Glycomic analysis of sialic acid linkages in glycans derived from blood serum glycoproteins.
ABSTRACT: A number of alterations to the normal glycomic profile have been previously described for a number of diseases and disorders, thus underscoring the medical importance of studying the glycans associated with proteins present in biological samples. An important alteration in cancer progression is an increased level of alpha2,6-sialylation, which aids in increasing the metastatic potential of tumor cells. Here we report a glycomic method that selectively amidates alpha2,6-linked sialic acids, while those that are alpha2,3-linked undergo spontaneous lactonization. Following subsequent permethylation, MALDI-TOF MS analysis revealed that many sialylated glycans present on glycoproteins found in blood serum featured increased levels of alpha2,6-sialylation in breast cancer samples. On the basis of the altered ratios of alpha2,3-linked to alpha2,6-linked sialic acids, many of these glycans became diagnostically relevant when they did not act as such indicators when based on traditional glycomic profiling alone.
Project description:Galectin-1, a beta-galactoside-binding protein highly expressed in the thymus, induces apoptosis of specific thymocyte subsets and activated T cells. Galectin-1 binds to N- and O-glycans on several glycoprotein receptors, including CD7, CD43, and CD45. Here we show that galectin-1 signaling through CD45, which carries both N- and O-glycans, is regulated by CD45 isoform expression, core 2 O-glycan formation and the balance of N-glycan sialylation. Regulation of galectin-1 T cell death by O-glycans is mediated through CD45 phosphatase activity. While galectin-1 signaling in cells expressing low molecular weight isoforms of CD45 requires expression of core 2 O-glycans (high affinity ligands for galectin-1), galectin-1 signaling in cells expressing a high molecular weight isoform of CD45 does not require core 2 O-glycans, suggesting that a larger amount of core 1 O-glycans (low affinity ligands for galectin-1) is sufficient to overcome lack of core 2 O-glycans. Furthermore, regulation of galectin-1 signaling by alpha2,6-sialylation of N-glycans is not solely dependent on CD45 phosphatase activity and can be modulated by the relative expression of enzymes that attach sialic acid in an alpha2,6- or alpha2,3-linkage. Thus, N- and O-glycans modulate galectin-1 T cell death by distinct mechanisms, and different glycosylation events can render thymocytes susceptible or resistant to galectin-1.
Project description:Sialyltransferases transfer sialic acid from cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to an acceptor molecule. Trans-sialidases of parasites transfer alpha2,3-linked sialic acid from one molecule to another without the involvement of CMP-NeuAc. Here we report another type of sialylation, termed reverse sialylation, catalyzed by mammalian sialyltransferase ST3Gal-II. This enzyme synthesizes CMP-NeuAc by transferring NeuAc from the NeuAcalpha2,3Galbeta1,3GalNAcalpha unit of O-glycans, 3-sialyl globo unit of glycolipids, and sialylated macromolecules to 5'-CMP. CMP-NeuAc produced in situ is utilized by the same enzyme to sialylate other O-glycans and by other sialyltransferases such as ST6Gal-I and ST6GalNAc-I, forming alpha2,6-sialylated compounds. ST3Gal-II also catalyzed the conversion of 5'-uridine monophosphate (UMP) to UMP-NeuAc, which was found to be an inactive sialyl donor. Reverse sialylation proceeded without the need for free sialic acid, divalent metal ions, or energy. Direct sialylation with CMP-NeuAc as well as the formation of CMP-NeuAc from 5'-CMP had a wide optimum range (pH 5.2-7.2 and 4.8-6.4, respectively), whereas the entire reaction comprising in situ production of CMP-NeuAc and sialylation of acceptor had a sharp optimum at pH 5.6 (activity level 50% at pH 5.2 and 6.8, 25% at pH 4.8 and 7.2). Several properties distinguish forward/conventional versus reverse sialylation: (i) sodium citrate inhibited forward sialylation but not reverse sialylation; (ii) 5'-CDP, a potent forward sialyltransferase inhibitor, did not inhibit the conversion of 5'-CMP to CMP-NeuAc; and (iii) the mucin core 2 compound 3-O-sulfoGalbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-benzyl, an efficient acceptor for ST3Gal-II, inhibited the conversion of 5'-CMP to CMP-NeuAc. A significant level of reverse sialylation activity is noted in human prostate cancer cell lines LNCaP and PC3. Overall, the study demonstrates that the sialyltransferase reaction is readily reversible in the case of ST3Gal-II and can be exploited for the enzymatic synthesis of diverse sialyl products.
