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1.
Howard S. Judelson Randall L. Dimond 《Differentiation; research in biological diversity》1988,37(1):7-13
Abstract. Changes during the development of Dictyostelium discoideum , in the abundance, synthesis, and cell-type-specific distribution of modifications on N-linked oligosaccharides, were measured using specific affinity probes for N-linked moeities. Total proteins and individual lysosomal enzymes were reacted with three monoclonal antibodies raised against Dictyostelium proteins (recognizing epitopes containing mannose 6-sulfate, sulfated N-acetylglucosamine, and an undefined but unsulfated N-linked group, respectively), the mammalian 215-kDa phosphomannosyl receptor, and Con A. Independent and dramatic changes in the reaction of the antibodies and phosphomannosyl receptor with protein were observed during development, whereas modest changes were observed in Con A binding. The two sulfated antigens, but not the other moeities, were reduced preferentially in prestalk and mature stalk cells. The lysosomal enzyme β-glucosidase, which is synthesized late in development, binds poorly to the phosphomannosyl receptor and contains little of the three antigens. The subcellular transport of lysosomal enzymes also changes during development, as most are not targeted to lysosomes as is normal, but are secreted in precursor form. 相似文献
2.
Hudson H. Freeze 《Molecular and cellular biochemistry》1986,72(1-2):47-65
This paper has two purposes. The first is to review the past studies on the structure, biosynthesis, and immunological properties of a class of glycoproteins, the lysosomal enzymes, in Dictyostelium discoideum. The second purpose is to present new data on the analysis of mutant strains altered in the biosynthesis of the lipid-linked precursor of N-linked oligosaccharides, and on the characterization of new carbohydrate antigenic determinants found on multiple proteins in Dictyostelium. We will also show how a combination of genetic, biochemical and immunochemical approaches have been used to unravel a portion of the glycosylation pathway in Dictyostelium.The long-term goal of these studies is to use Dictyostelium discoideum as a model system to understand the functions of a variety of glycoconjugates in a multicellular organism. The existence of a large number of mutant strains which are altered in a variety of cellular functions, development and the posttranslational modification of multiple proteins, offers a great opportunity to explore this area. 相似文献
3.
Mannose 6-sulfate is present in the N-linked oligosaccharides of lysosomal enzymes of Dictyostelium 总被引:4,自引:0,他引:4
H H Freeze 《Archives of biochemistry and biophysics》1985,243(2):690-693
The major N-linked, anionic oligosaccharide found on several lysosomal enzymes of Dictyostelium discoideum contains five charges, composed of three sulfate esters and two residues of Man-6-P in phosphodiester linkage. Most of the SO4 was found as Man-6-SO4. This novel sulfated sugar was detected and quantitated by measuring the appearance of 3,6-anhydromannitol following acid hydrolysis and reduction of base-treated, reduced oligosaccharides. If SO4 is removed by solvolysis prior to the base treatment, the anhydrosugar is not formed, indicating that its presence is not an artifact of the procedure. That these oligosaccharides are derived from standard high-mannose-type oligosaccharides indicates that only one or, at most, two Man residues are unsubstituted at the 6-position. 相似文献
4.
