Identification and quantification of N-linked oligosaccharides released from glycoproteins: an inter-laboratory study

As characterization of glycosylation is required for the licensing of recombinant glycoprotein therapeutics, technique comparability must be assessed. Eleven UK laboratories (seven industrial, two regulatory or government, two academic) participated in an inter-laboratory study to analyze N-glycans present in four mixtures prepared by PNGase F cleavage of commercial glycoproteins: human alpha1-acid glycoprotein (H alpha1), bovine alpha1-acid glycoprotein (B alpha1), bovine pancreatic ribonuclease B (RNaseB), and human serum immunoglobulin G (hIgG). Participants applied their routine glycan mapping methodology using predominantly chromatography and mass spectrometry to identify and quantify components. Data interpretation focused on the relative amounts of different glycan structures present, the degree of sialylation, antennary and the galactosylation profiles, fucosylation and bisecting GlcNAc content, and the number of glycan components identified. All laboratories found high levels of sialylation for H alpha1 and B alpha1 (Z-numbers 271 +/- 24 and 224 +/- 18, respectively), but varying ratios of di-, tri-, and tetra-antennary chains. The Z-score for hIgG glycans had high variability as values obtained from mass spectrometric and chromatographic methods clustered separately. The proportion of the major penta-mannosyl chain from RNaseB was between 29 and 62%. Proportions of fucosylated and bisected GlcNAc chains from hIgG were between 58 and 96% and 9 and 23%, respectively. Mass spectrometric approaches consistently identified more glycan species, especially when both N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac) were present. These data highlight the need for well-characterized reference standards to support method validation and regulatory guidance on selection of approaches. Pharmacopoeial specifications must acknowledge method variability.     

Histochemical analysis of glycoconjugates in the domestic cat testis

The localization and characterization of oligosaccharide sequences in the cat testis was investigated using 12 lectins in combination with the beta-elimination reaction, N-Glycosidase F and sialidase digestion. Leydig cells expressed O-linked glycans with terminal alphaGalNAc (HPA reactivity) and N-glycans with terminal/internal alphaMan (Con A affinity). The basement membrane showed terminal Neu5Acalpha2,6Gal/GalNAc, Galbeta1,3GalNAc, alpha/betaGalNAc, and GlcNAc (SNA, PNA, HPA, SBA, GSA II reactivity) in O-linked oligosaccharides, terminal Galbeta1,4GlcNAc (RCA120 staining) and alphaMan in N-linked oligosaccharides; in addition, terminal Neu5acalpha2,3Galbeta1,4GlcNac, Forssman pentasaccharide, alphaGal, alphaL-Fuc and internal GlcNAc (MAL II, DBA, GSA I-B4, UEA I, KOH-sialidase-WGA affinity) formed both O- and N-linked oligosaccharides. The Sertoli cells cytoplasm contained terminal Neu5Ac-Galbeta1,4GlcNAc, Neu5Ac-betaGalNAc as well as internal GlcNAc in O-linked glycans, alphaMan in N-linked glycoproteins and terminal Neu5Acalpha2,6Gal/ GalNAc in both O- and N-linked oligosaccharides. Spermatogonia exhibited cytoplasmic N-linked glycoproteins with alphaMan residues. The spermatocytes cytoplasm expressed terminal Neu5Acalpha2,3Galbeta1,4 GlcNAc and Galbeta1,3GalNAc in O-linked oligosaccharides, terminal Galbeta1,4GlcNAc and alpha/betaGalNAc in N-linked glycoconjugates. The Golgi region showed terminal Neu5Acalpha2,3Galbeta1,4GlcNac, Galbeta1,4GlcNAc, Forssman pentasaccharide, and alphaGalNAc in O-linked oligosaccharides, alphaMan and terminal betaGal in N-linked oligosaccharides. The acrosomes of Golgi-phase spermatids expressed terminal Galbeta1,3GalNAc, Galbeta1,4GlcNAc, Forssmann pentasaccharide, alpha/betaGalNAc, alphaGal and internal GlcNAc in O-linked oligosaccharides, terminal alpha/betaGalNAc, alphaGal and terminal/internal alphaMan in N-linked glycoproteins. The acrosomes of cap-phase spermatids lacked internal Forssman pentasaccharide and alphaGal, while having increased alpha/betaGalNAc. The acrosomes of elongated spermatids did not show terminal Galbeta1,3GalNAc, displayed terminal Galbeta1,4GlcNAc and alpha/betaGalNAc in N-glycans and Neu5Ac-Galbeta1,3GalNAc in O-linked oligosaccharides.     