Project description:Protein glycosylation mediates a wide range of cellular processes, affecting development and disease in mammals. Deciphering the "glycocodes" requires rapid, sensitive and in-depth characterization of diverse glycan structures derived from biological samples. In this study, we described a two-step derivatization strategy termed linkage-specific sialic acid permethylation (SSAP) consisting of dimethylamination and permethylation for the improved profiling of glycosylation by matrix-assisted laser desorption/ionization (MALDI) time-of-fight (TOF) mass spectrometry (MS). High linkage-specificity (?99%) of SSAP to both the two most common forms of sialic acid, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), permitted direct discrimination of ?2,3- and ?2,6-linked sialic acids in MALDI-TOF MS. The enhanced intensity (>10-fold) and increased detection limit (>10-fold) of derivatized glycans were valued for sensitive glycomics. Moreover, the good compatibility and reaction efficiency of the two steps of SSAP allowed rapid sample preparation (<2 h), benefiting robust analysis of glycans in a high-throughput manner. The SSAP strategy was further applied to investigate the protein glycosylation of human serum associated with rheumatoid arthritis (RA). It was demonstrated that the relative abundances of individual glycans were different in RA negative and RA positive samples, and meanwhile the RA patient/control ratios of both ?2,3- and ?2,6-sialylated glycans tended to elevate accompanied with the increase of sialylation. Those findings of the glycosylation changes occurred in human serum protein may contribute to the diagnosis of RA. Herein, SSAP derivatization combined with MALDI-TOF MS exhibits unique advantages for glycomic analysis and shows potential in glycosylation profiling of therapeutic proteins and clinical glycan biomarker discovery.
Project description:We previously showed that the expression of (Gal alpha 1-4Gal)-bearing glycoproteins among birds is related to their phylogeny. However, precise structures of (Gal alpha 1-4Gal)-containing N-glycans were only known for pigeon egg white glycoproteins and IgG. To compare structural features of (Gal alpha 1-4Gal)-containing N-glycans from other species, we analyzed N-glycans of gull egg white (GEW)-glycoproteins, ovomucoid, and ovotransferrin, and gull egg yolk IgG by HPLC, mass spectrometry (MS), and MS/MS analyses. GEW-glycoproteins included neutral, monosialyl, and disialyl N-glycans, and some of them contained Gal alpha 1-4Gal sequences. Bi-, tri-, and tetra-antennary oligosaccharides that lacked bisecting GlcNAc were the major core structures, and incomplete alpha-galactosylation and sialylation as well as the presence of diLacNAc on the branches generated microheterogeneity of the N-glycan structures. Moreover, unlike pigeon egg white glycoproteins, the major sialylation in GEW-glycoproteins is alpha2,3-, but not alpha2,6-linked sialic acids (NeuAc). In addition to the complex-type oligosaccharide, hybrid-type oligosaccharides that lack bisecting GlcNAc were also abundant in GEW-glycoproteins. Gull egg yolk IgG also contained Gal alpha 1-4Gal beta 1-4GlcNAc beta 1- sequences, but unlike pigeon IgG, no Gal alpha 1-4Gal beta 1-4Gal beta 1-4GlcNAc beta 1- sequence was detected. Bi- and tri-antennary complex-type oligosaccharides with bisecting GlcNAc and with core fucosylation as well as high-mannose-type oligosaccharides were the major structures in gull IgG. Our data indicated that some N-glycans from both GEW-glycoproteins and gull IgG contain the Gal alpha 1-4Gal beta 1-4GlcNAc beta 1- sequence, but the ratio of alpha-Gal-capped residues to non-alpha-Gal-capped residues in the nonreducing termini of N-glycans is much lower than that in those of pigeon glycoproteins.
Project description:Influenza virus entry is mediated by the receptor binding domain (RBD) of its spike, the hemagglutinin (HA). Adaptation of avian viruses to humans is associated with HA specificity for alpha2,6- rather than alpha2,3-linked sialic acid (SA) receptors. Here, we define mutations in influenza A subtype H5N1 (avian) HA that alter its specificity for SA either by decreasing alpha2,3- or increasing alpha2,6-SA recognition. RBD mutants were used to develop vaccines and monoclonal antibodies that neutralized new variants. Structure-based modification of HA specificity can guide the development of preemptive vaccines and therapeutic monoclonal antibodies that can be evaluated before the emergence of human-adapted H5N1 strains.
