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1.
A Herscovics A M Golovtchenko C D Warren B Bugge R W Jeanloz 《The Journal of biological chemistry》1977,252(1):224-234
Calf pancreas microsomes incorporated radioactive D-mannose from GDP-D-[14C]mannose into lipid-bound oligosaccharides extracted with chloroform/methanol/water (10/10/2.5, v/v). Several products, which probably differed in the size of the oligosaccharide moiety, were labeled. These could be partially resolved by thin layer chromatography and DEAE-cellulose chromatography. The labeled lipid-bound oligosaccharides were retained on DEAE-cellulose more strongly than synthetic dolichyl alpha-D-[14C]mannopyranosyl phosphate. They were stable to mild alkali, but labile to acid and hot alkali. Acid treatment yielded a neutral 14C-labeled oligosaccharide fraction which was estimated by gel filtration to have a minimum of 8 monosaccharide residues. Hot alkali treatment yielded a mixture of neutral and acidic 14C-labeled oligosaccharides which could be transformed into neutral products by alkaline phosphatase. The D-[14C]mannose residues were alpha-linked at the nonreducing terminus of the oligosaccharides since they could be removed completely with alpha-mannosidase. Most of the D-[14C]mannose-labeled oligosaccharides were retained on concanavalin A Sepharose and eluted with methyl alpha-D-mannopyranoside. Pancreatic dolichyl beta-D-[14C]mannopyranosyl phosphate incubated with calf pancreas microsomes in the presence of sodium taurocholate was efficiently utilized as donor of alpha-D-mannosyl residues in lipid-bound oligosaccharides. The products formed from dolichyl beta-D-[14C]mannopyranosyl phosphate were identical with those formed from GDP-D-[14C]mannose, and evidence was obtained to show that the dolichyl beta-D-[14C]mannopyranosyl phosphate was serving as donor without prior conversion to GDP-D-[14C]mannose. Transfer of mannose from dolichyl beta-D-[14C]mannopyranosyl phosphate to lipid-bound oligosaccharides took place at a pH optimum of 7.3, whereas transfer to the precipitate containing glycoproteins was greatest at pH 6.0 in Tris/maleate buffer. The addition of divalent cation was not required, but low concentrations of EDTA were extremely inhibitory. The carbohydrate composition of the lipid-bound oligosaccharides of microsomal membranes was investigated by gas-liquid chromatography and by reduction with sodium borotritide. A heterogeneous mixture of oligosaccharides containing N-acetyl-D-glucosamine, D-mannose, and D-glucose varying in proportions from approximately 1/2.5/0.5 to 1/5/1.5 was obtained with glucosamine at the reducing end. Acid treatment of the lipid-bound oligosaccharide fraction yielded dolichyl pyrophosphate, suggesting that at least some of the oligosaccharides were linked to dolichol through a pyrophosphate group. 相似文献
2.
Glycosylation in vitro of Semliki-Forest-virus and influenza-virus glycoproteins and its suppression by nucleotide-2-deoxy-hexose 总被引:3,自引:0,他引:3
Cell-free enzyme preparations from cultured fibroblasts infected with Semliki forest virus or fowl plague virus (an influenza A virus) incorporate [14C]-mannose into dolichol-phosphate-mannose, lipid-linked oligosaccharides and into endogenous virus-specific glycoproteins. When GDP-2-deoxy-D-[14C]glucose serves as substrate 2-deoxy-D-[14C]glucose is transferred to dolichol phosphate yielding dolichol-monophosphate-2-deoxy-D-[14C]glucose. UDP-2-deoxy-D-[14C]glucose gives rise also to a lipid which, however, is not a polyprenol derivative. The transfer of [14C]mannose to lipid-extractable fractions and glycoproteins in vitro is blocked by GDP-2-deoxy-D-glucose. It can be restored by exogenous dolichol monophosphate only with regard to the formation of dolichol-monophosphate-[14C]mannose-labelled oligosaccharides into glycoproteins. UDP-2-deoxy-D-glucose has no inhibitory effect on transfer reactions of [14C]mannose from GDP-[14C]mannose into various lipid fractions or into glycoprotein. It is concluded therefore, that the inhibition of glycosylation brought about by 2-deoxyglucose in vivo is caused by an interference of its GDP derivative with the formation of a correct lipid-oligosaccharide. 相似文献
3.