Structural analysis of trisialylated biantennary glycans isolated from mouse serum transferrin. Characterization of the sequence Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2)Man

Five variants of mouse serum transferrin (mTf, designated mTf-I to mTf-V) with respect to carbohydrate composition have been isolated by DEAE-cellulose chromatography in the following relative percentages: mTf-I: 0.55; mTf-II: 0.79; mTf-III: 71.80; mTf-VI: 21. 90 and mTf-V: 4.96. The primary structures of the major glycans from mTf-III and mTf-IV were determined by methylation analysis and 1H-nuclear magnetic resonance (NMR) spectroscopy. All glycans possessed a common trimannosyl-N,N'-diacetylchitobiose core. From the glycovariant mTf-III two isomers of a conventional biantennary N-acetyllactosamine type were isolated, in which two N-glycolylneuraminic acid (Neu5Gc) residues are linked to galactose either by a (alpha 2-6) or (alpha 2-3) linkage. A subpopulation of this glycovariant contains a fucose residue (alpha 1-6)-linked to GlcNAc-1. The structure of the major glycan found in variant mTf-IV contained an additional Neu5Gc and possessed the following new type of linkage: Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2 )Man(alpha 1-3). In addition to this glycan, a minor compound contained the same antennae linked to Man(alpha 1-6). In fraction mTf-V, which was found to be very heterogeneous by (1)H NMR analysis, carbohydrate composition and methylation analysis suggested the presence of tri'-antennary glycans sialylated by Neu5Gc alpha-2,6- and alpha-2, 3-linked to the terminal galactose residues. In summary, mTf glycans differed from those of other analyzed mammalian transferrins by the presence of Neu5Gc and by a Neu5Gc(alpha 2-6)GlcNAc linkage in trisialylated biantennary structures, reflecting in mouse liver, a high activity of CMP-Neu5Ac hydroxylase and (alpha 2-6)GlcNAc sialyltransferase.     

Identification of proteins bearing beta1-6 branched N-glycans in human melanoma cell lines from different progression stages by tandem mass spectrometry analysis

The common structural alterations in the cell-surface glycoproteins concern the highly elevated expression of tri- and tetra-antennary beta1-6-N-acetylglucosamine (beta1-6 GlcNAc) bearing N-glycans, which are recognised by Phaseolus vulgaris agglutinin (PHA-L). In this report we identified proteins bearing beta1-6 GlcNAc branched N-glycans in three human melanoma cell lines: WM35--from the primary tumour site, as well as WM239 and WM9 from different metastatic sites: the skin and the lymph node, respectively, by tandem mass spectrometry (MS/MS) on PHA-L agarose bound material, followed by immunochemical identification. Our results show that melanoma cell lines differ from each other in the number of N-glycoproteins bearing beta1-6 GlcNAc branched oligosaccharides. Among identified proteins the largest group consists of integrin subunits. In addition, L1-CAM, Mac-2 binding protein, melanoma cell adhesion molecule, intercellular adhesion molecule, melanoma associated antigen, tumour rejection antigen-1, melanoma-associated chondroitin sulfate proteoglycan 4 and lysosome-associated membrane protein (LAMP-1) were found. It was indicated that WM35 cell line showed the lowest number of proteins possessing beta1-6 GlcNAc branched N-glycans in comparison to metastatic WM9 and WM239 cell lines. Our data suggest that changes in the number of proteins being a substrate for GlcNAc-TV are better correlated with melanoma development and progression than with expression of cell adhesion molecules.     

Use of Wisteria floribunda agglutinin affinity chromatography in the structural analysis of the bovine lactoferrin N-linked glycosylation

Background

Over the years, the N-glycosylation of both human and bovine lactoferrin (LF) has been studied extensively, however not all aspects have been studied in as much detail. Typically, the bovine LF complex-type N-glycans include certain epitopes, not found in human LF N-glycans, i.e. Gal(α1-3)Gal(β1-4)GlcNAc (αGal), GalNAc(β1-4)GlcNAc (LacdiNAc), and N-glycolylneuraminic acid (Neu5Gc). The combined presence of complex-type N-glycans, with αGal, LacdiNAc, LacNAc [Gal(β1-4)GlcNAc], Neu5Ac (N-acetylneuraminic acid), and Neu5Gc epitopes, and oligomannose-type N-glycans complicates the high-throughput analysis of such N-glycoprofiles highly.

