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1.
Characterization of terminal NeuNAcalpha2-3Galbeta1-4GlcNAc sequence in lipooligosaccharides of Neisseria meningitidis 总被引:1,自引:0,他引:1
Group B and C Neisseria meningitidis are the major cause of meningococcal
disease in the United States and in Europe. N . meningitidis
lipooligosaccharide (LOS), a major surface antigen, can be divided into 12
immunotypes of which L1 through L8 were found among Group B and C
organisms. Groups B and C but not Group A may sialylate their LOSs with
N-acetylneuraminic acid (NeuNAc) at the nonreducing end because they
synthesize CMP-NeuNAc. Using sialic acid-galactose binding lectins as
probes in an ELISA format, six of the eight LOS immunotypes (L2, L3, L4,
L5, L7, and L8) in Groups B and C bound specifically to Maackia amurensis
leukoagglutinin (MAL), which recognizes NeuNAcalpha2- 3Galbeta1-4GlcNAc/Glc
sequence, but not to Sambucus nigra agglutinin, which binds
NeuNAcalpha2-6Gal sequence. The combination of SDS-PAGE and MAL-blot
analyses revealed that these six LOSs contained only the
NeuNAcalpha2-3Galbeta1-4GlcNAc trisaccharide sequence in their 4.1 kDa LOS
components, which have a common terminal lacto-N-neotetraose (LNnT,
Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) structure when nonsialylated as shown
by previous studies. The LOS-lectin binding was abolished when the LOSs
were treated with Newcastle disease viral neuraminidase which cleaves
alpha2-->3 linked sialic acid. Methylation analysis of a representative
LOS (L2) confirmed that NeuNAc is 2-->3 linked to Gal. Thus, these LOSs
structurally mimic certain glycolipids, i.e., paragloboside (LNnT-ceramide)
and sialylparagloboside and some glycoproteins in having LNnT and
N-acetyllactosamine sequences, respectively, with or without alpha2-->3
linked NeuNAc. The molecular mimicry of the LOSs may play a role in the
pathogenesis of N.meningitidis by assisting the organism to evade host
immune defenses in man.
相似文献
2.
El-Fasakhany FM Uchimura K Kannagi R Muramatsu T 《The Journal of biological chemistry》2001,276(29):26988-26994
Based on sequence homology with the previously cloned human cerebroside sulfotransferase (CST) cDNA, a novel sulfotransferase was cloned by screening a human fetal brain cDNA library. The novel sulfotransferase gene was present on human chromosome 11q13; the location was different from human CST and from that of the recently cloned human beta-Gal 3'-sulfotransferase (GP3ST). The isolated cDNA contained an open reading frame that encoded a predicted protein of 431 amino acid residues with type II transmembrane topology. The amino acid sequence showed 33% identity with that of human CST and 38% with that of human GP3ST. The recombinant enzyme expressed in Chinese hamster ovary cells catalyzed transfer of sulfate to position 3 of non-reducing beta-galactosyl residues in Galbeta1-4GlcNAc. Type 2 chains served as good acceptors, whereas type 1 chains served as poor acceptors, and intermediate activity was found toward Galbeta1-3GalNAc. Therefore, the substrate specificity was different from that of GP3ST. CST activity was not detected in the newly cloned enzyme. Northern blotting analysis showed that the sulfotransferase mRNA was strongly expressed in the thyroid and moderately expressed in the brain, heart, kidney, and spinal cord. Co-transfection of the enzyme cDNA and fucosyltransferase III into COS-7 cells resulted in expression of (SO(4)-3)Galbeta1-4(Fucalpha1-3)GlcNAc and a small amount of (SO(4)-3)Galbeta1-3(Fucalpha1-4)GlcNAc. These results indicated that the newly cloned enzyme is a novel Gal-3-O-sulfotransferase and is involved in biosynthesis of the (SO(4)-3)Galbeta1-4(Fucalpha1-3)GlcNAc structure. 相似文献
3.
Wakabayashi H Natsuka S Honda M Naotsuka M Ito Y Kajihara J Hase S 《Journal of biochemistry》2001,130(4):543-552
The sugar chains of human urinary thrombomodulin were studied. N- and O-linked sugar chains were simultaneously liberated by hydrazinolysis followed by N-acetylation and were tagged with 2-aminopyridine. Then the structures of the N- and O-linked pyridylamino (PA-) sugar chains were analyzed by two-dimensional sugar mapping combined with exoglycosidase digestion. The major N-linked sugar chains of human urinary thrombomodulin were found to be monosialo- and disialofucosylbiantennary chains, while the major O-linked sugar chain was +/-Siaalpha2-3Galbeta1-3(+/-Siaalpha2-6)GalNAc. Thrombomodulin also contained the reported structure SO4-3GlcAbeta1-3Galbeta1-3(+/-Siaalpha2-6)Galbeta1-4Xyl [H. Wakabayashi, S. Natsuka, T. Mega, N. Otsuki, M. Isaji, M. Naotsuka, S. Koyama, T. Kanamori, K. Sakai, and S. Hase (1999) J. Biol. Chem. 274, 5436-5442]. In addition to these sugar chains, a single Glc was linked to Ser 287. 相似文献
4.
