Carbohydrate binding module recognition of xyloglucan defined by polar contacts with branching xyloses and CH-Π interactions.

Engineering of novel carbohydrate-binding proteins that can be utilized in various biochemical and biotechnical applications would benefit from a deeper understanding of the biochemical interactions that determine protein-carbohydrate specificity. In an effort to understand further the basis for specificity we present the crystal structure of the multi-specific carbohydrate-binding module (CBM) X-2 L110F bound to a branched oligomer of xyloglucan (XXXG). X-2 L110F is an engineered CBM that can recognize xyloglucan, xylans and ß-glucans. The structural observations of the present study compared with previously reported structures of X-2 L110F in complex with linear oligomers, show that the π-surface of a phenylalanine, F110, allows for interactions with hydrogen atoms on both linear (xylopentaose and cellopentaose) and branched ligands (XXXG). Furthermore, X-2 L110F is shown to have a relatively flexible binding cleft, as illustrated in binding to XXXG. This branched ligand requires a set of reorientations of protein side chains Q72, N31, and R142, although these residues have previously been determined as important for binding to xylose oligomers by mediating polar contacts. The loss of these polar contacts is compensated for in binding to XXXG by polar interactions mediated by other protein residues, T74, R115, and Y149, which interact mainly with the branching xyloses of the xyloglucan oligomer. Taken together, the present study illustrates in structural detail how CH-π interactions can influence binding specificity and that flexibility is a key feature for the multi-specificity displayed by X-2 L110F, allowing for the accommodation of branched ligands.

α2,6 sialylation associated with increased beta 1,6-branched N-oligosaccharides influences cellular adhesion and invasion.

Expression of ß1,6-branched N-linked oligosaccharides have a definite association with invasion and metastasis of cancer cells. However, the mechanism by which these oligosaccharides regulate these processes is not well understood. Invasive variants of B16 murine melanoma, B16F10 (parent) and B16BL6 (highly invasive variant) cell lines have been used for these studies. We demonstrate that substitution of α2,6-linked sialic acids on multiantennary structures formed as a result of ß1,6-branching modulate cellular adhesion on both extracellular matrix (ECM) and basement membrane (BM) components. Removal of α2,6 sialic acids either by enzymatic desialylation or by stably down-regulating the ST6Gal-I (enzyme that catalyses the addition of α2,6-linked sialic acids on N-linked oligosaccharides) by lentiviral driven shRNA decreased the adhesion on both ECM and BM components and invasion through reconstituted BM matrigel.

Binding of N-acetylglucosamine (GlcNAc) ß1-6-branched oligosaccharide acceptors to ß4-galactosyltransferase I reveals a new ligand binding mode.

N-acetyllactosamine is the most prevalent disaccharide moiety in the glycans on the surface of mammalian cells and often found as repeat units in the linear and branched polylactosamines, known as i- and I-antigen, respectively. The ß1-4-galactosyltransferase-I (ß4Gal-T1) enzyme is responsible for the synthesis of the N-acetyllactosamine moiety. To understand its oligosaccharide acceptor specificity, we have previously investigated the binding of tri- and pentasaccharides of N-glycan with a GlcNAc at their nonreducing end and found that the extended sugar moiety in these acceptor substrates binds to the crevice present at the acceptor substrate binding site of the ß4Gal-T1 molecule. Here we report seven crystal structures of ß4Gal-T1 in complex with an oligosaccharide acceptor with a nonreducing end GlcNAc that has a ß1-6-glycosidic link and that are analogous to either N-glycan or i/I-antigen. In the crystal structure of the complex of ß4Gal-T1 with I-antigen analog pentasaccharide, the ß1-6-branched GlcNAc moiety is bound to the sugar acceptor binding site of the ß4Gal-T1 molecule in a way similar to the crystal structures described previously; however, the extended linear tetrasaccharide moiety does not interact with the previously found extended sugar binding site on the ß4Gal-T1 molecule. Instead, it interacts with the different hydrophobic surface of the protein molecule formed by the residues Tyr-276, Trp-310, and Phe-356. Results from the present and previous studies suggest that ß4Gal-T1 molecule has two different oligosaccharide binding regions for the binding of the extended oligosaccharide moiety of the acceptor substrate.

