共查询到20条相似文献,搜索用时 31 毫秒
1.
G Srikrishna NM Varki PC Newell A Varki HH Freeze 《Canadian Metallurgical Quarterly》1997,272(41):25743-25752
Core fucosylation of N-linked oligosaccharides (GlcNAcbeta1, 4(Fucalpha1,6)GlcNAcbeta1-Asn) is a common modification in animal glycans, but little is known about the distribution of core-fucosylated glycoproteins in mammalian tissues. Two monoclonal antibodies, CAB2 and CAB4, previously raised against carbohydrate epitopes of Dictyostelium discoideum glycoproteins (Crandall, I. E. and Newell, P. C. (1989) Development 107, 87-94), specifically recognize fucose residues in alpha1,6-linkage to the asparagine-bound GlcNAc of N-linked oligosaccharides. These IgG3 antibodies do not cross-react with glycoproteins containing alpha-fucoses in other linkages commonly seen in N- or O-linked sugar chains. CAB4 recognizes core alpha1,6 fucose regardless of terminal sugars, branching pattern, sialic acid linkage, or polylactosamine substitution. This contrasts to lentil and pea lectins that recognize a similar epitope in only a subset of these structures. Additional GlcNAc residues found in the core of N-glycans from dominant Chinese hamster ovary cell mutants LEC14 and LEC18 progressively decrease binding. These antibodies show that many proteins in human tissues are core-fucosylated, but their expression is localized to skin keratinocytes, vascular and visceral smooth muscle cells, epithelia, and some extracellular matrix-like material surrounding subpopulations of lymphocytes. The availability of these antibodies now allows for an extended investigation of core fucose epitope expression in development and malignancy and in genetically manipulated mice. 相似文献
2.
LEC18 and LEC14 cells are gain-of-function glycosylation mutants isolated from Chinese hamster ovary cells for resistance to pea lectin. Structural studies have shown that LEC18 cells synthesize complex N-glycans with a GlcNAc residue linked at the O-6 position of the core GlcNAc (Raju, T. S., Ray, M. K., and Stanley, P. (1995) J. Biol. Chem. 270, 30294-30302), whereas LEC14 cells synthesize complex N-glycans with a GlcNAc residue linked at the O-2 position of the core beta-linked Man residue (Raju, T. S., and Stanley, P. (1996) J. Biol. Chem. 271, 7484-7493). Both modifications are novel and have not been reported in glycoproteins from any other source. We now show that, in both LEC18 and LEC14 cells, GlcNAc transfer is mediated by a distinct N-acetylglucosaminyltransferase (GlcNAc-T) activity. The LEC18 activity, termed GlcNAc-TVIII, transfers GlcNAc to GlcNAcbeta1-O-pNP and to a GlcNAc-terminating, biantennary, complex N-glycan, with or without a core fucose. By contrast, the LEC14 transferase, termed GlcNAc-TVII, does not have significant activity with simple acceptors, and transfers GlcNAc preferentially to a GlcNAc-terminating biantennary glycopeptide that contains a core fucose residue. The acceptor specificities and other biochemical properties of GlcNAc-TVII and GlcNAc-TVIII differ from previously characterized GlcNAc-transferases including GlcNAc-TIII, indicating that they represent new members of the mammalian GlcNAc-T group of transferases. 相似文献
3.
