Glycoprotein Methods and Protocols: The Mucins

The mucins (mucus glycoproteins) have long been a complex corner of glycoprotein biology. While dramatic advances in the separation, structural an- ysis, biosynthesis, and degradation have marked the progress in general glycop- tein understanding, the mucins have lagged behind. The reasons for this lack of progress have always been clear and are only now being resolved. The mucins are very large molecules; they are difficult to separate from other molecules present in mucosal secretions or membranes; they are often degraded owing to natural protective functions or to isolation methodology and their peptide and oligos- charide structures are varied and complex. Understanding these molecules has demanded progress in several major areas. Isolation techniques that protect the intact mucins and allow dissociation from other adsorbed but discrete molecules needed to be developed and accepted by all researchers in the field. Improved methods for the study of very large molecules with regard to their aggregation and polymerization were also needed. Structural analysis of the peptide domains and the multitude of oligosaccharide chains was required for smaller sample sizes, for multiple samples, and in shorter time. In view of these problems it is perhaps not surprising that the mucins have remained a dilemma, of obvious biological importance and interest, but very difficult to analyze.

Table of Contents:
Purification of Mucin.- Isolation of Large Gel-Forming Mucins.- Preparation of Membrane Mucin.- Detection and Ouantitation of Mucin.- Histologically Based Methods for Detection of Mucin.- Detection and Quantitation of Mucins Using Chemical, Lectin, and Antibody Methods.- The Gastrointestinal Adherent Mucous Gel Barrier.- Quantitation of Biosynthesis and Secretion of Mucin Using Metabolic Labeling.- Separation, Identification, and Physical Characterization of Mucins.- Separation and Identification of Mucins and Their Glycoforms.- Heterogeneity and Size Distribution of Gel-Forming Mucins.- Rheology of Mucin.- Rheology of Mucin.- Mucin Peptide Analysis.- Amino Acid Analysis of Mucins.- Identification of Glycosylation Sites in Mucin Peptides by Edman Degradation.- Synthetic Peptides for the Analysis and Preparation of Antimucin Antibodies.- Mucin Domains to Explore Disulfide-Dependent Dimer Formation.- Carbohydrate Structural Analysis of Mucins.- Monosaccharide Composition of Mucins.- O-Linked Oligosaccharide Chain Release and Fractionation.- Structural Analysis of Mucin-Type O-Linked Oligosaccharides.- Measurement of Sulfate in Mucins.- Mucin Biosynthesis.- Biosynthesis of Mucin Cell and Organ Culture Methods for Biosynthetic Study.- Metabolic Labeling Methods for the Preparation and Biosynthetic Study of Mucin.- Identification of Mucins Using Metabolic Labeling, Immunoprecipitation, and Gel Electrophoresis.- Mucin Precursors Identification and Analysis of Their Intracellular Processing.- Inhibition of Mucin Glycosylation.- O-Linked Chain Glycosyltransferases.- Mucin Gene Detection.- Mucin cDNA Cloning.- Northern Blot Analysis of Large mRNAs.- Southern Blot Analysis of Large DNA Fragments.- In Situ Hybridization Techniques for Localizing Mucin mRNA.- Detection of Mucin Gene Polymorphism.- Preparation of Antimucin Antibodies.- Polyclonal and Monoclonal Techniques.- Monoclonal Antibodies to Mucin VNTR Peptides.- Enzymatic Degradation of Mucins.- Mucinase Activity.- Proteinase Activity.- Glycosidase Activity.- Assays for Bacterial Mucin-Desulfating Sulfatases.- Mucin-Bacterial Interactions.- Mucin-Bacterial Binding Assays.- Growth of Mucin Degrading Bacteria in Biofilms.- Cellular And Humoral Responses To Mucins.- Generation of MUC1 CytotoxicT-Cells in Mice and Epitope Mapping.- Analysis of the Frequency of MHC-Unrestricted MUC1 -Specific CytotoxicT-Cells in Peripheral Blood by Limiting Dilution Assay.- Expression of MUC1 in Insect Cells Using Recombinant Baculovirus.- Mucin Antigen Presentation Using Dendritic Cells.- Detection of Humoral Immune Responses to Mucins.