Human prostate cancer in a clinically relevant xenograft mouse model: identification of ß(1,6)-branched oligosaccharides as a marker of tumor progression.

PURPOSE: To establish xenograft mouse models of metastatic and nonmetastatic human prostate cancer and to apply these models to the search for aberrant glycosylation patterns associated with tumor progression in vivo and in patients. EXPERIMENTAL DESIGN: Prostate cancer cells (LNCaP, PC-3, LuCaP 23.1, and DU-145) were xenografted subcutaneously into immunodeficient pfp(-/-)/rag2(-/-) mice. Tumor growth and metastasis formation were quantified and as altered glycosylation patterns have been associated with metastasis formation in several other malignancies, prostate cancer cells were profiled by a quantitative real-time PCR (qRT-PCR) glycosylation array and compared with normal human prostate cells. The activity of upregulated glycosyltransferases was analyzed by their sugar residues end products using lectin histochemistry on primary tumors and metastases in the animal experiments and on 2,085 clinical samples. RESULTS: PC-3 cells produced the largest number of spontaneous lung metastases, followed by LNCaP and LuCaP 23.1, whereas DU-145 was nonmetastatic. qRT-PCR revealed an upregulation of ß1,6-N-acetylglucosaminyltransferase-5b (Mgat5b) in all prostate cancer cell lines. Mgat5b products [ß(1,6)-branched oligosaccharides] were predominantly detectable in metastatic xenografts as shown by increased binding of Phaseolus vulgaris leukoagglutinin (PHA-L). The percentage of prostate cancer patients who were PHA-L positive was 86.5. PHA-L intensity correlated with serum prostate-specific antigen and a cytoplasmic staining negatively affected disease-free survival. CONCLUSION: We show a novel xenograft mouse model for human prostate cancer respecting the complete metastatic cascade. Specific glycosylation patterns reveal Mgat5b products as relevant markers of both metastatic competence in mice and disease-free survival in patients. This is the first description of Mgat5b in prostate cancer indicating a significant biologic importance of ß(1,6)-branched oligosaccharides for prostate cancer progression.

Changes in oligosaccharide chains of autoantibodies to GRP78 expressed during progression of malignant melanoma stimulate melanoma cell growth and survival.

A correlation between expression of the glucose-regulated protein of 78 kDa (GRP78) in malignant melanoma tumors and poor patient survival is well established. In this study, in addition to demonstrating the expression of GRP78 in tumor tissue, we investigated the immune response against GRP78 in a group of patients with different progression stages of malignant melanoma. Furthermore, we analyzed the glycosylation status of GRP78 immunoglobulin (Ig) G autoantibodies at these stages and evaluated their capacities to affect the protein B-dependent protein kinase signaling pathway and unfolded protein response signaling mechanisms, all known to promote malignant melanoma cell proliferation and survival. We found that progression of disease correlates not only with enhanced expression of GRP78 in the tumor but also with an increase in GRP78 autoantibody serum titers in these patients. We also found that the glycosylation status of anti-GRP78 IgG changes as the disease progresses. The anti-GRP78 IgG is abnormally glycosylated in the Fc region and asymmetrically glycosylated in the Fab region. We demonstrate that hyperglycosylated anti-GRP78 IgGs stimulate cell proliferation through protein B-dependent protein kinase signaling pathways. They also mimic the effects of α2-macroglobulin on the upregulation of GRP78 and X-box binding protein 1, activating transcription factor 6 α, and serine/threonine-protein kinase/endoribonuclease precursor α as endoplasmic reticulum stress biomarkers and show no effect on expression or activation of caspases 3, 9, or 12. In conclusion, the anti-GRP78 IgG autoantibodies downregulate apoptosis and activate unfolded protein response mechanisms, which are essential to promote melanoma cell growth and survival.