Methods

For the structural analysis of enzymatically released N-glycan pools, containing both LacNAc and LacdiNAc epitopes, a prefractionation protocol based on Wisteria floribunda agglutinin affinity chromatography was developed. The sub pools were analysed by MALDI-TOF-MS and HPLC-FD profiling, including sequential exoglycosidase treatments.

Results

This protocol separates the N-glycan pool into three sub pools, with (1) free of LacdiNAc epitopes, (2) containing LacdiNAc epitopes, partially shielded by sialic acid, and (3) containing LacdiNAc epitopes, without shielding by sialic acid. Structural analysis by MALDI-TOF-MS and HPLC-FD showed a complex pattern of oligomannose-, hybrid-, and complex-type di-antennary structures, both with, and without LacdiNAc, αGal and sialic acid.

Conclusions

Applying the approach to bovine LF has led to a more detailed N-glycome pattern, including LacdiNAc, αGal, and Neu5Gc epitopes, than was shown in previous studies.

General significance

Bovine milk proteins contain glycosylation patterns that are absent in human milk proteins; particularly, the LacdiNAc epitope is abundant. Analysis of bovine milk serum proteins is therefore excessively complicated. The presented sub fractionation protocol allows a thorough analysis of the full scope of bovine milk protein glycosylation. This article is part of a Special Issue entitled Glycoproteomics.     

Structural analysis of trisialylated biantennary glycans isolated from mouse serum transferrin: Characterization of the sequence Neu5Gc(α2-3)Gal(β1-3)[Neu5Gc(α2-6)]GlcNAc(β1-2)Man

Five variants of mouse serum transferrin (mTf, designated mTf-I to mTf-V) with respect to carbohydrate composition have been isolated by DEAE-cellulose chromatography in the following relative percentages: mTf-I: 0.55; mTf-II: 0.79; mTf-III: 71.80; mTf-VI: 21.90 and mTf-V: 4.96. The primary structures of the major glycans from mTf-III and mTf-IV were determined by methylation analysis and ¹H-nuclear magnetic resonance (NMR) spectroscopy. All glycans possessed a common trimannosyl-N,N′-diacetylchitobiose core. From the glycovariant mTf-III two isomers of a conventional biantennary N-acetyllactosamine type were isolated, in which two N-glycolylneuraminic acid (Neu5Gc) residues are linked to galactose either by a (α2-6) or (α2-3) linkage. A subpopulation of this glycovariant contains a fucose residue (α1-6)-linked to GlcNAc-1. The structure of the major glycan found in variant mTf-IV contained an additional Neu5Gc and possessed the following new type of linkage: Neu5Gc(α2-3)Gal(β1-3)[Neu5Gc(α2-6)]GlcNAc(β1-2)Man(α1-3). In addition to this glycan, a minor compound contained the same antennae linked to Man(α1-6). In fraction mTf-V, which was found to be very heterogeneous by ¹H NMR analysis, carbohydrate composition and methylation analysis suggested the presence of tri′-antennary glycans sialylated by Neu5Gc α-2,6- and α-2,3-linked to the terminal galactose residues. In summary, mTf glycans differed from those of other analyzed mammalian transferrins by the presence of Neu5Gc and by a Neu5Gc(α2-6)GlcNAc linkage in trisialylated biantennary structures, reflecting in mouse liver, a high activity of CMP-Neu5Ac hydroxylase and (α2-6)GlcNAc sialyltransferase.     

The role of CMP-N-acetylneuraminic acid hydroxylase in determining the level of N-glycolylneuraminic acid in porcine tissues

The biosynthesis of the sialic acid N-glycolylneuraminic acid (Neu5Gc) occurs by the action of cytidine monophosphate-N-acetylneuraminate (CMP-Neu5Ac) hydroxylase. Previous investigations on a limited number of tissues suggest that the activity of this enzyme governs the extent of glycoconjugate sialylation with Neu5Gc. Using improved analytical procedures and a panel of nine porcine tissues, each expressing different amounts of Neu5Gc, we have readdressed the issue of the regulation of Neu5Gc incorporation into glycoconjugates. The following parameters were measured for each tissue: the molar ratio Neu5Gc/Neu5Ac, the activity of the hydroxylase, and the relative amount of hydroxylase protein, as determined by enzyme-linked immunosorbent assay (ELISA). A positive correlation between the activity of the hydroxylase and the molar ratio Neu5Gc/Neu5Ac was observed for each tissue. In addition, the hydroxylase activity correlated with the amount of enzyme protein, though in heart and lung disproportionately large amounts of immunoreactive protein were detected. Taken together, the results suggest that the incorporation of Neu5Gc into glycoconjugates is generally controlled by the amount of hydroxylase protein expressed in a tissue.     

