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MUC1 is displaced and altered in malignancies originating in epithelial tissues. As a consequence, a molecule with short carbohydrate side chains and exposed repetitive epitopes on its peptide core is shed into the interstitial space surrounding a tumor and gains access to the circulation (1-3), coming in contact with the immune system. Humoral immune responses to MUC1 have been described in healthy subjects and in patients with benign and malignant diseases (4-6). [ABSTRACT FROM AUTHOR]

The study of humoral and cellular responses to mucins requires many of the standard immunologic techniques, although working with molecules as large as mucins sometimes leads to logistic problems. This chapter focuses on some of the techniques that may be used to analyze the immune response to mucins using dendritic cells to present mucin peptides. [ABSTRACT FROM AUTHOR]

MUC1 mucin undergoes multistep posttranslational modifications before it is finally expressed on the apical surface of mammalian ductal epithelial cells. Two early precursor proteins are both N-glycosylated and differ in molecular weight owing to a proteolytic cleavage of a 20-kDa fragment. Proteolytically modified form is transported to the Golgi, where it undergoes extensive, although not complete, O-gly-cosylation on serine and threonine residues within the tandem repeat (TR) region. MUC1 is then transported to the cell surface. For additional glycosylation and sialylation, surface MUC1 is internalized and directed to trans-Golgi compartments. Mature form is again transported to the cell surface (1). [ABSTRACT FROM AUTHOR]

Mucins are highly glycosylated proteins present on the lumenal side of ductal epithelial cells. MUC1 is the only mucin with a transmembrane region anchoring it to the cell surface. The extracellular domain of MUC1 is composed of numerous tandem repeats of a 20-amino acid sequence (1). Normal cells produce a highly glycosylated and sialylated form of MUC1. The O-linked carbohydrate side chains of MUC1 on tumor cells are shorter and less abundant, exposing previously unrecognized antigenic sites on the polypeptide core of the molecule (2). This underglycosylated MUC1 molecule on tumor cells can be recognized as a tumor-specific antigen by T-cells. The major cytotoxic T-lymphocyte (CTL) response to tumor-specific MUC1 is T-cell receptor (TCR) mediated but major histocompatibility complex (MHC)-unrestricted. Owing to the high density of repeating antigenic epitopes extending along each MUC1 molecule, it is postulated that a large number of TCRs can be triggered simultaneously to activate the CTLs to kill tumor cells or proliferate (3-5). Antibodies specific for the TCR, or for the defined MUC1 epitope recognized by the TCR, inhibit CTL recognition. Antibodies against MHC molecules have no effect. [ABSTRACT FROM AUTHOR]

A successful vaccine for cancer immunotherapy, particularly for solid tumors, would require a suitable target antigen and the production of a cytotoxic T-cell response (1). In the mid- to late-1980s, there was a focus on monoclonal antibodies (MAbs) for the treatment of common cancers, such as those of the colon, breast, and lung. However, with the difficulties of using such agents, there is now a clear focus on cellular immunity for several reasons. First, using genetic engineering techniques, peptide epitopes have been identified and can be produced in large amounts, particularly as synthetic peptides and as recombinant molecules. Second, the description of many cytokines, combined with the knowledge of antigen processing and presentation by class I and class II pathways, has led to a degree of sophistication and knowledge in how to immunize to produce the desired response. These developmets are proving useful in the generation of new and improved vaccines and the future holds much promise for the production of effective vaccines to prevent, control, and possibly eradicate many diseases, including cancer. It is now theoretically possible to induce either antibodies or CTLs to defined polypeptides. However, it remains to be determined which will be the most effective. [ABSTRACT FROM AUTHOR]

Mucins are important sources of carbohydrate for bacteria growing in the human large intestine. As well as being produced by goblet cells in the colonic mucosa, salivary, gastric, biliary, bronchial, and small intestinal mucins also enter the colon in effluent from the small bowel. Particulate matter, such as partly digested plant cell materials, are entrapped in this viscoelastic gel, which must be broken down to facilitate access of intestinal microorganisms to the food residues. It is estimated that between 2 to 3 g of mucin enter the large bowel each day from the upper digestive tract (1), however, the rate of colonic mucus formation is unknown. Complex polymers, such as mucin must be degraded by a wide range of hydrolytic enzymes to smaller oligomers and their component sugars and amino acids before they can be assimilated by intestinal bacteria. [ABSTRACT FROM AUTHOR]

