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1.
The copolymeric structure of pig skin dermatan sulphate. Isolation and characterization of l-idurono-sulphate-containing oligosaccharides from copolymeric chains 总被引:3,自引:3,他引:0
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Lars-?ke Fransson Lars C?ster Birgitta Havsmark Anders Malmstr?m Ingrid Sj?berg 《The Biochemical journal》1974,143(2):379-389
Dermatan sulphate was degraded by testicular hyaluronidase and an oversulphated fraction was isolated by ion-exchange chromatography. This preparation, which contained fairly long segments derived from the non-reducing terminal portion of the molecule, was subjected to periodate oxidation under acidic conditions. The oxidized iduronic acid residues were cleaved by reduction-hydrolysis (Smith-degradation) (Fransson & Carlstedt, 1974) or by alkaline elimination. The oligosaccharides so obtained contained both GlcUA (glucuronic acid) and IdUA-SO(4) (sulphated iduronic acid) residues. Copolymeric oligosaccharides obtained after alkaline elimination were cleaved by chondroitinase-AC into disaccharide and higher oligosaccharides. Since the corresponding oligosaccharides obtained by Smith-degradation were unaffected by this enzyme, it was concluded that the carbohydrate sequences were GalNAc-(IdUA-GalNAc)(n)-GlcUA-GalNAc. The iduronic acid-containing sequences were resistant to digestion with chondroitinase-ABC. It was demonstrated that the presence of unsulphated N-acetylgalactosamine residues in these sequences could be responsible for the observed effect. This information was obtained in an indirect way. Chemically desulphated dermatan sulphate was found to be a poor substrate for the chondroitinase-ABC enzyme. Moreover, digestion with chondroitinase-ABC of chondroitinase-AC-degraded dermatan sulphate released periodate-resistant iduronic acid-containing oligosaccharides. It is concluded that copolymeric sequences of the following structure are present in pig skin dermatan sulphate: [Formula: see text] N-acetylgalactosamine moieties surrounding IdUA-SO(4) residues are unsulphated to a large extent. 相似文献
2.
Sulphation of proteins secreted by a human hepatoma-derived cell line. Sulphation of N-linked oligosaccharides on alpha 2HS-glycoprotein. 总被引:1,自引:1,他引:0
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Several human glycoproteins, including alpha 1-antitrypsin, alpha 1-acid glycoprotein, transferrin, caeruloplasmin and alpha 2HS-glycoprotein, synthesized by the hepatoma-derived cell line HepG2 were observed to contain covalently linked sulphate. These proteins were estimated to contain about 0.1 mol of sulphate/mol of protein. The most abundant of the sulphated glycoproteins, alpha 2HS-glycoprotein, was analysed in detail. All of the sulphate on this protein was attached to N-linked oligosaccharides which contained sialic acid and resisted release by endoglycosidase H. Several independent analytical approaches established that approx. 10% of the molecules of alpha 2HS-glycoprotein contained sulphate. Our results suggest that a number of human plasma proteins contain small amounts of sulphate linked to oligosaccharides. 相似文献
3.
The copolymeric structure of pig skin dermatan sulphate. Characterization of d-glucuronic acid-containing oligosaccharides isolated after controlled degradation of oxydermatan sulphate
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Lars-?ke Fransson Lars C?ster Anders Malmstr?m Ingrid Sj?berg 《The Biochemical journal》1974,143(2):369-378
Selective periodate oxidation of unsubstituted l-iduronic acid residues in copolymeric dermatan sulphate chains was followed by reduction-hydrolysis or alkaline elimination. By this procedure the glucuronic acid-containing periods were isolated in oligosaccharide form; general formula: [Formula: see text] Further degradation of these oligosaccharides with chondroitinase-AC yielded three types of products: (a) sulphated trisaccharide containing an unsaturated uronosyl moiety in the non-reducing terminal and a C(4) fragment in the reducing terminal, DeltaUA-GalNAc-(-SO(4))-R; (b) monosulphated, unsaturated disaccharide, DeltaUA-GalNAc-SO(4) when n is greater than or equal to 2; and (c) N-acetylgalactosamine with or without sulphate. Oligosaccharides containing a single glucuronic acid residue (n=1) comprised more than half of the glucuronic acid-containing oligosaccharides. The terminal N-acetylgalactosamine moiety of the shortest oligosaccharide was largely 4-sulphated, whereas higher oligosaccharides primarily contained 6-sulphated or unsulphated hexosamine moieties in the same position. Moreover, IdUA-SO(4)-containing oligosaccharides were encountered. These oligosaccharides were resistant to the action of chondroitinase-ABC. 相似文献
4.