Glycans in melanoma screening. Part 1. The role of ß1,6-branched N-linked oligosaccharides in melanoma.

Melanoma, which is one of the most aggressive human tumours, originates from melanin-producing melanocytes. As no effective systemic therapy exists for advanced-stage melanoma, the best chance of recovery remains surgical removal of thin early-stage melanoma. Aberrant glycosylation is a hallmark of malignancy and a well-studied class of ß1,6-branched oligosaccharides is associated with malignant transformation of rodent and human cells, and poor prognosis in cancer patients. It is evident that increased ß1,6 branching significantly contributes to the phenotype of melanoma cells, influencing the adhesion to extracellular matrix components and motility as well as invasive and metastatic potential. Despite the considerable success in establishing the role of ß1,6-branched N-linked oligosaccharides in melanoma biology, there is virtually no progress in using these glycans as a screening tool for the early diagnosis of the disease, or a target-specific therapeutic agent.

Terminal disialylated multiantennary complex-type N-glycans carried on acutobin define the glycosylation characteristics of the Deinagkistrodon acutus venom.

Glycosylation analysis of nonmammalian sources often springs surprises and conjures up intriguing views of evolutionary adaptation. Many of the constituents of snake venoms are known to be glycosylated and yet very few were fully characterized and accorded specific functions. In the process of glycomic screening through the venoms from Asian pit vipers, a partially O-acetylated NeuAcα2-8NeuAcα2-3Galß1-4GlcNAcß1-terminal epitope was found to be the predominant glycosylation characteristic of the snake venom produced by the monotypic Deinagkistrodon acutus, with acutobin, a highly specific fibrinogenase, being identified as a primary protein carrier. Full structural definition and glycosylation site mapping were completed through advanced mass spectrometry analyses at both the glycan and glycopeptide levels in conjunction with chemical and enzymatic cleavages. Although similar occurrence of such terminal disialyl cap on the N-glycans of several mammalian glycoproteins has been implicated, most of these correspond to only minor constituents of the full glycomic heterogeneity and remain poorly characterized. In contrast, each antennae of the hybrid- and complex-type N-glycans derived from acutobin was found to be rather homogeneously disialylated. With up to eight sialic acids evenly distributed on nonextended tetraantennary core structure, these unusual N-glycans are among those of highest sialic acid density ever identified without actually carrying polysialic acid chains. It remains to be tested whether they may serve as multivalent disialyl ligands for several of the human Siglecs and thus meddle with the natural immuno-recognition systems of snakebite victims, apart from affecting the general efficacy of acutobin as anticoagulant in biomedical applications.

High glucose disrupts oligosaccharide recognition function via competitive inhibition: a potential mechanism for immune dysregulation in diabetes mellitus.

Diabetic complications include infection and cardiovascular disease. Within the immune system, host-pathogen and regulatory host-host interactions operate through binding of oligosaccharides by C-type lectin. A number of C-type lectins recognise oligosaccharides rich in mannose and fucose - sugars with similar structures to glucose. This raises the possibility that high glucose conditions in diabetes affect protein-oligosaccharide interactions via competitive inhibition. Mannose-binding lectin, soluble DC-SIGN and DC-SIGNR, and surfactant protein D, were tested for carbohydrate binding in the presence of glucose concentrations typical of diabetes, via surface plasmon resonance and affinity chromatography. Complement activation assays were performed in high glucose. DC-SIGN and DC-SIGNR expression in adipose tissues was examined via immunohistochemistry. High glucose inhibited C-type lectin binding to high-mannose glycoprotein and binding of DC-SIGN to fucosylated ligand (blood group B) was abrogated in high glucose. Complement activation via the lectin pathway was inhibited in high glucose and also in high trehalose - a nonreducing sugar with glucoside stereochemistry. DC-SIGN staining was seen on cells with DC morphology within omental and subcutaneous adipose tissues. We conclude that high glucose disrupts C-type lectin function, potentially illuminating new perspectives on susceptibility to infectious and inflammatory disease in diabetes. Mechanisms involve competitive inhibition of carbohydrate binding within sets of defined proteins, in contrast to broadly indiscriminate, irreversible glycation of proteins.