In poorly differentiated hepatoma cells, a glycoprotein carrying lactosaminoglycans is identified, and the structure of its glycan moiety is proposed. After membrane solubilization, protein fractionation by gel filtration, and electroelution, this glycoprotein (GPIII) was identified by its affinity for Datura stramonium lectin and its content in large glycopeptides. As shown by PAGE, GPIII has an apparent molecular mass of 100 kDa and is highly glycosylated (36%). It appears as an integral membrane glycoprotein. It is absent from normal hepatocytes, in that no heavy glycopeptides could be detected that bound to Datura lectin or to specific antiserum. The glycan moiety of GPIII has been analyzed according to carbohydrate composition, glycosidase treatment, affinity chromatography on immobilized pokeweed, Datura and Griffonia lectins, and by NMR and methylation analyses. The glycan is a N-linked tetraantennary lactosaminoglycan of 6.6 kDa, containing Gal, GlcNAc, Man, and NeuNAc in a 16:14:3:4 molar ratio, with an average of three repeating units/branch. Its beta-Gal residues are in the penultimate position and are linked in beta1-4 at least in four structural elements (three peripheral and one internal). It contains a very branched structure with Gal alpha1-3Gal beta1-4GlcNAc side chains linked in the C6 position to an inner Gal residue in a main branch. Alpha-Gal and NeuNAc residues [mainly NeuNAc alpha(2-3) linkage] are expressed as the nonreducing terminal groups. A possible structural model is proposed for this heterogeneous lactosaminoglycan, although no definitive structure can be established. That this lactosaminoglycan-carrying glycoprotein GPIII is not expressed in hepatocytes suggests its expression to be linked to the undifferentiated and/or malignant state of this hepatoma. 相似文献
4.
YH Atkinson KJ Gogolin-Ewens EF Hounsell MJ Davies MR Brandon RF Seamark 《Canadian Metallurgical Quarterly》1993,268(35):26679-26685
The ovine binucleate cell-specific glycoproteins recognized by the monoclonal antibody SBU-3 first appear at the initiation of placentation, and their expression continues throughout gestation. These placenta-specific proteins have not been detected in any other adult or fetal sheep tissues and are specific to the materno-fetal interface. The SBU-3 monoclonal antibody recognizes the carbohydrate epitope common to a group of proteins ranging in molecular mass from 30 to 200 kDa whose function during pregnancy remains undefined. The biochemical properties of these uniquely expressed glycoproteins were investigated by analyzing both the carbohydrate and protein portion of the molecules. Analysis of phytohemagglutinin and concanavalin A binding to electrophoretically separated SBU-3 proteins revealed that the major proteins between 40 and 70 kDa bind phytohemagglutinin. In contrast, concanavalin A bound only to minor proteins in the SBU-3 glycoprotein preparation. Analysis of the carbohydrate conjugated to the SBU-3 glycoproteins revealed that the major chains are sialylated O-linked and complex partially sialylated multiple antennary N-linked chains. The presence of N-glycolylneuraminic acid in an N-linked structure indicates the unique nature of this carbohydrate epitope. The differential binding to phytohemagglutinin and concanavalin A provided a method for further purification and characterization of the major protein components with monoclonal antibody immunoaffinity-purified SBU-3 proteins being further separated by concanavalin A-Sepharose chromatography. Microsequence analysis of the major non-concanavalin A-binding proteins (69, 62, and 57 kDa) revealed partial homology to ovine and bovine pregnancy-associated glycoprotein and rabbit pepsinogen F. Immunoblot analysis of the SBU-3 proteins showed cross-reactivity with polyclonal antisera directed against ovine placental-associated glycoprotein and pregnancy-specific glycoprotein B. These results suggest that together these glycoproteins represent members of a binucleate cell-derived family of pregnancy-associated molecules in the ruminant placenta. 相似文献
5.
Electrospray ionization-ion trap mass spectrometry, with its capacity to perform multiple stages of fragmentation (MSn), is demonstrated as an effective method for the structural characterization of permethylated N-linked complex glycoprotein oligosaccharides. Complex glycan structural features, such as N-acetyllactosamine antenane, neuraminic acids, and nonreducing terminal GlcNAc monosaccharides, commonly suppress cross-ring and core saccharide cleavages in traditional MS/MS experiments. Using ion trap mass spectrometry, removal of these substituents permits determination of branching patterns and intersaccharide linkages by MS3 and MS4. Both sequence and linkage data are obtained for N-acetyllactosamine and sialyl-N-acetyllactosamine oligosaccharide antennae from biantennary glycans using MS3, and the location of a bisecting GlcNAc residue is also established after exposing the core pentasaccharide. Higher-order experiments further illustrate the potential of electrospray ionization-quadrupole ion trap mass spectrometry for carbohydrate analysis, as MS8 is used to produce significant and otherwise unobtainable branching information for an oligosaccharide from chicken ovalbumin. These studies constitute further evidence of the unique role that ion trap mass spectrometry can assume in the area of oligosaccharide analysis. 相似文献
6.