Dictyostelium discoideum synthesizes many highly immunogenic carbohydrates of unknown structure and function. We have used monoclonal antibodies prepared against one of these called CA1 to investigate its structure and the consequences of its loss. CA1 is preferentially expressed on lysosomal enzymes as a specific arrangement of mannose-6-SO4 residues on N-linked oligosaccharides. Mutant strains HL241 and HL243 do not express CA1, and synthesize a truncated lipid-linked oligosaccharide (LLO) precursor that lacks the critical mannose residues needed for expression. The lesion appears to result from the loss of mannosyl transferase activity involved in LLO biosynthesis. The truncated LLO is poorly transferred to an artificial peptide acceptor in a cell-free N-glycosylation assay, and this appears to result from improper topological localization of the LLO or to a lower affinity of the LLO for the oligosaccharyl transferase. Although both mutants share these lesions, they are biochemically and genetically distinct. Only HL243 is lower in N-glycosylation in intact cells, and this is not a result of an altered structure of the LLO. There are other differences between the strains. HL241 can form fruiting bodies at a slower rate than normal while HL243 cannot aggregate. Genetic analysis of defects shows that the CA1 lesion in HL241 is recessive, while the lesion in both CA1 and in development are dominant and co-segregate in HL243 and are, therefore, likely to be in the same gene. Lysosomal enzyme targeting is normal but enzyme processing proceeds at a 2-3 fold slower rate in HL241 and HL243 compared to wild-type. Strain HL244 does not express CA1 since it completely lacks protein sulfation, but lysosomal enzyme targeting and processing proceeds at a normal rate, showing that sulfate is not essential for these processes. Alterations in oligosaccharide structure can have individualized effects on the biosynthesis of lysosomal enzymes. The results presented here illustrate how this approach can be used to study both the structure and function of carbohydrate epitopes. 相似文献
5.
Structural analysis of N-linked oligosaccharides from glycoproteins secreted by Dictyostelium discoideum. Identification of mannose 6-sulfate 总被引:4,自引:0,他引:4
The N-linked oligosaccharides found on the lysosomal enzymes from Dictyostelium discoideum are highly sulfated and contain methylphosphomannosyl residues (Gabel, C. A., Costello, C. E., Reinhold, V. N., Kurtz, L., and Kornfeld, S. (1984) J. Biol. Chem. 259, 13762-13769). Here we report studies done on the structure of N-linked oligosaccharides found on proteins secreted during growth, a major portion of which are lysosomal enzymes. Cells were metabolically labeled with [2-3H]Man and 35SO4 and a portion of the oligosaccharides were released by a sequential digestion with endoglycosidase H followed by endoglycosidase/peptide N-glycosidase F preparations. The oligosaccharides were separated by anion exchange high performance liquid chromatography into fractions containing from one up to six negative charges. Some of the oligosaccharides contained only sulfate esters or phosphodiesters, but most contained both. Less than 2% of the oligosaccharides contained a phosphomonoester or an acid-sensitive phosphodiester typical of the mammalian lysosomal enzymes. A combination of acid and base hydrolysis suggested that most of the sulfate esters were linked to primary hydroxyl groups. The presence of Man-6-SO4 was demonstrated by the appearance of 3,6-anhydromannose in acid hydrolysates of base-treated, reduced oligosaccharides. These residues were not detected in acid hydrolysates without prior base treatment or in oligosaccharides first treated by solvolysis to remove sulfate esters. Based on high performance liquid chromatography quantitation of percentage of 3H label found in 3,6-anhydromannose, it is likely that Man-6-SO4 accounts for the majority of the sulfated sugars in the oligosaccharides released from the secreted glycoproteins. 相似文献
6.
Sulfated oligosaccharides block antibodies to many Dictyostelium discoideum acid hydrolases 总被引:3,自引:0,他引:3
H H Freeze R C Mierendorf R Wunderlich R L Dimond 《The Journal of biological chemistry》1984,259(16):10641-10643
The lysosomal hydrolases of the cellular slime mold, Dictyostelium discoideum, possess a common posttranslational modification which is extremely antigenic in rabbits and mice. Rabbit antisera and mouse monoclonal antibodies that recognize this determinant cross-react with a group of at least 40-50 highly negatively charged proteins which include most or all of the lysosomal enzymes. (Knecht, D. A., Dimond, R. L., Wheeler, S., and Loomis, W. F. (1984) J. Biol. Chem. 259, 10633-10640). The present study demonstrates that the determinant is found on certain N-linked oligosaccharides derived from one of these proteins. An esterified sulfate is absolutely required for antigenicity. 相似文献
7.
