Biomedical subjects

Inka Brockhausen

Publications and source records attributed to Inka Brockhausen.

10 recordsLinked to original sources

PubMed2005

N-Acyl derivatives of glucosamine as acceptor substrates for galactosyltransferase from bone and cartilage cells.

Glucosamine is commonly used as a nutraceutical by arthritis patients. However, its mode of action is still unknown, and there is controversy about its clinical efficacy. Synthetic N-acyl glucosamines (acyl group>2 carbons) comprise a new class of drugs. We examined these derivatives for their effect in bone and cartilage cells, and for their ability to serve as acceptor substrates for galactosyltransferase. With the exception of N-benzoylglucosamine, compounds of the series were good substrates for galactosyltransferases from bone and cartilage cells, and for purified enzyme from bovine milk. When N-butyrylglucosamine (GlcNBu) was added to the cell medium of primary bovine chondrocytes and human osteoblasts, small amounts were found to enter the cells and a radiolabeled metabolite appeared in the medium. However, GlcNBu did not appear to be incorporated directly into oligosaccharides. GlcNBu at 1 and 5mM concentrations in the glucose-free cell medium of primary human osteoblasts from osteoarthritis patients did not significantly alter cell proliferation or cell differentiation.

Animals↗

PubMed2005

Identification of a UDP-Gal: GlcNAc-R galactosyltransferase activity in Escherichia coli VW187.

A novel acceptor substrate for galactosyltransferase was synthesized containing GlcNAcalpha-pyrophosphate, covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc alpha-O-PO(3)-PO(3)-(CH(2))(11)-O-Phenyl). The new substrate was used to develop an assay for a galactosyltransferase activity from Escherichia coli strain VW187 that is involved in lipopolysaccharide synthesis and has not been studied by others. We showed that Gal was transferred from UDP-Gal to the novel acceptor substrate. This was a significant improvement over our previous preliminary assays of the enzyme using endogenous substrate, and showed that these synthetic substrates are useful for assaying enzymes that utilize lipid-bound substrates in O-chain synthesis in Gram-negative bacteria.

Escherichia coli Proteins↗

PubMed2004

The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal: GlcNAc{alpha}-pyrophosphate-R {beta}1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide.

In this work, we demonstrate that the wbbD gene of the O7 lipopolysaccharide (LPS) biosynthesis cluster in Escherichia coli strain VW187 (O7:K1) encodes a galactosyltransferase involved in the synthesis of the O7-polysaccharide repeating unit. The galactosyltransferase catalyzed the transfer of Gal from UDP-Gal to the GlcNAc residue of a GlcNAc-pyrophosphate-lipid acceptor. A mutant strain with a defective wbbD gene was unable to form O7 LPS and lacked this specific galactosyltransferase activity. The normal phenotype was restored by complementing the mutant with the cloned wbbD gene. To characterize the WbbD galactosyltransferase, we used a novel acceptor substrate containing GlcNAcalpha-pyrophosphate covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc alpha-O-PO(3)-PO(3)-(CH(2))(11)-O-phenyl). The WbbD galactosyltransferase had optimal activity at pH 7 in the presence of 2.5 mM MnCl(2). Detergents in the assay did not increase glycosyl transfer. Digestion of enzyme product by highly purified bovine testicular beta-galactosidase demonstrated a beta-linkage. Cleavage of product by pyrophosphatase and phosphatase, followed by HPLC and NMR analyses, revealed a disaccharide with the structure Gal beta1-3GlcNAc. Our results conclusively demonstrate that WbbD is a UDP-Gal: GlcNAcalpha-pyrophosphate-R beta1,3-galactosyltransferase and suggest that the novel synthetic glycolipid acceptor may be generally applicable to characterize other bacterial glycosyltransferases.

Carbohydrate Sequence↗

PubMed2004

Intestinal candyfloss.

In this issue of the Biochemical Journal, Robbe et al. have employed a state-of-the-art MS technique to re-examine the sweet and sticky sugar covering of human intestinal mucosa. The availability of highly sensitive MS methods has been instrumental in determining the complex structures of mucin oligosaccharides, and in demonstrating that there are variations in structure along the sections of the gut. In contrast to previous studies, these results described by Robbe et al. show some correlation with the activities of enzymes synthesizing mucin oligosaccharides in the human colon. Many questions still remain to be answered: for example, regarding the regulation of the relative amounts of sugar chains in individual mucins, and their roles in the homoeostasis of the intestinal mucosa. Structural studies are a solid basis for understanding the functions of sugar chains, and the mechanisms and significance of changes during the development of intestinal disease.

Gastric Mucins↗

PubMed2004

Detection of glycosyltransferases in the golden hamster (Mesocricetus auratus) oviduct and evidence for the regulation of O-glycan biosynthesis during the estrous cycle.