The all-transglycolytic synthesis of lacto-N-biose I (Galbeta1-3GlcNAc) and 3'-sialyl-lacto-N-biose I (NeuAcalpha2-3Galbeta1-3GlcNAc) was performed. The disaccharide lacto-N-biose I was obtained by use of p-nitrophenyl beta-D-galactopyranoside as the donor, 2-acetamido-2-deoxy-D-glucopyranose as the acceptor and Xanthomonas manihotis beta-D-galactosidase as the catalyst. The reaction was shown to be regiospecific, with a high molar yield (about 55%) with respect to the donor. Lacto-N-biose I obtained by this method was used as the acceptor for a subsequent enzymatic reaction catalyzed by Trypanosoma cruzi trans-sialidase in which 2'-(4-methylumbellyferyl)-alpha-D-N-acetylneuraminic was used as the donor of the N-acetylneuraminil moiety. The reaction generated the product, 3'-sialyl-lacto-N-biose I, regiospecifically and with a molar yield of about 35%. 相似文献
5.
Rapoport E Khaidukov S Baidina O Bojenko V Moiseeva E Pasynina G Karsten U Nifant'ev N LePendue J Bovin N 《European journal of cell biology》2003,82(6):295-302
A specific apoptotic glycosylation pattern may play an assistant or even a causative role in phagocytosis of apoptotic bodies. To elucidate the role of macrophages in lectin-mediated phagocytosis, an experimental system was used, where monocyte-derived THP-1 cells engulf the apoptotic bodies from the melanoma cell line MELJUSO. A flow cytometry assay was performed to reveal lectin expression and quantify the phagocytosis of apoptotic bodies. Taking into account that siglecs, a mannose receptor and galectins expressed on macrophages could be involved in engulfment of apoptotic bodies we studied their potential expression on THP-1 cells by means of polyacrylamide glycoconjugates. A strong binding of the cells to siglec ligands (3'SiaLac, 6'SiaLac, [Neu5Acalpha2-8]2) and galectin ligands (LacNAc, GalNAcbeta1 - 4GlcNAc, Galbeta1 - 3GalNAcbeta and asialoGM1) was observed. To reveal the corresponding targets on apoptotic bodies, the carbohydrate pattern of MELJUSO cells was analyzed. The apoptotic membrane was characterized by a high level of glycans terminated by galactose or sialic acid. To study lectin-mediated phagocytosis of apoptotic bodies by THP-1 cells, an inhibitory phagocytosis assay was performed. Binding of Galbeta1 - 3GalNAc- or LacNAc-specific reagents (lectins and antibodies) to apoptotic bodies abolished their engulfment by the THP-1 cells whereas blocking of Neu5Acalpha2 - 6 or Neu5Acalpha2 - 3 sites by the corresponding lectins was not effective. Furthermore, Galbeta1 - 3GalNAcbeta-PAA or asialoGM1-PAA binding to the THP-1 cells decreased phagocytosis, whereas two other potent THP-1-binding probes, LacNAc-PAA and GalNAcbeta1 - 4GlcNAc-PAA did not inhibit phagocytosis. Thus, Galbeta1 - 3GalNAcbeta-terminated chains represented on the apoptotic bodies but not the other tested galectin ligands appear to be a target for THP-1 cells. 相似文献
6.
