, and we have recently shown that these domains contain a series of 127-amino acid, cysteine-rich repeats. The aim of this study was to express a recombinant mucin polypeptide containing these repeats and investigate its lipid-binding and pronucleating properties. METHODS: A recombinant mucin polypeptide was expressed as a glutathione S-transferase fusion protein in Escherichia coli, purified by affinity chromatography, and compared with native bovine gallbladder mucin in lipid-binding and cholesterol crystal appearance time assays. RESULTS: The recombinant mucin polypeptide bound a hydrophobic fluorescent probe and cholesterol in a concentration-dependent manner. It accelerated the appearance of cholesterol crystals from lithogenic model bile, an effect that was both time and concentration dependent. CONCLUSIONS: The cysteine-rich repeats in the recombinant mucin polypeptide correspond to the protease-sensitive hydrophobic domains identified in earlier biochemical studies. Further delineation of the lipid-binding site(s) in these repeats will provide new insights into the mechanism of cholesterol crystal nucleation and stone growth.

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 amino-terminal sequence of MUC5B contains conserved multifunctional D domains: implications for tissue-specific mucin functions.

The MUC5B mucin gene product is expressed in a wide variety of secretory epithelia including the gallbladder, salivary glands, trachea, and colon. Previous studies by us and others have described the C-terminal region as well as the central tandem repeating domain of this mucin. In an effort to understand the functional role of MUC5B in diverse human tissues, the sequence encoding the N-terminal region of this mucin was determined from the sequences of exons in three overlapping genomic clones. Primer extension mapped the site of transcription initiation 25 bp downstream from a putative TATA box element. The N-terminal region of MUC5B contained 1321 amino acids organized into a signal peptide, a short serine-threonine rich region, and three von Willebrand factor-like D domains. Comparison of this sequence with the N-terminal sequences of MUC2 and MUC5AC revealed a much higher degree of identity (46-59%) than that observed in the C-terminal regions of these mucins (33%). The N-terminal sequence of MUC5B also contains a number of sequence motifs common to several groups of extracellular ligand binding and adhesion proteins not previously recognized in mammalian gel-forming mucins. The N-terminal D domains in MUC5B are likely to have important roles in both mucin assembly and in the protective functions of the secreted mucin.

Molecular characterization of a major high molecular weight mucin from human sublingual gland.

Human submandibular/sublingual gland secretions contain a multimeric high molecular weight mucin (MG1) and a monomeric low molecular weight mucin (MG2). MG2 is the product of the MUC7 gene, whereas the gene for MG1 has not been identified. Previously, we isolated a clone (pSM2-1) from a human sublingual gland cDNA expression library using an antibody against deglycosylated MG1 (Troxler et al., Biochem. Biophys. Res Commun., 217, 1112-1119, 1995). In order to identify the mucin gene from which pPM2-1 was derived, Northern blots of human submandibular and sublingual gland RNA were hybridized with a series of probes for tandem repeats found in the high molecular weight secreted mucins MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC6. The only known mucin expressed at high levels in sublingual gland was MUC5B, and no known mucin was expressed at high levels in submandibular gland. A series of overlapping clones was obtained by rescreening the sublingual gland cDNA library and by reverse transcriptase-polymerase chain reaction. The resulting clones connected pSM2-1 to a series of MUC5B tandem repeats at the 3' end of the repeat domain and provided the complete nucleotide and deduced amino sequence of the carboxyl terminal region of MG1. This region is enriched with respect to cysteine (approximately 10 mol %) and contained a D domain and a carboxyl terminal domain that could be aligned with the corresponding domains in human intestinal MUC2, human tracheobronchial MUC5AC, and human von Willebrand factor. The limited expression of known mucin genes, together with the considerable mucin synthesizing capacity of submandibular gland, suggests that a novel (previously not described) mucin gene is expressed in this gland and constitutes a portion of MG1 in salivary secretions.