Surface epithelia throughout the body are covered by mucus, a protective secretion that serves as a selective physical barrier between the epithelial cell plasma membrane and the extracellular environment. Mucin, the glycoprotein constituent of mucus, has been shown to bind bacteria at mucosal surfaces that line the lung, gut, bladder, oral cavity, and eye (1-7). Since bacterial binding to an epithelial cell surface is generally thought to be an important prerequisite for infection (8), the interaction between bacteria and mucin, together with normal mucosal clearance mechanisms, is believed to act as a defense against infection by inhibiting bacterial adherence to the underlying epithelial cell surface. [ABSTRACT FROM AUTHOR]

The regions of the gastrointestinal tract that are densely colonized by bacteria secrete mucus that stains as sulfomucus. A body of evidence suggests that the sulfation of mucins is protective against degradation by bacteria, and desulfation is one of the important rate-limiting steps in mucin degradation (1). Bacterial sulfatases that carry out mucin desulfation have been described, but undoubtedly a group of such enzymes will be discovered with distinctive specificities for the differently sulfated sugars found in mucins, and a combination of such enzymes will be required to completely desulfate mucins. [ABSTRACT FROM AUTHOR]

The glycosidases and associated hydrolytic enzymes acting on glycoconjugate oli-gosaccharides form part of the total mucinase activity. This chapter describes some assay methods for the determination of these enzymes. Our knowledge of the number of enzymes required for mucin degradation and their regulation in physiological situations is scanty (1). The degradation of both protein and carbohydrate domains require specific enzymes which are able to degrade mucin structure. Carbohydrate degradation may be dependent on prior or concomitant peptide cleavage in mucins. The issues that need to be addressed include the following: [ABSTRACT FROM AUTHOR]

Early studies concerning proteolytic degradation of mucins demonstrated that the protein core of mucins consisted of two distinct regions, glycosylated regions: protected from degradation by the densely packed carbohydrate side chains and nonglycosylated regions susceptible to proteases (1,2). Since the late 1980s, sequencing of mucin genes has underlined these studies and provided a firm molecular basis for these concepts (3). Gene-cloning studies have shown that the protein backbone of the subunits of secreted polymeric mucins can be up to 5000 amino acids in length (approx 20% by weight of the molecule) and consists of two major types of domain that alternate throughout the sequence (3). One type of domain, situated centrally accounts for about 50% of the protein core and is characterized by tandem repeat (TR) sequences, rich in threonine, serine, and proline, and the hydroxyl amino acids form the sites of attachment of the oligosaccharide chains (approx 80% by weight of the molecule). The other major type of domain, situated at the N- and C-terminals and between regions of TR sequences, is relatively poor in these three amino acids and relatively rich in cysteine (3). Some of these cysteine residues can form disulphide bridges with other mucin monomers (Mr 2-3 × 106) to form large polymeric mucins linked end to end (Mr ~ 107) and capable of forming gels (bi4-6). The tandem repeat domains are protected from proteolysis by the carbohydrate side chains that sterically inhibit proteinases from gaining access to the protein core, however, proteinases can hydrolyze the cysteine-rich regions of accessible nonglycosylated protein, thereby fragmenting the polymeric mucins (6). The soluble glycopeptides resistant to proteolysis remain of relatively high Mr (200-700 kD) and recent studies have suggested that individual mucin gene products may contain different types and lengths of glycosylated domains. For instance, analysis of high Mr glycopeptides produced by trypsin digestion of the MUC5B subunit indicated that it contained different types and lengths of glycosylated domains; one domain of Mr7.3 × 105, two domains of 5.2 × 105 and a third domain of 2 × 105 (7). Similarly rat small intestinal Muc2 mucin subunit contains two glycopeptides with an estimated mass of 650 and 335 kDa (8). The significance of the differences in size of these domains is unclear. [ABSTRACT FROM AUTHOR]