Kimura Y Tsumura K Kimura M Okihara K Sugimoto H Yamada H 《Bioscience, biotechnology, and biochemistry》2003,67(8):1852-1856
On a way of structural analysis of total N-glycans linked to glycoproteins in royal jelly (Kimura, Y. et al., Biosci. Biotechnol. Biochem., 64, 2109-2120 (2000), Kimura, M. et al., Biosci. Biotechnol. Biochem., 66, 1985-1989 (2002)), we found that some complex type N-glycans containing a beta1-3galactose residue occur on the insect glycoproteins. Up to date, it has been considered that naturally occurring insect glycoproteins do not bear the galactose-containing N-glycans, therefore, in this report we describe the structural analysis of the complex type N-glycans of royal jelly glycoproteins.By a combination of endo- and exo-glycosidase digestions, IS-MS analysis, and 1H-NMR spectroscopy, the structures of the beta1-3 galactose-containing N-glycan were identified as the following; GlcNAcbeta1-2Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc, Manalpha1-3Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc, and Manalpha1-6(Manalpha1-3)Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc. To our knowledge, this is the first report showing that the Galbeta1-3GlcNAcbeta1-4Man unit occurs in N-glycans of insect glycoproteins, indicating a beta1-3 galactosyl transferase and beta1-4GlcNAc transferase (GNT-IV) are expressed in the honeybee cells. 相似文献
7.
Jain RK; Piskorz CF; Huang BG; Locke RD; Han HL; Koenig A; Varki A; Matta KL 《Glycobiology》1998,8(7):707-717
The selectins interact in important normal and pathological situations with
certain sialylated, fucosylated glycoconjugate ligands containing sialyl
Lewisx(Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcN Ac). Much effort has gone
into the synthesis of sialylated and sulfated Lewisxanalogs as competitive
ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and
PSGL-1 carry sialyl Lewisxas part of a branched Core 2 O-linked structure,
we recently synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(SE-3Galbeta1++
+-3)GalNAc1alphaOMe and found it to be a moderately superior ligand for L
and P-selectin (Koenig et al. , Glycobiology 7, 79-93, 1997). Other studies
have shown that sulfate esters can replace sialic acid in some selectin
ligands (Yeun et al. , Biochemistry, 31, 9126-9131, 1992; Imai et al. ,
Nature, 361, 555, 1993). Based upon these observations, we hypothesized
that Neu5Acalpha2-3Galbeta1-3GalNAc might have the capability of
interacting with L- and P-selectin. To examine this hypothesis, we
synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Neu5Acalpha2++
+-3Galbeta1-3)- GalNAc alpha1-OB, which was found to be 2- to 3-fold better
than sialyl Lexfor P and L selectin, respectively. We also report the
synthesis of an unusual structure GalNAcbeta1-4(Fucalpha1-
3)GlcNAcbeta1-OMe (GalNAc- Lewisx-O-methyl glycoside), which also proved to
be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe.
Combining this with our knowledge of Core 2 branched structures, we have
synthesized a molecule that is 5- to 6-fold better at inhibiting L- and
P-selectin than sialyl Lewisx-OMe, By contrast to unbranched structures,
substitution of a sulfate ester group for a sialic acid residue in such a
molecule resulted in a considerable loss of inhibition ability. Thus, the
combination of a sialic acid residue on the primary (beta1-3) arm, and a
modified Lexunit on the branched (beta1-6) arm on an O-linked Core 2
structure generated a monovalent synthetic oliogosaccharide inhibitor
superior to SLexfor both L- and P-selectin.
相似文献
8.
The glycan shield comprised of multiple carbohydrate chains on the human immunodeficiency virus (HIV) envelope glycoprotein gp120 helps the virus to evade neutralizing antibodies. The present study describes a novel method for increasing immunogenicity of gp120 vaccine by enzymatic replacement of sialic acid on these carbohydrate chains with Galalpha1-3Galbeta1-4GlcNAc-R (alpha-gal) epitopes. These epitopes are ligands for the natural anti-Gal antibody constituting approximately 1% of immunoglobulin G in humans. We hypothesize that vaccination with gp120 expressing alpha-gal epitopes (gp120(alphagal)) results in in vivo formation of immune complexes with anti-Gal, which targets vaccines for effective uptake by antigen-presenting cells (APC), due to interaction between the Fc portion of the antibody and Fcgamma receptors on APC. This in turn results in effective transport of the vaccine to lymph nodes and effective processing and presentation of gp120 immunogenic peptides by APC for eliciting a strong anti-gp120 immune response. This hypothesis was tested in alpha-1,3-galactosyltransferase knockout mice, which produce anti-Gal. Mice immunized with gp120(alphagal) produced anti-gp120 antibodies in titers that were >100-fold higher than those measured in mice immunized with comparable amounts of gp120 and effectively neutralized HIV. T-cell response, measured by ELISPOT, was much higher in mice immunized with gp120(alphagal) than in mice immunized with gp120. It is suggested that gp120(alphagal) can serve as a platform for anti-Gal-mediated targeting of additional vaccinating HIV proteins fused to gp120(alphagal), thereby creating effective prophylactic vaccines. 相似文献
9.