N-glycolylneuraminic acid as a carbohydrate cancer biomarker

One of the forms of aberrant glycosylation in human tumors is the expression of N-glycolylneuraminic acid (Neu5Gc). The only known enzyme to biosynthesize Neu5Gc in mammals, cytidine-5′-monophosphate-N-acetylneuraminic acid (CMAH), appears to be genetically inactivated in humans. Regardless, low levels of Neu5Gc have been detected in healthy humans. Therefore, it is proposed that the presence of Neu5Gc in humans is from dietary acquisition, such as red meat. Notably, detection of elevated Neu5Gc levels has been repeatedly found in cancer tissues, cells and serum samples, thereby Neu5Gc-containing antigens may be exploited as a class of cancer biomarkers. Here we review the findings to date on using Neu5Gc-containing tumor glycoconjugates as a class of cancer biomarkers for cancer detection, surveillance, prognosis and therapeutic targets. We review the evidence that supports an emerging hypothesis of de novo Neu5Gc biosynthesis in human cancer cells as a source of Neu5Gc in human tumors, generated under certain metabolic conditions.     

Glycoengineering of therapeutic glycoproteins: in vitro galactosylation and sialylation of glycoproteins with terminal N-acetylglucosamine and galactose residues

Therapeutic glycoproteins produced in different host cells by recombinant DNA technology often contain terminal GlcNAc and Gal residues. Such glycoproteins clear rapidly from the serum as a consequence of binding to the mannose receptor and/or the asialoglycoprotein receptor in the liver. To increase the serum half-life of these glycoproteins, we carried out in vitro glycosylation experiments using TNFR-IgG, an immunoadhesin molecule, as a model therapeutic glycoprotein. TNFR-IgG is a disulfide-linked dimer of a polypeptide composed of the extracellular portion of the human type 1 (p55) tumor necrosis factor receptor (TNFR) fused to the hinge and Fc regions of the human IgG(1) heavy chain. This bivalent antibody-like molecule contains four N-glycosylation sites per polypeptide, three in the receptor portion and one in the Fc. The heterogeneous N-linked oligosaccharides of TNFR-IgG contain sialic acid (Sia), Gal, and GlcNAc as terminal sugar residues. To increase the level of terminal sialylation, we regalactosylated and/or resialylated TNFR-IgG using beta-1,4-galactosyltransferase (beta1,4GT) and/or alpha-2,3-sialyltransferase (alpha2,3ST). Treatment of TNFR-IgG with beta1,4GT and UDP-Gal, in the presence of MnCl(2), followed by MALDI-TOF-MS analysis of PNGase F-released N-glycans showed that the number of oligosaccharides with terminal GlcNAc residues was significantly decreased with a concomitant increase in the number of terminal Gal residues. Similar treatment of TNFR-IgG with alpha2,3ST and CMP-sialic acid (CMP-Sia), in the presence of MnCl(2), produced a molecule with an approximately 11% increase in the level of terminal sialylation but still contained oligosaccharides with terminal GlcNAc residues. When TNFR-IgG was treated with a combination of beta1,4GT and alpha2,3ST (either in a single step or in a stepwise fashion), the level of terminal sialylation was increased by approximately 20-23%. These results suggest that in vitro galactosylation and sialylation of therapeutic glycoproteins with terminal GlcNAc and Gal residues can be achieved in a single step, and the results are similar to those for the stepwise reaction. This type of in vitro glycosylation is applicable to other glycoproteins containing terminal GlcNAc and Gal residues and could prove to be useful in increasing the serum half-life of therapeutic glycoproteins.     

Synthesis of disaccharide Neu5Gcalpha(2-6)GalNAcalpha as a spacer glycoside

The first synthesis of the Neu5Gc analogue of SiaTn disaccharide, which can be detected in breast tumors by immunochemical methods, is reported. The regioselective sialylation of (3-trifluoroacetamidopropyl)-2-azido-2-deoxy-alpha-D-galactopyranoside with peracetate of the methyl ester of N-acetoxyacetylneuraminic acid beta-ethylthioglycoside in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid (or its trimethylsilyl ester) resulted in the derivatives of alpha- and beta-sialyl(2-->6)galactosaminide in 39 and 32% yields, respectively. The catalytic hydrogenolysis of the azido group and subsquent N- and O-acetylation of the alpha-anomer gave the trifluoroacetamidopropyl glycoside peracetate. Removal of the protective groups led to glycoside Neu5Gc alpha(2-->6)GalNAc alpha-O(CH2)3NH2. Using the Neu5Gc derivative with acetoxyacetyl groups at positions O9 and O4 as a donor increases the alpha-selectivity of sialylation to afford the alpha- and beta-anomers in 69 and 8% yields, respectively.     

