共查询到20条相似文献,搜索用时 15 毫秒
1.
Kouji Kuno Chie Baba Atsuko Asaka Chieko Matsushima Kouji Matsushima Ryuji Hosono 《The Journal of biological chemistry》2002,277(14):12228-12236
Remodeling of the extracellular matrix (ECM) is pivotal for various biological processes, including organ morphology and development. The Caenorhabditis elegans male tail has male-specific copulatory organs, the rays and the fan. Ray morphogenesis, which involves a rapid remodeling of the ECM, is an important model of morphogenesis, although its mechanism is poorly understood. ADAMTS (a disintegrin-like and metalloproteinase with thrombospondin type I motifs) is a novel metalloproteinase family that is thought to be an important regulator for ECM remodeling during development and pathological states. We report here that a new C. elegans ADAMTS family gene, adt-1, plays an important regulatory role in ray morphogenesis. Inactivation of the adt-1 gene resulted in morphological changes in the rays as well as the appearance of abnormal protuberances around the rays. In addition, mating ability was remarkably impaired in adt-1 deletion mutant males. Furthermore, we found that the green fluorescent protein reporter driven by the adt-1 promoter was specifically expressed throughout the rays in the male tail. We hypothesize that ADT-1 controls the ray extension process via remodeling of the ECM in the cuticle. 相似文献
2.
Hitomi Hoshino Tahmina Foyez Shiori Ohtake-Niimi Yoshiko Takeda-Uchimura Makoto Michikawa Kenji Kadomatsu Kenji Uchimura 《The journal of histochemistry and cytochemistry》2014,62(2):145-156
Keratan sulfate (KS) comprises repeating disaccharides of galactose (Gal) and N-acetylglucosamine (GlcNAc). Residues of Gal and GlcNAc in KS are potentially modified with sulfate at their C-6 positions. The 5D4 monoclonal antibody recognizes KS structures containing Gal and GlcNAc, both 6-sulfated, and has been used most extensively to evaluate KS expression in mammalian brains. We previously showed that GlcNAc6ST1 is an enzyme responsible for the synthesis of the 5D4 epitope in developing brain and in the adult brain, where it is induced after injury. It has been unclear which sulfotransferase is responsible for Gal-6-sulfation within the 5D4 KS epitope in developing brains. We produced mice deficient in KSGal6ST, a Gal-6-sulfotransferase. Western blotting and immunoprecipitation revealed that all 5D4-immunoreactivity to proteins, including phosphacan, were abolished in KSGal6ST-deficient postnatal brains. Likewise, the 5D4 epitope, expressed primarily in the cortical marginal zone and subplate and dorsal thalamus, was eliminated in KSGal6ST-deficient mice. Disaccharide analysis showed the loss of Gal-6-sulfate in KS of the KSGal6ST-deficient brains. Transfection studies revealed that GlcNAc6ST1 and KSGal6ST cooperated in the expression of the 5D4 KS epitope in HeLa cells. These results indicate that KSGal6ST is essential for C-6 sulfation of Gal within KS in early postnatal brains. 相似文献
3.
Expression of ram-5 in the structural cell is required for sensory ray morphogenesis in Caenorhabditis elegans male tail
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Tissue morphogenesis requires complex cellular interaction and communication. The sensory ray in the Caenorhabditis elegans male tail has a simple cellular make-up and a non-essential function, thus providing an ideal model for studying the mechanisms guiding morphogenesis. We present here the analysis of a novel gene, ram-5, mutations of which are characterized by abnormal lumpy rays in the male tail. Microscopic analysis and behavioral studies revealed that lumpy rays contain operational sensory neurons. However, abnormalities were observed in the hypodermis and structural cells as well as in appositions between these two cell types. Molecular cloning and expression studies revealed that the ram-5 gene encodes a transmembrane protein localized in sensory ray support cells, the structural cells. Expression of ram-5 in these cells is required for normal ray morphogenesis. ram-5-dependent cell-cell communication is implicated in organizing the structural cell and the hypodermis, potentially through adhesion at the structural cell-hypodermal cell border. 相似文献
4.