Combined transcriptome and proteome analysis of Bifidobacterium animalis subsp. lactis BB-12 grown on xylo-oligosaccharides and a model of their utilization.

Recent studies have demonstrated that xylo-oligosaccharides (XOS), which are classified as emerging prebiotics, selectively enhance the growth of bifidobacteria in general and of Bifidobacterium animalis subsp. lactis strains in particular. To elucidate the metabolism of XOS in the well-documented and widely used probiotic strain B. animalis subsp. lactis BB-12, a combined proteomic and transcriptomic approach was applied, involving DNA microarrays, real-time quantitative PCR (qPCR), and two-dimensional difference gel electrophoresis (2D-DIGE) analyses of samples obtained from cultures grown on either XOS or glucose. The analyses show that 9 of the 10 genes that encode proteins predicted to play a role in XOS catabolism (i.e., XOS-degrading and -metabolizing enzymes, transport proteins, and a regulatory protein) were induced by XOS at the transcriptional level, and the proteins encoded by three of these (ß-d-xylosidase, sugar-binding protein, and xylose isomerase) showed higher abundance on XOS. Based on the obtained results, a model for the catabolism of XOS in BB-12 is suggested, according to which the strain utilizes an ABC (ATP-binding cassette) transport system (probably for oligosaccharides) to bind XOS on the cell surface and transport them into the cell. XOS are then degraded intracellularly through the action of xylanases and xylosidases to d-xylose, which is subsequently metabolized by the d-fructose-6-P shunt. The findings obtained in this study may have implications for the design of a synbiotic application containing BB-12 and the XOS used in the present study.

Discrimination of porcine glycogen debranching enzyme isozymes by the ratios of their 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase activities.

Glycogen debranching enzyme (GDE) is a single-chain protein containing distinct active sites that exhibit 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase activities. The ratios of these two activities in porcine liver and muscle GDEs were compared using a set of homologous fluorogenic branched dextrins. For quantifying 4-alpha-glucanotransferase activity, 6(3)-O-alpha-maltotetraosyl-PA-maltooctaose (B3/84), 6(4)-O-alpha-maltotetraosyl-PA-maltooctaose (B4/84), 6(5)-O-alpha-maltotetraosyl-PA-maltooctaose (B5/84) and 6(6)-O-alpha-maltotetraosyl-PA-maltooctaose (B6/84) were used as substrates and maltohexaose (G6) as the acceptor. The substrate for amylo-alpha-1,6-glucosidase activity was 6(3)-O-alpha-glucosyl-PA-maltotetraose (B3/41). HPLC analysis of the fluorogenic branched dextrin digests in the presence of G6 revealed that GDE 4-alpha-glucanotransferases produce the corresponding 6-O-alpha-glucosyl-PA-maltooctaose (GG8PA) and maltononaose (G9). The ratios of the 4-alpha-glucanotransferase activity to amylo-alpha-1,6-glucosidase activity, for the liver and muscle enzymes were respectively 0.240 and 0.0840 for B3/84, 0.204 and 0.0788 for B4/84, 0.145 and 0.0592 for B5/84, and 0.109 and 0.0458 for B6/84. These data clearly indicate that porcine liver and muscle GDEs are different from each other. The ratios of porcine brain GDE were 0.155, 0.131, 0.0990 and 0.0745 for B3/84, B4/84, B5/84 and B6/84, respectively. These results indicate that porcine brain GDE is also unique from liver and muscle enzymes, suggesting that it is either a third enzyme, or a mixture of 45% liver and 55% muscle GDEs.

Stabilities of immobilized beta-galactosidase of Aspergillus sp. AF for the optimal production of galactooligosaccharides from lactose.