In our recent publication, we defined core aspects of the carbohydrate specificity of domain-I of recombinant tandem-repeat-type galectin-4 from rat gastrointestinal tract (G4-N), especially its potent interaction with the linear tetrasaccharide Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc (Ibeta1-3L). The assumed role of galectin-4 as a microvillar raft stabilizer/organizer and as a malignancy-associated factor in hepatocellular and gastrointestinal carcinomas called for further refinement of its binding specificity. Thus, the effects of polyvalency of glycotopes and natural modifications of human blood group ABH/Lewis sugars at the terminal Galbeta1-core saccharides were thoroughly examined by the enzyme-linked lectinosorbent and lectin-glycan inhibition assays. The results indicate that (a) a high-density of polyvalent Galbeta1-3/4GlcNAc (I/II), Galbeta1-3GalNAc (T) and/or GalNAcalpha1-Ser/Thr (Tn) strongly favors G4-N/glycoform binding. These glycans were up to 2.3 x 10(6), 1.4 x 10(6), 8.8 x 10(5), and 1.4 x 10(5) more active than Gal, GalNAc, monomeric I/II and T, respectively; (b) while lFuc is a poor inhibitor, its presence as alpha1-2 linked to terminal Galbeta1-containing oligosaccharides, such as H active Ibeta1-3L, markedly enhances the reactivities of these ligands; (c) when blood group A (GalNAcalpha1-) or B (Galalpha1-) determinants are attached to terminal Galbeta1-3/4GlcNAc (or Glc) oligosaccharides, the reactivities are also increased; (d) with lFucalpha1-3/4 linked to sub-terminal GlcNAc, the reactivities of these haptens are reduced; and (e) short chain Le(a)/Le(x)/Le(y) and the short chains of sialyl Le(a)/Le(x) are poor inhibitors. These distinct binding features of G4-N establish the important concept of affinity enhancement by high density polyvalencies of glycotopes (vs. multi-antennary I/II) and by introduction of an ABH key sugar to Galbeta1-terminated core glycotopes. The polyvalent ligand binding properties of G4-N may help our understanding of its crucial role for cell membrane raft stability and provide salient information for the optimal design of blocking substances such as anti-tumoral glycodendrimers. 相似文献
10.
Korekane H Taguchi T Sakamoto Y Honke K Dohmae N Salminen H Toivonen S Helin J Takio K Renkonen O Taniguchi N 《Glycobiology》2003,13(5):387-400
A rat intestinal beta1,6N-acetylglucosaminyltransferase (beta1-6GnT) responsible for the formation of the beta1,6-branched poly-N-acetyllactosamine structure has been purified to apparent homogeneity by successive column chromatographic procedures using an assay wherein pyridylaminated lacto- N-triose II (GlcNAcbeta1-3Galbeta1-4Glc-PA) was used as an acceptor substrate and the reaction product was GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4Glc-PA. The purified enzyme catalyzed the conversion of the polylactosamine acceptor GlcNAcbeta1-3'LacNAc into GlcNAcbeta1-3'(GlcNAcbeta1-6') LacNAc (dIGnT activity), but it could not transfer GlcNAc to LacNAcbeta1-3'LacNAc (cIGnT activity). This enzyme could also convert mucin core 1 and core 3 analogs, Galbeta1-3GalNAcalpha1-O-paranitrophenyl (pNP) and GlcNAcbeta1-3GalNAcalpha1-O-pNP, into Galbeta1-3(GlcNAcbeta1-6) GalNAcalpha1-O-pNP (C2GnT activity) and GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-O-pNP (C4GnT activity), respectively. Based on the partial amino acid sequences of the purified protein, the cDNA encoding this enzyme was cloned. The COS-1 cells transiently transfected with this cDNA had high dI/C2/C4GnT activities in a ratio of 0.34:1.00:0.90, compared with non- or mock-transfected cells. The primary structure shows a significant homology with human and viral mucin-type core 2 beta1-6GnTs (C2GnT-Ms), indicating that this enzyme is the rat ortholog of human and viral C2GnT-Ms. This is the first identification and purification of this enzyme as a major carrier of dIGnT activity in the small intestine. This rat ortholog should mostly be responsible for making distal I-branch structures on poly-N-acetyllactosamine sequences in this tissue, as well as making mucin core 2 and core 4 structures, given that it also has high C2/C4GnT activities. 相似文献
11.
