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Minor acidic glycans, such as sulfated and phosphorylated glycans, constitute only a small fraction of biological glycome, making their analysis a considerable challenge. In this study, we developed a technique to analyze minor acidic O -glycans in biological samples. First, efficient reaction conditions for the release of O -glycans from the proteins were determined. Next, a high-throughput method was established for the recovery of minor acidic glycans using NH 2 spin columns. The performance of the established method was evaluated using mucin samples, and sulfated O -glycans were successfully detected in bovine submaxillary gland mucin and porcine stomach mucin. We also analyzed the minor acidic O -glycans in cultured cancer cells. In addition to trifucosylated sulfated O -glycans and disulfated O -glycans, sulfated O -glycans with KDN were detected in LS174T cells. The relative amount of sulfated glycans in LS174T cells was almost 10-fold higher than that in the other cells. Moreover, a large polylactosamine-type sulfated O -glycan with a molecular weight >3500 was detected in MKN45 cells. Interestingly, phosphorylated ribose, possibly bound to serine/threonine, was observed in all the cells used in this study. Thus, our established analytical method allows for the analysis of minor acidic O -glycans that cannot be detected using existing glycomics methods.

We recently reported increased levels of urinary free-glycans in some cancer patients. Here, we focused on cancer related alterations in the levels of high molecular weight free-glycans. The rationale for this study was that branching, elongation, fucosylation and sialylation, which lead to increases in the molecular weight of glycans, are known to be up-regulated in cancer. Urine samples from patients with gastric cancer, pancreatic cancer, cholangiocarcinoma and colorectal cancer and normal controls were analyzed. The extracted free-glycans were fluorescently labeled with 2-aminopyridine and analyzed by multi-step liquid chromatography. Comparison of the glycan profiles revealed increased levels of glycans in some cancer patients. Structural analysis of the glycans was carried out by performing chromatography and mass spectrometry together with enzymatic or chemical treatments. To compare glycan levels between samples with high sensitivity and selectivity, simultaneous measurements by reversed-phase liquid chromatography-selected ion monitoring of mass spectrometry were also performed. As a result, three lactose-core glycans and 78 free-N-glycans (one phosphorylated oligomannose-type, four sialylated hybrid-type and 73 bi-, tri- and tetra-antennary complex-type structures) were identified. Among them, glycans with α1,3-fucosylation ((+/- sialyl) Lewis X), triply α2,6-sialylated tri-antennary structures and/or a (Man3)GlcNAc1-core displayed elevated levels in cancer patients. However, simple α2,3-sialylation and α1,6-core-fucosylation did not appear to contribute to the observed increase in the level of glycans. Interestingly, one tri-antennary free-N-glycan that showed remarkable elevation in some cancer patients contained a unique Glcβ1-4GlcNAc-core instead of the common GlcNAc2-core at the reducing end. This study provides further insights into free-glycans as potential tumor markers and their processing pathways in cancer., Competing Interests: The authors have declared that no competing interests exist.

We previously reported the precise structure of acidic free-glycans in human urine. In the present study, structural analysis of neutral free-glycans in urine was performed in fine detail. Urine samples were collected from 21 healthy volunteers and free-glycans extracted from the creatinine-adjusted urine and then fluorescently labeled with 2-aminopyridine. Neutral glycan profiling was achieved by a combination of high-performance liquid chromatography, mass spectrometry, enzymatic digestion, and periodate cleavage. A total of 79 glycans were identified. Because the ABO-blood group antigen containing urinary neutral glycans are major components, profiling patterns were similar between individuals of the same ABO-group. The neutral glycans were composed of lactose-core (Galβ1-4Glc) glycans, type-II N-acetyllactosamine-core (GlcNAcβ1-4Glc) glycans, hexose oligomers, N-glycans and to our surprise β1-3 galactosylglucose-core (Galβ1-3Glc) glycans. Although glycans with a β1-3 galactosylglucose-core were identified as major components in urine, comprising structurally simple isomers of a lactose-core, the core structure has not previously been reported. The major β1-3 galactosylglucose-core glycans were Fucα1-2Galβ1-3(Fucα1-4)Glc, GalNAcα1-3(Fucα1-2)Galβ1-3(Fucα1-4)Glc and Galα1-3(Fucα1-2)Galβ1-3(Fucα1-4)Glc, corresponding to H-, A-, and B-blood group antigens, respectively. Three lactosamine extended β1-3 galactosylglucose-core glycans were also detected as minor components. Elucidating the biosynthesis of β1-3 galactosylglucose will be crucial for understanding the in vivo function of these glycans., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Alterations to glycans in cancer patients have been used to identify novel tumor biomarkers. Most of these studies have focused on protein glycosylation but less attention has been paid to free-glycans. Here, we analyzed acidic free-glycans in the urine of cancer patients to identify novel tumor marker candidates. Specifically, urine samples were collected from patients with gastric cancer, pancreatic cancer and cholangiocarcinoma as well as normal controls. The free-glycans were extracted from creatinine-adjusted urine and fluorescently labeled with 2-aminopyridine. Initially, we performed profiling of urinary free-glycans by high-performance liquid chromatography and mass spectrometry with enzymatic and chemical degradation. More than 100 glycans, including novel structures, were identified. The chromatographic peaks suggested some of these glycans were present at elevated levels in cancer patients. To verify cancer-associated alterations, we compared the glycan levels between cancer patients and normal controls by selected reaction monitoring. Representative structures of glycans with elevated levels in cancer patients included the following: small glycans related to sialyllactose; sialyl Lewis X; lactose- and N-acetyllactosamine (LacNAc) type-II-core glycans with LacNAc (type-I or II)-extensions and modifications of α1,3/4-fucose and/or 6-sulfate on the Glc/GlcNAc; free-N-glycans containing sialylation or β1,6-branch of 6-sulfo Lewis X; novel NeuAcα2-3Galβ1-4(+/-Fucα1-3) Xylα1-3Glc glycans. Our results provide further insight into urinary free-glycans and suggest the potential utility of these compounds as tumor markers., (© The Author(s) 2020. Published by Oxford University Press.)

