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Werner Reutter

Publications and source records attributed to Werner Reutter.

At least 37 records · Page 2Linked to original sources
PubMed2004

Crystal structure of CD26/dipeptidyl-peptidase IV in complex with adenosine deaminase reveals a highly amphiphilic interface.

Dipeptidyl-peptidase IV (DPPIV or CD26) is a homodimeric type II membrane glycoprotein in which the two monomers are subdivided into a beta-propeller domain and an alpha/beta-hydrolase domain. As dipeptidase, DPPIV modulates the activity of various biologically important peptides and, in addition, DPPIV acts as a receptor for adenosine deaminase (ADA), thereby mediating co-stimulatory signals in T-lymphocytes. The 3.0-A resolution crystal structure of the complex formed between human DPPIV and bovine ADA presented here shows that each beta-propeller domain of the DPPIV dimer binds one ADA. At the binding interface, two hydrophobic loops protruding from the beta-propeller domain of DPPIV interact with two hydrophilic and heavily charged alpha-helices of ADA, giving rise to the highest percentage of charged residues involved in a protein-protein contact reported thus far. Additionally, four glycosides linked to Asn229 of DPPIV bind to ADA. In the crystal structure of porcine DPPIV, the observed tetramer formation was suggested to mediate epithelial and lymphocyte cell-cell adhesion. ADA binding to DPPIV could regulate this adhesion, as it would abolish tetramerization.

Adenosine Deaminase↗
PubMed2004

The homozygous M712T mutation of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase results in reduced enzyme activities but not in altered overall cellular sialylation in hereditary inclusion body myopathy.

Hereditary inclusion body myopathy (HIBM) is a neuromuscular disorder, caused by mutations in UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, the key enzyme of sialic acid biosynthesis. In Middle Eastern patients a single homozygous mutation occurs, converting methionine-712 to threonine. Recombinant expression of the mutated enzyme revealed slightly reduced N-acetylmannosamine kinase activity, in agreement with the localization of the mutation within the kinase domain. B lymphoblastoid cell lines derived from patients expressing the mutated enzyme also display reduced UDP-N-acetylglucosamine 2-epimerase activity. Nevertheless, no reduced cellular sialylation was found in those cells by colorimetric assays and lectin analysis, indicating that HIBM is not directly caused by an altered overall expression of sialic acids.

Amino Acid Substitution↗
PubMed2004

Increased expression of the selectin ligand sialyl-Lewis(x) by biochemical engineering of sialic acids.

Sialylation of glycoproteins and glycolipids plays an important role during development, regeneration and pathogenesis of several diseases. The precursor of all physiological sialic acids is N-acetyl-d-mannosamine. Using N-propanoyl mannosamine, a novel precursor of sialic acid, we showed earlier that sialic acids with a prolonged N-acyl side chain (e.g., N-propanoyl neuraminic acid) are incorporated into cell surface glycoconjugates. In this study, we report the structural and functional consequences of the incorporation of the nonphysiological sialic acid, N-propanoyl neuraminic acid, into glycoconjugates of HL60-I cells. These cells do not express UDP-GlcAc-2-epimerase, the key enzyme of the biosynthesis of N-acetyl-d-mannosamine. Therefore, they do not express sialyl-Lewis(x) structures and consequently do not bind to selectins. Application of N-acetyl-d-mannosamine leads to the expression of sialyl-Lewis(x) structures and to binding to selectins. Surprisingly, incorporation of N-propanoyl neuraminic acid into glycoconjugates of these cells leads to a dramatic increase of sialyl-Lewis(x) structures and to increased adhesion to selectins.

Cell Adhesion↗
PubMed2004

Cytoplasmic domain of NCAM 180 reduces NCAM-mediated neurite outgrowth.

The neural cell adhesion molecule (NCAM) is one of the best-characterized cell adhesion molecules of the immunoglobulin superfamily. In the nervous system, NCAM is involved in cell migration, axon fasciculation and in neurite outgrowth. Neurite outgrowth is mediated by homophilic NCAM-NCAM interactions. Alternative splicing generates three major isoforms of NCAM differing in their intracellular portion. Two of them, NCAM 180 and NCAM 140, are transmembrane proteins with large intracellular domains. The present study is concerned with novel details of the intracellular domains of NCAM 140 and NCAM 180. We expressed these NCAM isoforms consisting only of the transmembrane and intracellular domains (without extracellular domains) in PC12 cells and elaborated their function in neurite outgrowth assays. Our data demonstrate that membrane-associated NCAM 180 interferes with neurite outgrowth, whereas membrane-associated NCAM 140 promotes neurite outgrowth.

Animals↗
PubMed2004

Novel UDP-glycal derivatives as transition state analogue inhibitors of UDP-GlcNAc 2-epimerase.