The synthesis and secretion of chondroitin sulphate proteoglycan (CSPG) was examined in human muscle cultures during myogenesis prior to myoblast fusion and following myotube formation. Results from this study demonstrate that the major CSPG secreted into the medium had a Kav of 0.15 on Sephacryl 500 (exclusion limit of 10(7) Da) and contained predominantly unsulphated residues in mononucleated cell cultures but these became increasingly sulphated in postfusion cultures. Fibroblasts synthesised small amounts of a smaller molecular weight CSPG indicating that the Kav 0.15 proteoglycan is solely synthesised by cells of the myogenic lineage. These findings illustrate that sulphation of CSPG is developmentally regulated during myogenesis of human muscle cells grown under differentiating conditions. 相似文献
5.
Variable (Fv) domain N-glycosylation sites are found in approximately 20% of human immunoglobulin Gs (IgGs) in addition to the conserved N-glycosylation sites in the C(H)2 domains. The carbohydrate structures of the Fv glycans and their impact on in vivo half-life are not well characterized. Oligosaccharide structures in a humanized anti-Abeta IgG1 monoclonal antibody (Mab) with an N-glycosylation site in the complementary determining region (CDR2) of the heavy chain variable region were elucidated by LC/MS analysis following sequential exoglycosidase treatments of the endoproteinase Lys-C digest. Results showed that the major N-linked oligosaccharide structures in the Fv region have three characteristics (core-fucosylated biantennary oligosaccharides with one or two N-glycolylneuraminic acid [NeuGc] residues, zero or one alpha-linked Gal residue, and zero or one beta-linked GalNAc residue), whereas N-linked oligosaccharides in the Fc region contained typical Fc glycans (core-fucosylated, biantennary oligosaccharides with zero to two Gal residues). To elucidate the contribution of Fv glycans to the half-life of the antibody, a method that allows capture of the Mab and determination of its glycan structures at various time points after administration to mice was developed. Anti-Abeta antibody in mouse serum was immunocaptured by immobilized goat anti-human immunoglobulin Fc(gamma) antibody resin, and the captured material was treated with papain to generate Fab and Fc for LC/MS analysis. Different glycans in the Fc region showed the same clearance rate as demonstrated previously. In contrast to many other non-antibody glycosylated therapeutics, there is no strong correlation between oligosaccharide structures in the Fv region and their clearance rates in vivo. Our data indicated that biantennary oligosaccharides lacking galactosylation had slightly faster clearance rates than other structures in the Fv domain. 相似文献
6.
