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1.
S Soulier  P Gaye 《Biochimie》1981,63(7):619-628
The results of subcellular fractionation of sheep mammary gland membranes indicate that N-acetylgalactosaminyl polypeptide transferase and galactosyl-N-acetylgalactosaminyl transferase, which are involved in the assembly of disaccharide units of kappa-casein, are localized chiefly in Golgi membranes. The glycosyltransferase activities incorporating N-acetyl [1-14C] galactosamine and [U-14C] galactose from uridine diphosphate N-acetyl [1-14C] galactosamine and uridine diphosphate [U-14C] galactose, respectively, were measured after membrane solubilization with Triton X-100 either with unglycosylated caseinomacropeptide, or with this polypeptide containing the N-acetylgalactosamine side chain residues (desialylated and degalactosylated caseinomacropeptide). Radioactive N-acetylgalactosamine was incorporated in the unglycosylated acceptor peptide, and the glycosidic bonds in the product were alkali labile, suggesting that they were linked to the hydroxyamino acid residues. In addition radioactive N-acetylgalactosamine was released after alpha N-acetyl-D-galactosaminidase treatment of labelled caseinomacropeptide. [U-14C] galactose was incorporated in the desialylated and degalactosylated acceptor peptide. Reductive alkaline treatment of [U-14C] galactose peptide resulted in the release of a major product, the chromatographic properties of which in TLC were identical with authentic galactosyl (1 leads to 3) N-acetylgalactosaminitol. The structure of the labelled disacchariditol determined after periodate oxidation (two equivalents) by gas liquid chromatography-mass spectrometry revealed that the [U-14C] galactose was linked to position C-3 on the N-acetylgalactosaminyl-residue. The anomery of the galactose, as determined by a chemical method, indicates unambiguously a beta configuration.  相似文献   

2.
3.
Copolymers of acrylated derivatives of alpha-chymotrypsin and polyethylene glycol (PEG) have been prepared and used as biocatalysts for the synthesis of model peptides in organic solvent containing a low quantity of water. Other peptide couplings have been tried to point out the chemico- and stereoselectivity and examples of segment couplings are given.  相似文献   

4.
Synthetic peptides derived from a 45-kDa glycoprotein antigen of Mycobacterium tuberculosis were shown to function as glycosyltransferase acceptors for mannose residues in a mannosyltransferase cell-free assay. The mannosyltransferase activity was localized within both isolated membranes and a P60 cell wall fraction prepared from the rapidly growing mycobacterial strain, Mycobacterium smegmatis. Incorporation of radiolabel from GDP-[(14)C]mannose was inhibited by the addition of amphomycin, indicating that the glycosyl donor for the peptide acceptors was a member of the mycobacterial polyprenol-P-mannose (PPM) family of activated glycosyl donors. Furthermore, a direct demonstration of transfer from the in situ generated PP[(14)C]Ms was also demonstrated. It was also found that the enzyme activity was sensitive to changes in overall peptide length and amino acid composition. Because glycoproteins are present on the mycobacterial cell surface and are available for interaction with host cells during infection, protein glycosyltransferases may provide novel drug targets. The development of a cell-free mannosyltransferase assay will now facilitate the cloning and biochemical characterisation of the relevant enzymes from M. tuberculosis.  相似文献   

5.
Peptides containing a triprolyl sequence carboxyl to a threonine residue can be O-glycosylated by a crude Triton x-100 extract of porcine submaxillary glands (Young, J. D., Tsuchiya, D., Sandlin, D. E., and Holroyde, M. J. (1979) Biochemistry 18, 4444-4448). In the present paper, we have studied the characteristics of the O-glycosylating enzyme, UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosamine transferase, from a membrane extract of bovine submaxillary glands using 11 synthetic peptide substrates in which the Thr-Pro-Pro-Pro was varied. The effect of these changes was measured by determining the apparent Km and Vmax values of the substrates. The studies thus far reveal: threonine cannot be glycosylated without a carboxyl triprolyl sequence; the alpha amino acid group of the threonine must be blocked; the nature of the group NH2-terminal to the threonine affects the kinetics of the reaction; and one residue can be between the threonyl and the triprolyl sequence. The triprolyl sequence in a protein may be an important signal for O-glycosylation.  相似文献   