Beta-galactosidase of Aspergillus sp. AF crude homogenate was immobilized in Ca-alginate gel beads and used for the production of galactooligosaccharides (GOS) from lactose. Optimum pH and temperature, thermal and storage stability of the enzyme activity were investigated and compared with those of the free enzyme. The study on the improvement of mechanical strength of the alginate beads was carried out through various methods, which demonstrates that the hardening process, where the alginate beads were treated with 0.225 M CaCl(2) solution after three batches to compensate the lost of calcium in the beads, provided a high mechanical stability for repeated use in large-scale production. The experiment results show that GOS yield increased with the increase of lactose concentration, and also increased with excessive addition of lactose (exceeding its solubility) at the beginning of the reaction. The immobilization of beta-galactosidase of Aspergillus sp. AF crude homogenate is cheap in processing cost and easy to carry out, and the immobilized enzyme possesses high performance for industrial application.

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.

Melanophages reside in hypermelanotic, aberrantly glycosylated tumor areas and predict improved outcome in primary cutaneous malignant melanoma.

BACKGROUND: Previously, hypermelanotic regions of cutaneous malignant melanoma (CMM) were found to contain a mixture of highly melanized melanoma cells and melanophages. Both cell types produced beta1,6-branched oligosaccharides. These sugars are used for motility by myeloid cells and cancer cells alike and are associated with poor survival in carcinomas of the breast, colon and lung. This study further investigated associations between melanophages and beta1,6-branched oligosaccharides and their potential contributions to patient outcome. METHODS: Individual archival melanomas and high-throughput melanoma tissue microarrays were stained for melanophages with azure blue/S100 and for beta1,6-branched oligosaccharides with the lectin leukocytic phytohemagglutinin (LPHA, a selective marker for beta1,6-branched oligosaccharides). RESULTS: In primary CMM, melanophages were highly enriched in hypermelanotic, LPHA-positive tumor regions and correlated with improved outcome at 10- and 20-year follow ups. While the combination of melanophages, LPHA positivity and high pigmentation indicated better outcome, a subset of LPHA-positive cells not associated with melanophages indicated worse outcome. CONCLUSION: This is the first report of an anti-tumor role for the melanophage in melanoma biology. There appeared to be two classes of beta1,6-branched oligosaccharide-producing melanoma cells with opposing effects on outcome: one that attracted melanophages (better) and another that did not (worse). The findings disclose new aspects of the immune system and aberrant glycosylation in CMM.

N-glycan alterations are associated with drug resistance in human hepatocellular carcinoma.

BACKGROUND: Correlations of disease phenotypes with glycosylation changes have been analysed intensively in the tumor biology field. Glycoforms potentially associated with carcinogenesis, tumor progression and cancer metastasis have been identified. In cancer therapy, drug resistance is a severe problem, reducing therapeutic effect of drugs and adding to patient suffering. Although multiple mechanisms likely underlie resistance of cancer cells to anticancer drugs, including overexpression of transporters, the relationship of glycans to drug resistance is not well understood. RESULTS: We established epirubicin (EPI)--and mitoxantrone (MIT)--resistant cell lines (HLE-EPI and HLE-MIT) from the human hepatocellular carcinoma cell line (HLE). HLE-EPI and HLE-MIT overexpressed transporters MDR1/ABCB1 and BCRP/ABCG2, respectively. Here we compared the glycomics of HLE-EPI and HLE-MIT cells with the parental HLE line. Core fucosylated triantennary oligosaccharides were increased in the two resistant lines. We investigated mRNA levels of glycosyltransferases synthesizing this oligosaccharide, namely, N-acetylglucosaminyltransferase (GnT)-IVa, GnT-IVb and alpha1,6-fucosyltransferase (alpha1,6-FucT), and found that alpha1,6-FucT was particularly overexpressed in HLE-MIT cells. In HLE-EPI cells, GnT-IVa expression was decreased, while GnT-IVb was increased. Both GnT-IVs were downregulated in HLE-MIT cells. HLE-MIT cells also showed decreases in fucosylated tetraantennary oligosaccharide, the product of GnT-V. GnT-V expression was decreased in both lines, but particularly so in HLE-MIT cells. Thus both N-glycan and glycosyltransferase expression was altered as cells acquired tolerance, suggesting novel mechanisms of drug resistance. CONCLUSION: N-glycan and glycosyltransferase expression in HLE-EPI and HLE-MIT were analysed and presented that glycans altered according with acquired tolerance. These results suggested novel mechanisms of drug resistance.