Turnover of the mucous "barrier gel" overlying mucosal surfaces is essential for hydration, mechanical protection, the physical removal of contaminants and toxins, the generation of sacrificial binding ligands that prevent microbial penetration, and the provision of a suitable environment to renew other defensive molecules that are incorporated into mucus. Mucinase activity is crucial to this turnover process in locations such as the gut and the reproductive tract. Similar activity may also be of relevance at other mucosal surfaces that are not normally colonised by significant microbial populations, such as the eye and the respiratory tract. [ABSTRACT FROM AUTHOR]

One of the interesting technical aspects of working with mucins is that it is relatively easy to make antibodies to different mucin glycoproteins—mainly because the repeat sequences in the variable numbers of tandem repeat (VNTR) region are highly immunogenic. Indeed, all the mucin genes (MUC1-MUC8) (1,2) were originally cloned using polyclonal antisera and Escherichia coli DNA expressions systems, in which, because of the repeated sequences, the expressed cDNAs could be detected and cloned. We found this of particular interest because we had tried very hard in the early days of cloning to isolate lymphocyte surface antigens with monoclonal antibodies (MAbs)—all these efforts failed. Because the VNTRs are so highly immunogenic, immunization of mice with human tumors, mucin-containing materials such as the human milk fat globule membrane (HMFGM) (isolated from human milk), cell membranes or synthetic peptides, all lead to the production of MAbs. We have made numerous MAbs to human mucin 1, 2, 3, and 4 VNTRs; to variants, and to mouse muc1 (3-8). As will be described herein it is not difficult to make these antibodies, and, for the most part, these can be easily characterized and the antibodies recognize linear amino acids of peptides—whether the peptides are present in tissues or as native molecules (immunohistological detection), or the examination of synthetic peptides, whether they are bound to a solid support, in solution, on pins with one end tethered, or conjugated to other proteins, e.g., keyhole limpet hemocyanin (KLH). In almost all circumstances, the reactions obtained are clear-cut, which contrasts with many other antipeptide antibodies that react with nonlinear structures (requiring appropriate secondary or tertiary folding for detection), which makes detection erratic. We describe here the methods used to make the antibodies and the principles of their characterization. In addition, we summarize the properties of MAbs to human MUC1 VNTR peptide; to MUC1 variant peptides; to MUC2, MUC3, MUC4 VNTR peptides; and to mouse muc 1. [ABSTRACT FROM AUTHOR]

In the past twenty years, much progress in the study of mucins has resulted from the development of antibodies recognizing carbohydrate and peptide epitopes. Although antibodies are extremely useful reagents in biology, antibodies of ambiguous specificity, or antibodies whose specificity has been poorly characterized, can also add considerable confusion to any field of study. Whereas it is relatively straightforward to develop antibodies recognizing a known molecule or epitope, it is often much more difficult and time-consuming to establish the specificity of antibodies raised against complex molecular mixtures. Furthermore, because the universe of antigens cannot be systematically tested, it is extremely important to bear in mind that antibodies, whether polyclonal or monoclonal, raised against a known molecule may crossreact with unrelated molecules. The latter may share with cognate antigen chemical characteristics that are easy to identify (i.e., primary amino acid sequence), or lack apparent structural relatedness. Therefore, when using antibodies, a word of caution is always necessary. [ABSTRACT FROM AUTHOR]

The polypeptide backbones of mucins and mucin-type glycoproteins are each encoded by one of multiple genes . At least nine distinct genes (MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, MUC7, and MUC8) that encode mucin-type proteins expressed in epithelial cells have been reported in humans (1,2). The genes encoding mucins are dispersed in the human genome, although a family of four related genes—MUC2, MUC5AC, MUC5B, and MUC6—each of which encodes an apomucin expressed in specialized secretory cells, is found on chromosome 11p15.5 (1). The other genes appear to be rather different. MUC1, the first epithelial mucin gene to be identified, is located on chromosome 1q21, and encodes a relatively small molecule with a transmembrane anchor, which is widely expressed in epithelia and can be detected at low levels in certain other cells (3). MUC3 (7q22) and MUC4 (3q29) are extremely large and also have transmembrane anchors (5/2-7,7a-c). MUC3 and MUC4, like the 1 1p15.5 mucin genes, show a restricted tissue distribution, but are expressed in columnar cells as well as in specialized secretory cells (8,9). MUC7 (4q) encodes a very small secreted glycoprotein (MG2) expressed primarily in salivary glands (10,11), but there is little information about MUC8 (12q24.3) (2). [ABSTRACT FROM AUTHOR]