AH Khan AM Qazi DC Hoessli AP Torred-Duarte G Senaldi MH Qazi E Walker-Nasir Nasir-ud-Din 《Canadian Metallurgical Quarterly》1997,43(3):655-668
Metabolic labelling of Plasmodium falciparum parasites with [3H]GlcN, [3H]Man, [3H]Gal and [3H]ethanolamine, and subsequent purification by SDS-PAGE of the labelled material provided effective labelling of the MSP-1, 195 kDa, and MSP-2, 42-53 kDa, glycoproteins. Reductive beta-elimination of the MSP-2 released from the gel consisted of glycopeptides containing labelled sugars. Processing of the eliminated components and identification of the sugar residues demonstrated the presence of N-acetylglucosaminitol and N-acetylgalactosaminitol amongst other labelled sugars. Reductive beta-elimination with sodium hydroxide-sodium borotritide-borohydride showed the presence of glucosaminitol and alanine in the hydrolysis products. The MSP-2 was retained on solid phase wheat-germ agglutinin and was released from the lectin by treatment with GlcNAc. Upon treatment with O-glycanase the MSP-2 glycoprotein released labelled amino sugar, and derived oligosaccharides on treatment with exoglycosidases released labelled components corresponding to the metabolically incorporated sugars. Labelled Gal was incorporated into the MSP-2 glycoprotein using [3H]UDP-Gal and galactosyltransferase. The galactosylated glycoprotein released labelled Gal upon treatment with beta-galactosidase. The results of the present study suggest that the carbohydrate chains of the MSP-2 glycoprotein are attached to the protein backbone via GlcNAc- and GalNAc-serine/threonine in O-glycosyl linkage and the glycoprotein has terminal GlcNAc and Gal residues. The carbohydrate moieties of MSP-2, glycoprotein consist mainly of short chains linked to the protein core. 相似文献
7.
Oligosaccharides on invertase restricted to the endoplasmic reticulum (ER) in alg3,sec18 yeast at 37 degrees C were found to be 20% wild type Man8GlcNAc and 80% Man1 alpha-->2Man1 alpha-->2Man1 alpha-->3(Man1 alpha-->6)Man1 beta-->4GlcNAc2 (Verostek, M.F., Atkinson, P.H., and Trimble, R. B. (1991) J. Biol. Chem. 266, 5547-5551). These results suggested that alg3 was slightly leaky, but did not address whether the oligosaccharide-lipid Man9GlcNAc2 and Man5GlcNAc2 precursors were glucosylated in alg3 yeast. Therefore, an alg3,sec18,gls1 strain was constructed to delete the GLS1-encoded glucosidase I responsible for trimming the terminal alpha 1,2-linked glucose from newly transferred Glc3ManxGlcNAc2 oligosaccharides. Invertase activity was overexpressed 5-10-fold on transforming this strain with a multicopy plasmid (pRB58) carrying the SUC2 gene, and preparative amounts of the ER form of external invertase, derepressed and accumulated at 37 degrees C, were purified. The N-linked glycans were released by sequential treatment with endo-beta-N-acetylglucosaminidase H (endo H) and peptide-N4-N-acetyl-beta-glucosaminyl asparagine amidase. Oligosaccharide pools were sized separately on Bio-Gel P-4, which showed that endo H released about 17% of the carbohydrate as Glc3Man8GlcNAc, while peptide-N4-N-acetyl-beta-glucosaminyl asparagine amidase released the remainder as Hex8GlcNAc2 and Man5GlcNAc2 in a 1:4 ratio. Glycan structures were assigned by 500-MHz two-dimensional DQF-COSY 1H NMR spectroscopy, which revealed that the endo H-resistant Hex8GlcNAc2 pool contained Glc3Man5GlcNAc2 and Man8GlcNAc2 in a 6:4 ratio, the latter a different isomer from that formed by the ER alpha 1,2-mannosidase (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666). Recovery of Glc3Man8GlcNAc and not the ER form of Man8GlcNAc provided an internal control indicating the absence of glucosidase I, which was confirmed by incubation of [3H]Glc3[14C]Man9GlcNAc with solubilized membranes from either alg3,sec18,gls1 or alg3,sec18,GLS1 strains. Chromatographic analysis of the products showed that [3H]Glc was removed only in the presence of the GLS1 gene product. Thus, the vast majority of the N-linked glycosylation in the ER of alg3 yeast (> 75%) occurs by transfer of Man5GlcNAc2 without prior addition of the 3 glucoses normally found on the lipid-linked precursor. 相似文献
8.