The effects of altered N-linked oligosaccharide structures on maturation and targeting of lysosomal enzymes in Dictyostelium discoideum 总被引:2,自引:0,他引:2
H H Freeze P Koza-Taylor A Saunders J A Cardelli 《The Journal of biological chemistry》1989,264(32):19278-19286
We have examined the relationship of N-linked oligosaccharide structures to the proper targeting and proteolytic processing of two lysosomal enzymes, alpha-mannosidase and beta-glucosidase, in the slime mold Dictyostelium discoideum. Two different mutant strains, HL241 and HL243, each synthesize the same nonglucosylated, truncated, lipid-linked oligosaccharide precursor, Man6GlcNAc2. [3H]Mannose-labeled N-linked oligosaccharides were studied following their release from immunoprecipitated alpha-mannosidase and beta-glucosidase by digestion with peptide:N-glycosidase F. The oligosaccharides from both mutants resembled each other, but they were smaller and contained fewer anionic groups than those from the wild-type. The oligosaccharides from the mutants strains were reduced in sulfate and Man-6-P content, and all Man-6-P was in the form of acid-stable phosphodiesters. Pulse-chase radiolabeling experiments using [35S] methionine indicated that the precursor forms of both enzymes were smaller than wild-type, and that this difference was due solely to differences in N-linked oligosaccharides. The precursor forms of the enzymes were not over-secreted, but appeared to be proteolytically processed into mature forms at approximately 50% the rate of wild-type. This is mainly due to their prolonged retention in the rough endoplasmic reticulum, but, ultimately, both enzymes were properly targeted to lysosomes. These studies indicate that a reduction in the amount of sulfation, phosphorylation or size of the N-linked oligosaccharides in these mutants is not critical for the proteolytic processing and targeting of the lysosomal enzymes, but that these changes may influence their rate of exit from the rough endoplasmic reticulum. 相似文献
8.
Lysosomal enzymes in Dictyostelium discoideum contain high mannose oligosaccharides that contain mannose 6-phosphate and several unusual structures. The synthesis and distribution of these post-translational modifications were studied using probes for different carbohydrate groups. These probes include lectin-like antibodies directed to two distinct sulfated and one nonsulfated N-linked determinants, the lectin Con A, and the mammalian 215-kDa phosphomannosyl receptor. Only Con A binds to newly synthesized alpha-mannosidase present in the rough endoplasmic reticulum. The other modifications are acquired at different rates and are first detected on protein in light density Golgi-like membranes. Mutations which prevent protein transport to Golgi membranes block synthesis of these moieties, but inhibitors which prevent later transport steps have no effect. The majority of modified proteins are in lysosomes but significant amounts are delivered to nonlysosomal destinations. Different lysosomal proteins contain unequal amounts of each modification. 相似文献
9.
Production of hybrid glycoproteins and accumulation of oligosaccharides in the brain of sheep and pigs administered swainsonine or locoweed 总被引:5,自引:0,他引:5
D R Tulsiani H P Broquist L F James O Touster 《Archives of biochemistry and biophysics》1988,264(2):607-617
Swainsonine and swainsonine-containing plants produce biochemical and neurological changes in several mammalian species. The toxin is a potent inhibitor of liver lysosomal alpha-D-mannosidase and Golgi mannosidase II. The inhibition of the latter enzyme causes the production of abnormal glycoproteins containing hybrid oligosaccharides instead of complex types in a variety of cultured cells. In view of the widespread occurrence and biological importance of N-linked glycoproteins in the central nervous system, we initiated studies to determine the structure of oligosaccharides in glycoproteins prepared from the brain of control, swainsonine-fed, and locoweed-fed animals. The results presented here indicate that the feeding led to alteration in the structure of brain glycoproteins. Over 25% of the glycoproteins which presumably contained complex-type oligosaccharides were modified and now contained hybrid oligosaccharides. The structure of the N-linked oligosaccharide (glycopeptide) was established by (a) studying the binding properties of the glycopeptide to immobilized lectins of known sugar specificity, and (b) comparing the size of the glycopeptide before and after treatment with exo- and endoglycosidases. The production of hybrid oligosaccharides occurred despite the apparent absence of mannosidase II in brain. The relationships of the altered structure of brain glycoproteins, accumulation of mannose-rich oligosaccharides in the brain, and abnormal behavior of the animals administered swainsonine or locoweed are discussed. 相似文献
10.