Progress in understanding how mucosal surfaces are protected is closely related to the development of morphologic techniques to study the structure and secretory function of the mucosal epithelia. Morphologic methods have allowed characterization of mucus-secreting cells of the epithelia of the eye, and the respiratory, gastrointestinal (GI), and reproductive tracts. Characteristics of the mucus-secreting cells of these tissues vary, and many questions remain regarding special characteristics of mucus present over the differing mucosal surfaces. Recent progress in cloning and characterization of mucin genes has facilitated the use of in situ hybridization (ISH) to begin to characterize the mucin gene repertoires and specific functions of mucins expressed by the various epithelia, either those covering mucosal surfaces or glandular epithelia contributing to the mucous layer on the surface of the tissue. ISH has been a particularly valuable method in this regard, since antibodies to specific mucin proteins are often difficult to use on tissues or secretions without heroic methods to deglycosylate in order to make protein epitopes available. [ABSTRACT FROM AUTHOR]

Pulsed-field gel electrophoresis (PFGE) has been used successfully to generate physical maps of a large region from many genomes. In addition, PFGE is useful for determining the order of genes or markers more precisely than is possible with genetic linkage analysis. Since a book in the Methods in Molecular Biology series (1) has already been devoted to this subject, the aim of this chapter is to give protocols that were successfully used in our laboratory for the human mucin genes. Whenever possible, we refer to the relevant chapters of this book or other references in which strat-egies or techniques are discussed in detail. [ABSTRACT FROM AUTHOR]

Northern blot analysis has historically been one of the most common methods used to provide information on the number, length, and relative abundance of mRNAs expressed by a single gene. This technique also generates a record of the total mRNA content expressed by a cell culture or by a tissue, which can be analyzed and compared on the same specimens by successive hybridizations with specific probes. [ABSTRACT FROM AUTHOR]

Much information on the structure, organization, and expression of many genes has been acquired by using the powerful technique of cDNA cloning (1-5). cDNA clones differ from genomic DNA clones in that they represent a permanent copy of an mRNA and are representative of the parts of a gene that are expressed as mature RNA. This comparison of cDNA clones with their genomic homologs has resulted in the discovery of introns in most eukaryotic genes. Amino acid sequence of gene products can only be obtained from full-length cDNA sequences. cDNA clones can also be used to express the protein products of genes in prokaryotes. [ABSTRACT FROM AUTHOR]

The complex O-linked oligosaccharide chains (O-glycans) attached to the polypeptide backbone of mucins are assembled by glycosyltransferases. These enzymes act in the Golgi apparatus in a controlled sequence that is determined by their substrate specificities, their localization in Golgi compartments, and their relative catalytic activities (1). Activities are controlled by many factors, including the membrane environment, metal ions, concentrations of donor and acceptor substrates, cofactors, and, in some cases, posttranslational modifications of enzymes. Cloning of glycosyltransferases has revealed the existence of families of homologous glycosyltransferases with similar actions but encoded by different genes. Thus, many steps in the pathways of O-glycosylation appear to be catalyzed by several related glycosyltransferases that may show slight differences in properties and substrate specificities. The relative expression levels of these enzymes is cell typespecific and appears to be regulated during the growth and differentiation of cells and, during tissue development, and is altered in many disease states (2,3). [ABSTRACT FROM AUTHOR]

Mucins are secreted or membrane-bound large glycoproteins produced by epithelial cells of normal and malignant tissues. The secreted mucins are the major components of the mucous gel overlaying respiratory, gastrointestinal, or genital epithelia. Mucins constitute a family of extensively O-glycosylated glycoproteins (40-80% by weight) (1,2) encoded by a family of different MUC genes (3). The oligosaccharide side chains substitute threonine or serine residues of tandemly repeated sequences in the core of the molecule. [ABSTRACT FROM AUTHOR]

