Characterization of a Heparan Sulfate 3-O-Sulfotransferase-5, an Enzyme Synthesizing a Tetrasulfated Disaccharide

Mochizuki, Hiroyuki; Yoshida, Koichiro; Gotoh, Masao; Sugioka, Shigemi; Kikuchi, Norihiro; Kwon, Yeondae; Tawada, Akira; Maeyama, K.; Inaba, Niro; Hiruma, Toru; Kimata, Koji; Narimatsu, Hisashi

doi:10.1074/jbc.m301861200

Cited by 60 publications

(50 citation statements)

References 49 publications

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“…The elution profile is similar to that of our previous report in the case of 3OST-5 (Fig. 3E), and five peaks have been characterized as follows (27). Peaks 2, 4, and 5 are ⌬Di-(N,3,6)S, ⌬Di-(2,N,3)S, and ⌬Di-tetraS, respectively.…”

Section: Molecular Cloning and Expression Of 3ostsupporting

confidence: 59%

“…Construction and Purification of 3OST Family Enzymes Fused with FLAG Peptide-Recombinant 3OST family enzymes were expressed as a fusion protein with FLAG peptide and affinity-purified, based on the methods reported previously (27). DNA fragments coding the putative catalytic domain were amplified by PCR using the Marathon-Ready cDNA of human kidney or placenta as a template.…”

Section: Methodsmentioning

confidence: 99%

“…3OST-3-modified heparan sulfate specifically binds to glycoprotein D (gD) of herpes simplex virus type-1 (HSV-1) and also makes cells susceptible to HSV-1 entry (25,26). The reaction products of 3OST-5 have been determined to be HexA-GlcNS3S6S (Di-(N,3,6)S), HexA2S-GlcNS3S (Di-(2,N,3)S), and HexA2S-GlcNS3S6S (DitetraS) in our previous report (27). 3OST-5-modified heparan sulfate binds to both AT and gD (21).…”

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confidence: 99%

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Tetrasulfated Disaccharide Unit in Heparan Sulfate

Mochizuki

Yoshida

Shibata

et al. 2008

Journal of Biological Chemistry

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We previously reported that the heparan sulfate 3-O-sulfotransferase (3OST)-5 produces a novel component of heparan sulfate, i.e. the tetrasulfated disaccharide (Di-tetraS) unit (Mochizuki, H., Yoshida, K., Gotoh, M., Sugioka, S., Kikuchi, N., Kwon, Y.-D., Tawada, A., Maeyama, K., Inaba, N., Hiruma, T., Kimata, K., and Narimatsu, H. (2003) J. Biol. Chem. 278, 26780 -26787). In the present study, we investigated the potential of other 3OST isoforms to produce Di-tetraS with heparan sulfate and heparin as acceptor substrates. 3OST-2, 3OST-3, and 3OST-4 produce Di-tetraS units as a major product from both substrates. 3OST-5 showed the same specificity for heparin, but the production from heparan sulfate was very low. Di-tetraS production by 3OST-1 was negligible. We then investigated the presence of Di-tetraS units in heparan sulfates from various rat tissues. Di-tetraS was detected in all of the tissues analyzed. Liver and spleen contain relatively high levels of Di-tetraS, 1.6 and 0.95%, respectively. However, the content of this unit in heart, large intestine, ileum, and lung is low, less than 0.2%. We further determined the expression levels of 3OST transcripts by quantitative real time PCR. The 3OST-3 transcripts are highly expressed in spleen and liver. The 3OST-2 and -4 are specifically expressed in brain. These results indicate that the Di-tetraS unit is widely distributed throughout the body as a rare and unique component of heparan sulfate and is synthesized by tissue-specific 3OST isoforms specific for Di-tetraS production.

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Section: Molecular Cloning and Expression Of 3ostsupporting

confidence: 59%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Tetrasulfated Disaccharide Unit in Heparan Sulfate

Mochizuki

Yoshida

Shibata

et al. 2008

Journal of Biological Chemistry

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“…At the disaccharide level, 3-OST-4 recognizes heavily sulfated disaccharides and generates tri-and tetra-sulfated disaccharides. Tetra-sulfated disaccharide is the most heavily sulfated disaccharide in HS and is also found in 3-OST-5 generated products (23). The biological significance of this disaccharide is under further investigation.…”

Section: Discussionmentioning

confidence: 99%

Determining Heparan Sulfate Structure in the Vicinity of Specific Sulfotransferase Recognition Sites by Mass Spectrometry