N-Glycosylation of a mouse IgG expressed in transgenic tobacco plants 总被引:11,自引:0,他引:11
Cabanes-Macheteau M Fitchette-Lainé AC Loutelier-Bourhis C Lange C Vine ND Ma JK Lerouge P Faye L 《Glycobiology》1999,9(4):365-372
Since plants are emerging as an important system for the expression of recombinant glycoproteins, especially those intended for therapeutic purposes, it is important to scrutinize to what extent glycans harbored by mammalian glycoproteins produced in transgenic plants differ from their natural counterpart. We report here the first detailed analysis of the glycosylation of a functional mammalian glycoprotein expressed in a transgenic plant. The structures of the N-linked glycans attached to the heavy chains of the monoclonal antibody Guy's 13 produced in transgenic tobacco plants (plantibody Guy's 13) were identified and compared to those found in the corresponding IgG1 of murine origin. Both N-glycosylation sites located on the heavy chain of the plantibody Guy's 13 are N-glycosylated as in mouse. However, the number of Guy's 13 glycoforms is higher in the plant than in the mammalian expression system. Despite the high structural diversity of the plantibody N-glycans, glycosylation appears to be sufficient for the production of a soluble and biologically active IgG in the plant system. In addition to high-mannose-type N-glycans, 60% of the oligosaccharides N-linked to the plantibody have beta(1, 2)-xylose and alpha(1, 3)-fucose residues linked to the core Man3GlcNAc2. These plant-specific oligosaccharide structures are not a limitation to the use of plantibody Guy's 13 for topical immunotherapy. However, their immunogenicity may raise concerns for systemic applications of plantibodies in human. 相似文献
7.
T Oho F Rahemtulla B M?nsson-Rahemtulla A Hjerpe 《The International journal of biochemistry》1992,24(7):1159-1168
1. A glycosylated proline-rich protein (GPRP) was purified to homogeneity by subjecting parotid saliva to immunoaffinity, cation exchange, affinity and hydrophobic chromatography. 2. The purified GPRP had a molecular weight of 78 kDa as analyzed by SDS-PAGE. 3. The amino acid analysis revealed a preponderance of proline, glycine and glutamic acid/glutamine, which accounted for 77% of the total amino acids. 4. Cysteine, tyrosine or phenylalanine residues were not detected. 5. The glycoprotein contained 34% neutral sugars and the oligosaccharides were rich in mannose and N-acetylglucosamine, indicating that N-linked oligosaccharides were the predominant type of oligosaccharides in the molecule. 6. These observations were confirmed by treatment of the purified glycoprotein with specific N-glycosidase which removed the N-linked oligosaccharides leaving a core protein with an apparent molecular weight of 51 kDa. 7. The isoelectric point of GPRP was approx 7.0 and the molecule was not affected by reduction with 2-mercaptoethanol, indicating that no disulfide linkages were present. 8. The GPRP bound to hydroxyapatite and this binding could be partially inhibited by preincubation of the hydroxyapatite with parotid or submandibular saliva. 9. The purified GPRP also bound to a protein with an apparent molecular weight of 95 kDa present in submandibular saliva. 相似文献
8.
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
9.
《The Journal of cell biology》1987,105(6):2655-2664
The trans-Golgi has been recognized as having a key role in terminal glycosylation and sorting of proteins. Here we show that tyrosine sulfation, a frequent modification of secretory proteins, occurs specifically in the trans-Golgi. The heavy chain of immunoglobulin M (IgM) produced by hybridoma cells was found to contain tyrosine sulfate. This finding allowed the comparison of the state of sulfation of the heavy chain with the state of processing of its N-linked oligosaccharides. First, the pre-trans-Golgi forms of the IgM heavy chain, which lacked galactose and sialic acid, were unsulfated, whereas the trans-Golgi form, identified by the presence of galactose and sialic acid, and the secreted form of the IgM heavy chain were sulfated. Second, the earliest form of the heavy chain detectable by sulfate labeling, as well as the heavy chain sulfated in a cell-free system in the absence of vesicle transport, already contained galactose and sialic acid. Third, sulfate-labeled IgM moved to the cell surface with kinetics identical to those of galactose-labeled IgM. Lastly, IgM labeled with sulfate at 20 degrees C was not transported to the cell surface at 20 degrees C but reached the cell surface at 37 degrees C. The data suggest that within the trans-Golgi, tyrosine sulfation of IgM occurred at least in part after terminal glycosylation and therefore appeared to be the last modification of this constitutively secreted protein before its exit from this compartment. Furthermore, the results establish the covalent modification of amino acid side chains as a novel function of the trans-Golgi. 相似文献
10.