Gulberti S Lattard V Fondeur M Jacquinet JC Mulliert G Netter P Magdalou J Ouzzine M Fournel-Gigleux S 《The Journal of biological chemistry》2005,280(2):1417-1425
We determined whether the two major structural modifications, i.e. phosphorylation and sulfation of the glycosaminoglycan-protein linkage region (GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1), govern the specificity of the glycosyltransferases responsible for the biosynthesis of the tetrasaccharide primer. We analyzed the influence of C-2 phosphorylation of Xyl residue on human beta1,4-galactosyltransferase 7 (GalT-I), which catalyzes the transfer of Gal onto Xyl, and we evaluated the consequences of C-4/C-6 sulfation of Galbeta1-3Gal (Gal2-Gal1) on the activity and specificity of beta1,3-glucuronosyltransferase I (GlcAT-I) responsible for the completion of the glycosaminoglycan primer sequence. For this purpose, a series of phosphorylated xylosides and sulfated C-4 and C-6 analogs of Galbeta1-3Gal was synthesized and tested as potential substrates for the recombinant enzymes. Our results revealed that the phosphorylation of Xyl on the C-2 position prevents GalT-I activity, suggesting that this modification may occur once Gal is attached to the Xyl residue of the nascent oligosaccharide linkage. On the other hand, we showed that sulfation on C-6 position of Gal1 of the Galbeta1-3Gal analog markedly enhanced GlcAT-I catalytic efficiency and we demonstrated the importance of Trp243 and Lys317 residues of Gal1 binding site for enzyme activity. In contrast, we found that GlcAT-I was unable to use digalactosides as acceptor substrates when Gal1 was sulfated on C-4 position or when Gal2 was sulfated on both C-4 and C-6 positions. Altogether, we demonstrated that oligosaccharide modifications of the linkage region control the specificity of the glycosyltransferases, a process that may regulate maturation and processing of glycosaminoglycan chains. 相似文献
12.
Pykäri M Toivonen S Natunen J Niemela R Salminen H Aitio O Ekström M Parmanne P Välimäki M Alais J Augé C Lowe JB Renkonen O Renkonen R 《The Journal of biological chemistry》2000,275(51):40057-40063
We report here on in vitro acceptor and site specificity of recombinant alpha3-fucosyltransferase V (Fuc-TV) with 40 oligosaccharide acceptors. Galbeta1-4GlcNAc (LN) and GalNAcbeta1-4GlcNAc (LDN) reacted rapidly; Galbeta1-3GlcNAc (LNB) reacted moderately, and GlcNAcbeta1-4GlcNAc (N, N'-diacetyl-chitobiose) reacted slowly yet distinctly. In neutral and terminally alpha3-sialylated polylactosamines of i-type, the reducing end LN unit reacted rapidly and the distal (sialyl)LN group very slowly; the midchain LNs revealed intermediate reactivities. The data suggest that a distal LN neighbor enhances but a proximal LN neighbor reduces the reactivity of the midchain LNs. This implies that Fuc-TV may bind preferably the tetrasaccharide sequence Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc for transfer at the underlined monosaccharide. Terminal alpha3-sialylation of i-type polylactosamines almost doubled the reactivities of the LN units at all positions of the chains. We conclude that, in comparison with human Fuc-TIV and Fuc-TIX, Fuc-TV reacted with a highly distinct site specificity with i-type polylactosamines. The Fuc-TV reactivity of free LNB resembled that of LNBbeta1-3'R of a polylactosamine, contrasting strongly with the dissimilarity of the reactivities of the analogous pair of LN and LNbeta1-3'R. This observation supports the notion that LN and LNB may be functionally bound at distinct sites on Fuc-TV surface. Our data show that Fuc-TV worked well with a very wide range of LN-glycans, showing weak reactivity only with distal (sialyl)LN units of i-type polylactosamines, biantennary N-glycans, and I branches of polylactosamines. 相似文献
13.
Glycoconjugates with terminal Galalpha1-3Galbeta1-4GlcNAc sequences (alpha-galactosyl epitopes, natural xenoreactive antigens) are present on various tissues in pigs and are recognized by human anti-alphagalactosyl (alphaGal) antibodies1. Hence xenotransplantation (pig-to-human) would trigger immune reactions involving complement activation and lead to the hyperactute rejection of the graft. Xenoreactive antigens are often studied by using the lectin Griffonia simplicifolia 1 isolectin B4 (GS1 B4), which shows high affinity to galactose. We here estimate the specificity of GS1 B4 for detecting various galactosyl epitopes by measuring lectin binding to neoglycoproteins, thyroglobulin and pig skeletal muscle. Enzyme linked lectin assays confirmed that GS1 B4 was highly specific to alpha-galactosylated neoglycoproteins while the lectin did not detect a beta-galactosylated ligand. The length of the sugar chains influenced the lectin-carbohydrate interaction. A monosaccharide linked to serum albumin showed higher lectin affinity than did neoglycoproteins with di- and tri-alpha-galactosyl epitopes. When the carbohydrate was extended, as in the xenoreactive pentasaccharide (Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc), the carbohydrate- lectin interaction was meagre. Not only the terminal, but also the subterminal sugar affected the lectin binding because the GS1 B4 affinity to Galalpha1-3Gal was much stronger than to Galalpha1-3GalNAc. In bovine and porcine thyroglobulin most alphaGal epitopes appear to be cryptic, but are unmasked by a heat denaturation. In pig skeletal muscle there was lectin reaction not only in the muscle capillaries, but also in the connective tissue and intracellularly in muscle fibres. In Western blots of isolated proteins from pig muscle at least three bands were strongly stained after incubation with lectin. 相似文献
14.