The in vivo specificity for E-selectin binding to a panel of N-linked oligosaccharides containing a clustered array of one to four sialyl Lewisx (SLex; NeuAcalpha2-3Gal[Fucalpha1-3]beta1-4GlcNAc) determinants was studied in mice. Following intraperitoneal dosing with lipopolysaccharide, radioiodinated tyrosinamide N-linked oligosaccharides were dosed i.v. and analyzed for their pharmacokinetics and biodistribution. Specific targeting was determined from the degree of SLex oligosaccharide targeting relative to a sialyl oligosaccharide control. Oligosaccharides targeted the kidney with the greatest selectivity after a 4-h induction period following lipopolysaccharide dosing. Unique pharmacokinetic profiles were identified for SLex biantennary and triantennary oligosaccharides but not for monovalent and tetraantennary SLex oligosaccharides or sialyl oligosaccharide controls. Biodistribution studies established that both SLex biantennary and triantennary oligosaccharides distributed to the kidney with 2-3-fold selectivity over sialyl oligosaccharide controls, whereas monovalent and tetraantennary SLex oligosaccharides failed to mediate specific kidney targeting. Simultaneous dosing of SLex biantennary or triantennary oligosaccharide with a mouse anti-E-selectin monoclonal antibody blocked kidney targeting, whereas co-administration with anti-P-selectin monoclonal antibody did not significantly block kidney targeting. The results suggest that SLex biantennary and triantennary are N-linked oligosaccharide ligands for E-selectin and implicate E-selectin as a bivalent receptor in the murine kidney endothelium. 相似文献
11.
Cathepsin D is a bilobed lysosomal aspartyl protease that contains one Asn-linked oligosaccharide/lobe. Each lobe also contains protein determinants that serve as recognition domains for binding of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the first enzyme in the biosynthesis of the mannose 6-phosphate residues on lysosomal enzymes. In this study we examined whether the location of the protein recognition domain influences the relative phosphorylation of the amino and carboxyl lobe oligosaccharides. To do this, chimeric proteins containing either amino or carboxyl lobe sequences of cathepsin D substituted into a glycosylated form of the homologous secretory protein pepsinogen were expressed in Xenopus oocytes. The amino and carboxyl lobe oligosaccharides were then isolated from the various chimeric proteins and independently analyzed for their mannose 6-phosphate content. This analysis has shown that a phosphotransferase recognition domain located on either lobe of a cathepsin D/glycopepsinogen chimeric molecule is sufficient to allow phosphorylation of oligosaccharides on both lobes. However, phosphorylation of the oligosaccharide on the lobe containing the recognition domain is favored. We also found that the majority of the carboxyl lobe oligosaccharides of cathepsin D acquire two phosphates, whereas the amino lobe oligosaccharides only acquire one phosphate. 相似文献
12.
The mod A mutant of Dictyostelium discoideum is missing the alpha 1,3-glucosidase involved in asparagine-linked oligosaccharide processing 总被引:1,自引:0,他引:1
H H Freeze R Yeh A L Miller S Kornfeld 《The Journal of biological chemistry》1983,258(24):14880-14884
The recessive mutation, mod A, in the Dictyostelium discoideum strain M31 results in an alteration in the post-translational modification of lysosomal enzymes. We now report studies which indicate that mod A is deficient in glucosidase II, an enzyme which is involved in the processing of asparagine-linked oligosaccharides. [2-3H]Mannose-labeled glycopeptides were prepared from three purified mod A lysosomal enzymes and compared to the equivalent glycopeptides from parental enzymes. The mod A glycopeptides were deficient in high mannose oligosaccharides containing two phosphomannosyl residues and accumulated oligosaccharides with one phosphomannosyl residue. The phosphate was present in the form of an acid-stable phosphodiester in both instances. There was also an increase in the amount of nonphosphorylated high mannose oligosaccharides mod A and these were larger than the corresponding material from the parental enzymes. In addition, the nonphosphorylated oligosaccharides were only partially degraded by alpha-mannosidase, indicating the presence of a blocking moiety. In vitro enzyme assays demonstrated that the mod A cells cannot remove the inner 1 leads to 3-linked glucose from a glucosylated high mannose oligosaccharide. The cells are also deficient in membrane-bound neutral p-nitrophenyl-alpha-D-glucosidase activity. This activity has been attributed to glucosidase II in other systems. Removal of the outer 1 leads to 2-linked glucose from Glc3Man9Glc-NAc2 is normal, demonstrating the presence of glucosidase I activity. We conclude from these data that M31 cells are deficient in glucosidase II, the enzyme which removes the two inner glucose residues from the glucosylated oligosaccharides of newly glycosylated proteins. This defect can explain the mod A phenotype and is proposed to be the primary genetic defect in these cells. 相似文献
13.