Active site mapping of amylo-alpha-1,6-glucosidase in porcine liver glycogen debranching enzyme using fluorogenic 6-O-alpha-glucosyl-maltooligosaccharides.

Glycogen debranching enzyme (GDE) has two enzymatic activities, 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase. Products with 6-O-alpha-glucosyl structures formed from phosphorylase limit dextrin by the 4-alpha-glucanotransferase activity are hydrolyzed to glucose by the amylo-alpha-1,6-glucosidase activity. Here, we probed the active site of amylo-alpha-1,6-glucosidase in porcine liver GDE using various 6-O-alpha-glucosyl-pyridylamino (PA)-maltooligosaccharides, with structures (Glcalpha1-4)(m)(Glcalpha1-6)Glcalpha1-4(Glcalpha1-4)(n)GlcPA (GlcPA, 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol residue). Fluorogenic dextrins were prepared from 6-O-alpha-glucosyl-alpha-, beta-, or gamma-cyclodextrin through partial acid hydrolysis, followed by fluorescent tagging of the reducing-end residues of the hydrolysates and separation by gel filtration and reversed-phase HPLC. Porcine liver GDE hydrolyzed dextrins with the structure Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glc to glucose and the corresponding PA-maltooligosaccharides, whereas other dextrins were not hydrolyzed. Thus, substrates must have two glucosyl residues sandwiching the isomaltosyl moiety to be hydrolyzed. The rate of hydrolysis increased as m increased and reached maximum at m = 4. The rates were the highest when n = 1 but did not vary much with changes in n. Of the dextrins examined, Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glcalpha1-4GlcPA (6(3)-O-alpha-glucosyl-PA-maltoheptaose) was hydrolyzed most rapidly, suggesting that it fits the best in the amylo-alpha-1,6-glucosidase active site. It is likely that the active site accommodates 6(2)-O-alpha-glucosyl-maltohexaose and that the interactions of seven glucosyl residues with the active site allow the most rapid hydrolysis of the alpha-1,6-glucosidic linkage of the isomaltosyl moiety.

A structural factor responsible for substrate recognition by Bacillus sp. GL1 xanthan lyase that acts specifically on pyruvated side chains of xanthan.

Xanthan is a bacterial heteropolysaccharide composed of pentasaccharide repeating units, i.e., a cellobiose as a backbone and a trisaccharide consisting of two mannoses and one glucuronic acid as a side chain. Nonreducing terminal mannose residues of xanthan side chains are partially pyruvated. Bacillus sp. GL1 xanthan lyase, a member of polysaccharide lyase family 8, acts specifically on pyruvated side chains of xanthan and yields pyruvated mannose through a beta-elimination reaction by using a single Tyr255 residue as base and acid catalysts. Here we show structural factors for substrate recognition by xanthan lyase through X-ray crystallographic and mutational analyses. The enzyme accommodates mannose and pyruvated mannose at the -1 subsite, although both inhibitor and dissociation constants of the two monosaccharides indicated that the affinity of pyruvated mannose for xanthan lyase is much higher than that of mannose. The high affinity of pyruvated mannose is probably due to the formation of additional hydrogen bonds between the carboxyl group of pyruvated mannose and amino acid residues of Tyr315 and Arg612. Site-directed mutagenesis of the two residues demonstrated that Arg612 is a key residue in recognizing pyruvated mannose. Arg612 is located in the protruding loop covering the substrate, suggesting that the loop functions as a lid that is responsible for the proper accommodation of the substrate at the active site.

