Glycobiology最新文献

Heparan-6-O-endosulfatase 2 (Sulf-2) is a proteoglycan enzyme that modifies sulfation of heparan sulfate proteoglycans. Dysregulation of Sulf-2 is associated with various pathological conditions, including cancer, which makes Sulf-2 a potential therapeutic target. Despite the key pathophysiological roles of Sulf-2, inhibitors remain insufficiently developed. In previous work, a fucosylated chondroitin sulfate from the sea cucumber Holothuria floridana (HfFucCS) exhibited potent Sulf-2 inhibition. This study investigates the structural basis of HfFucCS-mediated Sulf-2 inhibition, examines the binding profile of HfFucCS to Sulf-2, and explores the mode of inhibition. Additionally, a structurally diverse library of sulfated poly/oligosaccharides, including common glycosaminoglycans and unique marine sulfated glycans, was screened for Sulf-2 inhibition. Results from a high-throughput arylsulfatase assay and specific 6-O-desulfation assay have proved that HfFucCS is the most potent among the tested sulfated glycans, likely due to the presence of the unique 3,4-disulfated fucose structural motif. HfFucCS demonstrated non-competitive inhibition, and inhibitory analysis of its low-molecular-weight fragments suggests a minimum length of ~7.5 kDa for effective inhibition. Surface plasmon resonance analyses revealed that Sulf-2 binds to surface heparin with high affinity (KD of 0.817 nM). HfFucCS and its derivatives effectively disrupt this interaction. Results from mass spectrometry-hydroxyl radical protein footprinting and repulsive scaling replica exchange molecular dynamics indicate similarities in the binding of heparin and HfFucCS oligosaccharides to both the catalytic and hydrophilic domains of Sulf-2. These findings reveal the unique inhibitory properties of a structurally distinct marine glycosaminoglycan, supporting its further investigation as a selective and effective inhibitor for Sulf-2-associated cancer events.

Glycosphingolipids are a unique class of bioactive lipids responsible for lateral membrane organization and signaling found in high abundance in the central nervous system. Using nanoflow MEA Chip Q/ToF mass spectrometry, we profiled the intact glycosphingolipids of the elderly human brain in a region-specific manner. By chromatographic separation of glycan and ceramide isomers, we determined gangliosides to be the highest source of heterogeneity between regions with the expression of a- and b-series glycan structures. Investigation of these trends showed that specific glycan structures were, in part, determined by the structure of their lipid backbone. This study provides insight into the dynamic process of membrane remodeling in the brain during aging.

Calreticulin (CRT), a chaperone that possesses both lectin and chaperone domains, is localized in the endoplasmic reticulum (ER). CRT has diverse functions and localizations; thus, CRT is a multifunctional protein. Particularly in the ER, CRT mainly aids in the proper folding of nascent glycoproteins as lectin chaperones. Approximately one-third of cellular proteins, including disease-related proteins, are synthesized in the ER. The lectin chaperones CRT and calnexin facilitate the correct folding of these glycoproteins; hence, these chaperones are essential for cells. Various CRT ligands have been reported, mainly composed of Glc1Man9GlcNAc2-type glycan. However, it remains problematic for the complicated synthesis and preparation, and it interacts with glycoprotein folding-related proteins in the ER other than CRT. This suggests that the development of CRT ligands still can be improved. In this study, we developed a hybrid binding concept, which encompasses concurrent binding of ligands to CRT lectin and chaperone domains. We synthesized a CRT-targeting glycan ligand with a glycan and hydrophobic aglycone for CRT lectin and chaperone domain binding, respectively. The thermal shift assay with the CRT-targeting glycan demonstrated that binding was enhanced by simultaneous glycan and hydrophobic aglycone binding. The affinity of the CRT-targeting ligand showed isothermal titration calorimetry approximately 50-fold stronger than that of the glycan alone, thereby supporting the hybrid binding concept. In addition, the CRT-targeting ligand inhibited chaperone function. Overall, these results indicate that the hybrid binding concept may be useful as a novel strategy for the development of CRT ligands and inhibitors.

Objective: This study aimed to identify distinct IgA1 N-glycan composition in patients with ankylosing spondylitis (AS) compared with healthy controls and to explore their associations with inflammatory markers and disease activity indices.

