Analytical approaches to the study of mucin glycosylation

Abstract

Mucins constitute the major protein component of the mucus layer, which covers the epithelial surfaces of the respiratory, the gastrointestinal and the genitourinary tracts. Mucins carry O-linked oligosaccharides which contribute to the molecule by 50-80 weight percent, and these can be highly diverse, with 50-150 different oligosaccharides found on a purified mucin population. Altered mucin glycosylation has been observed in mucus-related diseases, which implies the importance of the development of analytical methods and strategies for structural characterization and semiquantitative estimates of diverse mixtures of oligosaccharides.Oligosaccharides from mucin subpopulations from a patient with Cystic Fibrosis were characterized and compared regarding size, composition and sequence. Differences and similarities in the relative abundance of individual oligosaccharides were mapped using high temperature gas chromatography (GC), gas-chromatography/mass spectrometry (GC/MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) of subfractionated glycans. A similar approach was applied in a comparative study of the glycosylation on intestinal mucins from wild type and CFTR knock-out mice. Increased mucus production and an increased expression of the blood group H epitopes was found in the small intestine of CF mice. Increased mRNA levels for a fucosyltransferase Fut2 was identified by northern blot analysis on intestinal tissue sections from CF mice and compared to wild type mice, suggesting that this transferase could be responsible for the altered glycosylation.The two major mucin components in saliva, originally termed MG1 and MG2 were characterized and compared with respect to average oligosaccharide size, sequence, abundance and degree of diversity. 1H NMR spectroscopy, MALDI-TOF/MS and GC/MS of released oligosaccharides mixtures and subfractions of these showed that MG1 carried longer and more diverse glycans compared to MG2. 1H NMR spectroscopy of the glycans supported a higher diversity of peripheral fucose epitopes on MG1 compared to MG2, which mainly expressed Lex and sialyl-Lex blood group determinants. The two mucins shared few oligosaccharides in common, suggesting specific functions for each of these. Experiments confirming the binding of MG2 to L-selectin in in vitro studies proposed an involvement in the mediation of leukocyte interactions in the oral cavity.The use of liquid chromatography coupled to electrospray mass spectrometry (LC-ESI-MS) for analyzing mixtures of nonderivatized sulfated mucin oligosaccharides was explored. The chromatographic separation of this subclass of analytes was compared on two columns, on a porous graphitized carbon column and an amino-bonded column. The analysis of a diverse mixture of sulfated oligosaccharides from porcine large intestine with negative ion LC-ESI/MS/MS allowed the identification of 28 different oligosaccharides during a single chromatographic analysis. Sufficient fragmentation was obtained by LC/MS/MS to distinguish between sequence isomers.