A particulate preparation from 6-day-old seedlings of wheat (Triticum aestivum L.) was found to contain a xylosyltransferase (XylTase) which incorporated xylose (Xyl) from UDP-xylose into exogenous beta-(1-->4)-xylooligosaccharides with 2-aminopyridine-derivatized reducing end groups. High-performance liquid chromatographic analysis showed that the chain elongation of pyridylaminated beta-(1-->4)-xylotriose (Xyl3-PA) occurred by attachment of a series of one, two, or three xylosyl residues, depending on substrate concentrations and reaction times. Methylation analysis and beta-xylosidase digestion of the newly synthesized Xyl4-PA confirmed that the xylosyl residues were incorporated through beta-(1-->4)-linkages. The enzyme was maximally active at pH 6.8 and 20 degrees C, and required Triton X-100, which enhanced activity 5-fold at a concentration of 0.05-2%. Divalent ions, including Mn2+ and Mg2+, did not affect activity. Enzyme activity increased with increasing polymerization of xylosyl residues of the acceptor substrates: for instance, Xyl5-PA was almost 7 times as efficient as Xyl2-PA. The apparent Michaelis constants of the enzyme for Xyl3-PA and UDP-xylose were 13.5 and 7.9 mM, respectively. The enzyme also catalyzed incorporation of radioactive sugars (Xyl together with a small portion of L-arabinose) from UDP-[14C]xylose into higher beta-(1-->4)-xylooligosaccharides (degree of polymerization > 7) with or without (4-O-methyl-)glucuronosyl side chains at activities comparable to those observed for pyridylaminated xylooligosaccharides, and into several heteroxylans but with much lower efficiency. Enzymatic hydrolysis of the product with a beta-xylanase degraded it into mainly xylobiose, providing further evidence that the xylosyl residues are incorporated through beta-(1-->4)-linkages. 相似文献
15.
Chandrasekaran EV Lakhaman SS Chawda R Piskorz CF Neelamegham S Matta KL 《The Journal of biological chemistry》2004,279(11):10032-10041
Sulfated glycoconjugates regulate biological processes such as cell adhesion and cancer metastasis. We examined the acceptor specificities and kinetic properties of three cloned Gal:3-O-sulfotransferases (Gal3STs) ST-2, ST-3, and ST-4 along with a purified Gal3ST from colon carcinoma LS180 cells. Gal3ST-2 was the dominant Gal3ST in LS180. While the mucin core-2 structure Galbeta1,4GlcNAcbeta1,6(3-O-MeGalbeta1,3)GalNAcalpha-O-Bn (where Bn is benzyl) and the disaccharide Galbeta1,4GlcNAc served as high affinity acceptors for Gal3ST-2 and Gal3ST-3, 3-O-MeGalbeta1,4GlcNAcbeta1,-6(Galbeta1,3)GalNAcalpha-O-Bn and Galbeta1,3GalNAcalpha-O-Al (where Al is allyl) were efficient acceptors for Gal3ST-4. The activities of Gal3ST-2 and Gal3ST-3 could be distinguished with the Globo H precursor (Galbeta1,3GalNAcbeta1,3Galalpha-O-Me) and fetuin triantennary asialoglycopeptide. Gal3ST-2 acted efficiently on the former, while Gal3ST-3 showed preference for the latter. Gal3ST-4 also acted on the Globo H precursor but not the glycopeptide. In support of the specificity, Gal3ST-2 activity toward the Galbeta1,4GlcNAcbeta unit on mucin core-2 as well as the Globo H precursor could be inhibited competitively by Galbeta1,4GlcNAcbeta1,6(3-O-sulfoGalbeta1,3)GalNAcalpha-O-Bn but not 3-O-sulfoGalbeta1,-4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-Bn. Remarkably these sulfotransferases were uniquely specific for sulfated substrates: Gal3ST-3 utilized Galbeta1,4(6-O-sulfo)-GlcNAcbeta-O-Al as acceptor, Gal3ST-2 acted efficiently on Galbeta1,3(6-O-sulfo)GlcNAcbeta-O-Al, and Gal3ST-4 acted efficiently on Galbeta1,3(6-O-sulfo)GalNAcalpha-O-Al. Mg(2+), Mn(2+), and Ca(2+) stimulated the activities of Gal3ST-2, whereas only Mg(2+) augmented Gal3ST-3 activity. Divalent cations did not stimulate Gal3ST-4, although inhibition was noted at high Mn(2+) concentrations. The fine substrate specificities of Gal3STs indicate a distinct physiological role for each enzyme. 相似文献
16.