Binding properties of a mannose-specific lectin from the snowdrop (Galanthus nivalis) bulb 总被引:8,自引:0,他引:8
N Shibuya I J Goldstein E J Van Damme W J Peumans 《The Journal of biological chemistry》1988,263(2):728-734
Carbohydrate binding properties of a new plant lectin (GNA) isolated from snowdrop bulbs were studied using the technique of quantitative precipitation, hapten inhibition, and affinity chromatography on immobilized lectin. Purified GNA precipitated highly branched yeast mannans but did not react with most glucans. Hapten inhibition experiments showed that D-mannose is an inhibitor of GNA-mannan interaction but neither N-acetyl-D-mannosamine nor D-glucose is an inhibitor. Hapten inhibition with various sugars showed that GNA requires the presence of equatorial hydroxyl groups at the C-3 and C-4 positions and an axial group at the C-2 position of the D-pyranose ring. A nonreducing terminal D-mannose residue is necessary for the interaction of oligosaccharides, and oligosaccharides with terminal Man(alpha-1-3)Man units showed the highest inhibitory potency (10-30 times greater than D-mannose) among the manno-oligosaccharides tested. The presence of the hydrophobic p-nitrophenyl aglycone increased the affinity of D-mannose only slightly. Immobilized GNA bound yeast mannan but did not bind glycogen. The behavior of glycoproteins with high mannose type glycan chains depended on the density and the structure of their glycan chains. Glycopeptides which carry Man(alpha 1-3)Man units were retarded on the immobilized GNA column whereas those lacking this unit or with hybrid type glycan chains were not retarded on the column. 相似文献
4.
Yukihide Ohsumi Victor J Chen Sau-Chi Bettyyan Finn Wold Yuan Chuan Lee 《Glycoconjugate journal》1988,5(1):99-106
New types of neoglycoproteins, -caseins coupled with ovalbumin-derived asparagine oligosaccharides (AO), aspartate aminotransferase-phosphopyridoxylated AO complex (AAT-PG), and streptavidin-biotinylated AO complex (SA-BAO), were tested for their inhibitory effect on binding of bovine serum albumin derivatized with thiomannoside, Man-AI-BSA [Lee YC, Stowell CP, Krantz MJ (1976) Biochemistry 15:3956–63] by rabbit alveolar macrophages. The -casein derivatives and the AAT-PG complex increased binding affinity as the number of oligosaccharide chains attached was increased. Their inhibitory potencies were closely related to those of the Man-Al-BSA derivatives [Hoppe CA, Lee YC (1983) J Biol Chem 258:14193–99] on the basis of terminal mannose density. The SA-BAO complex containing three AO chains gave stronger inhibitory potency than the -casein derivative with three AO residues, suggesting that proper orientation of the oligosaccharides on the protein can affect the receptor-ligand interaction.Abbreviations BSA
bovine serum albumin
- Man43-BSA
BSA derivative containing on average 43 residues of Man linked through an amidino-linkage [7]
- AO
asparagine oligosaccharide (Man5-GlcNAc2-Asn) from ovalbumin
- AAT
aspartate aminotransferase
- PG
phosphopyridoxylated AO
- SA
streptavidin
- BAO
biotinylated AO 相似文献
5.
