Glycobiology最新文献

Coley's Toxin comprised a mixture of cell-free, heat-treated culture media from Streptococcus pyogenes (originally Streptococus erysipelatos) and Serratia marcescens (originally Bacillus prodigiosus). A 250 kDa tumor hemorrhage-inducing polysaccharide "PS1" is reported here secreted into culture medium by S. marcescens. Four h after PS1 is injected at 32 μg/kg (10pM) into the tail vein of Balb/C mice bearing C26 subcutaneous colon-derived tumors, tumor-specific capillary hemorrhage is exhibited in 90% of tumors. As a positive control, CM101, a similar tumor hemorrhagic polysaccharide from Streptococcus agalactica caused tumor hemorrhage in 75% of tumors in the Balb/C-C26 model at 7.5 μg/kg(2.5pM). CM101 has previously been safety tested in a Phase I clinical trial. These two polysaccharides have merit to be identified as the active principal ingredients (API's) of Coley'sToxin. Additional approaches to cancer therapy are a global need. No matter the level of wealth of victims, some cancers are still incurable. Recall in recent years the tragic early cancer deaths of Steve Jobs and Paul Allen among other luminaries. Streptococcal and Serratia bacterial extracts have unique tumor specific capillary destructive activity, with observations originating with sarcomas cured by nosocomial  erysipelas  infections in the 1860's. The active pharmaceutical ingredients (API's) in these extracts and Coley's Toxins are proposed to be polysaccharides.

N-glycosylation is a critical quality attribute to consider when expressing recombinant glycoproteins in eukaryotic cells including plant cells. N-acetylglucosaminyltransferase I (GnTI) initiates complex N-glycan maturation in the Golgi apparatus by transferring a single N-acetylglucosamine (GlcNAc) residue on the alpha1,3-arm of a Man5 N-glycan acceptor. This step is required for the processing of high mannose into hybrid and complex N-glycans. Therefore, Arabidopsis mutants lacking GnTI activity display accumulation of Man5 N-glycans instead of complex N-glycans and do not synthesise N-glycans containing core alpha1,3-fucose residue. In contrast, GnTI knockout cell line of Nicotiana tabacum BY-2 still displays a little core alpha1,3-fucose signal on western blotting. Here, we show that N. tabacum contains two alpha1,3-fucosyltransferase types, one of which is able to transfer a core alpha1,3-fucose on a Man5 substrate when no Man5Gn substrate is available such as in BY-2 GnTI knock-out cell lines.

The ephrin family of membrane proteins mediate intracellular signalling as ligands of transmembrane Eph tyrosine kinase receptors during cell-cell interactions. Ephrin/Eph signalling regulates processes like cell migration and angiogenesis and is of particular importance during embryonic development. Ephrins-A3 and -B3 can also bind to cell surface-associated and soluble heparan sulfate proteoglycans (HSPGs) that also play important roles during early development. Here we show that ephrins-B1, -B2, and -B3 all can bind in cis to cell surface HSPGs, while only ephrin-B1 interacts with cell surface HSPGs in a way that retards HSPG endocytosis. Expressing ephrin-B1 in HEK293T cells, using polyethyleneimine (PEI) as transfection agent, increased cell surface levels of HSPGs which were detected by an anti-heparan sulfate (HS) antibody or by ephrin-B3-Fc binding. Ephrin-B1 in the plasma membrane seemed to retard PEI-induced HSPG internalisation and degradation. Binding of HSPGs by ephrin-B1 was observed for the human, mouse, xenopus, and zebrafish homologs, and did not require the cytoplasmic tail of ephrin-B1 that contains tyrosines shown to be involved in intracellular signalling. Furthermore, ephrin-B1 could bind the HSPG variant of CD44 (CD44V3-10), a complex that could further associate with fibroblast growth factor receptors (1 and 4) after co-expression with one of these receptors. In summary, our data indicate that ephrin-B1 can regulate cellular HSPG turnover and is able to form complexes of potential biological importance with CD44V3-10 and fibroblast growth factor receptors.

Mucin-type O-linked glycosylation is initiated by the transfer of a single N-acetyl-D-galactosamine (GalNAc) to the hydroxyl group of either a serine (Ser) or threonine (Thr) residue. This process is catalysed by a portfolio of twenty isoenzymes, the UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts, GalNAc-Ts or GALNTs) to create the Thomsen nouvelle (Tn) antigen (GalNAcα1-O-Ser/Thr ). In healthy adult cells, Tn antigen is further elaborated by the action of specific glycosyltransferases to either form one of eight core structures, which themselves can be extended to form more complex glycans, or into sialyl Tn or sialyl core 1 (sialyl T), where sialylation terminates chain extension. These O-glycans, produced through mucin-type O-linked glycosylation, are a feature of many secreted and membrane-bound proteins, and are fundamental in a wide range of biological functions. Dysregulation of this process, often resulting in the exposure of usually cryptic truncated O-glycans including Tn antigen, is important in a wide range of pathologies and has been implicated in cancer metastasis. The regulation of mucin-type O-linked glycosylation, in health and disease, is highly complex and not fully understood. It is determined by a myriad of mechanisms, from transcriptional control, mutation, posttranslational control, stability of transferases, their relocation within the secretory pathway, and changes in the fundamental structure and environment of the Golgi apparatus. This review presents an overview of the evidence for these potential regulatory steps in the synthesis of truncated mucin-type O-linked glycans in cancer.

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.