The "epimerisation" of UDP-GlcNAc to ManNAc, the first step in the biosynthesis of sialic acids, is catalyzed by UDP-GlcNAc 2-epimerase. In this paper we report the synthesis of transition state based inhibitors of this enzyme. To mimic the assumed first transition state of this reaction (TS 1), we designed and synthesized the novel UDP-exo-glycal derivatives 1-4. We also report herein the synthesis of 5 and 6, the first C-glycosidic derivatives of 2-acetamidoglucal, and the synthesis of the ketosides 7 and 8, which were designed as bis-substrate analogue and bis- product analogue, respectively, to mimic the second step of the reaction via the assumed second transition state TS 2.

Carbohydrate Epimerases↗
PubMed2004

3D structure of the CD26-ADA complex obtained by cryo-EM and single particle analysis.

The specific binding of adenosine deaminase to the multifunctional membrane glycoprotein dipeptidyl peptidase IV is thought to be immunologically relevant for certain regulatory and co-stimulatory processes. In this study we present the 3D structure of the complete CD26-ADA complex obtained by single particle cryo-EM at 22A resolution. ADA binding occurs at the outer edges of the beta-propeller of CD26. Docking calculations of available CD26 and ADA crystal data into the obtained EM density map revealed that the ADA-binding site is stretched across CD26 beta-propeller blades 4 and 5 involving the outermost distal hydrophobic amino acids L294 and V341 but not T440 and K441 as suggested by antibody binding. Though the docking of the ADA orientation appears less significant due to the lack of distinct surface features, non-ambiguous conclusions can be drawn in the combination with earlier indirect non-imaging methods affirming the crucial role of the ADA alpha2-helix for binding.

Adenosine Deaminase↗
PubMed2004

PDGF-BB enhances alpha1beta1 integrin-mediated activation of the ERK/AP-1 pathway involved in collagen matrix remodeling by rat mesangial cells.

Platelet-derived growth factor-BB (PDGF-BB) has been implicated in the pathogenesis of progressive glomerulonephritis (GN). Previous studies have reported that PDGF-BB stimulates mesangial cells (MCs)-induced collagen matrix remodeling through enhancement of alpha1beta1 integrin-dependent migratory activity. To determine the cell signaling pathway responsible for abnormal MC-related mesangial matrix remodeling in progressive GN, we studied the involvement of the extracellular signal-regulated kinase (ERK)/activator protein-1 (AP-1) pathway in PDGF-BB-enhanced collagen gel contraction. Western blotting and gel shift assay revealed that MC-induced gel contraction resulted in ERK activation in parallel with that of AP-1 binding, peaking at 4 h and lasting at least for 24 h. Application of the MEK inhibitor, U0126, and the c-jun/AP-1 inhibitor, curcumin, inhibited gel contraction and AP-1 activity, respectively, dose dependently. PDGF-BB enhanced not only gel contraction but ERK phosphorylation and AP-1 activity by MCs. Marked inhibitory effects on PDGF-BB-induced gel contraction and ERK/AP-1 activity were observed in the presence of either function blocking anti-alpha1- or anti-beta1-integrin antibody or U0126. Consistently, AP-1-inactive MCs expressing a dominant-negative mutant of c-jun showed a significant decrease of PDGF-BB-induced gel contraction as compared with mock-transfected MCs. Finally, migration assay showed that ERK/AP-1 activity is required for PDGF-BB-stimulated alpha1beta1 integrin-dependent MC migration to collagen I. These results indicated that PDGF-BB enhances alpha1beta1 integrin-mediated collagen matrix reorganization through the activation of the ERK/AP-1 pathway that is crucial for MC migration. We conclude that the ERK/AP-1 pathway plays an important role in PDGF-BB-induced alpha1beta1 integrin-dependent collagen matrix remodeling; therefore, the inhibition of its pathway may provide a novel approach to regulate abnormal collagen matrix remodeling in progressive GN.

Animals↗
PubMed2004

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, functionally expressed in and purified from Escherichia coli, yeast, and insect cells.

UDP-GlcNAc 2-epimerase/ManNAc kinase is the key enzyme of sialic acid biosynthesis in mammals. Its functional expression is a prerequisite for early embryogenesis and for the synthesis of several cell recognition motifs in adult organism. This bifunctional enzyme is involved in the development of different diseases like sialuria or hereditary inclusion body myopathy. For a detailed understanding of the enzyme, large amounts of the pure active protein are needed. Different heterologous cell systems were therefore analyzed for the enzyme, which was found to be functionally expressed in Escherichia coli, the yeast strains Saccharomyces cerevisiae and Pichia pastoris, and insect cells. In all these cell types, the expressed enzyme displayed both epimerase and kinase activities. In E. coli, up to 2mg protein/l cell culture was expressed, in yeast cells only 0.4mg/L, while up to 100mg/L, were detected in insect cells. In all three cell systems, insoluble protein aggregates were also observed. Purification from E. coli resulted in 100microg/L pure and structurally intact protein. For insect cells, purification methods were established which resulted in up to 50mg/L pure, soluble, and active protein. In summary, expression and purification of the UDP-GlcNAc 2-epimerase/ManNAc kinase in Sf-900 cells can yield the milligram amounts of protein required for structural characterization of the enzyme. However, the easier expression in E. coli and yeast provides sufficient quantities for enzymatic and kinetic characterization.