Action of low-density lipoprotein and compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, on the synthesis of dolichol-linked oligosaccharides and low-density-lipoprotein receptor in human skin fibroblasts. 总被引:1,自引:1,他引:0
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To explore whether there is an inter-relationship between the rate of low-density (LD) lipoprotein binding to its receptor and the formation of dolichol-linked oligosaccharides, experiments were performed with human fibroblasts where the synthesis of lipoprotein receptor and dolichyl saccharides was under control of LD lipoprotein and compactin. Pretreatment of the cells with nonlabelled LD lipoprotein resulted in a suppression of both the binding of 125I-labelled LD lipoprotein to the receptor and the synthesis of dolichyl saccharides from [14C]acetate and [3H]mannose, but not from [3H]mevalonolactone. Compactin, in contrast, inhibited only the formation of dolichol-linked oligosaccharides. Mevalonolactone (1 microM) abolished the inhibitory effect of LD lipoprotein on dolichyl saccharide formation, but was not able to restore the receptor-binding capacity, thus suggesting that the synthesis of lipoprotein receptor is not coupled to the formation of dolichyl saccharides. 相似文献
6.
Nakamura T Takada N Tonozuka T Sakano Y Oguma K Nishikawa A 《Biochimica et biophysica acta》2007,1770(4):551-555
It has been reported that Clostridium botulinum type C 16S progenitor toxin (C16S toxin) first binds to the sialic acid on the cell surface of mucin before invading cells [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells, Biochem. Biophys. Res. Commun. 319 (2004) 327-333]. In this study we investigated the binding properties of the C16S toxin to glycoproteins. Although the toxin bound to membrane blotted mucin derived from the bovine submaxillary gland (BSM), which contains a lot of sialyl oligosaccharides, it did not bind to neuraminidase-treated BSM. The binding of the toxin to BSM was inhibited by N-acetylneuraminic acid, N-glycolylneuraminic acid, and sialyl oligosaccharides strongly, but was not inhibited by neutral oligosaccharides. Both sialyl alpha2-3 lactose and sialyl alpha2-6 lactose prevented binding similarly. On the other hand, the toxin also bound well to porcine gastric mucin. In this case, neutral oligosaccharides might play an important role as ligand, since galactose and lactose inhibited binding. These results suggest that the toxin is capable of recognizing a wide variety of oligosaccharide structures. 相似文献
7.
Toshio Nakamura Noriko Takada Takashi Tonozuka Yoshiyuki Sakano Keiji Oguma Atsushi Nishikawa 《Biochimica et Biophysica Acta (BBA)/General Subjects》2007
It has been reported that Clostridium botulinum type C 16S progenitor toxin (C16S toxin) first binds to the sialic acid on the cell surface of mucin before invading cells [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells, Biochem. Biophys. Res. Commun. 319 (2004) 327–333]. In this study we investigated the binding properties of the C16S toxin to glycoproteins. Although the toxin bound to membrane blotted mucin derived from the bovine submaxillary gland (BSM), which contains a lot of sialyl oligosaccharides, it did not bind to neuraminidase-treated BSM. The binding of the toxin to BSM was inhibited by N-acetylneuraminic acid, N-glycolylneuraminic acid, and sialyl oligosaccharides strongly, but was not inhibited by neutral oligosaccharides. Both sialyl α2–3 lactose and sialyl α2–6 lactose prevented binding similarly. On the other hand, the toxin also bound well to porcine gastric mucin. In this case, neutral oligosaccharides might play an important role as ligand, since galactose and lactose inhibited binding. These results suggest that the toxin is capable of recognizing a wide variety of oligosaccharide structures. 相似文献
8.