6.
Yeast and other fungal protein-expression hosts have been extensively used to produce industrial enzymes, and are often the expression system of choice when manufacturing costs are of primary concern. However, for the production of therapeutic glycoproteins intended for use in humans, yeast have been less useful owing to their inability to modify proteins with human glycosylation structures. Yeast N-glycosylation is of the high-mannose type, which confers a short half-life in vivo and thereby compromises the efficacy of most therapeutic glycoproteins. Several approaches to humanizing yeast N-glycosylation pathways have been attempted over the past decade with limited success. Recently however, advances in the glycoengineering of yeast and the expression of therapeutic glycoproteins with humanized N-glycosylation structures have shown significant promise - this review summarizes the most important developments in the field.  相似文献   

7.
Summary The molecular mechanism of reduced incorporation of radioactively labeled mannose into hamster liver glycoconjugates during the progression of vitamin A deficiency was investigated. In particular the in vivo incorporation of [2-3H]mannose into GDP-mannose, dolichyl phosphate mannose (Dol-P-Man), lipid-linked oligosaccharides, and glycopeptides of hamster liver was examined. Hamsters maintained on a vitamin A-free diet showed a reduction in the incorporation of mannose into GDP-mannose about 10 days before clinical signs of vitamin A deficiency could be observed. The decrease in [2-3H]mannose incorporated into GDP-mannose was accompanied by a reduction in label incorporated into Dol-P-Man, lipid linked oligosaccharides and glycopeptides, which became more severe with the progression of vitamin A deficiency. By the time they reached a plateau stage of growth, hamsters fed the vitamin A-free diet showed a 50% reduction in the amount of [2-3H]mannose converted to GDP-mannose, and the radioactivity associated with Dol-P-Man and glycopeptides was reduced by approximately 60% as compared to retinoic acid-supplemented controls. These results strongly indicate that the reduced incorporation of mannose into lipidic intermediates and glycoproteins observed during vitamin A deficiency is due to impaired GDP-mannose synthesis.Abbreviations Dol-P-Man Dolichyl Phosphate Mannose - Dol-P Dolichyl Phosphate  相似文献   

8.
Nine synthetic peptides containing sequences in the region of a threonine residue at position 98 of bovine basic myelin protein were prepared by the Merrifield solid-phase method and tested for their ability to be glycosylated with [14C]uridinediphospho-N-acetylgalactosamine and a crude detergent-solubilized preparation of uridinediphospho-N-acetylgalactosamine:mucin polypeptide N-acetylgalactosaminyltransferase obtained from porcine submaxillary glands. The tetrapeptide Thr-Pro-Pro-Pro and all larger peptides containing this sequence were glycosylated. The glycosylation was greater for peptides containing residues N-terminal to the Thr-Pro-Pro-Pro. Under the conditions used, the peptide Val-Thr-Pro-Arg-Thr-Pro-Pro-Pro was glycoslyated twice as much as bovine basic myelin protein. Thr-Pro and Thr-Pro-Pro, as well as 10 other synthetic peptides which did not contain the Thr-Pro-Pro-Pro sequence, were not glycosylated. Treatment of the glycopeptide of Phe-Lys-Asn-Leu-Val-Thr-Pro-Arg-Thr-Pro-Pro-Pro-Ser with an alpha-N-acetylgalactosaminidase released N-acetylgalactosamine from the peptide, indicating that the hexosamine was covalently bonded to the peptide in an alpha linkage.  相似文献   

9.
10.
Immunoglobulins (Igs) are the major molecules secreted by B lymphocytes during an adaptive immune response. They are glycoproteins with distinctive glycosylation patterns, resulting in wide variations in the number, type and location of their oligosaccharides in each isotype and subclass. The sugars play specific structural roles, maintaining and modulating effector functions of Igs. Aberrant glycosylation might contribute to disease pathogenesis. This review will focus on the glycosylation of IgG and IgA because they have been studied more extensively than other immunoglobulins. Rheumatoid arthritis and IgA nephritis are used to describe the association of glycosylation aberration and disease pathogenesis.  相似文献   