During studies on the fucosylation of endogenous proteins in parental (Pro5) and N-acetyl-D-glucosamine (GlcNAc) transferase I-deficient (Lec1) Chinese hamster ovary (CHO) cells, we observed that Lec1 cells incorporate approximately 10-fold less [3H]fucose into macromolecules than Pro5 cells. Interestingly, most of the labelled oligosaccharides from both cell types could be released from the macromolecules by digestion with peptide N-glycosidase F (PNGase F). This was unexpected for Lec1 cells because they do not synthesize complex- or hybrid-type N-glycans. Structural analyses of the fucosylated oligosaccharides from Lec1 cells showed the fucose to be in an alpha 1,6 linkage to the core GlcNAc of relatively small oligomannose N-glycans (Man4GlcNAc2 and Man5GlcNAc2, where Man is D-mannose). Comparing the sizes of oligomannose N-glycans from Pro5 and Lec1 cells demonstrated a much higher proportion of the small (Man4GlcNAc2 and Man5GlcNAc2) oligomannose species in Lec1 cells. These results suggest that the core alpha 1,6 fucosyltransferase will fucosylate small (Man4-Man5GlcNAc2), but not large (Man8-Man9GlcNAc2) oligomannose N-glycans. 相似文献
9.
KL Duffin GW Lange JK Welply R Florman PJ O'Brien A Dell AJ Reason HR Morris SJ Fliesler 《Canadian Metallurgical Quarterly》1993,3(4):365-380
The N-linked oligosaccharides of frog (Rana pipiens) rhodopsin were analysed by sequential exoglycosidase digestion and gel filtration chromatography, following reductive tritiation. In addition, selected tryptic glycopeptides obtained from frog retinal rod outer segment membranes were examined by electrospray mass spectrometry (ES-MS), fast atom bombardment mass spectrometry (FAB-MS), amino acid sequence and composition analysis, and carbohydrate composition analysis. The amino acid sequence data demonstrated that the glycopeptides were derived from rhodopsin and confirmed the presence of two N-glycosylation sites, at residues Asn2 and Asn15. The predominant glycan (approximately 60% of total) had the structure GlcNAc beta 1-2Man alpha 1-3(Man alpha 1-6) Man beta 1-4GlcNAc beta 1-4GlcNAc-(Asn), with the remaining structures containing 1-3 additional hexose residues, as reported previously for bovine rhodopsin. Unlike bovine rhodopsin, however, a sizable fraction of the total glycans of frog rhodopsin also contained sialic acid (NeuAc), with the sialylated oligosaccharides being present exclusively at the Asn2 site. FAB-MS analysis of oligosaccharides released from the Asn2 site gave, among other signals, an abundant quasimolecular ion corresponding to a glycan of composition NeuAc1Hex6HexNAc3 (where Hex is hexose and HexNAc is N-acetylhexosamine), consistent with a hybrid structure. The potential biological implications of these results are discussed in the context of rod outer segment membrane renewal. 相似文献
10.