We report the fine structure of a nearly contiguous series of N-glycans from the soil nematode Caenorhabditis elegans. Five major classes are revealed including high mannose, mammalian-type complex, hybrid, fuco-pausimannosidic (five mannose residues or fewer substituted with fucose), and phosphocholine oligosaccharides. The high mannose, complex, and hybrid N-glycan series show a high degree of conservation with the mammalian biosynthetic pathways. The fuco-pausimannosidic glycans contain a novel terminal fucose substitution of mannose. The phosphocholine oligosaccharides are high mannose type and are multiply substituted with phosphocholine. Although phosphocholine oligosaccharides are known immunomodulators in human nematode and trematode infections, C. elegans is unique as a non-parasitic nematode containing phosphocholine N-glycans. Therefore, studies in C. elegans should aid in the elucidation of the biosynthetic pathway(s) of this class of biomedically relevant compounds. Results presented here show that C. elegans has a functional orthologue for nearly every known enzyme found to be deficient in congenital disorders of glycosylation types I and II. This nematode is well characterized genetically and developmentally. Therefore, elucidation of its N-glycome, as shown in this report, may place it among the useful systems used to investigate human disorders of glycoconjugate synthesis such as the congenital disorders of glycosylation syndromes. 相似文献
9.
The isolation of phosphomannan-protein complexes from the viable cells of yeast (Y) and mycelial (M) forms of Candida albicans NIH B-792 strain was conducted by treatment with Zymolyase-100T followed by fractional precipitation with cetyltrimethylammonium bromide. The M-form complex was found to contain smaller amount of phosphate (1.3%) than that of the Y-form complex (1.6%). Proton magnetic resonance (PMR) spectra of these complexes indicated that the content of beta-1,2-linked oligomannosyl and nonreducing terminal alpha-1,3-linked mannopyranosyl residues in the M-form complex was lower than that of the Y-form complex. With hot 10 mM HCl, the Y-form complex released a mixture of oligosaccharides ranging from mannose to mannoheptaose, while the M-form complex produced lower oligosaccharides, from mannose to mannotetraose. Upon acetolysis, the acid-modified complex of the M form gave mainly mannotetraose, while that of the Y form produced mainly mannopentaose and mannohexaose in addition to mannotetraose. The average length of branching moieties of the mannan of Y-form cells was therefore longer than that of M-form cells. These results indicate that the Y to M transformation of this C. albicans strain accompanies the suppression of enzyme activity concerning the biosynthesis of mannan such as beta-1,2- and alpha-1,3-mannosyltransferases to synthesize the phosphomannan-protein complex containing mannan moiety with incomplete structure. 相似文献
10.
Feinberg H Castelli R Drickamer K Seeberger PH Weis WI 《The Journal of biological chemistry》2007,282(6):4202-4209
The dendritic cell surface receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR specifically recognize high mannose N-linked carbohydrates on viral pathogens. Previous studies have shown that these receptors bind the outer trimannose branch Manalpha1-3[Manalpha1-6]Manalpha present in high mannose structures. Although the trimannoside binds to DC-SIGN or DC-SIGNR more strongly than mannose, additional affinity enhancements are observed in the presence of one or more Manalpha1-2Manalpha moieties on the nonreducing termini of oligomannose structures. The molecular basis of this enhancement has been investigated by determining crystal structures of DC-SIGN bound to a synthetic six-mannose fragment of a high mannose N-linked oligosaccharide, Manalpha1-2Manalpha1-3[Manalpha1-2Manalpha1-6]Manalpha1-6Man and to the disaccharide Manalpha1-2Man. The structures reveal mixtures of two binding modes in each case. Each mode features typical C-type lectin binding at the principal Ca2+-binding site by one mannose residue. In addition, other sugar residues form contacts unique to each binding mode. These results suggest that the affinity enhancement displayed toward oligosaccharides decorated with the Manalpha1-2Manalpha structure is due in part to multiple binding modes at the primary Ca2+ site, which provide both additional contacts and a statistical (entropic) enhancement of binding. 相似文献
11.