Recently, we provided evidence that the glycosylation of hamster oviductin, a member of the mucin family of glycoproteins, is regulated during the estrous cycle. In order to further elucidate the glycosylation process of oviductal glycoproteins, we identified biosynthetic pathways involved in the assembly of mucin-type O-linked oligosaccharide (O-glycan) chains in the hamster oviduct. Our results demonstrated that the hamster oviduct has high activities of glycosyltransferases that synthesize O-glycans with core 1, 2, 3 and 4 structures as well as elongated structures. Oviduct therefore represents a typical mucin-secreting tissue. Our results also showed that specific glycosyltransferase activities are regulated during the estrous cycle. Mucin-type core 2 beta6-GlcNAc-transferase (C2GnT2) is responsible for synthesizing core 2 and core 4 structures in the oviduct. Specific assays for C2GnT2 revealed a cyclical pattern throughout the estrous cycle with high activity at the stages of proestrus and estrus and low activity at diestrus 1. Using semiquantitative RT-PCR, the mRNA levels for C2GnT2 in the estrous cycle stages could be correlated with the enzyme activities. An increase in glycosyltransferase activity in the hamster oviduct at the time of ovulation suggests that glycosylation of oviductal glycoproteins may be necessary for these proteins to exert their functions during the process of fertilization.

Animals↗

PubMed2004

The effect of TNF-alpha on glycosylation pathways in bovine synoviocytes.

Synoviocytes are fibroblastic cells that line joint cavities. These cells synthesize numerous cell-surface and extracellular-matrix glycoproteins that are required for maintenance of the joint. Joint inflammation, such as occurs in arthritis, has been shown to have major effects on synoviocyte proliferation and on the biosynthesis of glycoproteins. The structures of the carbohydrate moieties of glycoproteins, however, and the enzymes involved in their synthesis have not yet been described for synoviocytes. Therefore, to characterize the cell-surface glycoconjugates, synoviocytes were isolated from bovine ankles, and the cells were grown in primary cultures. Lectin-binding assays were used to identify exposed N- and O-glycan carbohydrate determinants on synoviocytes, and specific enzyme assays were used to identify some of the glycosyltransferases involved in the synthesis of the glycan chains. A number of the enzymes that synthesize N- and O-linked oligosaccharides were found to be active in cell-free extracts of synoviocytes, including those that synthesize core-1-based O-glycans and the more complex bi-antennary N-glycans. To understand the molecular events underlying the inflammatory response in the synovium of arthritis patients, we examined the effect of the inflammatory cytokine tumour necrosis factor alpha (TNF-alpha) on synoviocytes and on glycosylation profiles. TNF-alpha treatment, which induces apoptosis in synoviocytes, was accompanied by changes in lectin-binding patterns, indicating alterations in the expression of cell-surface oligosaccharides. Concurrently, changes in specific enzyme activities were observed in treated cells. Two enzymes potentially important to the inflammatory process, core 2 beta6-GlcNAc-transferase and beta4-Gal-transferase, increased after TNF-alpha treatment. This is the first study of glycoprotein biosynthesis in synoviocytes, and it shows that synoviocytes have a characteristic glycosylation phenotype that is altered in the presence of inflammatory cytokines.

Animals↗

PubMed2004

Sialylated core 1 based O-linked glycans enhance the growth rate of mammary carcinoma cells in MUC1 transgenic mice.

The MUC1 mucin, found on the luminal surface of simple epithelial cells is upregulated and aberrantly glycosylated in many carcinomas particularly breast and ovarian. MUC1 expressed by normal mammary epithelial cells, carries core 2 glycans but in breast carcinomas the simple core 1 based glycans are added. The binding of the monoclonal antibody SM3 to its peptide epitope in the tandem repeat of MUC1 is blocked by the branched core 2 glycans found on MUC1 expressed by normal cells. Thus SM3 does not bind to MUC1 expressed by normal mammary epithelial cells but reacts with more than 90% of breast carcinomas, suggesting that the loss of at least some core 2 glycans is a very common event in breast carcinogenesis. To determine if the change in glycosylation observed in breast carcinomas confers an advantage to cancer cells, murine mammary carcinoma cell lines were developed that express MUC1 carrying core 2 or core 1 linked glycans. The in vivo growth rate in wild-type and nude mice were identical regardless of the O-glycosylation patterns. However, the tumors that grew out of wild-type mice lost most of their MUC1 expression. In contrast, in MUC1 transgenic mice, where expression of MUC1 was retained by the tumor, a striking difference in growth rate was observed. In these mice, cells expressing core 1 glycans grew significantly faster than cells expressing core 2 glycans. These data suggest that MUC1 transgenic mice are more tolerant to core 1 expressing tumors than to tumors expressing core 2.

Animals↗

PubMed2003

Soluble human core 2 beta6-N-acetylglucosaminyltransferase C2GnT1 requires its conserved cysteine residues for full activity.