Project description:BACKGROUND:The aim of the present study was to determine whether stage of invasiveness of bladder cancer cell lines contributes to alterations in glycan pattern of their cadherins. RESULTS:Human non-malignant epithelial cell of ureter HCV29, v-raf transfected HCV29 line (BC3726) and transitional cell cancers of urine bladder Hu456 and T24 were grown in cell culture. Equal amounts of protein from each cell extracts were separated by SDS-PAGE electrophoresis and were blotted on an Immobilon P membrane. Cadherins were immunodetected using anti-pan cadherin mAb and lectin blotting assays were performed, in parallel. N-oligosaccharides were analysed by specific reaction with Galanthus nivalis agglutinin (GNA), Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Datura stramonium agglutinin (DSA), Aleuria aurantia agglutinin (AAA), Phaseolus vulgaris agglutinin (PHA-L) and wheat germ agglutinin (WGA). The cadherin from HCV29 cell line possessed bi- and/or 2,4-branched triantennary complex type glycans, some of which were alpha2,6-sialylated. The cadherin from BC3726 cell line exhibited exclusively high mannose type glycans. Cadherins from Hu456 and T24 cell lines expressed high mannose type glycans as well as beta1,6-branched oligosaccharides with poly-N-acetyllactosamine structures and alpha2,3-linked sialic acid residues. Additionally, the presence of fucose and alpha2,6-sialic acid residues on the cadherin from T24 cell line was detected. CONCLUSIONS:These results indicate that N-glycosylation pattern of cadherin from bladder cancer cell line undergoes modification during carcinogenesis.
Project description:A convenient chemoenzymatic strategy for synthesizing sialosides containing a C5-diversified sialic acid was developed. The alpha2,3- and alpha2,6-linked sialosides containing a 5-azido neuraminic acid synthesized by a highly efficient one-pot three-enzyme approach were converted to C5''-amino sialosides, which were used as common intermediates for chemical parallel synthesis to quickly generate a series of sialosides containing various sialic acid forms.
Project description:We previously described a transgenic insect cell line, Sfbeta4GalT/ST6, that expresses mammalian beta-1,4-galactosyltransferase and alpha2,6-sialyltransferase genes and produces glycoproteins with terminally sialylated N-glycans. The ability of these cells to produce sialylated N-glycans was surprising because insect cells contain only small amounts of sialic acid and no detectable CMP-sialic acid. Thus, it was of interest to investigate potential sources of sialic acids for sialoglycoprotein synthesis by these cells. We found that Sfbeta4GalT/ST6 cells can produce sialylated N-glycans when cultured in the presence but not in the absence of fetal bovine serum. The serum component(s) supporting N-glycan sialylation by Sfbeta4GalT/ST6 cells is relatively large-it was not removed by dialysis in a 50,000-molecular-weight cutoff membrane. Serum-free media supplemented with purified fetuin but not asialofetuin supported N-glycan sialylation by Sfbeta4GalT/ST6 cells. The terminally sialylated N-glycans isolated from fetuin also supported glycoprotein sialylation by Sfbeta4GalT/ST6 cells. Finally, serum-free medium supplemented with N-acetylneuraminic acid or N-acetylmannosamine supported glycoprotein sialylation by Sfbeta4GalT/ST6 cells but to a much lower degree than serum or fetuin. These results provide the first evidence of a sialic acid salvaging pathway in insect cells, which begins to explain how Sfbeta4GalT/ST6 and other transgenic insect cell lines can sialylate recombinant glycoproteins in the absence of a more obvious source of CMP-sialic acid.
Project description:Mass spectrometric glycomics was used as an innovative approach to identify biomarkers in serum and dialysate samples from peritoneal dialysis (PD) patients. PD is a life-saving treatment worldwide applied in more than 100,000 patients suffering from chronic kidney disease. PD treatment uses the peritoneum as a natural membrane to exchange waste products from blood to a glucose-based solution. Daily exposure of the peritoneal membrane to these solutions may cause complications such as peritonitis, fibrosis and inflammation which, in the long term, lead to the failure of the treatment. It has been shown in the last years that protein N-glycosylation is related to inflammatory and fibrotic processes. Here, by using a recently developed MALDI-TOF-MS method with linkage-specific sialic acid derivatisation, we showed that alpha2,6-sialylation, especially in triantennary N-glycans from peritoneal effluents, is associated with critical clinical outcomes in a prospective cohort of 94 PD patients. Moreover, we found an association between the levels of presumably immunoglobulin-G-related glycans as well as galactosylation of diantennary glycans with PD-related complications such as peritonitis and loss of peritoneal mesothelial cell mass. The observed glycomic changes point to changes in protein abundance and protein-specific glycosylation, representing candidate functional biomarkers of PD and associated complications.
Project description:Gastric carcinoma MKN45 cells stably transfected with the full-length ST3GAL4 gene were characterised by glycomic and sialoproteomic analysis. Complementary strategies were applied to assess the glycomic alterations induced by ST3GAL4 overexpression. The N- and O-glycome data were generated in two parallel structural analyzes, based on PGC-ESI-MS/MS. Data on glycan structure identification and relative abundance in ST3GAL4 overexpressing cells and respective mock control are presented. The sialoproteomic analysis based on titanium-dioxide enrichment of sialopeptides with subsequent LC-MS/MS identification was performed. This analysis identified 47 proteins with significantly increased sialylation. The data in this article is associated with the research article published in Biochim Biophys Acta "Glycomic analysis of gastric carcinoma cells discloses glycans as modulators of RON receptor tyrosine kinase activation in cancer" .