Natunen S Satomaa T Pitkänen V Salo H Mikkola M Natunen J Otonkoski T Valmu L 《Glycobiology》2011,21(9):1125-1130
The expression of the epitopes recognized by the monoclonal antibodies Tra-1-60 and Tra-1-81 is routinely used to assess the pluripotency status of human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells. Although it is known that the epitopes recognized by Tra-1-60 and Tra-1-81 are carbohydrates, the exact molecular identity of these epitopes has been unclear. Glycan array analysis with more than 500 oligosaccharide structures revealed specific binding of Tra-1-60 and Tra-1-81 to two molecules containing terminal type 1 lactosamine: Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc and Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6(Galβ1-3GlcNAcβ1-3)Galβ1-4Glc. The type 1 disaccharide in itself was not sufficient for binding, indicating that the complete epitope requires an extended tetrasaccharide structure where the type 1 disaccharide is β1,3-linked to type 2 lactosamine. Our mass spectrometric analysis complemented with glycosidase digestions of hESC O-glycans indicated the presence of the extended tetrasaccharide epitope on an O-glycan with the likely structure Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAc. Thus, the present data indicate that the pluripotency marker antibodies Tra-1-60 and Tra-1-81 recognize the minimal epitope Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc, which is present in hESCs as a part of a mucin-type O-glycan structure. The exact molecular identity of Tra-1-60 and Tra-1-81 is important for the development of improved tools to characterize the pluripotent phenotype. 相似文献
5.
6.
Relative accessibility of N-acetylglucosamine in trimers of the adenovirus types 2 and 5 fiber proteins.
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K G Mullis R S Haltiwanger G W Hart R B Marchase J A Engler 《Journal of virology》1990,64(11):5317-5323
Fiber is an adenovirus capsid protein responsible for virus attachment to the cell surface and contains O-linked N-acetylglucosamine (GlcNAc). Results of both amino acid analysis and Dionex chromatography indicated that 3 to 4 and 1.7 to 2.5 mol of GlcNAc are attached per mol of affinity-purified adenovirus type 2 (Ad2) and Ad5 fibers, respectively. Fiber shares an epitope with nuclear pore proteins containing O-linked GlcNAc, as shown by reactivity to monoclonal antibody RL2 directed against these pore proteins. GlcNAc on fiber was found to serve as an acceptor for the transfer of galactose from UDP-galactose by 4 beta-galactosyl-transferase in Ad2 and Ad5 but not in Ad7; quantitation by labeling with UDP-[U-14C]galactose in this reaction gave a 100-fold-lower estimate of the GlcNAc content of fiber, suggesting that these monosaccharides are buried within fiber trimers and are not accessible to the transferase. Affinity chromatography on lectin-bound Sepharose beads showed that Ad2 and Ad5 fibers bound weakly to wheat germ agglutinin and did not bind to ricin or concanavalin A; weak binding to wheat germ agglutinin suggests either that GlcNAc is not easily accessible or that there are not sufficient GlcNAcs for efficient binding. These data suggest that O-linked GlcNAc might be important for Ad2 and Ad5 fiber assembly or stabilization. 相似文献
7.
