Abstract
Bovine submaxillary mucin (BSM) is a gel-forming glycoprotein polymer, and Ser/Thr-linked glycans (O-glycans) are important in regulating BSM’s viscoelasticity and polymerization. However, details of O-glycosylation have not been reported. This study investigates the structural and quantitative characteristics of O-glycans and identifies O-glycosylation sites in BSM using liquid chromatography–tandem mass spectrometry. The O-glycans (consisting of di- to octa-saccharides) and their quantities (%) relative to total O-glycans (100%; 1.1 pmol per 1 μg of BSM) were identified with 14 major (>1.0%), 12 minor (0.1%–1.0%), and eight trace (<0.1%) O-glycans, which were characterized based on their constituents (sialylation (14 O-glycans; 81.9%, sum of relative quantities of each glycan), non-sialylation (20; 18.1%), fucosylation (20; 17.5%), and terminal-galactosylation (6; 3.6%)) and six core structure types [Gal-GalNAc, Gal-(GlcNAc)GalNAc, GlcNAc-GalNAc, GlcNAc-(GlcNAc)GalNAc, and GalNAc-GalNAc]. O-glycosylation sites were identified using O-glycopeptides (bold underlined; 56SGETRTSVI, 259SHSSSGRSRTI, 272GSPSSVSSAEQI, 307RPSYGAL, 625QTLGPL, 728TMTTRTSVVV, and 1080RPEDNTAVA) obtained from proteolytic BSM; these sites are in the four domains of BSM. The gel-forming mucins share common domain structures and glycosylation patterns; these results could provide useful information on mucin-type O-glycans. This is the first study to characterize O-glycans and identify O-glycosylation sites in BSM.
Highlights
Mucins are macromolecular glycoprotein constituents of mucus [1], and are highly modified with Ser/Thr-linked glycans (O-glycans) [2]
Glycopeptides from digested Bovine submaxillary mucin (BSM) were loaded onto a Sep-Pak C18 1cc Vac cartridge (Waters), and the peptides were eluted with 10% isopropanol in 5% acetic acid to selectively enrich glycopeptides
O-glycans were released from BSM by non-reductive β-elimination, and the reducing ends were labeled with AB or ProA
Summary
Mucins are macromolecular (molecular mass, 0.4–4 MDa) glycoprotein constituents of mucus [1], and are highly modified with Ser/Thr-linked glycans (O-glycans) [2]. The biosynthesis of mucin-type O-glycosylation is initiated by N-acetylgalactosamine (GalNAc) attached to the hydroxyl moiety of Ser or Thr, and it is usually extended to form one of several common core structures [4]. These mucin-type O-glycans consist of GalNAc, N-acetylglucosamine (GlcNAc), fucose (Fuc), galactose (Gal), and sialic acids (SAs), including N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). The functional roles of the identified mucin-type O-glycans and attachment sites in BSM domains are briefly discussed
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