Cell-free enzyme particles from mung bean seedlings catalyze the incorporation of mannose from GDP-[14C]mannose and GlcNAc from UDP-[3H]GlcNAc into glycolipids and into glycoprotein. The most rapidly labeled product from GDP-mannose was characterized as a mannosyl-phosphoryl-polyisoprenol, whereas that from UDP-GlcNAc was a mixture of GlcNAc-(pyro)phosphoryl-polyisoprenol and a disaccharide composed of two N-acetylglucosamine residues attached to the polyisoprenol by a phosphoryl or pyrophosphoryl linkage. Radioactivity from GDP-mannose and UDP-GlcNAc was also incorporated into more polar lipids which have been partially characterized as a series of oligosaccharide-(pyro)phosphoryl-lipids. The mannose-labeled oligosaccharides released from these lipids by mild acid hydrolysis were found to contain GlcNAc at their reducing end indicating that these oligosaccharides contain both GlcNAc and mannose. Both the GlcNAc-labeled and the mannose-labeled oligosaccharides gave multiple radioactive peaks upon paper chromatography indicating that they are composed of a series of different sized oligosaccharides. Finally, radioactivity from GDP-[14C]mannose and UDP-[3H]GlcNAc is incorporated into an insoluble component. Ten percent of the mannose label and all of the GlcNAc label in this insoluble material could be solubilized by digestion with Pronase. The glycopeptides released by Pronase digestion appeared to be approximately the same size as the oligosaccharides from the lipid-linked oligosaccharides based on gel filtration chromatography on Sephadex G-50. The results are consistent with a mechanism for glycoprotein synthesis involving lipid-linked oligosaccharide intermediates. 相似文献
12.
Glycoprotein biosynthesis in plants. Demonstration of lipid-linked oligosaccharides of mannose and N-acetylglucosamine. 总被引:13,自引:0,他引:13
Previous studies from this laboratory have shown that particulate preparations from maturing cotton fibers catalyze the transfer of mannose from GDP-[14C]mannose into mannosylphosphorylpolyisoprenol (Forsee, W. T., and Elbein,A. D. (1973) J. Biol. Chem. 248, 2858-2867). In this report, we show that these particulate preparations also catalyze the inocoporation of mannose from GDP-[14C]mannose into lipid-linked oligosaccharides and into glycoprotein. The oligosaccharide-lipids were treated with dilute acid to liberate the water-soluble oligosaccharides and these oligosaccharides could then be separated into seven or eight distinct radioactive peaks by paper chromatography in isobutyric acid/NH4OH/H2betaO (57/4/39). The smallest of the oligosaccharides appears to be a trisaccharide with the structure Man leads to GlcNAc-GlcNAc. Thus the oligosaccharides attached to the lipids apparently range in size from those having 3 glycose units to those having approximately 8 to 10 glycose units. The radioactivity in the smaller-sized oligosaccharide-lipids could be chased into the larger oligosaccharide-lipids by a second incubation in the presence of unlabeled GDP-mannose. The sugar at the reducing ends of the oligosaccharides was identified as GlcNAc while some mannose (20 to 30%) was present in alpha linkages at the nonreducing ends... 相似文献
13.
Both the Entamoeba histolytica lectin, a virulence factor for the causative
agent of amebiasis, and the mammalian hepatic lectin bind to
N-acetylgalactosamine (GalNAc) and galactose (Gal) nonreducing termini on
oligosaccharides, with preference for GalNAc. Polyvalent GalNAc-
derivatized neoglycoproteins have >1000-fold enhanced binding affinity
for both lectins (Adler,P., Wood,S.J., Lee,Y.C., Lee,R.T., Petri,W.A.,Jr.