Lech

Beeler

et al. 2004

Journal of Biological Chemistry

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Sulfated motifs on heparan sulfate (HS) are involved in various extracellular processes from cell signaling to enzymatic regulation, but the structures of these motifs are obscure. We have developed a strategy to determine the structure of sulfotransferase recognition sites which constitute these motifs. Stable isotope is first introduced into specific sites on HS with HS sulfotransferases and the modified HS is then digested into oligosaccharides of differing sizes. The overlapping oligosaccharides containing the introduced stable isotope are identified by changes in the m/z profiles by mass spectrometry, and their relationships are elucidated. In this way, the HS structures in the vicinity of the sulfotransferase recognition site are quickly determined and groups on precursor structures of HS that direct the action of HS sulfotransferases are pinpointed.Heparan sulfate (HS) 1 is one major polysaccharide found on proteoglycans. HS chains are first synthesized in the Golgi apparatus as repeated units of the disaccharide of a glucuronic acid and an N-acetylated glucosamine (GlcA-GlcNAc) n (1). The glucuronic acid can be epimerized to iduronic acid (IdoA). Incomplete sulfation at the 2-O positions of the uronic acids, and the 3-O, 6-O, and N positions of the glucosamine by various sulfotransferases results in structural diversity within HS (1, 2). The sulfate groups usually cluster in small regions and form sulfated motifs.The sulfated motifs can bind to growth factors, cytokines, and morphogens as well as proteases in the extracellular matrix and regulate their activities (3); thus HS plays critical roles in organ development (4 -6), morphogenesis (7, 8), angiogenesis (2), blood coagulation (2), inflammation (1), wound healing, and cancer progression (9). These sulfated motifs also play roles in pathological processes, such as functioning as receptors for viral entry (10 -13).Although HS plays important roles in various biological processes, the structures of these biological motifs are obscure, due to the difficulties involved in obtaining homogeneous components and determining their structures. Cloning, expressing, and sequencing biopolymers have tremendously advanced our understanding of DNA and protein, but no similar methods are available for studying HS.In this report, we describe a novel strategy that permits us to quickly determine specific HS structures in the vicinity of the sulfotransferase recognition site for 3-OST-4. HS 3-OSTs are rare modification enzymes that helps to generate binding sites for proteins such as antithrombin III (2) and herpes simplex virus glycoprotein D (11). 3-OST-4 is specifically expressed in brain tissues and may play a role in neuronal development (14). The delineation of sulfated motifs on the HS chain of proteoglycans will shed light on how cells interact with extracellular proteins, respond to extracellular signals, and initiate signaling cascade beginning on the cell membrane (15). This strategy relies on introducing a stable isotope of sulfate into specific sites on the...

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“…Indeed, several mammalian 3-OST isoforms are expressed in the brain (Mochizuki et al, 2003;Shworak et al, 1999); however, it is unknown if any of these isoforms exhibit neuronal expression. The identification of the major neuronal 3-OST isoforms would enable investigations into whether these critical regulators of HS motifs are involved in developmental, physiological and pathological aspects of neurobiology.…”

Section: Introductionmentioning

confidence: 99%

The principal neuronal gD-type 3-O-sulfotransferases and their products in central and peripheral nervous system tissues☆

et al. 2007

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Within the nervous system, heparan sulfate (HS) of the cell surface and extracellular matrix influences developmental, physiologic and pathologic processes. HS is a functionally diverse polysaccharide that employs motifs of sulfate groups to selectively bind and modulate various effector proteins. Specific HS activities are modulated by 3-O-sulfated glucosamine residues, which are generated by a family of seven 3-O-sulfotransferases (3-OSTs). Most isoforms we herein designate as gD-type 3-OSTs because they generate HS gD+ , 3-O-sulfated motifs that bind the gD envelope protein of herpes simplex virus 1 (HSV-1) and thereby mediate viral cellular entry. Certain gD-type isoforms are anticipated to modulate neurobiologic events, because a Drosophila gD-type 3-OST is essential for a conserved neurogenic signaling pathway regulated by Notch. Information about 3-OST isoforms expressed in the nervous system of mammals is incomplete. Here, we identify the 3-OST isoforms having properties compatible with their participation in neurobiologic events. We show that 3-OST-2 and 3-OST-4 are principal isoforms of brain. We find these are gD-type enzymes, as they produce products similar to a prototypical gD-type isoform, and they can modify HS to generate receptors for HSV-1 entry into cells. Therefore, 3-OST-2 and 3-OST-4 catalyze modifications similar or identical to those made by the Drosophila gD-type 3-OST that has a role in regulating Notch signaling. We also find that 3-OST-2 and 3-OST-4 are the predominant isoforms expressed in neurons of the trigeminal ganglion, and 3-OST-2/4-type 3-O-sulfated residues occur in

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Characterization of a Heparan Sulfate 3-O-Sulfotransferase-5, an Enzyme Synthesizing a Tetrasulfated Disaccharide

Cited by 60 publications

References 49 publications

Tetrasulfated Disaccharide Unit in Heparan Sulfate

Tetrasulfated Disaccharide Unit in Heparan Sulfate

Determining Heparan Sulfate Structure in the Vicinity of Specific Sulfotransferase Recognition Sites by Mass Spectrometry

The principal neuronal gD-type 3-O-sulfotransferases and their products in central and peripheral nervous system tissues☆

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