Methods: Serum samples were collected from 36 patients with AS and 35 healthy controls. The diagnosis of AS was based on the New York criteria. Clinical assessments included inflammatory markers (ESR, CRP, and IgA) and disease activity indices (BASDAI, ASDAS-ESR, and ASDAS-CRP). IgA1 was isolated using affinity purification and gel filtration chromatography, followed by mass spectrometry to identify N-glycans.

Results: Among the 23 detected N-glycan patterns, significant differences were observed in 13 of the 18 N-glycans at the N144 site and in all five N-glycans at the N340 site between patients with AS and controls. Notably, the glycans HexNAc3Hex4NeuAc1, HexNAc4Hex4NeuAc1 and HexNAc5Hex5NeuAc1 at N144 demonstrated strong associations with all three inflammatory markers, including ESR, CRP, and IgA (P < 0.001). Levels of HexNAc4Hex4NeuAc1 were significantly elevated in patients with AS compared with those in the healthy controls. Increased sialylation and galactosylation, along with decreased fucosylation, were noted at N144 of IgA1 in patients with AS. Conversely, no glycans at N340 showed a correlation with all inflammatory markers simultaneously or with any disease activity indicators.

Conclusion: IgA1 from patients with AS exhibited distinct glycosylation traits compared with controls, with elevated levels of HexNAc₄Hex₄NeuAc₁ at N144 associated with inflammatory markers. These findings suggested that differential glycosylation patterns of IgA1 may play a role in the pathogenesis of AS.

Mucin type O-glycan core elongation is typically performed by the C1GALT1, B3GNT6, and ST6GalNAc-I/-II O-glycosyltransferases. These enzymes target the Tn antigen (GalNAc-O-Thr/Ser) dictating the fate of O-glycan elongation, playing important roles in health and disease. Changes in transferase expression and glycan structure are commonly associated with diseases such as cancer, Tn-syndrome, and ulcerative colitis. Despite their significance, their substrate specificities and their biological roles remain elusive. Here, we examine the roles of flanking glycopeptide substrate charge using a library of differently charged glycopeptides and a small library of PSGL-1 Thr57 based charged glycopeptides. We found that C1GALT1 was most influenced by flanking charge preferring negatively charged substrates, while B3GNT6 and ST6GalNAc-II were less influenced, showing unique N- and C-terminal charge preferences. Interestingly, ST6GalNAc-I was not influenced by flanking charge. These charge specificities were further maintained against the charged PSGL-1 glycopeptides, although ST6GalNAc-I showed an increased preference towards a remote N-terminal positive charge. The observed charge preferences were to a large part driven by substrate interactions with the electrostatic surface of the transferase. We propose that negative flanking charge may assist C1GALT1 in targeting key glycosites such as in PSGL-1 and podoplanin. Our findings are consistent with a Golgi hierarchy, where the cis-Golgi localized GalNAc-Ts and C1GALT1 determine the site and thus fate of glycosylation, while the trans-Golgi less-specific ST6GalNAc-I provides a final capping function. This characterization of substrate charge preference furthers our understanding of how these enzymes select their substrates and may contribute to our understanding of their biological roles.

Accumulating evidences have shown that unfolded protein response (UPR) contributes to the increased survival of tumor cells under endoplasmic reticulum (ER) stress conditions. Malectin is an ER-resident lectin that selectively traps misfolded glycoproteins in ER for degradation, and its expression is upregulated upon ER stress. However, contribution of malectin to malignant behavior of tumor has not been reported. Here, we revealed that malectin expression is aberrantly up-regulated in human hepatocellular carcinoma (HCC) tissues and HCC cell lines compared to their matched normal tissues and cell lines. Knockout of malectin in two HCC cell lines HepG2 and QGY-7703 using CRISPR-Cas9 technology had no obvious effects on cell proliferation, but significantly suppressed cell colony formation, migration and invasion. Consistently, subcutaneously implanted malectin-deficient HCC cells in nude mice also showed an obvious decrease in tumor growth. These results indicate that malectin might play an oncogenic role in HCC tumorigenesis and development.