Site-specific N-glycosylation of chicken serum IgG 总被引:2,自引:0,他引:2
Avian serum immunoglobulin (IgG or IgY) is functionally equivalent to mammalian IgG but has one additional constant region domain (CH2) in its heavy (H) chain. In chicken IgG, each H-chain contains two potential N-glycosylation sites located on CH2 and CH3 domains. To clarify characteristics of N-glycosylation on avian IgG, we analyze N-glycans from chicken serum IgG by derivatization with 2-aminopyridine (PA) and identified by HPLC and MALDI-TOF-MS. There were two types of N-glycans: (1) high-mannose-type oligosaccharides (monoglucosylated 26.8%, others 10.5%) and (2) biantennary complex-type oligosaccharides (neutral, 29.9%; monosialyl, 29.3%; disialyl, 3.7%) on molar basis of total N-glycans. To investigate the site-specific localization of different N-glycans, chicken serum IgG was digested with papain and separated into Fab [containing variable regions (VH + VL) + CH1 + CL] and Fc (containing CH3 + CH4) fragments. Con A stained only Fc (CH3 + CH4) and RCA-I stained only Fab fractions, suggesting that high-mannose-type oligosaccharides were located on Fc (CH3 + CH4) fragments, and variable regions of Fab contains complex-type N-glycans. MS analysis of chicken IgG-glycopeptides revealed that chicken CH3 domain (structurally equivalent to mammalian CH2 domain) contained only high-mannose-type oligosaccharides, whereas chicken CH2 domain contained only complex-type N-glycans. The N-glycosylation pattern on avian IgG is more analogous to that in mammalian IgE than IgG, presumably reflecting the structural similarity to mammalian IgE. 相似文献
11.
Oligosaccharide units of lysosomal cathepsin D from porcine spleen. Amino acid sequence and carbohydrate structure of the glycopeptides 总被引:5,自引:0,他引:5
The amino acid sequences near the glycosylation sites and the oligosaccharide structures have been determined for the lysosomal protease cathepsin D from porcine spleen. Cathepsin D light and heavy chains were separately digested with proteases and the glycopeptides were purified. A single sequence was constructed from the amino acid sequence of the light chain glycopeptides which is: Tyr-Asn-Ser-Gly-Lys-Ser-Ser-Thr-Tyr-Val-Lys-Asn(CH2O)-Gly-Thr-Thr-Phe. A single glycopeptide sequence was also obtained for the heavy chain: Lys-Gly-Ser-Leu-Asp-Tyr-His-Asn(CH2O)-Val-Thr-Arg-Lys-Ala-Tyr. The light chain sequence is homologous with the sequence of porcine pepsin from residues 56 to 71. The heavy chain sequence is homologous with the pepsin sequence from residues 176 to 189. Thus, the 2 oligosaccharide-linked asparagines in cathepsin D correspond to residues 67 and 183 in pepsin and other homologous aspartyl proteases. These positions are located on the surface of the crystal structures of aspartyl proteases. Five oligosaccharides linked to Asn-67 were separated and their structures determined with proton NMR. Four major oligosaccharides are structural variants from the high mannose-type having 3, 5, 6, and 7 mannoses, respectively. A minor structure contained a third GlcNAc. Three oligosaccharide structures were found linked to Asn-183. Two major oligosaccharides are of the high mannose-type each with 5 mannose residues. One of the two contains a fucose linked to a GlcNAc. A third, very minor oligosaccharide contains galactose. 相似文献
12.
Types of oligosaccharide sulphation, depending on mucus glycoprotein source, corpus or antral, in rat stomach.