We have recently shown that binding of Helicobacter pylori to sialylated carbohydrates is dependent on the presence of the carboxyl group and the glycerol chain of Neu5Ac. In this work we investigated the importance of GlcNAc in the binding trisaccharide Neu5Acalpha3Galbeta4GlcNAc and the role of the N-acetamido groups of both Neu5Ac and GlcNAc. An important part of the project was epitope dissection, that is chemical derivatizations of the active carbohydrate followed by binding studies. In addition we used a panel of various unmodified carbohydrate structures in the form of free oligosaccharides or glycolipids. These were tested for binding by hemagglutination inhibition assay, TLC overlay tests, and a new quantitative approach using radiolabeled neoglycoproteins. The studies showed that the N-acetamido group of Neu5Ac is important for binding by H. pylori, whereas the same group of GlcNAc is not. In addition, Fuc attached to GlcNAc, as tested with sialyl-Lewis x, did not affect the binding. Free Neu5Ac was inactive as inhibitor, and Neu5Acalpha3Gal turned out to be active. The binding preference for neolacto structures was confirmed, although one strain also was inhibited by lacto chains. The combined results revealed that an intact Neu5Ac is crucial for the interactions with H. pylori. Parts of Gal also seem to be necessary, whereas the role of the GlcNAc is secondary. GlcNAc does influence binding, however, primarily serving as a guiding carrier for the binding epitope rather than being a part of the binding structure. 相似文献
8.
N-acetylglucosamine 6-O-sulfotransferase-1 regulates expression of L-selectin ligands and lymphocyte homing 总被引:2,自引:0,他引:2
Uchimura K Kadomatsu K El-Fasakhany FM Singer MS Izawa M Kannagi R Takeda N Rosen SD Muramatsu T 《The Journal of biological chemistry》2004,279(33):35001-35008
Lymphocyte homing is initiated by the binding of L-selectin on lymphocytes to its ligands on high endothelial venules (HEV). Sialyl 6-sulfo Lewis X is a major capping group of L-selectin ligands. N-Acetylglucosamine (GlcNAc) 6-sulfation is essential for the ligand activity, and is catalyzed by GlcNAc 6-O-sulfotransferases (GlcNAc6STs) of which GlcNAc6ST-1 and GlcNAc6ST-2 are expressed in HEV. Here, we report that mice deficient in GlcNAc6ST-1 were impaired in the elaboration of sialyl 6-sulfo Lewis X in HEV and that an epitope of L-selectin ligands recognized by the MECA-79 anti-body was greatly reduced or abolished in the abluminal aspect of HEV. Lymphocyte homing to peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches was significantly reduced in GlcNAc6ST-1 null mice. These results demonstrate that GlcNAc6ST-1 is involved in lymphocyte homing in vivo, and indicate that GlcNAc6ST-1 and -2 play complementary roles. The importance of GlcNAc6ST-1 is particularly high-lighted by its involvement in lymphocyte homing to Peyer's patches where GlcNAc6ST-2 expression is undetectable. 相似文献
9.
Isolation of MUC5AC mucins from the gastric mucosa from two secretor individuals (one from normal mucosa from a patient with gastric cancer and one from a control) showed different abilities to bind and induce the proliferation of the Helicobacter pylori strain J99. Analysis of the released O-linked oligosaccharides by LC-MS from these individuals showed a very heterogeneous mixture of species from the cancer patient containing both neutral and sialylated structures, whereas the normal sample showed dominating neutral blood group H terminating structures as well as neutral structures containing the di-N-acetyllactosamine (lacdiNAc) unit GalNAcβ1-4GlcNAcβ1- on the C-6 branch of the reducing end GalNAc. The linkage configuration of these epitopes were determined using C-4-specific fragmentation for the GalNAcβ1-4GlcNAcβ1- glycosidic linkage, comparison of the MS(3) fragmentation with standards for linkage configuration and N-acetylhexosamine type as well as exoglycosidase treatment. It was also shown that the lacdiNAc epitope is present in both human and porcine gastric mucins, indicating that this is an epitope preserved between species. We hypothesize that the termination on gastric MUC5AC with lacdiNAc is in competition with complex glycosylation such as the Le(b) and H type 1 as well as complex sialylated structures. These are epitopes known to bind the H. pylori BabA and SabA adhesins. 相似文献
10.
