Branching pattern of gluco-oligosaccharides and 1.5kDa dextran grafted by the α-1,2 branching sucrase GBD-CD2.

GBD-CD2, an engineered sucrose-acting enzyme of glycoside hydrolase family 70, transfers D-glucopyranosyl (D-Glcp) units from sucrose onto dextrans or gluco-oligosaccharides (GOS) through the formation of α-(1→2) linkages leading to branched products of interest for health, food and cosmetic applications. Structural characterization of the branched products obtained from sucrose and pure GOS of degree of polymerization (DP) 4 or DP 5 revealed that highly α-(1→2) branched and new molecular structures can be synthesized by GBD-CD2. The formation of α-(1→2) branching is kinetically controlled and can occur onto vicinal α-(1→6)-linked D-Glcp residues. To investigate the mode of branching of 1.5 kDa dextran, simulations of various branching scenarios and resistance to glucoamylase degradation were performed. Analysis of the simulation results suggests that the branching process is stochastic and indicates that the enzyme acceptor site can accommodate both linear and poly-branched acceptors. This opens the way to the design of novel enzyme-based processes yielding carbohydrate structures varying in size and resistance to hydrolytic enzymes.

Surface-exposed glycoproteins of hyperthermophilic Sulfolobus solfataricus P2 show a common N-glycosylation profile.

Cell surface proteins of hyperthermophilic Archaea actively participate in intercellular communication, cellular uptake, and energy conversion to sustain survival strategies in extreme habitats. Surface (S)-layer glycoproteins, the major component of the S-layers in many archaeal species and the best-characterized prokaryotic glycoproteins, were shown to have a large structural diversity in their glycan compositions. In spite of this, knowledge on glycosylation of proteins other than S-layer proteins in Archaea is quite limited. Here, the N-glycosylation pattern of cell-surface-exposed proteins of Sulfolobus solfataricus P2 were analyzed by lectin affinity purification, HPAEC-PAD, and multiple mass spectrometry-based techniques. Detailed analysis of SSO1273, one of the most abundant ABC transporters present in the cell surface fraction of S. solfataricus, revealed a novel glycan structure composed of a branched sulfated heptasaccharide, Hex4(GlcNAc)2 plus sulfoquinovose where Hex is d-mannose and d-glucose. Having one monosaccharide unit more than the glycan of the S-layer glycoprotein of S. acidocaldarius, this is the most complex archaeal glycan structure known today. SSO1273 protein is heavily glycosylated and all 20 theoretical N-X-S/T (where X is any amino acid except proline) consensus sequence sites were confirmed. Remarkably, we show that several other proteins in the surface fraction of S. solfataricus are N-glycosylated by the same sulfated oligosaccharide and we identified 56 N-glycosylation sites in this subproteome.

Glycomic analysis of alpha-fetoprotein L3 in hepatoma cell lines and hepatocellular carcinoma patients.

The N-glycan structures of the Lens culinaris agglutinin (LCA)-reactive fraction of alpha-fetoprotein (AFP-L3), a tumor marker of hepatocellular carcinomas (HCC), were analyzed in relationship to glycosyltransferases and LCA-affinity electrophoresis. Using HPLC and MALDI-TOF MS, we determined the N-glycan structures of AFP from HCC cell lines, and demonstrated they were affected by N-acetylglucosaminyltransferase III and fucosyltransferase VIII, but not by N-acetylglucosaminyltransferase V. Moreover, we identified the N-glycan structures of AFP in HCC patients.

Novel fucogangliosides found in human colon adenocarcinoma tissues by means of glycomic analysis.

The structures of acidic glycosphingolipids in colon adenocarcinoma have been analyzed extensively using a number of conventional methods, such as thin-layer chromatography and methylation analysis, and a variety of acidic glycosphingolipids present in the tissues have been reported. However, because of a number of limitations in the techniques used in previous studies in terms of resolution, quantification, and sensitivity, we employed a different method that could be applied to small amounts of tissue. In this technique, the carbohydrate moieties of acidic glycosphingolipids from approximately 20mg of colon adenocarcinoma were released by endoglycoceramidase II and were labeled by pyridylamination. They were separated and structurally characterized by a two-dimensional HPLC mapping technique, electrospray ionization tandem mass spectrometry (ESI-MS/MS), and enzymatic cleavage. A total of 22 major acidic glycosphingolipid structures were identified, and their relative quantities were revealed in detail. They are composed of 1 sulfated (SM3), 1 lacto-series (SLe(a)), 6 kinds of ganglio-series, and 14 kinds of neolacto-series glycosphingolipids. They include most of the acidic glycosphingolipids previously reported to be present in the tissues and two previously unknown fucogangliosides sharing the same terminal structure: NeuAcalpha2-6(Fucalpha1-2)Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc, and NeuAcalpha2-6(Fucalpha1-2)Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3-Galbeta1-4Glc. Thus, this highly sensitive, high-resolution analysis enabled the identification of novel structures of acidic glycosphingolipids from small amounts of already comprehensively studied cancerous tissues. This method is a powerful tool for microanalysis of glycosphingolipid structures from small quantities of cancerous tissues and should be applicable to different types of malignant tissues.