Accumulation of free Neu5Ac-containing complex-type N-glycans in human pancreatic cancers

We have analyzed the structures of glycosphingolipids and intracellular free glycans in human cancers. In our previous study, trace amounts of free N-acetylneuraminic acid (Neu5Ac)-containing complex-type N-glycans with a single GlcNAc at each reducing terminus (Gn1 type) was found to accumulate intracellularly in colorectal cancers, but were undetectable in most normal colorectal epithelial cells. Here, we used cancer glycomic analyses to reveal that substantial amounts of free Neu5Ac-containing complex-type N-glycans, almost all of which were α2,6-Neu5Ac-linked, accumulated in the pancreatic cancer cells from three out of five patients, but were undetectable in normal pancreatic cells from all five cases. These molecular species were mostly composed of five kinds of glycans having a sequence Neu5Ac-Gal-GlcNAc-Man-Man-GlcNAc and one with the following sequence Neu5Ac-Gal-GlcNAc-Man-(Man-)Man-GlcNAc. The most abundant glycan was Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3Manβ1-4GlcNAc, followed by Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6Manβ1-4GlcNAc. This is the first study to show unequivocal evidence for the occurrence of free Neu5Ac-linked N-glycans in human cancer tissues. Our findings suggest that free Neu5Ac-linked glycans may serve as a useful tumor marker.     

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: MECHANISMS UNDERLYING GASTROINTESTINAL INCORPORATION OF THE NON-HUMAN SIALIC ACID XENO-AUTOANTIGEN N-GLYCOLYLNEURAMINIC ACID

Although N-acetyl groups are common in nature, N-glycolyl groups are rare. Mammals express two major sialic acids, N-acetylneuraminic acid and N-glycolylneuraminic acid (Neu5Gc). Although humans cannot produce Neu5Gc, it is detected in the epithelial lining of hollow organs, endothelial lining of the vasculature, fetal tissues, and carcinomas. This unexpected expression is hypothesized to result via metabolic incorporation of Neu5Gc from mammalian foods. This accumulation has relevance for diseases associated with such nutrients, via interaction with Neu5Gc-specific antibodies. Little is known about how ingested sialic acids in general and Neu5Gc in particular are metabolized in the gastrointestinal tract. We studied the gastrointestinal and systemic fate of Neu5Gc-containing glycoproteins (Neu5Gc-glycoproteins) or free Neu5Gc in the Neu5Gc-free Cmah(-/-) mouse model. Ingested free Neu5Gc showed rapid absorption into the circulation and urinary excretion. In contrast, ingestion of Neu5Gc-glycoproteins led to Neu5Gc incorporation into the small intestinal wall, appearance in circulation at a steady-state level for several hours, and metabolic incorporation into multiple peripheral tissue glycoproteins and glycolipids, thus conclusively proving that Neu5Gc can be metabolically incorporated from food. Feeding Neu5Gc-glycoproteins but not free Neu5Gc mimics the human condition, causing tissue incorporation into human-like sites in Cmah(-/-) fetal and adult tissues, as well as developing tumors. Thus, glycoproteins containing glycosidically linked Neu5Gc are the likely dietary source for human tissue accumulation, and not the free monosaccharide. This human-like model can be used to elucidate specific mechanisms of Neu5Gc delivery from the gut to tissues, as well as general mechanisms of metabolism of ingested sialic acids.     