and Schnaar,R.L.,1995, J. Biol. Chem ., 270, 5164-5171). Substructural
specificity studies revealed that the 3-OH and 4-OH groups of GalNAc were
required for binding to both lectins, whereas only the E.histolytica lectin
required the 6-OH group. Whereas GalNAc binds with 4-fold lower affinity to
the E.histolytica lectin than to the mammalian hepatic lectin,
galactosamine and N-benzoyl galactosamine bind with higher affinity to the
E. histolytica lectin. Therefore, a synthetic scheme for converting
polyamine carriers to poly-N-acyl galactosamine derivatives (linked through
the galactosamine primary amino group) was developed to test whether such
ligands would bind the E.histolytica lectin with high specificity and high
affinity. Contrary to expectations, polyvalent derivatives including
GalN6lys5, GalN4desmosine, GalN4StarburstTMdendrimer, and
GalN8StarburstTMdendrimer demonstrated highly enhanced binding to the
mammalian hepatic lectin but little or no enhancement of binding to the
E.histolytica lectin. We propose that the mammalian hepatic lectin binds
with greatest affinity to GalNAc "miniclusters," which mimic branched
termini of N-linked oligosaccharides, whereas the E.histolytica lectin
binds most effectively to "maxiclusters," which may mimic more widely
spaced GalNAc residues on intestinal mucins.
相似文献
14.
V Pimpaneau P Midoux G Durand P De Baetselier M Monsigny A -C Roche 《Glycoconjugate journal》1989,6(4):561-574
Macrophages from various origins are known to express membrane lectins that mediate the endocytosis of mannose-bearing glycoconjugates. Most macrophage tumor cell-lines lack such receptors. In this paper we show by flow cytometry analysis that a newly generated macrophage hybridoma (2C11–12), which displays several macrophage characteristics, also expresses mannose membrane lectins, resulting in the internalization of fluoresceinylated neoglycoproteins into acidic compartments.Thioglycolate elicited mouse peritoneal macrophages and the 2C11–12 hybridomas were compared by flow cytometry with regard to the binding and endocytosis of 1-acid glycoprotein (AGP) variants separated by affinity chromatography on immobilized concanavalin A. AGP C eluted specifically with methyl -mannopyranoside, which contains two bi-antennary oligosaccharides, was endocytosed as mannosylated serum albumin (Man-BSA). In both types of macrophages, the fluoresceinylated ligands were internalized in acidic compartments as demonstrated by the fluorescence intensity increase upon monensin post-incubation. However the behaviour of the internalized ligands was found to be quite different. AGP C and Man-BSA were rapidly degraded by thioglycolate elicited peritoneal macrophages and excreted in the medium as small peptide fragments; conversely they remained a longer time in the 2C11–12 hybridoma. 相似文献
15.
Specificity of the mannosyltransferase which initiates outer chain formation in Saccharomyces cerevisiae. 总被引:1,自引:0,他引:1
The in vitro specificity of the alpha 1-6 mannosyltransferase that initiates outer chain formation in Saccharomyces cerevisiae (Romero and Herscovics, J. Biol. Chem., 264, 1946-1950, 1989) was reassessed by fast atom bombardment mass spectrometry (FAB-MS). A particulate fraction from the mnn1 mutant was incubated with GDP-mannose and either Man9GlcNAc (M9T) isolated from thyroglobulin or Man8GlcNAc (M8Y) obtained by treatment of the M9T with the yeast specific mannosidase. The Man10GlcNAc (M10Y) and Man9GlcNAc (M9Y) oligosaccharides thus obtained, and the substrate oligosaccharides, were peracetylated or perdeuteroacetylated and submitted to FAB-MS using meta-nitrobenzylalcohol as the matrix. The latter was chosen as the matrix because it enhances the abundance of high-mass-fragment ions of peracetylated oligosaccharides and thereby facilitates the assignment of branching patterns. The results indicate that the alpha 1-6 mannosyltransferase catalyses the addition of mannose to the alpha 1-3 mannose residue, and thus provide additional new evidence to support the revised structure of yeast mannoproteins proposed by Hernandez et al. (J. Biol. Chem., 264, 11849-11856, 1989). [formula: see text] where Gn is N-acetylglucosamine, M is mannose and M is mannose added by the enzyme. 相似文献
16.