Totani K Kubota T Kuroda T Murata T Hidari KI Suzuki T Suzuki Y Kobayashi K Ashida H Yamamoto K Usui T 《Glycobiology》2003,13(5):315-326
Highly water-soluble glycopolymers with poly(alpha-L-glutamic acid) (PGA) backbones carrying multivalent sialyl oligosaccharides units were chemoenzymatically synthesized as polymeric inhibitors of infection by human influenza viruses. p-Aminophenyl disaccharide glycosides were coupled with gamma-carboxyl groups of PGA side chains and enzymatically converted to Neu5Acalpha2-3Galbeta1-4GlcNAcbeta-, Neu5Acalpha2-6Galbeta1-4GlcNAcbeta-, Neu5Acalpha2-3Galbeta1-3GalNAcalpha-, and Neu5Acalpha2-3Galbeta1-3GalNAcbeta- units, respectively, by alpha2,3- or alpha2,6-sialytransferases. The glycopolymers synthesized were used for neutralization of human influenza A and B virus infection as assessed by measurement of the degree of cytopathic inhibitory effect in virus-infected MDCK cells. Among the glycopolymers tested, alpha2,6-sialo-PGA with a high molecular weight (260 kDa) most significantly inhibited infection by an influenza A virus, strain A/Memphis/1/71 (H3N2), which predominantly binds to alpha2-6 Neu5Ac residue. The alpha2,6-sialo-PGA also inhibited infection by an influenza B virus, B/Lee/40. The binding preference of viruses to terminal sialic acids was affected by core determinants of the sugar chain, Galbeta1-4GlcNAcbeta- or Galbeta1-3GalNAcalpha/beta- units. Inhibition of infection by viruses was remarkably enhanced by increasing the molecular weight and sialic acid content of glycopolymers. 相似文献
17.
Sakaguchi H Watanabe M Ueoka C Sugiyama E Taketomi T Yamada S Sugahara K 《Journal of biochemistry》2001,129(1):107-118
The glycosaminoglycan (GAG)-protein linkage regions of various proteoglycans share the common tetrasaccharide GlcA-Gal-Gal-Xyl-attached to Ser residues in the core proteins. In previous analysis we demonstrated unique modifications by epimerization, sulfation and phosphorylation of the component sugars. Here we developed a sensitive analytical method for the linkage region oligosaccharides to detect or monitor structural variations and changes. This will be useful for investigation of their biological roles, which are largely unknown, but they have been implicated in biosynthesis. A variety of linkage region-derived hexasaccharides was first prepared as reducing sugar chains from peptide chondroitin/dermatan sulfate of whale cartilage, shark cartilage, and bovine aorta by means of chondroitinase digestion in conjunction with beta-elimination in the absence of reducing reagents, but involving a mild alkali, 0.5 M LiOH, at 4 degrees C to prevent peeling reactions. The structures of these oligosaccharides were determined by the combination of HPLC, enzymatic digestion, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and (1)H NMR spectroscopy, which revealed eleven different hexasaccharides including a novel structure, DeltaHexAalpha1-3GalNAcbeta1-4IdoAalpha1-3Gal(4-O-sulfate)beta1-3Galbeta1-4Xyl (DeltaHexA and IdoA represent unsaturated hexuronic acid and L-iduronic acid, respectively). These oligosaccharides were labeled with a fluorophore, 2-aminobenzamide, to prepare analytical probes using the recently developed procedure [Kinoshita and Sugahara (1999) Anal. Biochem. 269, 367-378]. The fluorophore-tagged hexasacharides of low picomoles were well separated by HPLC and successfully analyzed by MALDI-TOF mass spectrometry. The principle of the method should be applicable to the analysis of the linkage region oligosaccharides derived from heparin and heparan sulfate as well. 相似文献
18.