Animals↗
PubMed2004

[Activation of mouse macrophages by the alkali-extracted polysaccharide from spore of Ganoderma lucidum].

AIM: To investigate the activation of mouse macrophages by the alkali-extracted polysaccharides from the spore of Ganoderma lucidum (LZSBS). METHODS: The mouse macrophages cultured in-vitro were stimulated by LZSBS. IL-1beta and TNF-alpha in the culture supernatants were detected by ELISA. NO production was detected by Griess assay. The percentage of phagocytosis of latex beads by mouse macrophages was counted under microscope. RESULTS: The mouse macrophages stimulated by LZSBS increased in volume and darkened in appearance under phase-contrast microscope. LZSBS-activated mouse macrophages secreted IL-1beta and TNF-alpha produced a large amount of NO. The percentage of phagocytosis of latex beads by mouse macrophages was also significantly increased in the presence of LZSBS. CONCLUSION: LZSBS can activate markedly the mouse macrophages.

Animals↗
PubMed2003

[Human lectins and their correspondent glycans in cell biology and clinical medicine].

Lectins are phylogenetically ancient proteins that have specific recognition and binding functions for complex carbohydrates of glycoconjugates, i. e., of glycoproteins, proteoglycans/glycosaminoglycans and glycolipids. This class of proteins mediates important processes of adhesion and communication both inside and outside cells. A large variety of lectins are expressed in the human organism. This article reviews the current knowledge of human lectins with a focus on biochemistry and pathobiochemistry (principles of protein glycosylation and defects of glycosylation as a basis of disease) and cell biology (protein sorting, exocytosis and endocytosis, apoptosis, cell adhesion, cell differentiation, and malignant transformation). The clinical significance of lectin-glycoconjugate interactions is described by example of inflammatory diseases, defects of immune defense, autoimmunity, infectious diseases, and tumor invasion/metastasis. Moreover, therapeutic perspectives of novel drugs that interfere with lectin-carbohydrate interactions are discussed.

Animals↗
PubMed2003

The neural cell adhesion molecule is associated with major components of the cytoskeleton.

The neural cell adhesion molecule (NCAM) is a member of the immunoglobulin superfamily. Two of the three major isoforms (NCAM 140 and NCAM 180) are transmembrane glycoproteins, which differ in their intracellular domains. The present study is concerned with the identification of novel intracellular binding partners of NCAM. We expressed and purified both cytoplasmic domains of NCAM. Using ligand affinity chromatography followed by peptide mass fingerprinting, we could identify several novel binding partners of the cytoplasmic domains of NCAM 140 and 180. We present data that alpha- and beta-tubulin as well as alpha-actinin 1 are associated with both NCAM 140 and 180. In contrast, beta-actin, tropomyosin, microtubuli-associated protein MAP 1A, and rhoA-binding kinase-alpha preferentially bind to NCAM 180. Furthermore, we demonstrate that inhibition of rhoA-binding kinase-alpha stimulates neurite outgrowth independently from NCAM.

Actinin↗
PubMed2003

Epigenetically mediated loss of UDP-GlcNAc 2-epimerase/ManNAc kinase expression in hyposialylated cell lines.

The bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase is the key enzyme in sialic acid biosynthesis. Loss of UDP-GlcNAc 2-epimerase activity results in a hyposialylated phenotype as shown for two human hematopoietic cell lines that lack the specific mRNA. We found that treatment with the DNA methylation inhibitor 5'-aza-2'-deoxycytidine (5-aza-dC) restored the UDP-GlcNAc 2-epimerase/ManNAc kinase mRNA, as well as enzyme activity and cell surface sialylation. Increase of UDP-GlcNAc 2-epimerase activity by 5-aza-dC treatment was also found for a rat Morris hepatoma cell line. These results indicate a regulation of UDP-GlcNAc 2-epimerase/ManNAc kinase expression on the transcriptional level by DNA methylation.

Animals↗
PubMed2003

The 3D structure of rat DPPIV/CD26 as obtained by cryo-TEM and single particle analysis.