Human UDP-GlcNAc: Galbeta1-3GalNAc- (GlcNAc to GalNAc) beta1,6-GlcNAc-transferase (C2GnT1) is a member of a group of beta6-GlcNAc-transferases that belongs to CAZy family 14. One of the striking features of these beta6-GlcNAc-transferases is the occurrence of nine completely conserved cysteine residues that are located throughout the catalytic domain. We have expressed the soluble catalytic domain of human C2GnT1 in insect cells, and isolated active enzyme as a secreted protein. beta-Mercaptoethanol (beta-ME) and dithiothreitol (DTT) were found to stimulate the enzyme activity up to 20-fold, indicating a requirement for a reduced sulfhydryl for activity. When the enzyme was subjected to nonreducing PAGE, the migration of the protein was identical to the migration in reducing gels, demonstrating the absence of intermolecular disulfide bonds. This suggested that the monomer is the active form of the enzyme. Sulfhydryl reagents such as 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and N-ethylmaleimide (NEM) inactivated the enzyme, and the inactivation was partially prevented by prior addition of donor or acceptor substrate and by sulfhydryl reducing agents. We therefore investigated the role of all nine conserved cysteine residues in enzyme stability and activity by site-directed mutagenesis where individual cysteine residues were changed to serine. All of the mutants were expressed as soluble proteins. Seven of the Cys mutants were found to be inactive, while C100S and C217S mutants had 10% and 41% activity, respectively, when compared to the wild-type enzyme. Wild-type and C217S enzymes had similar K(M) and V(max) values for acceptor substrate Galbeta1-3GalNAcalpha-p-nitrophenyl (GGApnp), but the K(M) value for UDP-GlcNAc was higher for C217S than for the wild-type enzyme. In contrast to wild-type enzyme, C217S was not stimulated by reducing agents and was not inhibited by sulfhydryl specific reagents. These results suggest that Cys-217 is a free sulfhydryl in active wild-type enzyme and that Cys-217, although not required for activity, is in or near the active site of the protein. Since seven of the mutations were totally inactive, it is likely that these seven Cys residues play a role in maintaining an active conformation of soluble C2GnT1 by forming disulfide bonds. These bonds are only broken at high concentrations of disulfide reducing agents.

Amino Acid Sequence↗

PubMed2003

Glycodynamics of mucin biosynthesis in gastrointestinal tumor cells.

Glycoproteins found in the secretions and on the surfaces of cancer cells include mucins and mucin-like glycoproteins. These molecules have been shown to carry antigens that are characteristically expressed on cancer cells, including Tn and T antigens and Lewis epitopes. The structures of O-glycans are often abnormal in gastrointestinal tumors, or else are present in abnormal amounts, and these structures greatly contribute to the phenotype and biology of cancer cells. It has been shown that glycans of cancer cells have functional importance in cell adhesion, invasion and metastasis. The possible mechanisms leading to these cancer-specific changes in carbohydrate structures (termed glycodynamics) involve altered mRNA expression and catalytic activities of glycosyltransferases and sulfotransferases found in tissues and cells of gastrointestinal tumors. In a number of cases it has been possible to correlate enzyme changes with oligosaccharide structures. Different mechanisms have been suggested leading to the synthesis of cancer-specific Lewis, T and Tn antigens, but the regulation of cancer mucin antigens generally appears to be very complex and is poorly understood. The expression levels of specific mucin antigens and enzymes in gastro-intestinal tumors have diagnostic as well as prognostic value. These antigens also have potential for cancer immunotherapy. However, we first need to unravel the complexity of the control of glycosylation in cancer cells. Most importantly, studies of the functional implications of the glycodynamics in cancer cells, as related to cell adhesion and impact on the immune system will provide promising directions for future research.

Animals↗

PubMed2002

Glycoprotein biosynthesis in porcine aortic endothelial cells and changes in the apoptotic cell population.

Porcine aortic endothelial cells (PAECs) produce glycoproteins with important biological functions, such as the control of cell adhesion, blood clotting, blood pressure, the immune system, and apoptosis. Cell surface glycoproteins play important roles in these biological activities. To understand the control of cell surface glycosylation, we elucidated biosynthetic pathways leading to N- and O-glycans in PAECs. Based on the enzyme activities, PAECs should be rich in complex biantennary N-glycans. In addition, the enzymes synthesizing complex O-glycans with core 1 and 2 structures are present in PAECs. The first enzyme of the O-glycosylation pathway, polypeptide GalNAc-transferase, was particularly active. Its specificity toward synthetic peptide substrates was found to be similar to that of purified bovine colostrum enzyme T1. A significant fraction of PAECs treated with tumour necrosis factor alpha or human serum detached from the culture plate, and most of these cells were apoptotic. The apoptotic cell population exhibited decreased core 2 beta 6-GlcNAc-transferase activity. In contrast, the activities of core 1 beta 3-Gal-transferase, which synthesizes O-glycan core 1, and of alpha 3-sialyltransferase (O), which sialylates core 1, were increased in apoptotic PAECs. Thus, apoptotic PAECs are predicted to have fewer complex O-glycans and a higher proportion of short, sialylated core 1 chains.

Amino Acid Sequence↗