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Radiolabelled mucus glycoprotein was obtained from tissue and a culture medium each of the corpus and antrum of rat stomach incubated with [35S]sulphate in vitro. Gel-filtration analysis of oligosaccharides liberated by alkaline-borohydride treatment from glycoproteins indicated that 35S-labelled oligosaccharides from the corpus vary considerably with respect to chain length whereas those from antral mucus glycoprotein are composed of small oligosaccharides. Examination of the reduced radiolabelled products obtained by HNO2 cleavage of the hydrazine-treated oligosaccharides indicated sulphate esters of N-acetylglucosamine to be present at three locations on a carbohydrate unit: [35S]sulphated monosaccharide (2,5-anhydromannitol 6-sulphate), [35S]sulphated disaccharide [galactosyl(beta 1-4)-2,5-anhydromannitol 6-sulphate] and [35S]sulphated trisaccharide [fucosyl(alpha 1-2)-galactosyl(beta 1-4)-2,5-anhydromannitol 6-sulphate]. Sulphated disaccharide and trisaccharide, possibly originating from the N-acetyl-lactosamine and fucosyl-N-acetyl-lactosamine sequences respectively, were detected in the corpus, especially as large oligosaccharides, but were present in the antrum in only very small amounts. The sulphated monosaccharide, however, most probably originating from 6-sulphated N-acetylglucosamine residues at non-reducing termini, was present in all oligosaccharide fractions in both the corpus and antrum. 相似文献
13.
R Doi K Inoue M Kogire S Sumi M Yun S Futaki N Fujii H Yajima T Tobe 《Biochemical and biophysical research communications》1988,153(3):1209-1213
To evaluate the role of the sulphated tyrosine residue in position 27 in human cholecystokinin-33, parallel bioassay of the sulphated form of human cholecystokinin-33 and the unsulphated form of human cholecystokinin-33 was performed on the pancreatic protein secretion. Both peptides increased the protein output in a dose-related manner. However, the sulphated form possessed a considerably higher activity than the sulphated form. The relative potency of the unsulphated human cholecystokinin-33 compared to that of the sulphated human cholecystokinin-33 (taken as 1.0) was 0.08. From the results, it was suggested that the sulphated tyrosine may play an important role in controlling the activity of the longer molecular forms as well as that of the smaller forms of cholecystokinin. 相似文献
14.
Hannele Merivuori Jeffrey A. Sands Bland S. Montenecourt 《Applied microbiology and biotechnology》1985,23(1):60-66
Summary The effects of tunicamycin, an inhibi-tor of N-asparagine linked glycosylation, on the synthesis, secretion, and activities
of the cellulases produced byTrichoderma reesei wild type QM6a and hypersecrefing mutant RL-P37 were studied. Neither the level of secreted cellutase nor the total amount
of secreted protein was affected by the drug at a concentration (5 μg/ml) that slightly in-hibited growth. SDS-polyacrylamide
gel electro-phoretic mobilities of proteins secreted during growth in tunicamycin were similar to those of proteins from control
cultures that had their N-linked oligosaccharides removed by endoglycosi-dase H. Isoelectric focusing patterns of secreted
proteins were also altered by growth in the pres-ence of tunicamycin. All of the bands stained with Schiff’s reagent, indicating
that the secreted cellu-lases contained O-linked oligosaccharides in ad-dition to N-linked sugars. Endoglucanase activity
in culture broths from tunicamycin grown mycelia was more thermolabile and protease-sensitive than the same activity from
control cultures. Thus, N-asparagine linked oligosaccharides do not appear to be necessary forT. reesei cellulase secretion or activity, but do seem to contribute to the stability of the enzymes. The role of O-finked oligosaccharides
is being investigated. 相似文献
15.