We present the three-dimensional structure of rat DPPIV/CD26, as determined by cryo-TEM and single particle analysis at a resolution of approximately 14A. The reconstruction confirms that the protein exists as a dimer, as predicted earlier. Since there are structural analogies to the serine peptidase POP, docking calculations of the two structures were performed. Although the docking showed a similar spatial organization (catalytic domain, beta-propeller, distal opening, central cavity), the detailed comparison revealed clear discrepancies. The most marked difference is a second (lateral) opening in DPPIV/CD26, which would enable direct access to the catalytic site. We therefore assume that substrate selectivity and binding rate are most probably driven by different mechanisms in DPPIV/CD26 and POP.

Animals↗
PubMed2003

Sialic acid biosynthesis: stereochemistry and mechanism of the reaction catalyzed by the mammalian UDP-N-acetylglucosamine 2-epimerase.

The bifunctional enzyme, UDP-N-acetylglucosamine 2-epimerase/ManNAc kinase, catalyzes the first two steps in the biosynthesis of the sialic acids in mammals. The epimerase domain converts UDP-GlcNAc into ManNAc and UDP. This paper demonstrates that alpha-ManNAc is the first formed anomer and therefore the reaction proceeds with a net retention of configuration at C-1. Studies in deuterated buffer show that solvent-derived deuterium is quantitatively incorporated into the C-2 position of the product during catalysis, but it is not incorporated into the remaining pool of substrate. This indicates that the inversion of stereochemistry is ultimately brought about by the removal and replacement of a proton at C-2 and is consistent with a two-base mechanism. Studies with (18)O-labeled UDP-GlcNAc show that the anomeric oxygen of the glycosyl phosphate bond departs with the UDP product and therefore the net hydrolysis reaction involves C-O bond cleavage. Incubation of the putative intermediate, 2-acetamidoglucal, with the enzyme resulted in a slow hydration reaction to give the product, ManNAc. Additional kinetic isotope effect and positional isotope exchange (PIX) experiments address the nature of the rate-determining step of the reaction and show that C-H bond cleavage is not rate limiting. Overall, these results support a reaction mechanism involving an anti-elimination of UDP to give 2-acetamidoglucal, followed by a syn-addition of water.

Animals↗
PubMed2003

Deficiency of CD26 results in a change of cytokine and immunoglobulin secretion after stimulation by pokeweed mitogen.

To investigate the role of CD26 in the immune system, CD26 gene knockout mice with C57BL/6 background were used to study the immune response after stimulation with PWM. CD26(-/-) mice display an apparently normal phenotype. However, in their spleen lymphocyte population the percentage of CD4(+) T cells is lower, and that of NK cells is higher, than that in CD26(+/+) mice. In their peripheral blood, CD26(-/-) mice present a conspicuously decreased proportion of CD4(+) NKT lymphocytes. In vitro, the PWM-stimulated IL-4 production was decreased by 60-80% in the supernatants of spleen lymphocytes of CD26(-/-) mice compared to that of CD26(+/+) mice, whereas levels of IL-10 and IFN-gamma were increased. No significant differences were found in the production of IL-2, IL-5, IL-6 and IL-13 between knockout and wild-type mice. After immunization of mice with PWM in vivo, serum levels of total IgG, IgG1, IgG2a and IgE were markedly lower in CD26(-/-) mice than those in CD26(+/+) mice, while no difference was found in IgM production. Further analysis of cytokine levels in vivo revealed a reduced IL-4, IL-2 and delayed IFN-gamma production in sera of CD26(-/-) mice upon immunization with PWM. These results indicate that CD26 contributes to the regulation of development, maturation and migration of CD4(+) T, NK and NKT cells, cytokine secretion, T cell-dependent antibody production and immunoglobulin isotype switching of B cells.

Animals↗
PubMed2002

Biochemical engineering of cell surface sialic acids stimulates axonal growth.

Sialylation is essential for development and regeneration in mammals. Using N-propanoylmannosamine, a novel precursor of sialic acid, we were able to incorporate unnatural sialic acids with a prolonged N-acyl side chain (e.g., N-propanoylneuraminic acid) into cell surface glycoconjugates. Here we report that this biochemical engineering of sialic acid leads to a stimulation of neuronal cells. Both PC12 cells and cerebellar neurons showed a significant increase in neurite outgrowth after treatment with this novel sialic acid precursor. Furthermore, also the reestablishment of the perforant pathway was stimulated in brain slices. In addition, we surprisingly identified several cytosolic proteins with regulatory functions, which are differentially expressed after treatment with N-propanoylmannosamine. Because sialic acid is the only monosaccharide that is activated in the nucleus, we hypothesize that transcription could be modulated by the unnatural CMP-N-propanoylneuraminic acid and that sialic acid activation might be a general tool to regulate cellular functions, such as neurite outgrowth.

Animals↗