AS Couto LL Soprano M Landoni M Pourcelot DM Acosta L Bultel J Parente MR Ferrero M Barbier C Dussouy MI Esteva J Kovensky VG Duschak 《The FEBS journal》2012,279(19):3665-3679
Cruzipain (Cz), the major cysteine proteinase of Trypanosoma?cruzi, is a glycoprotein that contains sulfated high-mannose-type oligosaccharides. We have previously determined that these sulfate groups are targets of specific immune responses. In order to evaluate the structural requirements for antibody recognition of Cz, a systematic structure-activity study of the chemical characteristics needed for antibody binding to the Cz sulfated epitope was performed by immunoassays. With this aim, different synthesized molecules were coupled to the proteins BSA and aprotinin and confronted with (a) mouse sera specific for Cz and its carboxy-terminal (C-T) domain, (b) antibodies raised in rabbits immunized with Cz and its C-terminal domain and (c) IgGs purified from human Chagas disease sera. Our results indicate that a glucosamine containing an esterifying sulfate group in position O-6 and an N-acetyl group was the preferred epitope for the immune recognition of sera specific for Cz and its C-T domain. Although to a minor extent, other anionic compounds bearing sulfate groups in different positions and number as well as different anionic charged groups including carboxylated or phosphorylated monosaccharides, disaccharides and oligosaccharides were recognized. In conclusion, we found that synthetic anionic sugar conjugates containing N-acetyl d-glucosamine-6-sulfate sodium salt (GlcNAc6S) competitively inhibit the binding of affinity purified rabbit anti-C-T IgG to the C-T extension of Cz. Extending these findings to the context of natural infection, immune assays performed with Chagas disease serum confirmed that the structure of synthetic GlcNAc6S mimics the N-glycan-linked sulfated epitope displayed in the C-T domain of Cz. 相似文献
11.
Rendić D Klaudiny J Stemmer U Schmidt J Paschinger K Wilson IB 《The Biochemical journal》2007,402(1):105-115
12.
Regulation of the expression of Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipids in kidney 总被引:2,自引:0,他引:2
S P Hendricks P He C L Stults B A Macher 《The Journal of biological chemistry》1990,265(29):17621-17626
Previous studies (Galili, U., Clark, M. R., Shohet, S. B., Buehler, J., and Macher, B. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1369-1373; Galili, U., Shohet, S. B., Korbrin, E., Stults, C. L. M., and Macher, B. A. (1988) J. Biol. Chem. 263, 17755-17762) have established that there is a unique evolutionary distribution of glycoconjugates carrying the Gal alpha 1-3Gal beta 1-4GlcNAc epitope. These glycoconjugates are expressed by cells from New World monkeys and non-primate mammals, but not by cells from humans, Old World monkeys, or apes. The lack of expression of this epitope in the latter species appears to result from the suppression of gene expression for the enzyme UDP-galactose:nLc4Cer alpha 1-3-galactosyltransferase (alpha 1-3GalT) (Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J., and Shaper, N. L. (1989) J. Biol. Chem. 264, 14290-14297). Although many non-primate species are known to express this carbohydrate epitope, the nature (i.e. glycoprotein or glycosphingolipid) of the glycoconjugate carrying this epitope is only known for a few tissues in a few animal species. Furthermore, it is not known whether all animal species express this epitope in the same tissues. We have investigated these questions by analyzing the glycosphingolipids in kidney from several non-primate animal species. Immunostained thin layer chromatograms of glycosphingolipids from sheep, pig, rabbit, cow, and rat kidney with the Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipid-specific monoclonal antibody, Gal-13, demonstrated that kidney from all of these species except rat contained Gal alpha 1-3Gal beta 1-4GlcNAc neutral glycosphingolipids. A lack of expression of Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipids in rat may be due to the lack of expression of the enzyme (alpha 1-3GalT) which catalyzes the formation of the Gal alpha 1-3Gal nonreducing terminal sequence of these compounds or to the lack of expression of glycosyltransferases which are necessary for the synthesis of the neolacto core structure of these compounds. These possibilities were evaluated in two ways. First, the three enzymes (UDP-N-acetylglucosamine:LacCer beta 1-3-N-acetyl-glucosaminyltransferase, UDP-galactose:Lc3Cer beta 1-4-galactosyltransferase, and alpha 1-3GalT) involved in the synthesis of the Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipids were assayed using an enzyme-linked immunosorbent assay-based assay system and carbohydrate sequence-specific monoclonal antibodies. Second, TLC immunostaining was done to determine if the glycosphingolipid precursors (i.e. Lc3Cer and nLc4Cer) are expressed in rat kidney. Interestingly, rat kidney had a relatively high level of alpha 1-3GalT activity compared with the other animals tested.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
13.