Glycomic survey mapping of zebrafish identifies unique sialylation pattern

Functional genomics and proteomics studies of the developmental glycobiology of zebrafish are greatly hampered by the current lack of knowledge on its glycosylation profile. To furnish the requisite structural basis for a more insightful functional delineation and genetic manipulation, we have initiated a survey mapping of the possible expression of stage-specific glycoconjugates in zebrafish. High-sensitivity mass spectrometry (MS) analysis in conjunction with the usual array of enzymatic and chemical derivatization was employed as the principal method for rapid differential mapping of the glycolipids and sequentially liberated N- and O-glycans from the total extracts. We demonstrated that all developmental stages of the zebrafish under investigation, from fertilized eggs to hatched embryos, synthesize oligomannosyl types of N-glycans, as well as complex types with additionally beta4-galactosylated, Neu5Ac/Neu5Gc monosialylated Lewis x termini. A combination of collision-induced dissociation (CID)-MS/MS and nuclear magnetic resonance (NMR) analyses led to the identification of an abundant and unusual mucin-type O-glycosylation, based on a novel sequence Fucalpha1-3GalNAcbeta1-4(Neu5Ac/Neu5Gcalpha2-3)Galbeta1-3GalNAc. This core structure may be further oligosialylated, but exclusively in the earlier development stages. Similarly, MS and MS/MS analyses of the extracted glycolipid fraction revealed the presence of a heterogeneous family of oligosialylated lactosylceramide compounds. In contrast to the O-glycans, these glycolipids only appear in the later development stages, suggesting a complex pattern of regulation for sialyltransferase activities during zebrafish embryogenesis.     

Glycoproteomics and glycomics investigation of membrane N-glycosylproteins from human colon carcinoma cells

Aberrant glycosylation of proteins is known to profoundly affect cellular adhesion or motility of tumoral cells. In this study, we used HT-29 human colon epithelial cancer cells as a cellular model of cancer progression, as they can either proliferate or differentiate into enterocyte phenotype. A glycoproteomic approach based on Con A lectin-affinity chromatography, SDS-PAGE and MS analysis, allowed the identification of membrane N-glycoproteins from Triton X-100-solubilized proteins from membrane preparation. Among them, 65% were membrane proteins, and 45% were known to be N-glycosylated, such as alpha chains integrin and dipeptidyl isomerase IV. By lectin-blot analysis, significant changes of alpha-2,3- and alpha-2,6-sialylation of membrane glycoproteins were observed between proliferating and differentiated HT-29 cells. From these results, nano-LC-MS/MS analysis of the tryptic digests of the corresponding bands was performed and led to the identification of several transmembrane glycoproteins, like members of the solute carrier family and adhesion proteins. Finally, we compared N-glycans profiles and monosaccharide composition of proliferating and enterocyte-like HT-29 cells using MALDI-MS and GC-MS analyses of permethylated derivatives. This glycomic approach allowed to underscore significant changes in N-glycans structure, in particular the expression of atypical N-acetylglucosamine (GlcNAc)-ended N-glycans in enterocyte-like HT-29 cells.     

Identification of proteins bearing β1–6 branched N-glycans in human melanoma cell lines from different progression stages by tandem mass spectrometry analysis

The common structural alterations in the cell-surface glycoproteins concern the highly elevated expression of tri- and tetra-antennary β1–6-N-acetylglucosamine (β1–6 GlcNAc) bearing N-glycans, which are recognised by Phaseolus vulgaris agglutinin (PHA-L). In this report we identified proteins bearing β1–6 GlcNAc branched N-glycans in three human melanoma cell lines: WM35 — from the primary tumour site, as well as WM239 and WM9 from different metastatic sites: the skin and the lymph node, respectively, by tandem mass spectrometry (MS/MS) on PHA-L agarose bound material, followed by immunochemical identification. Our results show that melanoma cell lines differ from each other in the number of N-glycoproteins bearing β1–6 GlcNAc branched oligosaccharides. Among identified proteins the largest group consists of integrin subunits. In addition, L1-CAM, Mac-2 binding protein, melanoma cell adhesion molecule, intercellular adhesion molecule, melanoma associated antigen, tumour rejection antigen-1, melanoma-associated chondroitin sulfate proteoglycan 4 and lysosome-associated membrane protein (LAMP-1) were found. It was indicated that WM35 cell line showed the lowest number of proteins possessing β1–6 GlcNAc branched N-glycans in comparison to metastatic WM9 and WM239 cell lines. Our data suggest that changes in the number of proteins being a substrate for GlcNAc-TV are better correlated with melanoma development and progression than with expression of cell adhesion molecules.     