MUC-type mucins are generally very large glycoproteins. They are encoded by very large mRNAs, and possess polypeptides between 200 and more than 900 kDa(l). The only notable exception is MUC7, which is considerably smaller, i.e. thepolypeptide is only 39 kDa (1). Without exception however, mucins are very heavily Oglycosylated: Up to 50-80% of their molecular mass is due to O-glycosylation (1,2). Moreover, potential N-glycosylation sites are found in virtually all mucin sequences, and in several MUCs N-glycosylation is actually demonstrated (1,2). Human MUC2 for instance contains 30 potential N-glycosylation sites, and if these are all used, the N-glycans together would constitute a molecular mass of about 60 kDa. It is only the very large size of the mature mucins, that makes the amount of N-glycosylation seem insignificant (3). Generally, the sizes of the mature mucins are difficult to estimate; The approximations run from 1 to 20 MDa for single mucin molecules, which hampers many forms of biochemical analysis (3). Also, the extensive glycosylation of mucins results in an intrinsically very heterogeneous population of mature mucins. [ABSTRACT FROM AUTHOR]

Metabolic labeling of mucins is a powerful method for two reasons: (1) it lowers the detection limits of the mucins and their precursors considerably, and (2) it provides data on the actual synthesis of mucins in living cells. The produced radioactive mucins can be isolated and studied using biochemical methods, as described in Chapter 19, but these techniques apply basically to the study of mature mucins. In this chapter, we outline the methods for the immunoprecipitation of mucins, i.e., the immunoisolation of the mature mucins as well as their corresponding precursors. By applying metabolic labeling using amino acids and immunoprecipitation with the proper antibodies against the mucin polypeptide, it becomes possible to detect the earliest mucin precursor in the rough endoplasmic reticulum, to follow its subsequent complex conversion into a mature mucin, and to observe its storage and eventual secretion (1-3). Moreover, this antibody-based technique has the required specificity to discriminate the primary translation-product of each mucin gene. How mucin precursors can be distinguished is described in detail for each of the MUC-type mucins in Chapter 21. [ABSTRACT FROM AUTHOR]

Radiolabeling methods have been introduced into the study of the biology of mucin for several reasons (1-3). In many instances, the biochemical analysis of mucins in any form may be limited owing to the small amounts of mucosal tissue available, of cells from culture systems, and the difficulty in obtaining normal material for comparison (3-6). The use of radiolabeling in direct assessment of the biochemistry of the metabolism of mucins, in particular their biosynthesis, is well suited to these techniques in the same way they have been adopted for other proteins and glycoconjugates. It is currently of special interest in evaluating the different stages in the maturation, aggregation, and secretion of mucin. [ABSTRACT FROM AUTHOR]

The study of the biosynthesis has been greatly assisted by the use of cultured cells and tissue explants in short-term culture. Cells are available from a wide range of tissue sources, and this chapter focuses on the use of intestinal cells and tissue. Human colonic cell lines have been widely used in biosynthetic studies and the relationship of some lines to stages in the adenoma-carcinoma sequence is of particular interest, allowing study of the expression of mucin during the development and progression of disease (1-3). Recently the importance of proliferation, differentiation, and apoptosis has attracted attention to the use of culture systems for the study of cell behavior in normal and disease processes (4,5). In the same way, tissue obtained from patients at surgery or as biopsies can be placed in short-term primary or organ culture to study similar changes in disease (6,7). [ABSTRACT FROM AUTHOR]

The sulfation of the terminal sugar residues of mucins is a common and extensive posttranslational event that greatly influences the ultimate viscoelastic properties of mucin. Highly sulfated and/or sialylated mucins comprise a considerable proportion of the mucous layers of the gastrointestinal, respiratory, and reproductive tracts, and have been demonstrated to be associated with some pathological conditions and patho-genesis (1-4). The precise biological roles for glycan sulfation are largely unknown; however, several groups have demonstrated discrete biological roles for specific instances of glycan sulfation of non-mucin glycoproteins. These roles include the control of the circulatory half-life of human luteinizing hormone, symbiotic interactions of leguminous plants and nitrogen-fixing bacteria, and the targeting of lymphocytes to lymph nodes (5). [ABSTRACT FROM AUTHOR]