Stable BHK-21 cell lines were constructed expressing the Golgi membrane-bound form and two secretory forms of the human alpha1, 3/4-fucosyltransferase (amino acids 35-361 and 46-361). It was found that 40% of the enzyme activity synthesized by cells transfected with the Golgi form of the fucosyltransferase was constitutively secreted into the medium. The corresponding enzyme detected by Western blot had an apparent molecular mass similar to those of the truncated secretory forms. The secretory variant (amino acids 46-361) was purified by a single affinity-chromatography step on GDP-Fractogel resin with a 20% final recovery. The purified enzyme had a unique NH2 terminus and contained N-linked endo H sensitive carbohydrate chains at its two glycosylation sites. The fucosyltransferase transferred fucose to the O-4 position of GlcNAc in small oligosaccharides, glycolipids, glycopeptides, and glycoproteins containing the type I Galbeta1-3GlcNAc motif. The acceptor oligosaccharide in bovine asialofetuin was identified as the Man-3 branched triantennary isomer with one Galbeta1-3GlcNAc. The type II motif Galbeta1-4GlcNAc in bi-, tri-, or tetraantennary neutral or alpha2-3/alpha2-6 sialylated oligosaccharides with or without N-acetyllactosamine repeats and in native glycoproteins were not modified. The soluble forms of fucosyltransferase III secreted by stably transfected cells may be used for in vitro synthesis of the Lewisa determinant on carbohydrates and glycoproteins, whereas Lewisx and sialyl-Lewisx structures cannot be synthesized. 相似文献
11.
A Apffel JA Chakel WS Hancock C Souders T M'Timkulu E Pungor 《Canadian Metallurgical Quarterly》1996,732(1):27-42
The application of high-performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and selective enzymatic deglycosylation treatments is demonstrated in the analysis of glycosylation patterns in recombinant Desmodus salivary plasminogen activator, a heterogeneous glycoprotein. The sample was initially digested with a proteolytic enzyme (endoproteinase Lys-C) and then further treated with either PNGase F to remove N-linked carbohydrates or a combination of neuraminidase and O-glycosidase to remove sialic acid and O-linked carbohydrates. By comparison of the LC-ESI-MS peptide maps for the fully glycosylated and deglycosylated samples, it was possible to unambiguously identify the sites of N-linked glycosylation as well a number of N-linked glycopeptides. The O-link glycopeptides, which are present at low level ( < 1%), were not detected prior to the deglycosylation, nor could changes in peptide elution in the map following deglycosylation be correlated with potential O-linked glycosylation sites. 相似文献
12.
Carbohydrate binding specificity of the B-cell maturation mitogen from Artocarpus integrifolia seeds
Artocarpin, a mannose-specific lectin, is a homotetrameric protein (M(r) 65,000) devoid of covalently attached carbohydrates and consists of four isolectins with pI in the range 5-6.5. Investigations of its carbohydrate binding specificity reveal that among monosaccharides, mannose is preferred over glucose. Among mannooligosaccharides, mannotriose (Man alpha 1-3[Man alpha 1-6]Man) and mannopentaose are the strongest ligands followed by Man alpha 1-3Man. Extension of these ligands by GlcNAc at the reducing ends of mannooligosaccharides tested remarkably improves their inhibitory potencies, while substitution of both the alpha 1-3 and alpha 1-6 mannosyl residues of mannotriose and the core pentasaccharide of N-linked glycans (Man alpha 1-3[Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc) by GlcNAc or N-acetyllactosamine in beta 1-2 linkage diminishes their inhibitory potencies. Sialylated oligosaccharides are non-inhibitory. Moreover, the substitution of either alpha 1-3 or alpha 1-6 linked mannosyl residues of M5Gn or both by mannose in alpha 1-2 linkage leads to a considerable reduction of their inhibitory power. Addition of a xylose residue in beta 1-2 linkage to the core pentasaccharide improves the inhibitory activity. Considering the fact that artocarpin has the strongest affinity for the xylose containing hepasaccharide from horseradish peroxidase, which differs significantly from all the mannose/glucose-specific lectins, it should prove a useful tool for the isolation and characterization of glycoproteins displaying such structure. 相似文献
13.