The tapered sensory rays of the male Caenorhabditis elegans are important for successful male/hermaphrodite copulation. A group of ram (ray morphology abnormal) genes encoding modifying enzymes and transmembrane protein have been reported as key regulators controlling ray morphogenesis. Here we report the characterization of another component essential for this morphogenetic process encoded by mab-7. This gene is active in the hypodermis, structural cells, the body seam and several head neurons. It encodes a novel protein with a hydrophobic region at the N-terminus, an EGF-like motif, an ShKT motif and a long C-terminal tail. All these domains are shown to be critical to MAB-7 activity except the EGF-like domain, which appears to be regulatory and dispensable. MAB-7 is shown to be a type II membrane protein, tethered on the cell surface by the N-terminal transmembrane domain with the remainder of the protein exposed to the extracellular matrix. Since ectopic mab-7 expression in any ray cell or even in touch neurons of non-ray lineage can rescue the mutant phenotype, mab-7 is probably acting non-autonomously. It may facilitate intercellular communication among ray cells to augment normal ray morphogenesis. 相似文献
14.
W Y Choi J H Youn H W Nam W S Kim W K Kim S Y Park Y W Oh 《The Korean journal of parasitology》1989,27(3):217-223
Four females and a male nematode isolated from 2 patients who visited eye clinics in Seoul were identified as Thelazia callipaeda and their ultrastructures were observed by scanning electron microscopy (SEM). General features of the worms were slender and attenuated at both ends. Vaginal opening was located at 0.27 mm from the anterior end, and in front of the esophago-intestinal junction. In the body cuticle transverse striations varied characteristically through the body. The number of cuticular transverse striations was 400-650/mm at head portion, 250/mm at middle portion and 300-350/mm at tail portion. The SEM observation of the mouth part of the females showed 6 cord-like cuticular thickenings in hexagonal arrangement and an amphid was observed. A lateral line, a vaginal opening, a pair of phasmids, and an anus were identified in the body portion. A pair of papillae and 6 cord-like cuticular thickenings were on the mouth part of the male. It was difficult to observe structures at the tail of the male except wrinkle-like structures. Most of the larvae isolated from the uterus of a female worm were sheathed and thus cuticular striations were not seen. Others were un-sheathed and revealed cuticular striations. The oval membrane which encysted sheathed larvae was also observed. These are the 18th and 19th record of human thelaziasis in Korea as the literature are concerned. 相似文献
15.
The intermediate filament protein IFA-2 is essential for the structural integrity of the Caenorhabditis elegans epidermis. It is one of the major components of the fibrous organelle, an epidermal structure comprised of apical and basal hemidesmosomes linked by cytoplasmic intermediate filaments that serve to transmit force from the muscle to the cuticle. Mutations of IFA-2 result in epidermal fragility and separation of the apical and basal epidermal surfaces during postembryonic growth. An IFA-2 lacking the head domain fully rescues the IFA-2 null mutant, whereas an IFA-2 lacking the tail domain cannot. Conversely, an isolated IFA-2 head was able to localize to fibrous organelles whereas the tail was not. Taken together these results suggest that the head domain contains redundant signals for IF localization, whereas non-redundant essential functions map to the IFA-2, tail, although the tail is unlikely to be directly involved in fibrous organelle localization. 相似文献
16.