The carbohydrate moiety of mucin is characterized by the presence of oligosaccharides linked to the peptide backbone by an O-glycosidic linkage between an N-acetylgalactosamine residue and a hydroxylated amino acid (serine or threonine). These linkages are alkali labile and the carbohydrate chains can be released as oligosaccharide-alditols by a β-elimination, with NaOH in the presence of NaBH4. The structures of carbohydrate chains found in mucins can be as simple as the disaccharide NeuAc α2 →6GalNAc in ovine submaxillary mucin and as complex as the ones found in human respiratory or salivary mucins, in which several hundred different carbohydrate chains exist (1,2). This diversity is generated (1) by the different monosaccharides constituting the glycans, generally fucose, galactose, N-acetylgalactosamine, N-acetylglucosamine, and N-acetyl neuraminic acid, but also other monosaccharides such as ketodesoxynonulosonic acid or N-glycolylneuraminic acid, and, finally the occurance of sulfation of galactose and N-acetylglucosamine (3,4); and (2) by the difference in length, in branching, and by the occurrence of all the different possible linkages between the constituting monosaccharides. The diversity of mucin-type oligosaccharides can be extreme. For example, 88 oligosaccharides have been isolated from the respiratory mucins of a single individual (5-10) and more than 150 have been isolated from the jelly coat from eggs of different species of amphibians (11-15). [ABSTRACT FROM AUTHOR]

O-linked chains of glycoproteins have classically been released by alkaline boro-hydride degradation, in which mild alkali (0.05 M OH-) is used to cause β-elimination from the β carbon of serine (R-H) or threonine (R-CH3) in the protein backbone. [ABSTRACT FROM AUTHOR]

Mucin oligosaccharides are constructed by monosaccharide addition to form common cores. This architecture limits the number of constituent monosaccharides. Monosaccharides commonly found in mucins may be divided into neutral (galactose [Gal]; fucose [Fuc]), hexosamines (N-acetylgalactosamine [GalNAc]; N-acetyl-glucosamine [GlcNAc]), and acidic compounds (sialic acids [NeuAc]). Additive heterogeneity comes from the possible substitution with aglycone residues such as sulfate, phosphate, or acetate groups. Prior to their analysis, monosaccharides must be released from the oligosaccharide chain by acidic hydrolysis. Monosaccharide composition can also be achieved on free oligosaccharide-alditols released from the native glycoprotein by reductive alkaline treatment (β-elimination). In this case, GalNAc is converted into N-acetylgalactosaminitol (GalNAc-ol). Different methods are available for the analysis of monosaccharides depending mainly on the amount of material available. Several techniques, such as gas-liquid chromatography (GLC) or high-performance liquid chromatography (HPLC), allow both quantitative and qualitative analysis of monosaccharide mixtures. Other chromatographic or electrophoretic procedures are described herein, but these only allow a rapid qualitative analysis of samples. Single separated monosaccharides may be further identified by physicochemical methods such as mass spectrometry (MS) or nuclear magnetic resonance. [ABSTRACT FROM AUTHOR]

The viscoelastic properties needed for the protective functions of secretory mucins are in part conditional on the capacity of mucin macromolecules to form linear polymers stabilized by disulfide bonds. The individual mucin monomers have a distinctive structure, consisting of a long central peptide region of tandem repeat sequences, flanked by cysteine-rich regions at each end, which are presumed to mediate polymerization. Secretory mucins contain approx 60-80% carbohydrate, with extensive O-glycosylation in the central tandem repeat regions, and N-linked oligosaccharides in the peripheral regions (1). [ABSTRACT FROM AUTHOR]

Since the mid-1980s, the family of high molecular weight glycoproteins known as mucins have evoked considerable interest among those in the field of cancer research. Mucins, which are constituents of mucus, have a lubricating and protective function in normal epithelial tissue (1). However, expression of mucin by the cancer cell is often highly disorganized and upregulated, sometimes to the extent that mucin can be detected in the circulation of the cancer patient. These changes in expression of mucin observed in neoplasia have led to the exploitation of some members of the mucin family as circulating tumor markers (2,3) or targets for diagnostic imaging (4-6) and therapy of cancer. [ABSTRACT FROM AUTHOR]