Glycoproteins often display a complex isoelectric focusing profile because of the presence of negatively charged carbohydrates, such as sialic acid, phosphorylated mannose, and sulfated GalNAc. Until now, understanding the role of these charged carbohydrates in determining the isoelectric focusing profile has been limited to observing pattern shifts following complete removal of the sugars in question. We have developed a simple and sensitive method for analyzing N-linked oligosaccharides from the individual isoelectric focusing bands of a glycoprotein using recombinant human thyroid-stimulating hormone as a model system. N-linked oligosaccharides were released and profiled from individual bands following electroblotting of isoelectric focusing gels. As might be predicted, high-pH anion-exchange chromatography-pulsed amperometric detection and matrix-assisted laser desorption/ionization-time of flight analyses indicated that the bands that migrated closer to the positive electrode contained more sialylated N-linked oligosaccharides. The sialic acid content of these bands correlated with that predicted from the corresponding oligosaccharide analyses. 相似文献
16.
The structure of the keratan sulphate chains attached to fibromodulin from human articular cartilage
The small keratan sulphate proteoglycan, fibromodulin, has been isolated from pooled human articular cartilage. The main chain
repeat region and the chain caps from the attached N-linked keratan sulphate chains have been fragmented by keratanase II
digestion, and the oligosaccharides generated have been reduced and isolated. Their structures and abundance have been determined
by high pH anion-exchange chromatography.
These regions of the keratan sulphate from human articular cartilage fibromodulin have been found to have the following general
structure:
Significantly, both α(2-6)- and α(2-3)-linked N-acetyl-neuraminic acid have been found in the capping oligosaccharides. Fucose,
which is α(1-3)-linked as a branch to N-acetylglucosamine, has also been found along the length of the repeat region and in
the capping region. The chains, which have been found to be very highly sulphated, are short; the length of the repeat region
and chain caps is ca. nine disaccharides.
These data demonstrate that the structure of the N-linked keratan sulphate chains of human articular cartilage fibromodulin
is similar, in general, to articular cartilage derived O-linked keratan sulphate chains. Further, the general structure of
the keratan sulphate chains attached to human articular cartilage fibromodulin has been found to be generally similar to that
of both bovine and equine articular cartilage fibromodulin.
Abbreviations: KS, keratan sulphate; IEC, ion-exchange chromatography; ELISA, enzyme linked immunosorbent assay; Gal, β-D-galactose;
Fuc, α-L-Fucose; GlcNAc, N-acetylglucosamine (2-acetamido-β-D-glucose); GlcNAc-ol, N-acetylglucosaminitol (2-acetamido-D-glucitol);
NeuAc, N-acetyl-neuraminic acid; 6S/(6S), O-ester sulphate group on C6 present/sometimes present; NMR -nuclear magnetic resonance;
HPAE, high pH anion-exchange; PED, pulsed electrochemical detection; HPLC, high performance liquid chromatography
This revised version was published online in November 2006 with corrections to the Cover Date. 相似文献
17.
Degradation of human gastrin and CCK by endopeptidase 24.11: differential behaviour of the sulphated and unsulphated peptides 总被引:1,自引:0,他引:1
S Pauwels T Najdovski R Dimaline C M Lee M Deschodt-Lanckman 《Biochimica et biophysica acta》1989,996(1-2):82-88
The degradation of human sulphated heptadecapeptide gastrin (G17s) by human endopeptidase 24.11 was studied in vitro. The products of degradation were characterized by HPLC, region-specific gastrin radioimmunoassay and amino acid analysis. The enzyme cleaved G17s at four sites, Trp4-Leu5, Ala11-Tyr12, Gly13-Trp14 and Asp16-Phe17. The patterns of fragments produced when sulphated and unsulphated G17s are hydrolysed by endopeptidase 24.11 indicate that the enzyme cleaves both substrates at the same four bonds. However, the sulphated G17 was 3-times less rapidly degraded than the unsulphated G17 (G17ns). In contrast, the rate of cleavage of the octapeptide cholecystokinin (CCK8) was faster when the peptide was sulphated. The kinetic data of endopeptidase 24.11 indicated similar Km values for sulphated or unsulphated gastrin and CCK; sulphated CCK8 exhibited a 2-fold higher kcat/Km value compared to unsulphated CCK8, whereas G17s exhibited a 2-fold lower kcat/Km value compared to G17ns. The results indicate that the presence of a sulphate group causes a marked reduction in the rate of hydrolysis of gastrin by endopeptidase 24.11, whereas sulphation enhances cholecystokinin degradation by the same enzyme. They also suggest that endopeptidase 24.11 may be responsible for the difference in metabolism of sulphated and unsulphated G17, previously observed in human circulation. 相似文献
18.