Molecular cloning of a major human gall bladder mucin: complete C-terminal sequence and genomic organization of MUC5B.

Gall bladder mucin has been shown to play a central role in the pathogenesis of cholesterol gallstone disease. While cloning and sequencing studies have provided a wealth of information on the structure of other gastrointestinal and respiratory mucins, nothing is known about the primary structure of human gall bladder mucin. In this study, we show that the tracheobronchial mucin MUC5B is a major mucin gene product expressed in the gall bladder. Antibodies directed against deglycosylated human gall bladder mucin were used to screen a gall bladder cDNA expression library, and most of the isolated clones contained repetitive sequences nearly identical with those in the tandem repeat region of MUC5B. An additional clone (hGBM2-3) contained an open reading frame coding for a 389 residue cysteine-rich sequence. The arrangement of cysteine residues in this sequence was very similar to that in the C-terminal regions of MUC2, MUC5AC and human von Willebrand factor. This cysteine-rich sequence was connected to a series of degenerate MUC5B tandem repeats in a 7.5 kb HincII genomic DNA fragment. This fragment, with ten exons and nine introns, contained MUC5B repeats in exon 1 and a 469 residue cysteine-rich sequence in exons 2-10 that provided a 152 nucleotide overlap with cDNA clone hGBM2-3. Interestingly, the exon-intron junctions in the MUC5B genomic fragment occurred at positions equivalent to those in the D4 domain of human von Willebrand factor, suggesting that these proteins evolved from a common evolutionary ancestor through addition or deletion of exons encoding functional domains.

The pronase resistance of cholesterol-nucleating glycoproteins in human bile.

BACKGROUND & AIMS: Many putative pronucleating proteins have been isolated from the biliary concanavalin A (con A)-binding fraction. The pronase resistance of the overall nucleating-promoting activity was almost never taken into consideration. The aim of this study was to identify the major pronase-resistant con A-binding glycoproteins. METHODS: Pronase-treated and -untreated con A-binding glycoproteins were separated on a Superose 12 gel permeation column (Pharmacia, Uppsala, Sweden) and tested in a crystal growth assay. Proteins were identified by amino-terminal sequencing. RESULTS: Con A-binding pronucleating activity eluted in two peaks on the Superose column. This activity was unaltered after pronase treatment. Activity peak I contained too little protein to allow amino-terminal sequencing. In activity peak II, the major pronase-resistant con A-binding glycoproteins were identified as alpha 1-antitrypsin and alpha 1-antichymotrypsin. The 130-kilodalton nucleation promoter was identified as aminopeptidase N, but the full pronase resistance of this protein, reported earlier, was not confirmed. Immunoabsorptive removal of alpha 1-antitrypsin and alpha 1-antichymotrypsin and immunopurification showed that only alpha 1-antichymotrypsin had pronucleating activity. CONCLUSIONS: The pronase resistance of the nucleating-promoting activity of the con A-binding glycoprotein fraction was confirmed. An important part of this activity could be attributed to alpha 1-antichymotrypsin. It is an acute-phase protein, as are many other pronucleating proteins, which might indicate a general mechanism of action in gallstone formation.

Molecular cloning of a novel high molecular weight mucin (MG1) from human sublingual gland.

A human sublingual gland cDNA library was screened with a polyclonal antiserum against deglycosylated MG1 and a positive clone, pSM2-1, was isolated which codes for 196 amino acids in the carboxyl-terminal region of this mucin. This region is cysteine-rich and contains a C2-like domain upstream of the extreme carboxyl-terminal domain in which the arrangement of cysteines is nearly identical to that in human von Willebrand factor, human intestinal mucin MUC2, human tracheobronchial mucin MUC5 and porcine and bovine submaxillary gland mucins. Northern analyses with pSM2-1 showed MG1 transcripts are abundant in sublingual gland and barely detectable in submandibular gland. This study provides the first primary sequence data on human salivary mucin MG1 and the significance of the results is discussed with respect to the biosynthesis and differential expression of MG1 in human salivary glands.