Although it is possible to determine and characterize the total carbohydrate profile after release of the mucin oligosaccharides (usually by β-elimination), it is challenging to assign the sites of glycosylation (macroheterogeneity) and the carbohydrate heterogeneity at a given glycosylation site (microheterogeneity). Typically, the characterization of macro- and microheterogeneity has been dependent on the isolation of small peptides with only one glycosylation site. However, this is not possible with high molecular weight, heavily glycosylated domains such as those found in mucins. [ABSTRACT FROM AUTHOR]

Amino acid analysis is a commonly used technique that provides quantitative estimation of the amounts of proteins/amino acids present in a sample and/or qualitative information on the amino acid composition of a protein. For protein analysis, the technique essentially involves acid hydrolysis of amino acid peptide bonds within the protein; chemical derivatization of hydrolysate (amino acids) of the protein; and high-performance liquid chromatography (HPLC) separation, detection, and analysis of those derivatized amino acids. [ABSTRACT FROM AUTHOR]

Mucins are secreted from epithelial cells of the gastrointestinal (GI), genitourinary, and respiratory tracts to form mucous gels protecting mucosal surfaces from damaging effects and agents (11,2). Mucous gels are not pure mucin but contain other components secreted into the gel, e.g., IgA and protein, lipid, and nucleic acid from shed epithelial cells, and particularly, in the lower GI tract from bacteria. When measuring the rheological properties of mucous/mucin gels, it is essential to consider the effect of these nonmucin components. Mucin alone is the gel-forming component of mucus; other components can inhibit gel formation and strength. Thus, cellular debris has been shown to reduce markedly gel strength and even inhibit gel formation (3,4). [ABSTRACT FROM AUTHOR]

The rheological properties of human mucus are dominated by the physical properties of large secreted O-linked glycoproteins often referred to as gel-forming mucins. These molecules share the ability to assemble into long oligomeric structures via the agency of disulfide bonds. There is evidence that at least four mucins—MUC2, MUC5AC, MUC5B, and MUC6 (1-4)—are gel-forming mucins, and it is possible that there are others. In isolating a mixture of mucins from mucus, there is considerable scope for heterogeneity in their mass and length owing to the presence of different gene products that may themselves show polymorphism, different glycoforms of the various gene products (seeChapter 7), and variable numbers of subunits contributing to the final polymer. It is possible that heterogeneity may be an important biological property of gel-forming mucins and a key comparative characteristic when studying the change of mucous properties through the course of disease. [ABSTRACT FROM AUTHOR]

This chapter describes a strategy for the separation and identification of the mucins present in mucous secretions or from cell culture, focusing primarily on those mucins involved in gel formation. At present, the mucins MUC2, MUC5AC, MUC5B, and MUC6 are known to be gel-forming molecules (1-4). These mucins share common features in that they are oligomeric in nature and consist of a variable number of monomers (subunits) linked in an end-to-end fashion via the agency of disulfide bonds. In addition, their polypeptides comprise regions of dense glycosylation interspersed with "naked" cysteine-rich domains (4-7). [ABSTRACT FROM AUTHOR]

Most epithelial mucins are secretory glycoproteins. The mucin-producing cells are characterized by large intracellular stores of these very large and complex glycoproteins (1,2). These secretory mucins form mucous layers on the apical side of the cells, protecting the vulnerable epithelium, while allowing selective interactions with the apical environments, which is typically the lumen of an organ that is continuous with the outer world. Secretion from mucin-producing cells is regulated. Normally, mucins are constitutively secreted in relatively low amounts, which are sufficient under normal conditions to sustain the thickness of the mucous layer. On acute threats, the accumulated mucins may be secreted in bulk amounts to provide mucus as an effective, yet temporary, means of epithelial protection (1,2). Both types of secretion require synthesis of mucin: constitutive secretion demands a continuous low level of biosynthesis, whereas stimulated secretion requires massive synthesis to replenish the diminished resources. [ABSTRACT FROM AUTHOR]