We have recently discovered unusual sugar chains [xylose-glucose and (xylose)2-glucose] linked to a serine residue in the first epidermal growth factor (EGF)-like domains of human and bovine coagulation factors VII, IX, and protein Z. The sequence surrounding this serine residue has a common -Cys-X-Ser-X-Pro-Cys- structure. Since one (residues 533-538) of the three EGF-like domains found in human thrombospondin contains the conserved sequence, we examined the presence of such O-linked sugar chains in bovine thrombospondin (bTSP) and its 210-kDa fragment. Component sugar analysis after pyridylamination (PA) of the acid hydrolysates of the S-aminoethylated proteins revealed that the proteins contain glucose (Glc) and xylose (Xyl). The oligosaccharide moieties released from intact bTSP by hydrazinolysis followed by pyridylamination were separated into two PA-oligosaccharides by high performance liquid chromatography (HPLC). Component sugar analysis of these PA-oligosaccharides indicated that they consist of Glc and Xyl in molar ratios of 1:1 and 1:2 (or 1:3). The reducing ends of both PA-sugar chains were found to be PA-Glc, as judged from the retention time of the HPLC peak of their hydrolysates. The presence of these PA-sugar chains in bTSP was confirmed by HPLC mapping with two different columns, using standard PA-di- or PA-trisaccharide derived from coagulation factors. From these results, we concluded that bTSP contains O-linked sugar chains consisting of Glc and Xyl in one of its three EGF-like domains. 相似文献
19.
The proteoglycans of animal cells typically contain one or more heparan sulfate or chondroitin sulfate chains. These glycosaminoglycans assemble on a tetrasaccharide primer, -GlcAbeta1, 3Galbeta1,3Galbeta1,4Xylbeta-O-, attached to specific serine residues in the core protein. Studies of Chinese hamster ovary cell mutants defective in the first or second enzymes of the pathway (xylosyltransferase and galactosyltransferase I) show that the assembly of the primer occurs by sequential transfer of single monosaccharide residues from the corresponding high energy nucleotide sugar donor to the non-reducing end of the growing chain. In order to study the other reactions involved in linkage tetrasaccharide assembly, we have devised a powerful selection method based on induced resistance to a mitotoxin composed of basic fibroblast growth factor-saporin. One class of mutants does not incorporate 35SO4 and [6-3H]GlcN into glycosaminoglycan chains. Incubation of these cells with naphthol-beta-D-xyloside (Xylbeta-O-Np) resulted in accumulation of linkage region intermediates containing 1 or 2 mol of galactose (Galbeta1, 4Xylbeta-O-Np and Galbeta1, 3Galbeta1, 4Xylbeta-O-Np) and sialic acid (Siaalpha2,3Galbeta1, 3Galbeta1, 4Xylbeta-O-Np) but not any GlcA-containing oligosaccharides. Extracts of the mutants completely lacked UDP-glucuronic acid:Galbeta1,3Gal-R glucuronosyltransferase (GlcAT-I) activity, as measured by the transfer of GlcA from UDP-GlcA to Galbeta1,3Galbeta-O-naphthalenemethanol (<0.2 versus 3.6 pmol/min/mg). The mutation most likely lies in the structural gene encoding GlcAT-I since transfection of the mutant with a cDNA for GlcAT-I completely restored enzyme activity and glycosaminoglycan synthesis. These findings suggest that a single GlcAT effects the biosynthesis of common linkage region of both heparan sulfate and chondroitin sulfate in Chinese hamster ovary cells. 相似文献
20.
Palacpac NQ Kimura Y Fujiyama K Yoshida T Seki T 《Bioscience, biotechnology, and biochemistry》1999,63(1):35-39
The structures of N-linked sugar chains of glycoproteins expressed in tobacco BY2 cultured cells are reported. Five pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the glycoproteins by reversed-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were identified by two-dimensional PA-sugar chain mapping, ion-spray MS/MS analysis, and exoglycosidase digestions. The five structures fell into two categories; the major class (92.5% as molar ratio) was a xylose containing-type (Man3Fuc1 Xyl1GlcNAc2 (41.0%), GlcNAc2Man3Fuc1Xyl1GlcNAc2 (26.5%), GlcNAc1Man3Fuc1Xyl1GlcNAc2 (21.7%), Man3 Xyl1GlcNAc2 (3.3%)), and the minor class was a high-mannose type (Man5GlcNAc2 (7.5%)). This is the first report to show that alpha(1-->3) fucosylation of N-glycans does occur but beta(1-->4) galactosylation of the sugar chains does not in the tobacco cultured cells. 相似文献