Lactoferrin (LF) is an iron-binding glycoprotein present in the cytoplasmic granules of neutrophils and in external secretions of mammals. Although the biological role of human and bovine lactoferrin has been extensively studied, there is still uncertainty as to the nature and function of lactoferrin receptors. We recently determined that methyl-alpha-D-mannopyranoside given intraperitoneally (i.p.) could suppress the adjuvant activity of LF in the generation of delayed-type hypersensitivity (DTH) to ovalbumin (OVA). We concluded that the lactoferrin effects in DTH are mediated by carbohydrate-recognizing receptors like the mannose receptor (MR). This study indicates that subcutaneous (s.c.) administration of very small doses of the Man-bovine serum albumin (Man-BSA) complex, together with a sensitizing dose of the antigen, gives the same effects as i.p. administration of methyl-alpha-D-mannopyranoside. The latter is also a blocker of MR, although of a much lower affinity to the receptor than Man-BSA. The blocking of the adjuvant effect of LF by the Man-BSA complex (when given together with the sensitising dose of antigen) suggests that the function of antigen-presenting cells in the skin (presumably immature dendritic cells expressing MR) is inhibited. The results of our study indicate that a receptor with an affinity for mannose is essential for the mediation of adjuvant lactoferrin function in the generation of DTH. 相似文献
17.
Studies were initiated to determine whether the formation of lipid-linked oligosaccharides was coupled to the synthesis of protein. Canine kidney cells were grown with [2-3H]mannose or [3H]leucine in the presence of cycloheximide or puromycin and the effect of these inhibitors on the synthesis of proteins and lipid-linked oligosaccharides was measured. In all cases, the inhibition of protein synthesis resulted in a substantial inhibition in the incorporation of mannose into the lipid-linked oligosaccharides, although the synthesis of mannosyl-phosphoryl-dolichol was only slightly inhibited. Cycloheximide had no effect on the in vitro incorporation of mannose into lipid-linked oligosaccharides when GDP-[14C]mannose was incubated with aorta microsomal preparations. The inhibition of lipid-linked oligosaccharides was apparently not due to a decrease in the amount of glycosyltransferases as a result of protein degradation in the absence of protein synthesis, nor was it the result of a more rapid degradation of lipid-linked oligosaccharides. The inhibition also did not appear to be due to limitations in the available dolichyl-phosphate. The results suggest that the formation of lipid-linked oligosaccharides may be regulated by end product inhibition. 相似文献
18.
Protein determinants impair recognition of procathepsin L phosphorylated oligosaccharides by the cation-independent mannose 6-phosphate receptor 总被引:3,自引:0,他引:3
Cathepsin L, a lysosomal cysteine protease, is the major excreted protein of transformed mouse NIH 3T3 cells. Previous studies have shown that asparagine-linked oligosaccharides associated with the secreted hydrolase contain mannose 6-phosphate (Man 6-P), the recognition marker for transport of newly synthesized acid hydrolases to lysosomes. To investigate the mechanism by which cathepsin L evades targeting to lysosomes, we determined the structure of the enzyme's oligosaccharides and analyzed its interaction with the cation-independent mannose 6-phosphate (Man 6-PCl) receptor. Oligosaccharides associated with procathepsin L isolated from the medium of [3H]mannose-labeled J774 cells were remarkably homogeneous; all of the radiolabeled structures were high mannose-type units that contained two phosphomonoesters and 7 mannose residues. Both the alpha 1,3- and alpha 1,6-branches of the oligosaccharides were phosphorylated. Oligosaccharides released by endoglycosidase H from [3H]mannose-labeled procathepsin L bound to a Man 6-PCl receptor affinity column. Despite the high affinity binding of these oligosaccharides, the intact glycoprotein was not a good ligand for the Man 6-PCl receptor. Procathepsin L was internalized poorly by Man 6-P receptor-mediated endocytosis and the purified acid protease interacted weakly with a Man 6-PCl affinity column. In contrast, pro-beta-glucuronidase (another acid hydrolase produced by J774 cells) was an excellent ligand for the Man 6-PCl receptor as judged by the endocytosis and affinity chromatographic assays. Phosphorylated oligosaccharides associated with the J774-secreted pro-beta-glucuronidase were heterogeneous and contained both mono- and diphosphorylated species. Tryptic glycopeptides generated from [3H]mannose-labeled procathepsin L, unlike the intact protein, were excellent ligands for the Man 6-PCl receptor. The results indicate that oligosaccharides associated with procathepsin L are processed uniformly to diphosphorylated species that bind with high affinity to the Man 6-PCl receptor. Protein determinants inherent within the intact acid hydrolase, however, inhibit the high affinity binding of these oligosaccharides and, as a result, impair the interaction of procathepsin L with the receptor. 相似文献
19.