Oligosaccharide structures of von Willebrand factor and their potential role in von Willebrand disease.

Oligosaccharides make up approximately 20% of the mass of VWF and although their structures are well established, their functional role remains unclear. Modification of the VWF oligosaccharide structures has been shown to result in increased plasma clearance of the protein. A mutation which alters cell type-specific expression of the Galgt2 glycosyltransferase gene in the RIIIS/J mouse results in an autosomal dominant partial quantitative deficiency of VWF. Increased plasma clearance of VWF has been demonstrated in some individuals with a partial quantitative deficiency of the protein and it is possible that variation in VWF glycosylation may contribute towards this. ABH antigens occur within the oligosaccharide component of VWF and may account for the variation in plasma VWF:Ag levels observed between individuals of different ABO blood groups. The structures and functional roles of the oligosaccharide side chains of VWF and possible pathogenetic mechanisms by which they may contribute towards VWD are reviewed in this article.

Molecular cloning and characterization of Arabidopsis thaliana Golgi alpha-mannosidase II, a key enzyme in the formation of complex N-glycans in plants.

N-glycosylation is one of the major post-translational modifications of proteins in eukaryotes; however, the processing reactions of oligomannosidic N-glycan precursors leading to hybrid-type and finally complex-type N-glycans are not fully understood in plants. To investigate the role of Golgi alpha-mannosidase II (GMII) in the formation of complex N-glycans in plants, we identified a putative GMII from Arabidopsis thaliana (AtGMII; EC 3.2.1.114) and characterized the enzyme at a molecular level. The putative AtGMII cDNA was cloned, and its deduced amino acid sequence revealed a typical type II membrane protein of 1173 amino acids. A soluble recombinant form of the enzyme produced in insect cells was capable of processing different physiologically relevant hybrid N-glycans. Furthermore, a detailed N-glycan analysis of two AtGMII knockout mutants revealed the predominant presence of unprocessed hybrid N-glycans. These results provide evidence that AtGMII plays a central role in the formation of complex N-glycans in plants. Furthermore, conclusive evidence was obtained that alternative routes in the conversion of hybrid N-glycans to complex N-glycans exist in plants. Transient expression of N-terminal AtGMII fragments fused to a GFP reporter molecule demonstrated that the transmembrane domain and 10 amino acids from the cytoplasmic tail are sufficient to retain a reporter molecule in the Golgi apparatus and that lumenal sequences are not involved in the retention mechanism. A GFP fusion construct containing only the transmembrane domain was predominantly retained in the ER, a result that indicates the presence of a motif promoting ER export within the last 10 amino acids of the cytoplasmic tail of AtGMII.

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.

High expression of shMDG1 gene is associated with low metastatic potential of tumor cells.

Metastasis is the primary cause of mortality associated with cancer. Molecular mechanisms leading to metastatic spread are poorly studied. To get a better understanding of this process, we compared the gene expression pattern of two isogenic cell lines, HET-SR and HET-SR1 (Rous Sarcoma Virus-transformed embryo hamster fibroblasts) with different metastatic activity using the differential display technique. A novel cDNA of hamster gene shMDG1 (Syrian hamster homologue of microvascular differentiation gene 1), which had 94% homology with rat MDG1 gene, was identified. Expression of shMDG1 was increased in low metastatic HET-SR cell line in comparison to high metastatic HET-SR1. Sequence analysis of the ORF of shMDG1 gene showed that it belongs to the DnaJ/heat-shock proteins of 40 kDa (HSP40) chaperones family, considered to function as a cochaperone of HSP70 family. In order to confirm involvement of shMDG1 in metastasis, we injected parental and shMDG1 overexpressed cells into animals. We showed that overexpression of the shMDG1 gene significantly diminished the metastatic activity of both HET-SR and HET-SR1 cells. The shMDG1-induced repression of metastasis was not connected with alterations in cell proliferation and motility in vitro, but correlated well with a decrease in content of the Asn-linked beta1-6 branched oligosaccharides on cell surface.