Structural diversity of cytosolic free oligosaccharides in the human hepatoma cell line, HepG2.

It is thought that free oligosaccharides in the cytosol are an outcome of quality control of glycoproteins by endoplasmic reticulum-associated degradation (ERAD). Although considerable amounts of free oligosaccharides accumulate in the cytosol, where they presumably have some function, detailed analyses of their structures have not yet been carried out. We isolated 21 oligosaccharides from the cytosolic fraction of HepG2 cells and analyzed their structures by the two-dimensional high-performance liquid chromatography (HPLC) sugar-mapping method. Sixteen novel oligosaccharides were identified in the cytosol in this study. All had a single N-acetylglucosamine at their reducing-end cores and could be expressed as (Man)n (GlcNAc)1. No free oligosaccharide with N,N'-diacetylchitobiose was detected in the cytosolic fraction of HepG2 cells. This suggested that endo-beta-N-acetylglucosaminidase was a key enzyme in the production of cytosolic free oligosaccharides. The 21 oligosaccharides were classified into three series--series 1: oligosaccharides processed from Manalpha1-2Manalpha1-6 (Manalpha1-2Manalpha1-3)Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3) Manbeta1-4GlcNAc (M9A') and Manalpha1-2Manalpha1-6(Manalpha1-3) Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc (M8A') by digestion with cytosolic alpha-mannosidase; series 2: oligosaccharides processed with Golgi alpha-mannosidases in addition to endoplasmic reticulum (ER) and cytosolic alpha-mannosidases; and series 3: glucosylated oligosaccharides produced from Glc1Man9GlcNAc1 by hydrolysis with cytosolic alpha-mannosidase. The presence of the series "2" oligosaccharides suggests that some of the misfolded glycoproteins had been processed in pre-cis-Golgi vesicles and/or the Golgi apparatus. When the cells were treated with swainsonine to inhibit cytosolic alpha-mannosidase, the amounts of M9A' and M8A' increased remarkably, suggesting that these oligosaccharides were translocated into the cytosol. Four oligosaccharides of series "2" also increased. In contrast, there were obvious reductions in Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc (M5B'), the end product from M9A' by digestion with cytosolic alpha-mannosidase, and Manalpha1-6(Manalpha1- 2Manalpha1-3)Manbeta1-4GlcNAc, derived from series "2" oligosaccharides by digestion with cytosolic alpha-mannosidase. Our data suggest that (1) some of the cytosolic oligosaccharides had been processed with Golgi alpha-mannosidases, (2) the major oligosaccharides translocated from the ER were M9A' and M8A', and (3) M5B' and Glc1M5B' were maintained at relatively high concentrations in the cytosol.

Isolation and structural analyses of positional isomers of 6(1),6(n)-di-O-(N-acetyl-beta-D-glucosaminyl)cyclomaltoheptaose (n=2, 3, and 4) and 6-O-[6-O-(N-acetyl-beta-D-glucosaminyl)-N-acetyl-beta-D-glucosaminyl]cyclomaltoheptaose.

6-O-[6-O-(N-acetyl-beta-D-glucosaminyl)-N-acetyl-beta-D-glucosaminyl]cyclomaltoheptaose (beta CD) and three positional isomers of 6(1),6(n)-di-O-(N-acetyl-beta-D-glucosaminyl)cyclomaltoheptaose (n=2, 3, and 4) in a mixture of products from beta CD and N-acetylglucosamine by the reversed reaction of beta-N-acetylhexosaminidase from jack bean were isolated and purified by HPLC. The structures of four isomers of di-N-acetylglucosaminyl-beta CDs were determined by FABMS and NMR spectroscopy. The degree of polymerization of the branched oligosaccharides produced by enzymatic degradation with bacterial saccharifying alpha-amylase (BSA) was established by LC-MS methods.