M Heffernan R Lotan B Amos M Palcic R Takano JW Dennis 《Canadian Metallurgical Quarterly》1993,268(2):1242-1251
beta-All-trans-retinoic acid (RA)-induced endodermal differentiation of mouse F9 teratocarcinoma cells is accompanied by changes in glycoprotein glycosylation, including expression of i antigen (i.e. polylactosamine) and leukophytohemagglutinin-reactive oligosaccharides (i.e. -GlcNAc beta 1-6Man alpha 1-6-branched N-linked). We have used the F9 teratocarcinoma cells as a model to study developmental regulation of glycosyltransferase activities which are responsible for the biosynthesis of beta 1-6GlcNAc-branched N- and O-linked oligosaccharides and polylactosamine. Growth of F9 cells in the presence of 10(-6) M RA for 4 days increased core 2 GlcNAc transferase and GlcNAc transferase V activities by 13- and 6-fold, respectively, whereas the activities of GlcNAc transferase I, beta 1-3GlcNAc transferase (i), beta 1-4Gal transferase, and beta 1-3Gal transferase increased 2-4-fold. Induction of glycosyltransferase activities by RA was dose-dependent and showed a biphasic response with approximately half of the increase observed 3 days after RA treatment and the remainder occurred by day 4. PYS-2, a parietal endoderm cell line, showed levels of glycosyltransferase activities similar to those of RA-treated F9 cells. Glycosyltransferase activities in the RA-resistant F9 cell line (RA-3-10) were low and showed only a small induction by RA. These observations suggest that differentiation of F9 cells is closely associated with induction of multiple glycosyltransferase activities, with most pronounced increases in GlcNAc transferase V and 2',5'-tetradenylate (core 2) GlcNAc transferase. The increase in GlcNAc transferase V was also reflected by the 4-6-fold increase in the binding of 125I-leukophytohemagglutinin to several cellular glycoproteins, which occurred after 3 days of RA treatment. The endo-beta-galactosidase-sensitive polylactosamine content of membrane glycoproteins and, in particular, the LAMP-1 glycoprotein was markedly increased after RA treatment of F9 cells. Consistent with these observations, fucosylated polylactosamine (i.e. dimeric Lex) was also increased in RA-treated cells. Analysis of the aryl oligosaccharides produced by F9 cells cultured in the presence of aryl alpha-D-GalNAc showed that RA treatment enhanced the synthesis of disialyl core 2 O-linked oligosaccharides and increased the polylactosamine content of the aryl oligosaccharides by > 20-fold. The results suggest that differentiation of F9 cells into endoderm is closely associated with increased GlcNAc transferase V and core 2 GlcNAc transferase activities, enzymes which control the level of beta 1-6GlcNAc-branched N- and O-linked oligosaccharides, the preferred substrates for polylactosamine addition. 相似文献
14.
SM Haslam GC Coles EA Munn TS Smith HF Smith HR Morris A Dell 《Canadian Metallurgical Quarterly》1996,271(48):30561-30570
Structural studies on the N-linked oligosaccharides of Haemonchus contortus, an economically important nematode that parasitizes domestic ruminants, have revealed core fucosylation of a type not previously observed in any eukaryotic glycoprotein. Mass spectrometric analyses were performed on detergent extracts of homogenized adult H. contortus and on purified H11, a glycoprotein isolated from intestinal brush borders which has been previously shown to be an effective vaccine antigen. The major N-linked glycans identified in the present study have up to three fucose residues attached to their chitobiose cores. The fucoses are found at the 3- and/or 6-positions of the proximal GlcNAc and at the 3-position of the distal GlcNAc. The latter substitution is unique in N-glycans. Most anti-H11 monoclonal antibodies are known to recognize carbohydrate epitopes, and it is possible that the newly discovered multifucosylated core structures are highly immunogenic in this glycoprotein. 相似文献
15.
P Mattila H Salminen L Hirvas J Niittym?ki H Salo R Niemel? M Fukuda O Renkonen R Renkonen 《Canadian Metallurgical Quarterly》1998,273(42):27633-27639
In the present experiments the cDNA coding for a truncated form of the beta1,6N-acetylglucosaminyltransferase responsible for the conversion of linear to branched polylactosamines in human PA1 cells was expressed in Sf9 insect cells. The catalytic ectodomain of the enzyme was fused to glutathione S-transferase, allowing effective one-step purification of the glycosylated 67-74-kDa fusion protein. Typically a yield of 750 microg of the purified protein/liter of suspension culture was obtained. The purified recombinant protein catalyzed the transfer of GlcNAc from UDP-GlcNAc to the linear tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc, converting the acceptor to the branched pentasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 GlcNAc as shown by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, degradative experiments, and 1H NMR spectroscopy of the product. By contrast, the recombinant enzyme did not catalyze any reaction when incubated with UDP-GlcNAc and the trisaccharide GlcNAcbeta1-3Galbeta1-4GlcNAc. Accordingly, we call the recombinant beta1,6-GlcNAc transferase cIGnT6 to emphasize its action at central rather than peridistal galactose residues of linear polylactosamines in the biosynthesis of blood group I antigens. Taken together this in vitro expression of I-branching enzyme, in combination with the previously cloned enzymes, beta1,4galactosyltransferase and beta1, 3N-acetylglucosaminyltransferase, should allow the general synthesis of polylactosamines based totally on the use of recombinant enzymes. 相似文献
16.