Glycomics and proteomics analyses of mouse uterine luminal fluid revealed a predominance of Lewis Y and X epitopes on specific protein carriers

Sperm motility and maturation are known to be affected by a host of factors encountered en route in both male and female genital tracts prior to fertilization. Using a concerted proteomics and glycomics approach with advanced mass spectrometry-based glycan sequencing capability, we show in this work that 24p3, an abundant mouse uterine luminal fluid (ULF) glycoprotein also called lipocalin 2 (Lcn2), is highly fucosylated in the context of carrying multiple Lewis X and Y epitopes on complex type N-glycans at its single glycosylation site. The predominance of Lewis X/Y along with Neu5Acalpha2-6 sialylation was found to be a salient feature of the ULF glycome, and several other protein carriers were additionally identified including the highly abundant lactotransferrin, which is N-glycosylated at two sites, both with a similar range of highly fucosylated N-glycans. A comparative glycomics analysis of the male genital tract fluids revealed that there is a gradient of glycomic complexity from the cauda to caput regions of the epididymis, varying from high mannose to sialylated complex type N-glycans but mostly devoid of fucosylation. The seminal vesicle fluid glycome, on the other hand, carries equally abundant multimeric Lewis X structures but is distinctively lacking in additional fucosylation of the terminal galactose to give the Lewis Y epitope typifying the glycome of female ULF. One-dimensional shotgun proteomics analysis identified over 40 proteins in the latter, many of which are reported for the first time, and a majority are notably involved in immune defense and antigen processing. Further sperm binding and motility assays suggest that the Lewis X/Y epitopes do contribute to the sperm motility-enhancing activity of 24p3, whereas lactotransferrin is largely inactive in this context despite being similarly glycosylated. These findings underline the importance of glycoproteomics in delineating both the specific glycan structures and their carriers in assigning glycobiological functions.     

N-glycan structures of pigeon IgG: a major serum glycoprotein containing Galalpha1-4 Gal termini.

We had shown previously that all major glycoproteins of pigeon egg white contain Galalpha1-4Gal epitopes (Suzuki, N., Khoo, K. H., Chen, H. C., Johnson, J. R., and Lee, Y. C. (2001) J. Biol. Chem. 276, 23221-23229). We now report that Galalpha1-4Gal-bearing glycoproteins are also present in pigeon serum, lymphocytes, and liver, as probed by Western blot with Griffonia simplicifolia-I lectin (specific for terminal alpha-Gal) and anti-P1 (specific for Galalpha1-4Galbeta1-4GlcNAcbeta1-) monoclonal antibody. One of the major glycoproteins from pigeon plasma was identified as IgG (also known as IgY), which has Galalpha1-4Gal in its heavy chains. High pressure liquid chromatography, mass spectrometric (MS), and MS/MS analyses revealed that N-glycans of pigeon serum IgG included (i) high mannose-type (33.3%), (ii) disialylated biantennary complex-type (19.2%), and (iii) alpha-galactosylated complex-type N-glycans (47.5%). Bi- and tri-antennary oligosaccharides with bisecting GlcNAc and alpha1-6 Fuc on the Asn-linked GlcNAc were abundant among N-glycans possessing terminal Galalpha1-4Gal sequences. Moreover, MS/MS analysis identified Galalpha1-4Galbeta1-4Galbeta1-4GlcNAc branch terminals, which are not found in pigeon egg white glycoproteins. An additional interesting aspect is that about two-thirds of high mannose-type N-glycans from pigeon IgG were monoglucosylated. Comparison of the N-glycan structures with chicken and quail IgG indicated that the presence of high mannose-type oligosaccharides may be a characteristic of these avian IgG.     

Histochemical study of glycoconjugates in the toadfish Halobatrachus didactylus oesophagus epithelium