Terada M Khoo KH Inoue R Chen CI Yamada K Sakaguchi H Kadowaki N Ma BY Oka S Kawasaki T Kawasaki N 《The Journal of biological chemistry》2005,280(12):10897-10913
Mannan-binding protein (MBP) is a C-type serum lectin and activates complement through the lectin pathway when it binds to ligand sugars such as mannose, N-acetylglucosamine, and fucose on microbes. In addition, the vaccinia virus carrying the human MBP gene was shown to exhibit potent growth inhibitory activity toward human colorectal carcinoma, SW1116, cells in nude mice. We have proposed calling this activity MBP-dependent cell-mediated cytotoxicity (MDCC) (Ma, Y., Uemura, K., Oka, S., Kozutsumi, Y., Kawasaki, N., and Kawasaki, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 371-375). In this study, the MBP ligands on the surface of SW1116 cells were characterized. Initial experiments involving plant lectins and anti-Lewis antibodies as inhibitors of MBP binding to SW1116 cells indicated that fucose plays a crucial role in the interaction. Subsequently, Pronase glycopeptides were prepared from whole cell lysates, and oligosaccharides were liberated by hydrazinolysis. After being tagged by pyridylamination, MBP ligand oligosaccharides were isolated with an MBP affinity column, and then their sequences were determined by mass spectrometry and tandem mass spectrometry after permethylation, in combination with endo-beta-galactosidase digestion and chemical defucosylation. The MBP ligands were shown to be large, multiantennary N-glycans carrying a highly fucosylated polylactosamine type structure. At the nonreducing termini, Le(b)/Le(a) or tandem repeats of the Le(a) structure prevail, a substantial proportion of which are attached via internal Le(x) or N-acetyllactosamine units to the trimannosyl core. The structures characterized are unique and distinct from those of other previously reported tumor-specific carbohydrate antigens. It is concluded that MBP requires clusters of tandem repeats of the Le(b)/Le(a) epitope for recognition. 相似文献
20.
We recently reported that tri- and tetraantennary complex type oligosaccharides with nonreducing terminal galactose residues and the triantennary asialofetuin glycopeptide can bind and precipitate certain galactose specific lectins (L. Bhattacharyya, and C.F. Brewer (1986) Biochem. Biophys. Res. Commun. 141, 963-967; L. Bhattacharyya, M. Haraldsson, and C.F. Brewer (1988) Biochemistry 27, 1034-1041). The present study investigates the binding interactions of two of these lectins, those from Erythrina indica and Ricinus communis (Agglutinin I), with mono-, bi-, and triantennary synthetic cluster glycosides, which have little structural resemblance to complex type oligosaccharides other than they possess nonreducing terminal galactose residues (R.T. Lee, P. Lin, and Y.C. Lee (1984) Biochemistry 23, 4255-4261). The enhanced affinities of the bi- and triantennary glycosides relative to the monoantennary glycoside for the two lectins are consistent with an increase in the probability of binding due to multiple binding residues in the multiantennary glycosides. The triantennary glycoside is capable of precipitating the two lectins, and quantitative precipitation data indicate that it is a trivalent ligand. The results show that the binding and precipitation activities of complex type oligosaccharides with these lectins is due solely to the presence of multiple terminal galactose residues and not to the overall structures of the oligosaccharides. 相似文献