Between 30 and 50% of pig lymphocyte plasma membrane vesicles were not bound by concanavalin A (Con A)-Sepharose. Various results suggest that the Con A-unretarded fraction represents "inside-out" membrane vesicles. First, an alternative cell surface ligand, anti-lymphocytic serum, gave a similar fractionation to Con A. Second, lack of binding by Con A was not due to lack of carbohydrate or to masking of carbohydrate by extraneous protein, because the unfractionated membrane and the unretarded fraction had similar carbohydrate and polypeptide compositions. Third although the carbohydrate of the unretarded membrane vesicles was accessible to 125I-labelled Con A and to release by soluble trypsin, it was not accessible to ferritin-Con A or trypsin-Sepharose. Fourth, antisera against the external surface of the Con A-unretarded vesicles strongly agglutinated the unretarded membrane, but caused negligible agglutination of whole lymphocytes. When attached to Sepharose these antisera bound all of the Con A-unretarded fraction, but failed to bind the membrane that adhered to Con A-Sepharose. 相似文献
17.
PA Prieto RD Larsen M Cho HN Rivera A Shilatifard JB Lowe RD Cummings DF Smith 《Canadian Metallurgical Quarterly》1997,272(4):2089-2097
The human H(O) blood group is specified by the structure Fucalpha1-2Galbeta1-R, but the factors regulating expression of this determinant on cell surface glycoconjugates are not well understood. To learn more about the regulation of H blood group expression, cDNA encoding the human H-type GDPFuc:beta-D-galactoside alpha1, 2-fucosyltransferase (alpha1,2FT) was stably transfected into Chinese hamster ovary (CHO) cells. The new cell line, designated CHO(alpha1,2)FT, expressed surface neoglycans containing the H antigen. The structures of the fucosylated neoglycans in CHO(alpha1, 2)FT cells and the distribution of these glycans on glycoproteins were characterized. Seventeen percent of the [3H]Gal-labeled glycopeptides from CHO(alpha1,2)FT cells bound to the immobilized H blood group-specific lectin Ulex europaeus agglutinin-I (UEA-I), whereas none from parental CHO cells bound to the lectin. The glycopeptides from CHO(alpha1,2)FT cells binding to UEA-I contained polylactosamine [3Galbeta1-4GlcNAcbeta1-]n with the terminal sequence Fucalpha1-2Galbeta1- 4GlcNAc-R. Fucosylation of the polylactosamine sequences on complex-type N-glycans in CHO(alpha1, 2)FT cells caused a decrease in both sialylation and length of polylactosamine. Unexpectedly, only small amounts of terminal fucosylation was found in diantennary complex-type N-glycans. The O-glycans and glycolipids were not fucosylated by the H-type alpha1, 2FT. Two major high molecular weight glycoproteins, one of which was shown to be the lysosome-associated membrane glycoprotein LAMP-1, preferentially contained the H-type structure and were bound by immobilized UEA-I. These results demonstrate that in CHO cells the expressed H-type alpha1,2FT does not indiscriminately fucosylate terminal galactosyl residues in complex-type N-glycans, but it favors glycans containing polylactosamine and dramatically alters their length and sialylation. 相似文献
18.
