Substrate Specificity of the Heparan Sulfate Hexuronic Acid 2-<i>O</i>-Sulfotransferase

Rong, Jianhui; Habuchi, Hiroko; Kimata, Koji; Lindahl, Ulf; Kusche-Gullberg, Marion

doi:10.1021/bi002926p

Cited by 95 publications

(96 citation statements)

References 55 publications

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“…The 2-Osulfated glucuronic acid residue is a rare constituent of HS, and was found in HS isolated from the adult human cerebral cortex, a nuclear fraction from hepatocytes, HS from bovine kidney, and heparin from porcine intestines (41)(42)(43). This residue is synthesized by HS 2-O-sulfotransferase, although the enzyme preferably generates 2-O-sulfated iduronic acid (IdoUA2S) (44). To this end, it is still not known whether the presence of a 2-O-sulfated glucuronic acid, or possibly a 2-O-sulfated iduronic acid, residue is essential for gD binding.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Heparan Sulfate Octasaccharide That Binds to Herpes Simplex Virus Type 1 Glycoprotein D

Liu

Shriver

Pope

et al. 2002

Journal of Biological Chemistry

153

116

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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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Section: Discussionmentioning

confidence: 99%

Characterization of a Heparan Sulfate Octasaccharide That Binds to Herpes Simplex Virus Type 1 Glycoprotein D

Liu

Shriver

Pope

et al. 2002

Journal of Biological Chemistry

153

116

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“…Therefore, epimerization starts after N-deacetylation and N-sulfation but before 6-Oand 3-O-sulfation (38). The iduronic acid residue may then be 2-O-sulfated by the 2-O-sulfotransferase, which acts on both IdoA and GlcA units but prefers the former (39,40). The percentage of 2-O-sulfation of the GlcA units is very low (32), and GlcA2S-GlcNAc disaccharide units have not been reported in HS/heparin.…”

Section: Discussionmentioning

confidence: 99%

Detection of 2-O-Sulfated Iduronate and N-Acetylglucosamine Units in Heparan Sulfate by an Antibody Selected against Acharan Sulfate (IdoA2S-GlcNAc)

Dam¹,

Westerlo²,

Smetsers³

et al. 2004

Journal of Biological Chemistry

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The snail glycosaminoglycan acharan sulfate (AS) is structurally related to heparan sulfates (HS) and has a repeating disaccharide structure of ␣-D-N-acetylglucosaminyl-2-O-sulfo-␣-L-iduronic acid (GlcNAc-IdoA2S) residues. Using the phage display technology, a unique antibody (MW3G3) was selected against AS with a V H 3, DP 47, and a CDR3 amino acid sequence of QKKRPRF. Antibody MW3G3 did not react with desulfated, N-deacetylated or N-sulfated AS, indicating that reactivity depends on N-acetyl and 2-O-sulfate groups. Antibody MW3G3 also had a high preference for (modified) heparin oligosaccharides containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues. In tissues, antibody MW3G3 identified a HS oligosaccharide epitope containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues as enzymatic N-deacetylation of HS in situ prevented staining, and 2-O-sulfotransferase-deficient Chinese hamster ovary cells were not reactive. An immunohistochemical survey using various rat organs revealed a distinct distribution of the MW3G3 epitope, which was primarily present in the basal laminae of most (but not all) blood vessels and of some epithelia, including human skin. No staining was observed in the glycosaminoglycan-rich tumor matrix of metastatic melanoma. In conclusion, we have selected an antibody that identifies HS oligosaccharides containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues. This antibody may be instrumental in identifying structural alterations in HS in health and disease.Acharan sulfate (AS) 1 is a glycosaminoglycan (GAG) abundantly present in the giant African snail Achatina fulica (1). It largely consists of the repeating disaccacharide structure of 34)-␣-D-2-acetamido-2-deoxyglucopyranose(134)-␣-L-idopyranosyluronic acid-2-sulfate(13 and is closely related to heparan sulfates. AS is located in large granules present in the outer surface of the snail and is secreted onto the surface as mucus. This mucus consists of 26% proteins, and the GAG moiety is entirely formed by AS. Whether AS is present as a proteoglycan is unclear (2). The pattern of adjacent N-acetylglucosamine and 2-sulfoiduronic acid residues is unusual and suggests interesting biological activities. Among the proposed biological functions of AS in snails are binding, uptake, and transport of divalent cations and functioning as an antidesiccant (1). AS inhibits the mitogenic activity induced by basic fibroblast growth factor (3), angiogenesis (4), and tumor growth (5).To study the cell biological importance of specific HS epitopes and their location in tissue, single chain antibodies have been generated as described by our group (6 -9). These antibodies were selected against HS from various sources and stained differentially in tissues sections from various organs. Defined HS oligosaccharides were used to reveal details of the epitopes recognized by the antibodies (6). Although some chemical groups in HS essential for antibody reactivity could be determined, the oligosaccharide sequence...

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“…R ecently, substrate specificity investigations along with specificity constant (k cat /K M ) measurements have provided significant insights into a number of biologically important enzymes [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]. Specificity studies of an enzyme for different natural or synthetic substrates can help determine the enzyme structure and physiological functions [1, 4 -7] as well as address the key residues involved in substrate binding and catalysis [2,8].…”

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confidence: 99%

“…Traditionally, the general method of evaluating the specificity of a given enzyme is to measure the specificity constant of a group of substrates individually followed by comparison of measured values, regardless of the assay used for measurement [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]. This requires large amounts of material and considerable effort to study substrate libraries.…”

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confidence: 99%

Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

Leary

2004

J. Am. Soc. Mass Spectrom.

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The traditional method used to investigate the reaction specificity of an enzyme with different substrates is to perform individual kinetic measurements. In this case, a series of varied concentrations are required to study each substrate and a non-regression analysis program is used several times to obtain all the specificity constants for comparison. To avoid the large amount of experimental materials, long analysis time, and redundant data processing procedures involved in the traditional method, we have developed a novel strategy for rapid determination of enzyme substrate specificity using one reaction system containing multiple competing substrates. In this multiplex assay method, the electrospray ionization mass spectrometry (ESI-MS) technique was used for simultaneous quantification of multiple products and a steady-state kinetics model was established for efficient specificity constant calculation. The system investigated was the bacterial sulfotransferase NodH (NodST), which is a host specific nod gene product that catalyzes the sulfate group transfer from 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS) to natural Nod factors or synthetic chitooligosaccharides. Herein, the reaction specificity of NodST for four chitooligosaccharide acceptor substrates of different chain length (chitobiose, chitotriose, chitotetraose, and chitopentaose) was determined by both individual kinetic measurements and the new multiplex ESI-MS assay. The results obtained from the two methods were compared and found to be consistent. The multiplex ESI-MS assay is an accurate and valid method for substrate specificity evaluation, in which multiple substrates can be evaluated in one assay. (J Am Soc Mass Spectrom 2004, 15, 233-243)

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Substrate Specificity of the Heparan Sulfate Hexuronic Acid 2-O-Sulfotransferase

Cited by 95 publications

References 55 publications

Characterization of a Heparan Sulfate Octasaccharide That Binds to Herpes Simplex Virus Type 1 Glycoprotein D

Characterization of a Heparan Sulfate Octasaccharide That Binds to Herpes Simplex Virus Type 1 Glycoprotein D

Detection of 2-O-Sulfated Iduronate and N-Acetylglucosamine Units in Heparan Sulfate by an Antibody Selected against Acharan Sulfate (IdoA2S-GlcNAc)

Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

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