Membrane proteins (such as ion channels, transporters, and receptors) and secreted proteins are essential for cellular activities. N-linked glycosylation is involved in stability and function of these proteins and occurs at Asn residues. In several organs, profiles of N-glycans have been determined by comprehensive analyses. Nevertheless, the cochlea of the mammalian inner ear, a tiny organ mediating hearing, has yet to be examined. Here, we focused on the stria vascularis, an epithelial-like tissue in the cochlea, and characterised N-glycans by liquid chromatography with mass spectrometry. This hypervascular tissue not only expresses several ion transporters and channels to control the electrochemical balance in the cochlea but also harbours different transporters and receptors that maintain structure and activity of the organ. Seventy-nine N-linked glycans were identified in the rat stria vascularis. Among these, in 55 glycans, the complete structures were determined; in the other 24 species, partial glycosidic linkage patterns and full profiles of the monosaccharide composition were identified. In the process of characterisation, several sialylated glycans were subjected sequentially to two different alkylamidation reactions; this derivatisation helped to distinguish α2,3-linkage and α2,6-linkage sialyl isomers with mass spectrometry. These data should accelerate elucidation of the molecular architecture of the cochlea.

Zebrafish is a model organism suitable for studying vertebrate development. We analyzed the N-glycan structures of zebrafish embryos and their alterations during zebrafish embryogenesis to obtain basic data for studying the roles of N-glycosylation. Multiple modes of high-performance liquid chromatography and multistage mass spectrometry were used for structural analysis of N-glycans. The N-glycans from deyolked embryos at 36 hours postfertilization, a mid-pharyngula stage, contained relatively higher amounts of complex- and hybrid-type glycans with LacNAc (Galβ1-4GlcNAc) and/or sialyl LacNAc without additional β1,4-Gal, which are commonly found in mammalian tissues, as well as abundant oligomannose-type glycans. Some of the complex- and hybrid-type glycans possessed various extended LacNAc structures, such as Galβ1-4LacNAc, LacNAc-repeat or unique (+/- dHex)-GalNAcα1-GlcNAcβ1-LacNAc. In contrast, the yolk of the embryo contains predominant oligomannose-type glycans and complex-type glycans with Galβ1-4(Siaα2-3)Galβ1-4(Fucα1-3)GlcNAc antennae. N-Glycan profiles obtained from deyolked embryos at different stages showed stage-dependent variation of complex- and hybrid-type glycans. At gastrula and early segmentation stages, complex- and hybrid-type glycans were minor components, and their antenna structures were mainly sialyl LacdiNAc (Siaα2-6GalNAcβ1-4GlcNAc). From the mid-segmentation to pharyngula stages, those with LacNAc and/or α2,6-sialyl LacNAc antenna structures increased remarkably, and those with α2,3-sialyl LacNAc antenna, core α1,6-Fuc and bisecting GlcNAc modifications increased gradually. These results suggest the presence of mechanisms for regulating the antenna structures of complex/hybrid N-glycan biosynthesis in the phylotypic stage of vertebrate development., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Unfractionated heparin (UFH) is the standard drug for the initial treatment of pulmonary embolism (PE) and deep vein thrombosis (DVT) in Japan, whereas fondaparinux is the standard drug in Europe and the United States. Here, we examine the efficacy and safety of fondaparinux in Japanese patients., Methods and Results: In 2 randomized, open-label, multicenter studies, 80 Japanese patients with acute PE or DVT received either subcutaneous fondaparinux or intravenous UFH as a non-comparative reference, in a 3:1 ratio, for 5-10 days. Concomitant warfarin therapy was continued until Day 90. Multidetector-row computed tomography-based assessment showed that 57.9% and 45.9% of the patients with acute PE and acute proximal DVT had proximal DVT and PE as a complication, respectively. There was no recurrence of symptomatic venous thromboembolism. In the fondaparinux group, the respective improvement rates at the end of the initial treatment and follow-up periods were 71.4% and 86.8% for 42 patients with PE, and 57.8% and 83.3% for 46 patients with DVT; similar results were noted in the UFH group. One patient in the fondaparinux group experienced major bleeding during the initial treatment, but no such episode in the UFH group., Conclusions: Once-daily, subcutaneous fondaparinux is as effective and safe without monitoring as adjusted-dose intravenous UFH for the initial treatment of acute PE and DVT in Japanese patients.