Interaction of bovine gallbladder mucin and calcium-binding protein: effects on calcium phosphate precipitation.

BACKGROUND & AIMS: Gallstones consist of calcium salts and cholesterol crystals, arrayed on a matrix of gallbladder mucin (GBM), and regulatory proteins like calcium-binding protein (CBP). To determine if interactions between CBP and GBM follow a biomineralization scheme, their mutual binding and effects on CaHPO4 precipitation were studied. METHODS: Binding of CBP to GBM was assessed by inhibition of the fluorescence of the complex of GBM with bis-1,8-anilinonaphthalene sulfonic acid (bis-ANS). The effects of the proteins on precipitation of CaHPO4 were assessed by nephelometry and gravimetry. Precipitates were analyzed for calcium, phosphate, and protein. RESULTS: CBP and bis-ANS competitively displaced each other from 30 binding sites on mucin, with a 1:1 stoichiometry and similar affinity. The rate of precipitation of CaHPO4 was retarded by mucin and CBP. Precipitate mass was unaffected by GBM alone but decreased with the addition of CBP. Complexing CBP with GBM abolished or moderated this latter effect, altered precipitate morphology, and changed the stoichiometric ratios of Ca to PO4 in the precipitates from 1:1 to 3:2. Mucin and CBP were incorporated into the precipitates. CONCLUSIONS: These studies suggest that the formation of calcium-containing gallstones is a biomineralization process regulated by both GBM and CBP.

Mucin-vesicle interactions in model bile: evidence for vesicle aggregation and fusion before cholesterol crystal formation.

Nucleation of cholesterol monohydrate crystals from bile is a critical step in the formation of cholesterol gallstones. Measurement of nucleation in model bile system and the characteristics of the initial nucleus have proven elusive. In this study we have used three separate physical chemical techniques to examine vesicle aggregation and fusion, including dynamic light scattering (DLS), transmission electron microscopy (TEM), and fluorescent biochemical assays. These assays enabled us to quantify the effect of biliary proteins, such as gallbladder mucin, on vesicle fusion and aggregation. In the absence of mucin, fusion is a relatively slow process occurring over 24 hours, whereas physiological concentrations of mucin are able to accelerate almost complete fusion of vesicles within 6 hours. Vesicle fusion and aggregation as characterized by TEM result in the formation of aggregates of multilamellar vesicles and giant fusion bodies associated with a background of mucin. These mucin-vesicle aggregate bodies may represent true nuclei and precede cholesterol monohydrate crystal nucleation. In future studies, these vesicle fusion assays can be used to quantitatively examine the effect of putative pro- and anti-nucleating proteins on the earliest steps of cholesterol crystal nucleation.

Bovine gall-bladder mucin contains two distinct tandem repeating sequences: evidence for scavenger receptor cysteine-rich repeats.

Gall-bladder mucin is a densely glycosylated macromolecule which is the primary secretory product of the gall-bladder epithelium. It has been shown to bind cholesterol and other biliary lipids and to promote cholesterol crystal nucleation in vitro. In order to understand the molecular basis for mucin-lipid interactions, bovine gall-bladder mucin cDNAs were identified by expression cloning and were isolated and sequenced. The nucleotide sequences of these cDNAs revealed two distinct tandem repeating domains. One of these domains contained a 20-amino acid tandem repeating sequence enriched in threonine, serine and proline. This sequence was similar to, but not identical with, the short tandem repeating sequences identified previously in other mammalian mucins. The other domain contained a 127-amino acid tandem repeating sequence enriched in cysteine and glycine. This repeat displayed considerable sequence similarity to a family of receptor- and ligand-binding proteins containing scavenger receptor cysteine-rich repeats. By analogy with other proteins containing these cysteine-rich repeats, it is possible that, in gall-bladder mucin, this domain serves as a binding site for hydrophobic ligands such as bilirubin, cholesterol and other biliary lipids.