Oligosaccharide heterogeneity of glycoproteins sulfated during the vegetative growth of Dictyostelium discoideum 总被引:1,自引:0,他引:1
S J Davis 《Journal of cellular biochemistry》1988,38(2):77-86
Macromolecules are sulfated during the vegetative growth of Dictyostelium discoideum. A characterisation of the structures of sulfated oligosaccharides associated with these macromolecules indicates that the oligosaccharides are heterogeneous. Endoglycosidase and pronase digestion were used with gel-filtration chromatography to obtain two different oligosaccharide fractions and a glycopeptide fraction; these were further characterised by ion-exchange and lectin-affinity chromatography and by acid hydrolysis. The data indicate that up to 43% of the sulfate is associated with typical N-linked oligosaccharides, that up to 5% is associated with N-linked oligosaccharides that are either very large or extremely highly charged, and that the remaining sulfate is associated with oligosaccharides non-N-linked to protein. Each fraction was also shown to be heterogeneous at most other structural levels. Electrophoretic analyses following the endoglycosidase and pronase treatments indicated that all of the macromolecules are glycoproteins and suggested further that at least two of the oligosaccharide fractions are located on different groups of glycoproteins. 相似文献
14.
Lipovitellin II (Lv II), the major yolk protein of the anomuran crab Emerita asiatica, was purified using heparin-sepharose affinity column chromatography. The purified Lv II was a glycoprotein as it was stainable with periodic acid-Schiff's reagent. Quantitative analysis of sugars showed the presence of fucose, mannose, galactosamine, N-linked oligosaccharides, as well as O-linked oligosaccharides containing N-acetyl hexosamine as the terminal residue. The amount of N-linked oligosaccharides is higher than that of the O-linked oligosaccharides. Biogel P-4 column chromatographic separation of the radiolabeled oligosaccharides of Lv II showed the presence of five different O-linked oligosaccharides and four different N-linked oligosaccharide species. HPTLC separation of the neoglycolipids prepared from the O-linked oligosaccharides also showed the presence of five different O-linked oligosaccharide species. N-linked oligosaccharides contain significant quantities of mannose. Unisil column chromatographic purification in conjunction with HPTLC separation revealed three neutral glycolipid species such as monoglycosylceramide, diglycosylceramide, and triglycosylceramide in the Lv II. The functional significance of these carbohydrate components of the major yolk protein during embryogenesis of the sand crab is discussed. 相似文献
15.
A mucus-secreting human colonic cancer cell line. Purification and partial characterization of the secreted mucins.
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J J Maoret J Font C Augeron P Codogno C Bauvy M Aubery C L Laboisse 《The Biochemical journal》1989,258(3):793-799
A stably differentiated clonal derivative (Cl.16E) of the human colonic adenocarcinoma cell line HT29 secretes in culture high-Mr glycoproteins that were purified from the serum-free conditioned medium by preparative SDS/polyacrylamide-gel electrophoresis. Analysis of the oligosaccharides released from the [3H]glucosamine-labelled high-Mr glycoproteins by alkaline-borohydride treatment showed that this material consisted of O-linked oligosaccharides (without any detectable N-linked oligosaccharides) that were eluted as three fractions from Bio-Gel P-6 columns. The main oligosaccharide fraction obtained after such treatment and desialylation was eluted together with a six-unit glucose polymer from a Bio-Gel P-4 column. Polyclonal antibodies were raised against the high-Mr glycoproteins, and in immunoblot analysis they reacted specifically with the high-Mr glycoproteins present in the conditioned medium. Furthermore, immunohistochemical staining of sections in paraffin wax revealed that these antibodies labelled normal human gastrointestinal mucins. We conclude that (1) the high-Mr glycoproteins prepared by SDS/polyacrylamide-gel electrophoresis are pure mucus glycoproteins on the basis of sensitivity to alkaline-borohydride treatment, monosaccharide composition and immunochemical and immunohistological findings, and (2) these mucins have antigenic determinants in common with the normal human gastrointestinal mucins. 相似文献
16.