Vervecken W Kaigorodov V Callewaert N Geysens S De Vusser K Contreras R 《Applied and environmental microbiology》2004,70(5):2639-2646
The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an alpha-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and beta-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides. 相似文献
19.
1. A multienzyme system capable of degrading keratosulphates to yield galactose, N-acetylglucosamine and sulphate was found in the liver extract of a marine gastropod, Charonia lampas. 2. During the degradation, neither oligosaccharides nor sulphated sugars were produced. 3. It is suggested that the degradation could be attributed to the concerted action of beta-galactosidase, beta-N-acetylglucosaminidase and a sulphatase (sulphohydrolase), tentatively designated keratosulphatase. 4. Two forms of keratosulphatase (I and II) were separated by DEAE-Sephadex column chromatography. Both forms could release all the sulphate from keratosulphates and neither appeared to be identical with glycosulphatase or chondrosulphatase, both of which are also present in Charonia lampas. 5. beta-Galactosidase and beta-N-acetylglucosaminidase could degrade keratopolysulphate to a greater extent in the presence of keratosulphatase than in its absence. 6. It is suggested that keratosulphate was first desulphated by the action of keratosulphatase, and the desulphated polymer was then degraded to galactose and N-acetylglucosamine by the action of beta-galactosidase and beta-N-acetylglucosaminidase. 7. beta-Galactosidase alone released a small amount of galactose from shark cartilage keratopolysulphate, but beta-N-acetylglucosaminidase alone did not release N-acetylglucosamine. This indicates that unsulphated galactose residues occupy all the non-reducing terminal positions in keratopolysulphate chains. 相似文献
20.
Very-high-field n.m.r. studies of bovine lung heparan sulphate tetrasaccharides produced by nitrous acid deaminative cleavage. Determination of saccharide sequence, uronate composition and degrees of sulphation. 总被引:2,自引:2,他引:0
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Tetrasaccharides with the general structure UA-GlcNAc-GlcUA-aManOH (where UA represents uronate, GlcNAc N-acetylglucosamine, GlcUA glucuronate and aManOH anhydromannitol) were prepared from low-sulphated heparan sulphates of bovine lung origin by complete nitrous acid deaminative cleavage followed by reduction and fractionated by gel filtration. Ion-exchange chromatography of the tetrasaccharides yielded three major fractions in approximate yields of 37%, 45% and 14%. These were shown to be non-, mono- and di-sulphated respectively. Complete structural characterization of the tetrasaccharide fractions by quantitative high-field n.m.r. spectroscopy showed that each fraction contained only two discrete species and led to the following observations. (1) All of the uronate residues in the tetrasaccharides (and in larger oligosaccharides) are unsulphated, and hence sulphated iduronate [IdUA(2SO3)] must occur exclusively within -GlcNSO3-IdUA(2SO3)-GlcNSO3- sequences (where GlcNSO3 represents N-sulpho-glucosamine) in the parent polymers. (2) The GlcNAc residues in the tetrasaccharides are more highly C-6-O-sulphated than are the aManOH residues, and furthermore sulphation on the aManOH appears to occur only where the GlcNAc is also sulphated. (3) Where the GlcNAc is C-6-O-sulphated, iduronate is the major non-reducing terminal residue, whereas glucuronate predominates in this position if the GlcNAc is unsulphated. The quantitative data obtained are used to determine the degree of C-6-O-sulphation of glucosamine residues in specific sequences within the parent heparan sulphates. 相似文献