Three phases of production of mucin can be identified in the gastrointestinal (GI) tract on the basis of their location in vivo: the stored, presecreted, intracellular mucin; the gel phase adherent to the epithelial surfaces; and the viscous, mobile mucin, which is largely in soluble form and mixes with the luminal contents. The layer of adherent mucous gel that lines the epithelial surfaces throughout the gut from the stomach to the colon marks the interface between the mucosal epithelium and the fluid luminal environment, which is teeming with nutrients, bacteria, destructive hydrolases, foreign compounds and so on. The adherent mucous gel thus provides a protective barrier and a stable unstirred layer with its own microenvironment, between the mucosal surface and the lumen (1,2). In the mouth, where salivary mucins are secreted, and the esophagus, there is no discernible adherent mucous gel layer. [ABSTRACT FROM AUTHOR]

Detection and quantitation of mucins can be important in both the research and clinical settings. Applications may range from detection of potentially novel mucins present during purification from mucus, to quantitation of specific mucin core proteins or carbohydrate moieties present in clinical samples. This chapter discusses procedures and limitations of several different strategies available to detect and quantify these glycoproteins from biological samples, with a view to providing guidelines from which to select the best applicable techniques. Example protocols are then provided to give a starting point for development of a technique. Refer to Chapter 3 for detection of mucins in histological preparations (1); note, however, that many of the principles for selection of detection tools discussed herein are applicable to histological detection. [ABSTRACT FROM AUTHOR]

Morphologically based studies on mucins allow structural characterization to be linked to specific sites of synthesis and secretion. The histochemical approach to the study of mucin is therefore highly informative. There is a correspondingly large body of literature documenting the tissue distribution of mucins as demonstrated by mucin histochemistry, lectin histochemistry, and immunohistochemistry (and various combinations of these methods). Two principal issues need to be considered in order to maximize the potential value of morphologically based methodologies: (1) nature and limitations of the individual techniques, and (2) interpretation and reporting of mucin staining. [ABSTRACT FROM AUTHOR]

The first task in this chapter is to define the term membrane mucin. In a classical sense, the term is an oxymoron, because mucins were defined as the major glycoproteins of mucous secretions. However, the recognition of the importance of mucinous tumor cell surface glycoproteins and their prominence in the early work on the cloning of mucin has led to a shift in usage (1,2), in which both secreted and membrane components are recognized as mucins. This usage has led to another complication, in which membrane components with highly O-glycosylated mucinlike domains are called mucins (3,4). Such mucinous domains are present in many cell surface molecules, most of which have few of the characteristics of other mucins (4). For the purpose of this chapter I have assumed a simple definition of membrane mucins. They must exhibit two characteristics: (1) they must be strongly bound to the membrane, and (2) they must have a large, highly O-glycosylated domain of mucin. Eventually this definition should evolve to require that membrane mucins contain a defined membrane-binding domain, such as a hydrophobic transmembrane sequence, and mucin repeat sequences. However, application of that requirement at present would exclude epiglycanin, the first membrane mucin to be discovered, for which such information is not available, because it has not been cloned. The present definition still restricts the number of membrane mucins to four examples: epiglycanin, MUC1, sialomucin complex ([SMC], also ASGP-1/ASGP-2 and MUC4), and rat MUC3. Another aspect of these mucins needs to be considered. All three, which have been sufficiently characterized, are found in soluble forms as well as membrane forms. SMC is found in goblet cell secretory granules in the intestine and is secreted via a regulated mechanism (5). Thus, the term membrane mucin is somewhat of a misnomer, although it remains the best descriptor until functional descriptive names become feasible. [ABSTRACT FROM AUTHOR]

The large gel-forming mucins, which form the major macromolecular components of mucous secretions, are members of the mucin "superfamily." Nine mucin genes (MUC1-MUC4, MUC5AC, MUC5B, and MUC6-MUC8) have been identified (for reviews seerefs. 1 and 2, with each gene showing expression in several tissues. Only the MUC1, MUC2, MUC4, MUC5, and MUC7 mucins have been sequenced completely (3-11) although large stretches of MUC5AC (12-15) as well as the C-terminal sequences of MUC3 (16) and MUC6 (17) are now known. [ABSTRACT FROM AUTHOR]