S Masutani N Miyazawa S Fujii A Nishikawa H Matsukawa T Shimano T Mori N Taniguchi 《Analytical biochemistry》1990,188(1):149-154
Two monoclonal antibodies to an N-linked oligosaccharide, MT-5 and MT-9, have been prepared by immunization with a pyridylaminated, asialylated, galactosylated, fucosylated, bisected biantennary sugar. The reactivity of these antibodies was monitored by their reaction with human asialoglycophorin in a solid-phase enzyme-linked immunosorbent assay. Both antibodies reacted with the sugar chains of various human glycoproteins such as immunoglobulin G, transferrin, gamma-glutamyl transpeptidase, alpha 1-acid glycoprotein, and alpha-fetoprotein. Treatment of asialoglycophorin with beta-N-acetylhexosaminidase or alpha-mannosidase resulted in reduction of the binding to these antibodies. The reactivity of MT-5 to asialoglycophorin was slightly inhibited by D-mannose and N-acetylglucosamine, whereas that of MT-9 was inhibited by D-mannose, N-acetyl-D-glucosamine, chitobiose, and L-fucose. The epitope specificity of MT-5 appears to be a sugar chain containing biantennary N-acetyl-D-glucosamine residues, the bisected N-acetyl-D-glucosamine residue, and a trimannosyl core. The epitope to which MT-9 is directed may be a complex made up of beta-mannose, chitobiose, and L-fucose. These studies indicate that immunization with pyridylaminated sugars can produce antibodies that recognize N-linked oligosaccharides. Monoclonal/polyclonal antibodies to the N-linked sugar chains of glycopeptides would be useful in such studies of proteins. 相似文献
17.
Structural study of asparagine-linked oligosaccharide moiety of taste-modifying protein, miraculin 总被引:3,自引:0,他引:3
N Takahashi H Hitotsuya H Hanzawa Y Arata Y Kurihara 《The Journal of biological chemistry》1990,265(14):7793-7798
The structures of the N-linked oligosaccharides of miraculin, which is a taste modifying glycoprotein isolated from miracle fruits, berries of Richadella dulcifica, are reported. Asparagine-linked oligosaccharides were released from the protein by glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by high performance liquid chromatography (HPLC) on an ODS-silica column. More than five kinds of oligosaccharide fractions were separated by the one chromatographic run. The structure of each oligosaccharide thus isolated was analyzed by a combination of sequential exoglycosidase digestion and another kind of HPLC with an amidesilica column. Furthermore, high resolution proton nuclear magnetic resonance (1H NMR) measurements were carried out. It was found that 1) five oligosaccharides obtained are a series of compounds with xylose-containing common structural core, Xyl beta 1----2 (Man alpha 1----6) Man beta 1----4-GlcNAc beta 1----4 (Fuca1----3)GlcNAc, 2) a variety of oligosaccharide structures are significant for two glycosylation sites, Asn-42 and Asn-186, and 3) two new oligosaccharides, B and D, with unusual structures containing monoantennary complex-type were characterized. (formula; see text) 相似文献
18.