The carbohydrate expression in the epithelium lining the oesophagus of the toadfish Halobatrachus didactylus was studied by means of conventional and lectin histochemistry. The stratified epithelium was constituted by basal cells, polymorphous cells in the intermediate layer, pyramidal and flattened cells in the outer layer and contained two types of large secretory cells: goblet cells and sacciform cells. PAS, Alcian blue pH 2.5 and pH 1.0 stained very strongly the goblet cells, weakly the surface of the other epithelial cells but did not stain the sacciform cells. The goblet cells cytoplasm contained oligosaccharides with terminal Galbeta1,3GalNAc, alpha/betaGalNAc, Galbeta1,4GlcNAc, alphaL-Fuc and internal betaGlcNAc residues (PNA, SBA, RCA120, UEA I, LTA and KOH-sialidase-WGA affinity). Galbeta1,4GlcNAc, alphaL-Fuc and internal betaGlcNAc were also found in the glycocalyx. The sacciform cells expressed sialyloligosaccharides terminating with Neu5Acalpha2,3Galbeta1,4GlcNac, Neu5Acbeta2,6Gal/GalNAc, Neu5AcForssman pentasaccharide (MAL II, SNA, KOH-sialidase-DBA staining) as well as asialo-glycoconjugates with terminal/internal alphaMan (Con A affinity) and with terminal Galbeta1,3GalNAc, Forssman pentasaccharide, Galbeta1,4GlcNAc, GalNAc (HPA and SBA reactivity), alphaGal (GSA I-B4 reactivity), D-GlcNAc (GSA II labelling), alphaL-Fuc. The basal cells cytoplasm exhibited terminal/internal alphaMan and terminal Neu5Acalpha2,6Gal/GalNAc, Galbeta1,4GlcNAc, alpha/betaGalNAc, alphaGal, GlcNAc, alphaL-Fuc. Intermediate cells showed oligosaccharides with terminal/internal alphaMan and/or terminating with Neu5Acalpha2,6Gal/GalNAc, Galbeta1,4GlcNAc in the cytoplasm and with Neu5Acalpha2,3Galbeta1,4GlcNac, alpha/betaGalNAc, alphaGal, GlcNAc, alphaL-Fuc in the glycocalyx. The pyramidal cells expressed terminal/internal alphaMan and terminal Neu5Acalpha2,6Gal/GalNAc, alpha/betaGalbeta1,4NAc, alphaGal, alphaL-Fuc in the entire cytoplasm, terminal Neu5Acalpha2,3Galbeta1,4GlcNac and Forssman pentasaccharide in the apical extension, internal betaGlcNAc and/or terminal alphaL-Fuc in the luminal surface, Neu5Acalpha2,3Galbeta1,4GlcNac, Neu5Acalpha2,6Gal/GalNAc, Galbeta1,4GlcNAc, alphaGal in the basolateral surface. The flattened cells displayed glycans with terminal/internal alphaMan and terminal Neu5Acalpha2,6Gal/GalNAc, alpha/betaGalNAc, alphaGal, D-GlcNAc in the entire cytoplasm, glycans terminating with Galbeta1,3GalNAc and/or internal betaGlcNAc in the sub-nuclear cytoplasm.     

MS-based glycomic strategies for probing the structural details of polylactosaminoglycan chain on N-glycans and glycoproteomic identification of its protein carriers

Most MS-based glycomic and glycoproteomic analyses focus on identifying changes in terminal glyco-epitopes represented by sialylation and fucosylation at specific positions of the terminal N-acetyllactosamine units. Much less attention was accorded to the underlying linear or branched poly-N-acetyllactosamine extension from the N-glycan trimannosyl core other than a simple inference of its presence due to mass data and hence glycosyl compositional assignment. Using the EA.hy926 cell line derived from human umbilical vein endothelial cells (HUVEC), we have systematically investigated the MALDI- and ESI-MS-based methodologies for probing the structural details of endothelial polylactosaminoglycans at both MS and MS(2) levels in conjunction with the use of endo-β-galactosidase to identify branching motifs and initiation sites. We showed that the polylactosaminoglycan chains on the N-glycans of EA.hy926 were less sialylated and fucosylated but more extended and branched than those of human umbilical vein endothelial cells, thus demonstrating a fundamental glycomic difference. For EA.hy926 that was investigated in more details, its polylactosaminoglycan chains were shown to be not restricted to extending from a specific antenna including the biologically important 6-arm position. Finally, experimental conditions for glycopeptide enrichment by tomato lectin were further optimized, which led to identification of over 40 candidate endothelial membrane protein carriers of polylactosaminoglycans by proteomic analysis.     

Quantitative analysis of total serum glycome in human and mouse

Model mice are frequently used in drug discovery research. Knowledge of similarities and differences between the mouse and human glycomes is critical when model mice are used for the discovery of glycan‐related biomarkers and targets for therapeutic intervention. Since few comparative glycomic studies between human and mouse have been conducted, we performed a comprehensive comparison of the major classes of glycans in human and mouse sera using mass spectrometric and liquid chromatographic analyses. Up to 131 serum glycans, including N‐glycans, free oligosaccharides (fOSs), glycosaminoglycans, O‐glycans, and glycosphingolipid (GSL)‐glycans, were quantified. In both serum samples, N‐glycans were the most abundant in the total serum glycome, while fOSs were the least abundant. As expected, the diversity of sialic acid (i.e. Neu5Ac vs. Neu5Gc) was the major species difference between human and mouse in terms of N‐ and O‐glycosylation, while GSL‐glycomic profiles were completely different, even when the sialic acid diversity was taken into consideration. Furthermore, total serum glycomics of STAM mouse were unveiled as an initial step to identify novel biomarkers of liver diseases, with which we could identify several glycans with expression significantly increased or decreased expression.