Samples of heparan sulfate, isolated from bovine aorta, lung, intestine, and kidney, were degraded by digestion with a mixture of heparitinases or by treatment with nitrous acid, with or without previous Ndeacetylation. Analysis of the resulting oligosaccharides showed that the various heparan sulfate samples all contained regions of up to 8 or 9 consecutive Nacetylated glucosamine residues, as well as contiguous N-sulfated sequences. L-Iduronic acid accounted for a remarkably constant proportion, 50 -60%, of the total hexuronic acid units within the latter structures. Of the total iduronic acid units, 36 -55% were located outside the contiguous N-sulfated regions, presumably in sequences composed of alternating N-acetylated and Nsulfated disaccharide residues. While most of the iduronic acid units within the N-sulfated blocks were 2-Osulfated, those located outside were almost exclusively nonsulfated. The heparan sulfate preparations differed markedly with regard to the content of 6-O-sulfated glucosamine units, more than half of which were located outside the N-sulfated block regions. These findings suggest that the formation of iduronic acid residues and their subsequent 2-O-sulfation are coupled within but not outside the contiguous N-sulfated regions of the heparan sulfate chains and, furthermore, that the 2-Oand 6-O-sulfotransferase reactions are differentially regulated during heparan sulfate biosynthesis.
Membrane fusion is an essential step in the encounter of two nuclei from sex cells-sperm and egg-in fertilization. However, aside from the involvement of two molecules, CD9 and Izumo, the mechanism of fusion remains unclear. Here, we show that spermegg fusion is mediated by vesicles containing CD9 that are released from the egg and interact with sperm. We demonstrate that the CD9 ؊/؊ eggs, which have a defective sperm-fusing ability, have impaired release of CD9-containing vesicles. We investigate the fusion-facilitating activity of CD9-containing vesicles by examining the fusion of sperm to CD9 ؊/؊ eggs with the aid of exogenous CD9-containing vesicles. Moreover, we show, by examining the fusion of sperm to CD9 ؊/؊ eggs, that hamster eggs have a similar fusing ability as mouse eggs. The CD9-containing vesicle release from unfertilized eggs provides insight into the mechanism required for fusion with sperm.
We previously cloned heparan sulfate 6-O-sulfotransferase (HS6ST) (Habuchi, H., Kobayashi, M., and Kimata, K. (1998) J. Biol. Chem. 273, 9208 -9213). In this study, we report the cloning and characterization of three mouse isoforms of HS6ST, a mouse homologue to the original human HS6ST (HS6ST-1) and two novel HS6STs (HS6ST-2 and HS6ST-3). The cDNAs have been obtained from mouse brain cDNA library by cross-hybridization with human HS6ST cDNA. The three cDNAs contained single open reading frames that predicted type II transmembrane proteins composed of 401, 506, and 470 amino acid residues, respectively. Amino acid sequence of HS6ST-1 was 51 and 57% identical to those of HS6ST-2 and HS6ST-3, respectively. HS6ST-2 and HS6ST-3 had the 50% identity. Overexpression of each isoform in COS-7 cells resulted in about 10-fold increase of HS6ST activity. The three isoforms purified with anti-FLAG antibody affinity column transferred sulfate to heparan sulfate and heparin but not to other glycosaminoglycans. Each isoform showed different specificity toward the isomeric hexuronic acid adjacent to the targeted N-sulfoglucosamine; HS6ST-1 appeared to prefer the iduronosyl N-sulfoglucosamine while HS6ST-2 had a different preference, depending upon the substrate concentrations, and HS6ST-3 acted on either substrate. Northern analysis showed that the expression of each message in various tissues was characteristic to the respective isoform. HS6ST-1 was expressed strongly in liver, and HS6ST-2 was expressed mainly in brain and spleen. In contrast, HS6ST-3 was expressed rather ubiquitously. These results suggest that the expression of these isoforms may be regulated in tissue-specific manners and that each isoform may be involved in the synthesis of heparan sulfates with tissue-specific structures and functions.Heparan sulfate proteoglycans (HSPGs) 1 are ubiquitously present on cell surface and in extracellular matrix including basement membrane and have divergent structures and functions (1-3). The heparan sulfate (HS) chains in HSPGs are known to interact with a variety of proteins such as heparinbinding growth factors, extracellular matrix components, protease inhibitors, protease, and lipoprotein lipase (4 -8). These interactions are implicated not only in various dynamic cellular behaviors including cell proliferation, differentiation, adhesion, migration, and morphology during development (9 -16), but also in various physiological phenomena such as inflammation (17), blood coagulation (18 -20), and tumor cell invasion and malignancy (21-23). Moreover, the pathogens such as bacteria, parasites, and viruses are known to infect host cells through the interactions between the cell surface HS on host cell and the coat proteins or cell surface proteins of pathogens (24,25).Recently, genetic screens and analyses are suggesting not only in Drosophila but also in mammals that these interactions also play pivotal roles in embryonic development. For example, the sugarless mutant (10 -12), which is deficient in UDP-glucose dehydrogenas...