11.
Endo-beta-xylosidase from the mid-gut gland of the molluscus Patinopecten is an endo-type glycosidase that hydrolyzes the xylosyl serine linkage between a core protein and a glycosaminoglycan (GAG) chain, releasing the intact GAG chain from proteoglycan. In this study, we investigated GAG chain transfer activity of this enzyme, in order to develop a method for attaching GAG chains to peptide. Peptidochondroitin sulfate (molecular mass of sugar chain, 30 kDa) from bovine tracheal cartilage as a donor and butyloxycarbonyl-leucyl-seryl-threonyl-arginine-(4-methylcoumaryl-7-amide) as an acceptor were incubated with endo-beta-xylosidase. As a result, a reaction product with the same fluorescence as the acceptor peptide was observed. High pressure liquid chromatography analysis, cellulose acetate membrane electrophoresis, and enzymatic digestion showed that this reaction product had the chondroitin sulfate (ChS) from the donor. Furthermore, the acceptor peptide was released from this reaction product after hydrolysis by endo-beta-xylosidase. Therefore, it was confirmed that the ChS chain released from the donor was transferred to the acceptor peptide by the GAG chain transfer reaction of endo-beta-xylosidase. The optimal pH for hydrolysis by this enzyme was found to be about 4.0, whereas that for this reaction was about 3.0. Not only the ChS but also the dermatan sulfate and the heparan sulfate were transferred to the acceptor peptide by this reaction. By using this reaction, the GAG chain could be attached to the peptide in one step. The GAG chain transfer reaction of endo-beta-xylosidase should be a significant glycotechnological tool for the artificial synthesis of proteoglycan.  相似文献   

12.
To compare the site specificity of O-glycosylation in lower and higher eukaryotes, we expressed human granulocyte/macrophage colony-stimulating factor (hGM-CSF) in the yeast Saccharomyces cerevisiae and in COS-1 cells. Analyses of specific hGM-CSF mutants secreted by yeast led to the conclusion that efficient O-glycosylation in yeast requires residues S9 and T10. However, only S9 is used as an attachment point for an extended O-glycosyl chain in a 15.5-kDa hGM-CSF form. A 14.5-kDa hGM-CSF form, secreted by yeast, appears substituted by single mannosyl residues at both positions S9 and T10, indicating that O-glycosylation at T10 inhibits extension of the O-glycosyl chain attached to S9. As in yeast cells, the addition of O-glycosyl chains to hGM-CSF secreted by COS-1 cells requires the presence of S9 and T10 residues. These results demonstrate that, inspite of different biosynthetic routes, the selection of O-glycosylation sites is similar between lower and higher eukaryotes.  相似文献   

13.
Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is O-glycosylated at residues Ser9 and Thr10 during secretion by yeast and COS-1 cells [Ernst, J.F., Mermod, J.-J. and Richman, L.I. (1992) Eur. J. Biochem. 203, 663-667]. Two types of octapeptides encompassing residues 4-11 (peptide 4-11) and variants thereof, or residues 8-15 (peptide 8-15) of hGM-CSF were tested as substrates for in vitro O-glycosylation using dolichyl-phosphate- D-mannose: protein O-D-mannosyltransferase (Man-transferase) of the yeast Saccharomyces cerevisiae, or UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) of rat liver cells. Peptide 8-15 was found to be O-glycosylated at residues Ser9 and Thr10 by GalNAc-transferase and, with reduced efficiency, also by Man-transferase. Peptide 4-11 was a good substrate for yeast Man-transferase, leading to mannosylation of only Thr10, whereas it was very poorly O-glycosylated at positions Ser5 and Ser7 by GalNAc-transferase. The observed differences in peptide-acceptor activities indicate that the site of O-glycosylation depends on similar, but not identical protein structural features in yeast and mammalian cells.  相似文献   

14.
Trans-4-hydroxy-L-proline has been converted to four imino- and carboxyl-blocked derivatives which are suitable for the synthesis of 4-O-glycosyl conjugates. Reaction of these derivatives with 2,3,5-tri-O-benzyl-alpha-L-arabinofuranosyl chloride in the presence of a silver zeolite promoter yielded the blocked beta-furanosyl amino-acid conjugates. Deprotection gave trans-4-(beta-L-arabinofuranosyloxy)-L-proline which was characterised as its crystalline isopropyl ester. 13C-NMR Data are presented for the compounds described.  相似文献   

15.
Vasoactive intestinal peptide is an amino acceptor and donor substrate for tissue transglutaminase (TGase) in vitro. This peptide contains a single glutamine residue, Gln16, which was identified as the amino acceptor substrate. Different gamma(glutamyl16)amine derivatives of vasoactive intestinal peptide were synthesized enzymatically in vitro. The modification is very fast when compared with that of many native substrates of TGase. The analogs 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine, glycine ethyl ester and mono-dansylcadaverine of the peptide were purified by high-performance liquid chromatography on a reverse-phase column and were analyzed by electrospray mass spectrometry. When amines were absent in the assay mixture as an external amino donor, lysine residue occurring in the peptide was an effective amino donor site for TGase. Only one of the three lysine residues of vasoactive intestinal peptide, namely Lys21, was demonstrated to be involved in both inter- and intramolecular cross-link formation.  相似文献   