Production and characterization of transgenic mice systemically expressing endo-beta-galactosidase C
Watanabe S Misawa M Matsuzaki T Sakurai T Muramatsu T Yokomine TA Sato M 《Glycobiology》2008,18(1):9-19
The alphaGal epitope (Galalpha1-3Gal) is a sugar structure expressed on the cell surface of almost all organisms except humans and old-world-monkeys, which express natural anti-alphaGal antibodies. The presence of these antibodies elicits a hyper acute rejection (HAR) upon xenotransplantation of cellular materials, such as from pigs to human beings. Endo-beta-galactosidase C (EndoGalC), an enzyme isolated from Clostridium perfringens, removes the alphaGal epitope by cleaving the Galbeta1-4GlcNAc linkage in the Galalpha1-3Galbeta1-4GlcNAc sequence. To explore the possibility that cells or organs from transgenic pigs systemically expressing EndoGalC might be suitable for xenotransplantation, we first introduced the EndoGalC transgene into the mouse genome via pronuclear injection. The progeny of the resulting transgenics expressed EndoGalC mRNA and protein. Flow cytometry and histochemical analyses revealed a dramatic reduction in the expression of the alphaGal epitope in these mice. They also exhibited abnormal phenotypes, such as occasional death immediately after birth, growth retardation, and transient skin lesions. Interestingly, the phenotypic abnormalities seen in these transgenics were similar to those observed in beta1,4-galactosyltransferase 1 (beta4GalT-1) knockout (KO) mice. Most probably, these phenotypes were caused by exposure of the internal N-acetylglucosamine residue at the end of the sugar chain on the cell surface. The present findings also provide some basis for evaluating possible application of the transgenic approach for xenotranplantation. 相似文献
17.
S Koyota Y Ikeda S Miyagawa H Ihara M Koma K Honke R Shirakura N Taniguchi 《The Journal of biological chemistry》2001,276(35):32867-32874
The down-regulation of the alpha-Gal epitope (Galalpha1,3Galbeta-R) in swine tissues would be highly desirable, in terms of preventing hyperacute rejection in pig-to-human xenotransplantation. In an earlier study, we reported that the introduction of the beta1,4-N-acetylglucosaminyltransferase (GnT) III gene into swine endothelial cells resulted in a substantial reduction in the expression of the alpha-Gal epitope. In this study, we report on the mechanism for this down-regulation of the alpha-Gal epitope by means of structural and kinetic analyses. The structural analyses revealed that the amount of N-linked oligosaccharides bearing the alpha-Gal epitopes in the GnT-III-transfected cells was less than 10% that in parental cells, due to the alteration of the terminal structures as well as a decrease in branch formation. In addition, it appeared that the addition of a bisecting GlcNAc, which is catalyzed by GnT-III, leads to a more efficient sialylation rather than alpha-galactosylation. In vitro kinetic analyses showed that the bisecting GlcNAc has an inhibitory effect on alpha-galactosylation, but does not significantly affect the sialylation. These results suggest that the bisecting GlcNAc in the core is capable of modifying the biosynthesis of the terminal structures via its differential effects on the capping glycosyltransferase reactions. The findings may contribute to the development of a novel strategy to eliminate carbohydrate xenoantigens. 相似文献
18.
B M Nilsen K Sletten B S Paulsen M O'Neill H van Halbeek 《The Journal of biological chemistry》1991,266(4):2660-2668
The glycoprotein allergen Art v II, from the pollen of mugwort (Artemisia vulgaris L.) was treated with peptide:N-glycosidase F (PNGase F) to release asparagine-linked oligosaccharides. The oligosaccharides were isolated by gel permeation chromatography and their structures determined by 500-MHz 1H NMR spectroscopy, fast atom bombardment-mass spectrometry, and high-pH anion-exchange chromatography. The high-mannose oligosaccharides Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 were present in the ratios 2:49:19:24:6 and accounted for all the asparagine-linked oligosaccharides released from Art v II by PNGase F. The NH2-terminal amino acid sequences of Art v II and of four peptides generated by cyanogen bromide (CNBr) cleavage of deglycosylated Art v II were determined. The first 30 amino acid residues of Art v II did not contain any potential N-glycosylation sites. One potential N-glycosylation site was identified in one of the CNBr fragments. The native protein conformation was shown by enzyme-linked immunosorbent assay inhibition assays to be essential for the binding of rabbit IgG to Art v II and for the binding of human IgE to the major IgE-binding epitope(s) in this allergen. At least one minor IgE-binding epitope still bound IgE after denaturation of the allergen. Removal of the high-mannose chains from denatured Art v II had no significant effect on the binding of human IgE to the minor IgE-binding epitope(s). 相似文献
19.