Site specificity of the chorionic gonadotropin N-linked oligosaccharides in signal transduction 总被引:12,自引:0,他引:12
The role of the human chorionic gonadotropin (hCG) N-linked oligosaccharides in receptor binding and signal transduction was analyzed using site-directed mutagenesis and transfection studies. hCG derivatives with alterations at individual glycosylation sites were expressed in Chinese hamster ovary cells. Receptor binding studies showed that absence of any or all of the hCG N-linked oligosaccharides had only a minor effect on the receptor affinity of the derivatives. Similarly, absence of the N-linked oligosaccharides from the beta subunit or a single oligosaccharide from Asn-78 of alpha had no effect on the production of cAMP or on steroidogenesis. However, the absence of carbohydrate at Asn-52 of alpha decreases both the steroidogenic and cAMP responses. Furthermore, absence of this critical oligosaccharide unit on alpha unmasks differences in the two N-linked oligosaccharides on beta; the beta Asn-13 oligosaccharide but not the beta Asn-30 oligosaccharide plays a more important role in steroidogenesis. Dimers containing deglycosylated beta subunit and an alpha subunit lacking either the Asn-52 oligosaccharide or both oligosaccharides fail to stimulate cAMP or steroid formation. Moreover, these derivatives bind to receptor and behave as competitive antagonists. The use of site-directed mutagenesis was critical in uncovering site-specific functions of the hCG N-linked oligosaccharides in signal transduction and reveals the importance of the Asn-52 oligosaccharide in this process. 相似文献
19.
Monoclonal antibodies specific for oligosaccharides prepared by partial nitrous acid deamination of heparin 总被引:5,自引:0,他引:5
G Pejler U Lindahl O Larm E Scholander E Sandgren A Lundblad 《The Journal of biological chemistry》1988,263(11):5197-5201
Monoclonal antibodies were raised against a conjugate between heparin oligosaccharides and human serum albumin. The oligosaccharides were prepared by partial nitrous acid degradation of heparin and were coupled to human serum albumin by reductive amination. Characterization of the antibodies secreted by one of the resulting clones showed that they recognize a determinant present in the oligosaccharide antigen, but not in intact heparin, nor in a variety of related polysaccharides. Degradation of heparin by nitrous acid generates a 2,5-anhydro-D-mannose residue at the reducing end of the resulting oligosaccharides, and it is concluded that this structure is essential for interaction with the antibodies. Reduced oligosaccharides (containing a terminal anhydromannitol residue) are also active. After gel chromatography of partially degraded heparin, the smallest components capable of binding to the antibodies were found in a tetrasaccharide fraction. Affinity chromatography on immobilized monoclonal antibodies separated this tetrasaccharide fraction into distinct populations of binding and nonbinding species. Structural analysis showed that the tetrasaccharide fraction that bound to the monoclonal antibodies contained one single component with the structure IdoA(2-OSO3)-GlcNSO3 (6-OSO3)-IdoA(2-OSO3)-aManR(6-OSO3), whereas the fraction that did not bind to the antibodies contained a mixture of different structures. 相似文献
20.
The N-linked oligosaccharides on three lysosomal enzymes in Dictyostelium discoideum were found to contain mannose 6-phosphomethyl residues. We have identified and partially characterized a novel S-adenosylmethionine-dependent methyltransferase that is probably responsible for the synthesis of this unusual diester from Man-6-P. The enzyme selectively methylates the phosphate group of Man-6-P (Km 4.3 mM). Glucose-6-P and fructose-1-P are relatively poor acceptors; however, the enzyme is inactive against a broad array of other phosphorylated compounds. Using model di-, tri-, and pentasaccharide acceptors that include portions of the three different branches of high mannose-type oligosaccharides, we found that the enzyme prefers terminal alpha 1----2-linked Man-6-P residues (Km 0.15-1.25 mM) found on the known phosphorylated branches. The enzyme is membrane bound, has a neutral pH optimum and cofractionates on sucrose gradients with GlcNAc-1-P transferase, which resembles its mammalian counterpart, and is, presumably, the first enzyme in the phosphorylation pathway. Based on the substrate specificity and colocalization with GlcNAc-1-P transferase, the phosphate methyltransferase is likely to be responsible for the generation of mannose-6-phosphomethyldiester on Dictyostelium oligosaccharides. 相似文献