3-O-Sulfation of glucosamine by heparan sulfate D-glucosaminyl 3-O-sulfotransferase (3-OST-1) is the key modification in anticoagulant heparan sulfate synthesis.However, the heparan sulfates modified by 3-OST-2 and 3-OST-3A, isoforms of 3-OST-1, do not have anticoagulant activity, although these isoforms transfer sulfate to the 3-OH position of glucosamine residues. In this study, we characterize the substrate specificity of purified 3-OST-3A at the tetrasaccharide level. The 3-OST-3A enzyme was purified from Sf9 cells infected with recombinant baculovirus containing 3-OST-3A cDNA. Two 3-OST-3A-modified tetrasaccharides were purified from the 3-O-35 S-sulfated heparan sulfate that was digested by heparin lyases. These tetrasaccharides were analyzed using nitrous acid and enzymatic degradation combined with matrix-assisted laser desorption/ionization-mass spectrometry. Two novel tetrasaccharides were discovered with proposed structures of ⌬UA2S-GlcNS-IdoUA2S- HS act contains defined antithrombin-binding sites with the structure -GlcNSorAc6S-GlcUA-GlcNS3SϮ6S-IdoUA2S-Glc-NS6S-(12, 13). Within the pentasaccharide, 3-O-sulfation to form GlcNS3SϮ6S is one of the critical modifications that confers antithrombin binding affinity (14). This critical 3-Osulfation is performed by 3-OST-1 (EC 2.8.2.23) (10, 15). By using purified 3-OST-1, we found that there are six antithrombin-binding sites in a single HS act chain, suggesting that the synthesis of HS act is a highly organized process requiring a specific biosynthetic pathway (16).The different isoforms of 3-O-sulfotransferase sulfate unique disaccharides and generate HS with different biological functions (17). These isoforms have more than 60% homology in the sulfotransferase domain (18). The isoforms are expressed at different levels in various human tissues (18), suggesting their importance in making tissue-specific HS.Given that 3-O-sulfation of glucosamine made by 3-OST-1 is critical for synthesizing HS act and is a rare modification in any given HS (10, 15, 19), we are interested in determining the biological functions of 3-OST-3 and 3-OST-2 modified HS. In * This work was supported by National Institutes of Health Grants 5.P01.HL41484 (to R. D. R.) and R01.GM57073 (to R. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ** To whom correspondence and reprint requests should be addressed: 68-480, Massachusetts Institute of Technology, 31 Ames St., Cambridge, MA 02139. Tel.: 617-253-8803; Fax: 617-258-6553. 1 The abbreviations used are: HS, heparan sulfate; PAPS, 3Ј-phosphoadenosine 5Ј-phosphosulfate; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; ⌬UA, ⌬ 4,5 -unsaturated uronic acid; GlcUA, D-glucuronic
Herpes simplex virus type 1 utilizes cell surface heparan sulfate as receptors to infect target cells. The unique heparan sulfate saccharide sequence offers the binding site for viral envelope proteins and plays critical roles in assisting viral infections. A specific 3-O-sulfated heparan sulfate is known to facilitate the entry of herpes simplex virus 1 into cells. The 3-O-sulfated heparan sulfate is generated by the heparan sulfate D-glucosaminyl-3-O-sulfotransferase isoform 3 (3-OST-3), and it provides binding sites for viral glycoprotein D (gD). Here, we report the purification and structural characterization of an oligosaccharide that binds to gD. The isolated gDbinding site is an octasaccharide, and has a binding affinity to gD around 18 M, as determined by affinity coelectrophoresis. The octasaccharide was prepared and purified from a heparan sulfate oligosaccharide library that was modified by purified 3-OST-3 enzyme. The molecular mass of the isolated octasaccharide was determined using both nanoelectrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. The results from the sequence analysis suggest that the structure of the octasaccharide is a heptasulfated octasaccharide. The proposed structure of the octasaccharide is ⌬UA-GlcNSIdoUA2S-GlcNAc-UA2S-GlcNS-IdoUA2S-GlcNH 2 3S6S. Given that the binding of 3-O-sulfated heparan sulfate to gD can mediate viral entry, our results provide structural information about heparan sulfate-assisted viral entry.Heparan sulfates (HS), 1 highly sulfated polysaccharides, are present on the surface of mammalian cells and in the extracellular matrix in large quantities. HS play critical roles in a variety of biological interactions, including assisting viral infection, regulating blood coagulation and embryonic development, suppressing tumor growth, and controlling the eating behavior of mice by interacting with specific regulatory proteins (1-5). HS is initially synthesized as a copolymer of glucuronic acid and N-acetylated glucosamine by D-glucuronyl and N-acetyl-D-glucosaminyl transferase, followed by various modifications (6). These modifications include C 5 -epimerization of glucuronic acid to form iduronic acid residues, 2-O-sulfation of iduronic and glucuronic acid, N-deacetylation and N-sulfation of glucosamine, as well as 6-O-sulfation and 3-O-sulfation of glucosamine. Numerous HS biosynthetic enzymes have been cloned and characterized (for review, see Esko and Lindahl (7)).The specific sulfated saccharide sequences play critical roles in determining the functions of HS. A recent report suggests that the expression levels of various isoforms of each class of HS biosynthetic enzyme contribute to the synthesis of specific saccharide sequences in specific tissues (8). HS N-deacetylase/ N-sulfotransferase, 3-O-sulfotransferase, and 6-O-sulfotransferase are present in multiple isoforms, and each isoform is believed to recognize the saccharide sequence around the modification site to generate a specific sulfated saccharid...
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