Precise structural analysis of O-linked oligosaccharides in human serum

Abstract

O-glycans are suitable targets as novel and useful tumor markers. The structures of O-glycans in human sera from four healthy controls were precisely analyzed to obtain the reference O-glycan database. O-glycans were prepared from sera by hydrazine treatment followed by fluorescent labeling with aminopyridine and identified using two-dimensional mapping, enzymatic digestion and mass spectrometry (MS) together with methanolysis and the use of newly synthesized sulfated oligosaccharides as standards. O-glycans, present at more than 0.01% of the total O-glycans, were analyzed, and 18 kinds of acidic and 2 kinds of neutral glycans were identified. NeuAcα2-3Galβ1-3N-acetylgalactosamine (GalNAc) (61-64%), NeuAcα2-3Galβ1-3(NeuAcα2-6)GalNAc (15-26%) and Galβ1-3GalNAc (6-14%) were major components while other sialylated glycans, Galβ1-3(NeuAcα2-6)GalNAc, Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-3)GalNAc and NeuAcα2-3Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-3)GalNAc were relatively minor components, accounting for ∼1-2%. Very minor glycans accounting for ∼0.01-0.1% of the total include (i) the neutral glycan, Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAc, (ii) sialylated glycans, having sialyl Tn antigen, agalacto and trisialylated structures, (iii) fucosylated glycans forming blood type H antigen, blood type A antigen, blood type B antigen, Lewis X antigen and sialyl Lewis X antigen and (iv) sulfated glycans, having 6-sulfo and 3'-sulfo structures. Two kinds of clinically applied tumor markers namely sialyl Tn antigen and sialyl Lewis X antigen in healthy controls sera were revealed to be present at ∼0.1-0.2% of the total. However, other markers such as CA19-9 and DU-PAN-2 were not found, suggesting the relative amounts of these glycans to be <0.01%. These detailed O-glycan profiles will help to find novel carbohydrate tumor markers.

O-glycosylation sites identified from mucin core-1 type glycopeptides from human serum

“…In the SP2 workflow, glycopeptides were isolated from a human serum tryptic digest by sequential lectin affinity chromatography, first using wheat germ agglutinin (WGA) for general glycopeptide enrichment, followed by Jacalin for capturing O-glycopeptides. In addition, the WGA eluant was treated with neuraminidase to eliminate interference by sialic acids modifying the 6-OH of the core GalNAcα (disialo structures represent ~20% of the O-glycan pool [37]). Isolated glycopeptide mixtures were analyzed by data-dependent LC-MS/MS.…”

“…Our lectin selection can be further justified by the composition of the human serum O-glycan pool. The dominance of the monosialylated mucin core-1 O-glycan, NeuAcα2–3Galβ1–3GalNAcα in human serum O-glycoproteins has been demonstrated by two independent studies [36, 37]. This structure represents more than 60% of the glycans, followed by the diasialo and asialo variants, at approximately 20% and 10%, respectively [37].…”

“…We aimed at the isolation of glycopeptides modified with mucin-type core-1 structures, as this glycoform has been reported to be the most abundant in human serum [36]: NeuAcα2–3Galβ1–3GalNAcα (~60%), NeuAcα2–3Galβ1–3(NeuAcβ2–6)GalNAcα (~20%), and Galβ1–3GalNAcα (~10%) represent ~90% of the total O-glycan pool [37]. Considering the wide dynamic range of serum protein levels, and the expected heterogeneity of O-glycosylation, we followed multistep enrichment strategies.…”

Characterizing the O-glycosylation landscape of human plasma, platelets, and endothelial cells

“…1,3 The major O-glycan structures in humans are the sialylated Core 1 O-glycans-sialyl T and disialyl-T-along with the lesser represented Core 2 glycans (Figure 1). 4,5 These can be further elongated and modified, generating a large set of O-glycan structures. [4][5][6] Historically, O-glycosylation has been considered a relatively rare, densely clustered, posttranslational modification (PTM) occurring in mucins and mucin-like proteins.…”

“…4,5 These can be further elongated and modified, generating a large set of O-glycan structures. [4][5][6] Historically, O-glycosylation has been considered a relatively rare, densely clustered, posttranslational modification (PTM) occurring in mucins and mucin-like proteins. Such O-glycosylated regions are thought to attract water molecules, stabilize protein structure, extend the polypeptide backbone, and protect from proteolytic cleavage.…”