Document Type

Dissertation - University Access Only

Award Date

2003

Degree Name

Doctor of Philosophy (PhD)

Department / School

Dairy Science

First Advisor

A. K. Erickson

Abstract

Mucins are a diverse family of secretory and membrane-bound glycoproteins characterized by their high-carbohydrate content, consisting primarily of O-linked glycans. In the current study, we examined the hypothesis that milk mucins act as receptor decoys that prevent gastrointestinal (GI) pathogens from binding to their receptors on epithelial cells lining the GI tract. To begin to test this hypothesis, we devised the main objective of the current study which was to compare the mucins found in cow and sow milk with those found on epithelial cells in the porcine GI tract. At first, we compared staining methods for detection of mucins on both SOS-PAGE gels and on polyvinylidene difluoride (PVDF) membranes. Our results indicate that a modified silver staining technique, first described by Morrissey [Anal. Biochem. l 981.117(2):307-31 0], is the most sensitive detection method for staining mucins on SDS-P AGE gels, and biotinylated Jacalin staining is the most sensitive detection method for staining mucins on PVDF membranes. We then identified both MUCX and MUCl from bovine and porcine milk. Our results indicate that bovine and porcine MUCl have polymorphic appearance on SDS-PAGE gels and are present in both milk fat globule membrane (MFGM) and skim milk. Porcine MUCX has polymorphic appearance, while bovine MUCX is a nonpolymorphic single band, on SDS-PAGE gels. Bovine and porcine MUCX are present in skim milk only. Both bovine and porcine milk mucins are N-glycosylated (more for MUCl), and contain abundant terminal sialic acid (Sia) residues and some terminal galactose (Gal) residues. Other carbohydrate structures on milk mucins are expressed in mucin type-specific (MUCX versus MUCl) and species-specific (bovine versus porcine) manner. We cloned partial porcine MUCl cDNA (606 bp; GenBank accession number: AY24508) and quantified porcine MUCl mRNA expression level along the porcine GI tract. Our results indicate that MUCl mRNA expression is abundant in stomach and mammary gland, moderate in duodenum and colon, and not detectable in jejunum and ileum. We then identified the major high-molecular-weight membrane-bound mucins in porcine intestinal brush border (IBB) preparations using silver staining. Our results indicate that, based on staining behavior and density, the major small intestinal membrane-bound mucins are intestinal mucin-type glycoproteins (IMTGPs) previously described by Erickson et al. [Infect. Immun.1992. 60(3):983-988; 1994.62(12):5404- 5410]. The IMTGPs exhibit age-related glycosylation patterns, and milk MUCl carries similar glycan moieties to those of IMGTPs in newborn animals but not those of IMGTPs in adult animals, which provides support for the hypothesis that milk MUCl acts as a receptor decoy for preventing binding of pathogens to the GI tract of newborn animals. We also identified a number of proteins in both porcine MFGM and skim milk that are capable of binding biotinylated Escherichia coli expressing any of the three K88 fimbrial adhesin variants (K88ab, K88ac, and K88ad) and K88 fimbrial adhesins. The number and intensity of these K88-binding proteins was high for K88ab, intermediate for K88ac, and low for K88ad, which directly correlated with the ability of E. coli expressing these variants to form aggregates with MFGs and skim milk.

Library of Congress Subject Headings

Mucins.
Brush border membrane.
Milk.
Gastrointestinal system -- Diseases -- Prevention.

Publisher

South Dakota State University

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Rights Statement

In Copyright