Biosynthesis of L-selectin ligands: sulfation of sialyl Lewis x-related oligosaccharides by a family of GlcNAc-6-sulfotransferases 总被引:5,自引:0,他引:5
The leukocyte adhesion molecule L-selectin mediates lymphocyte homing to secondary lymphoid organs and to certain sites of inflammation. The cognate ligands for L-selectin possess the unusual sulfated tetrasaccharide epitope 6-sulfo sialyl Lewis x (Siaalpha2-->3Galbeta1-->4[Fucalpha1-->3][SO(3)-->6]GlcNAc). Sulfation of GlcNAc within sialyl Lewis x is a crucial modification for L-selectin binding, and thus, the underlying sulfotransferase may be a key modulator of lymphocyte trafficking. Four recently discovered GlcNAc-6-sulfotransferases are the first candidate contributors to the biosynthesis of 6-sulfo sLex in the context of L-selectin ligands. Here we report the in vitro activity of the four GlcNAc-6-sulfotransferases on a panel of synthetic oligosaccharide substrates that comprise structural motifs derived from sialyl Lewis x. Each enzyme preferred a terminal GlcNAc residue, and was impeded by the addition of a beta1,4-linked Gal residue (i.e., terminal LacNAc). Surprisingly, for three of the enzymes, significant activity was observed with sialylated LacNAc, and two of the enzymes were capable of detectable sulfation of GlcNAc in the context of sialyl Lewis x. On the basis of these results, we propose possible pathways for 6-sulfo sialyl Lewis x biosynthesis and suggest that sulfation may be an early committed step. 相似文献
20.
Moriguchi K Takemoto T Aoki T Nakakita S Natsuka S Hase S 《Journal of biochemistry》2007,142(2):213-227
We previously reported that zebrafishalpha1-3fucosyltrasferase 1 (zFT1) was expressed in embryos at the segmentation period, and was capable of synthesizing the Lewis x epitope [Gal beta1-4(Fuc alpha1-3)GlcNAc] [Kageyama et.al, J. Biochem., 125, 838-845 (1999)]. In the current study, we attempted to detect the enzyme products of zFT1 in zebrafish embryos. Oligosaccharides were prepared from the zebrafish embryos at 12, 18 and 48 h after fertilization and labelled with a fluorophore, 2-aminopyridine, for highly sensitive detections. Pyridylamino (PA)-oligosaccharides that were alpha1-3/4fucosidase sensitive and time-dependently expressed at 18 h after fertilization were identified as candidates for the in vivo products synthesized by zFT1. Structures of these oligosaccharides were determined by a combination of exoglycosidase digestions and two-dimensional HPLC sugar mapping to be the biantennary complex-type structures with two Lewis x epitopes: (Gal beta1-4)(0,1,2)-{Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-6[Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-3]}Man beta1-4GlcNAc, and (Gal beta1-4)(0,1)-{Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-6[Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-3]} Man beta1-4GlcNAc beta1-4GlcNAc. The presence of Lewis x structure of these oligosaccharides together with their expression time suggests that they are products of zFT1. Remarkably, most of these oligosaccharides were free form. Furthermore, we detected an endo-beta-N-acetylglucosaminidase activity in the 18 h embryo. These results suggest that the oligosaccharides synthesized by zFT1 are present in the embryo at the segmentation period in free form, owing to the liberation from glycoproteins with endo-beta-N-acetylglucosaminidase(s) and/or glycoamidase(s). 相似文献