Cell electrophoretic mobility of cultured melanoma cells or rat erythrocytes decreased with time after X-irradiation. Addition of tetravalent concanavalin A or divalent succinyl-concanavalin A before (not after) irradiation, completely blocked the mobility reduction in greater concentrations than 5 mug/l. At 5 mug/l only 3.7 - 10(3) concanavalin A molecules bound to receptors per cell, while 4.18 - 10(7) molecules/cell bound at saturating concentrations. Preincubation with concanavalin A at 37 degrees C was effective even when the cells were treated with alpha-methylmannoside immediately after irradiation. At low temperature, however, concanavalin A was not effective despite a sufficient amount of bound 125I-labelled concanavalin A. Treatment with alpha-methylmannoside following the binding of concanavalin A at 37 degrees C before irradiation inhibited the concanavalin A effect depending on temperature. The residual amount of bound lectin could not account for the temperature dependence. The amount of sialic acid (the main charged substance) was not altered by X-irradiation with or without the lectin. Divalent succinyl-concanavalin A was also effective in blocking the radiation effect on electrophoretic mobility. These results seem to suggest that binding of a very small amount of concanavalin A without causing cell agglutination or clustering of its receptors, induces some alteration in the conformation of receptor glycoprotein, which blocks the internalization of acidic sugar residues by subsequent irradiation. 相似文献
19.
Galactosyltransferase, sialyltransferase, and fucosyltransferase were used to create a panel of complex oligosaccharides that possess multiple terminal sialyl-Le(x) (NeuAc alpha 2-3Gal[Fuc alpha 1-3] beta 1-4GlcNAc) and GalNAc-Le(x) (GalNAc[Fuc alpha 1-3]beta 1-4GlcNAc). The enzymatic synthesis of tyrosinamide biantennary, triantennary, and tetraantennary N-linked oligosaccharides bearing multiple terminal sialyl-Le(x) was accomplished on the 0.5 mumol scale and the purified products were characterized by electrospray MS and 1H NMR. Likewise, biantennary and triantennary tyrosinamide oligosaccharides bearing multiple terminal GalNAc-Le(x) determinants were synthesized and similarly characterized. The transfer kinetics of human milk alpha 3/4-fucosyltransferase were compared for biantennary oligosaccharide acceptor substrates possessing Gal beta 1-4GlcNAc, GalNAc beta 1-4GlcNAc, and NeuAc alpha 2-3Gal beta 1-4GlcNAc which established NeuAc alpha 2-3Gal beta 1-4GlcNAc as the most efficient acceptor substrate. The resulting complex oligosaccharides were chemically tethered through the tyrosinamide aglycone to the surface of liposomes containing phosphatidylthioethanol, resulting in the generation of glycoliposomes probe which will be useful to study relationships between binding affinity and the micro- and macro-clustering of selectin ligand. 相似文献
20.
Two-dimensional 1H nuclear magnetic resonance spectroscopy has been used to examine the complexes formed in solution between hen egg-white lysozyme and N-acetylglucosamine (GlcNAc) oligosaccharides. Changes in chemical shift have been measured for resonances of the majority of residues of lysozyme on binding the monomer, dimer and trimer of GlcNAc. The three inhibitors induce very similar changes in chemical shift, and these increase slightly with the length of the oligosaccharide. The largest changes are confined principally to the vicinity of site C in the active site cleft of the enzyme. These changes in chemical shift have been compared with differences in the ring current chemical shifts calculated from the crystal structures of unbound and GlcNAc3 bound lysozyme. This comparison suggests that the major conformational changes of residues in the vicinity of site C of the enzyme, that are caused by the binding of GlcNAc3, observed in the diffraction studies are at least consistent with the changes that occur in solution. Small changes in chemical shift are observed in the enzyme in regions remote from the active site, which indicate that the effects of inhibitor binding are felt throughout the enzyme. These changes in chemical shift correlate to a lesser extent than those near site C with the changes in chemical shift predicted from changes in conformation observed in the crystal structures. The results illustrate that chemical shifts are useful in assessing the significance of small conformational changes in proteins, although the usefulness of this approach will be limited by the resolution of the crystallographic structures, as well as the uncertainties in the origins of the chemical shift. Although conformational changes in site C account for many of the changes in the NMR spectrum of lysozyme, evidence is, however, presented for multiple binding sites for the GlcNAc oligosaccharides in solution, perhaps involving partial occupancy of site D. 相似文献