Structural diversity and specific distribution of O-glycans in normal human mucins along the intestinal tract

Abstract

Purified human mucins from different parts of the intestinal tract (ileum, cecum, transverse and sigmoid colon and rectum) were isolated from two individuals with blood group ALe(b) (A-Lewis(b)). After alkaline borohydride treatment the released oligosaccharides were structurally characterized by nano-ESI Q-TOF MS/MS (electrospray ionization quadrupole time-of-flight tandem MS) without prior fractionation or derivatization. More than 100 different oligosaccharides, with up to ten monosaccharide residues, were identified using this technique. Oligosaccharides based on core 3 structures, GlcNAc(beta1-3)GalNAc (where GlcNAc is N-acetyl-D-glucosamine and GalNAc is N-acetylgalactosamine), were widely distributed in human intestinal mucins. Core 5 structures, GalNAc(alpha1-3)GalNAc, were also recovered in all fractions. Moreover, a comparison of the oligosaccharide repertoire, with respect to size, diversity and expression of glycans and terminal epitopes, showed a high level of mucin-specific glycosylation: highly fucosylated glycans, found specifically in the small intestine, were mainly based on core 4 structures, GlcNAc-(beta1-3)[GlcNAc(beta1-6)]GalNAc, whereas the sulpho-Le(X) determinant carrying core 2 glycans, Gal(beta1-3)[GlcNAc(beta1-6)]-GalNAc (where Gal is galactose), was recovered mainly in the distal colon. Blood group H and A antigenic determinants were present exclusively in the ileum and cecum, whereas blood group Sd(a)/Cad related epitopes, GalNAc(beta1-4)[NeuAc(alpha2-3)]Gal (where NeuAc is N-acetylneuraminate), were found to increase along the length of the colon. Our findings suggest that mucins create an enormous repertoire of potential binding sites for micro-organisms that could explain the regio-specific colonization of bacteria in the human intestinal tract.

Figure 1. Mass spectra of the total oligosaccharides, acquired in the negative ion mode [M−H]⁻, from ileum, cecum, transverse colon, sigmoid colon and rectum of donor 1

Signals marked with asterisks referr to dicharged ions [M−2H]²⁻; ions at m/z 551, 584, 613, 657, 685 and 807 correspond respectively to the [M−H]⁻ ions at m/z 1104, 1169, 1227, 1315, 1372 and 1615.

Figure 2. MS/MS spectrum of a core 4 oligosaccharide at m/z 1163, recorded in the positive ion mode [M+Na]⁺

Figure 3. MS/MS spectrum, recorded in the negative ion mode, of the dicharged ion [M−2H]²⁻ corresponding to a core 2 oligosaccharide at m/z 1055 with two sulphate residues

Figure 4. MS/MS spectrum of the [M−H]⁻ ion at m/z 1081 corresponding to an oligosaccharide carrying a blood group Sd^a/Cad antigen, isolated from human sigmoid mucins

Figure 5. MS/MS spectrum of the [M+Na]⁺ ion at m/z 1397 corresponding to an O-glycan with a blood group ALe^b determinant

Figure 6. MS/MS spectrum of the [M−H]⁻ ion at m/z 813 corresponding to an O-glycan carrying a Le^x determinant