16.
17.
D-Glucose diethyl dithioacetal (1), its penta-O-acetyl derivative (2), penta-O-acetyl-aldehydo-D-glucose (3), L-xylo-hexulose phenylosotriazole (4), 1,2:5,6-di-O-isopropylidene-D-mannitol (5), 1,2:4,5-di-O-isopropylidene-β-D-fructopyranose (6), 1,2-O-isopropylidene-α-D-glucofuranose (7) and its triacetate (8), 1,6-anhydro-β-D-galactopyranose (9) and its triacetate (10), D-glucopyranose (11), methyl β-D-glucopyranoside tetraacetate (12), 1-thio-β-D-glucopyranose pentaacetate (13), β-D-fructofuranose pentaacetate (14), and raffinose hendecaacetate (15) have been examined by chemical-ionization mass-spectrometry with both isobutane and ammonia as ionizing intermediates. Extreme simplicity characterizes these spectra, and, in most instances, molecular-weight data are available from intact, protonor NH4+capture ions; the limited fragmentation that occurs corresponds in large measure to simple dehydration or substituent-cleavage processes, and is strongly dependent upon the groups present, so that considerable information about the substituent groups in the sugar molecule may be inferred.  相似文献   

18.
Dolichyl monophosphate and its sugar derivatives in plants.   总被引:5,自引:5,他引:5       下载免费PDF全文
A glucose acceptor was isolated from soya beans by extraction with chloroform/methanol (2:1, v/v), followed by DEAE-cellulose column chromatography of the extract. This acceptor could not be distinguished from liver dolichyl monophosphate by t.l.c. It could replace dolichyl monophosphate as a mannose acceptor with a liver enzyme and its glucosylated derivative could replace dolichyl monophosphate glucose as a glucose donor in the same system. These results, together with those already reported [Pont Lezica, Brett, Romero Martinez & Dankert (1975) Biochem, Biophys. Res. Commun. 66, 980-987], indicate that the acceptor from soya bean is a dolichyl monophosphate. Gel filtration of its glucosylated derivative on Sephadex G-75 in the presence of sodium deoxycholate indicated that the acceptor contained 17 or 18 isoprene units. An enzyme preparation from pea seedlings was shown to use endogenous acceptors to form lipid phosphate sugars containing mannose and N-acetylglucosamine from GDP-mannose and UDP-N-acetylglucosamine. Chromatographic and degradative techniques indicated that the compounds formed were lipid monophosphate mannose, lipid pyrophosphate N-acetylglucosamine, lipid pyrophosphate chitobiose and a series of lipid pyrophosphate oligosaccharides containing both mannose and N-acetylglucosamine. None of these compounds was degraded by catalytic hydrogenation, and so the lipid moiety in each case was probably an alpha-saturated polyprenol. The endogenous acceptors for mannose and N-acetylglucosamine in peas may therefore be dolichyl monophosphate, as has been found in mammalian systems.  相似文献   

19.
The biological activities of two synthetic oligopeptides (His-Trp-Leu-Gln-Leu and Trp-Leu-Gln-Leu), which represent part of the primary structure of the mating hormone alpha factor from Saccharomyces cerevisiae, were studied. The peptides did not exhibit hormonal activity by themselves. However, both intensified the mating-type-specific inhibitory effect of native alpha factor on the division of haploid cells of mating type a. Random peptides or mixtures of the corresponding amino acids did not stimulate alpha factor activity. Likewise, a synthetic peptide representing another part of the alpha factor sequence was ineffective. In addition, the activity of a factor, the mating hormone produced by a cells, was not influenced by the synthetic peptides, indicating that the compounds specifically affect the interaction between alpha factor and its target cells. The analysis of the utilization of the tetrapeptide as a source of amino acids for auxotrophic a strains suggested an extracellular site of action for the observed enhancement of alpha factor activity.  相似文献   

20.
Yeast has been increasingly used as a host for the expression of enzymes. Compared to other expression systems, the yeast expression system has many advantages including its suitability for large-scale fermentation and its ability to modify enzymes. When expressed in yeast, many recombinant enzymes are N-glycosylated, and this may play an important role in their activity, thermostability and secretion. Although the mechanism underlying this process is not clear, the regulation of N-glycosylation by introducing or eliminating N-glycosylation at specific sites has developed into an important strategy for improving the production or catalytic properties of recombinant enzymes. In this review, we summarize the recent advances in understanding the effects of N-glycosylation on the expression and characteristics of recombinant enzymes, and discuss novel strategies for regulating N-glycosylation in yeast. We hope that this review will help improve the understanding of the expression and the catalytic properties of N-glycosylated proteins.  相似文献   

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