Biosynthesis of 3-O-sulfated heparan sulfate: unique substrate specificity of heparan sulfate 3-O-sulfotransferase isoform 5

Jinghua Chen2, Michael B. Duncan2, Kevin Carrick3, R. Marshall Pope3 and Jian Liu1,2

2 Division of Medicinal Chemistry and Natural Products, School of Pharmacy, CB#7360, University of North Carolina, Chapel Hill, NC 27599; and 3 Proteomic Core Facility, Department of Biochemistry, University of North Carolina, Chapel Hill, NC 27599

Received on June 5, 2003; revised on July 9, 2003; accepted on July 10, 2003


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Heparan sulfate 3-O-sulfotransferase transfers sulfate to the 3-OH position of a glucosamine to generate 3-O-sulfated heparan sulfate (HS), which is a rare component in HS from natural sources. We previously reported that 3-O- sulfotransferase isoform 5 (3-OST-5) generates both an antithrombin-binding site to exhibit anticoagulant activity and a binding site for herpes simplex virus 1 glycoprotein D to serve as an entry receptor for herpes simplex virus. In this study, we characterize the substrate specificity of 3-OST-5 using the purified enzyme. The enzyme was expressed in insect cells using the baculovirus expression approach and was purified by using heparin-Sepharose and 3',5'-ADP- agarose chromatographies. As expected, the purified enzyme generates both an antithrombin binding site and a glycoprotein D binding site. We isolated IdoUA-AnMan3S and IdoUA-AnMan3S6S from nitrous acid–degraded 3-OST-5-modified HS (pH 1.5), suggesting that 3-OST-5 enzyme sulfates the glucosamine residue that is linked to an iduronic acid residue at the nonreducing end. We also isolated a disaccharide with a structure of {Delta}UA2S-GlcNS3S and a tetrasaccharide with a structure of {Delta}UA2S-GlcNS-IdoUA2S-GlcNH23S6S from heparin lyases–digested 3-OST-5-modified HS. Our results suggest that 3-OST-5 enzyme sulfates both N-sulfated glucosamine and N-unsubstituted glucosamine residues. Taken together, the results indicate that 3-OST-5 has broader substrate specificity than those of 3-OST-1 and 3-OST-3. The unique substrate specificity of 3-OST-5 serves as an additional tool to study the mechanism for the biosynthesis of biologically active HS.

Key words: antithrombin / anticoagulation / heparan sulfate / heparan sulfate sulfotransferase / herpes simplex virus


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Heparan sulfates (HSs), highly sulfated polysaccharides, are present on the surface of mammalian cells and in the extracellular matrix in large quantities. HSs play critical roles in a variety of biological processes, 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 (Alexander et al., 2000Go; Bernfield et al., 1999Go; Liu and Thorp, 2002Go; Reizes et al., 2001Go; Rosenberg et al., 1997Go). The disaccharide repeating units of HS consist of 1-4-linked sulfated glucosamine and sulfated glucuronic/iduronic acid residues. The specific sulfated saccharide sequences within HS determine the functions (Sasisekharan et al., 2002Go).

One approach to understanding the relationship between the functions of HS and the saccharide sequences is to investigate the mechanism for the biosynthesis of HS. HS is initially synthesized as a copolymer of glucuronic acid and N-acetylated glucosamine by D-glucuronyl and N-acetyl-D-glucosaminyltransferase, followed by various modifications in the Golgi apparatus (Lindahl et al., 1998Go). These modifications include N-deacetylation and N-sulfation of glucosamine, C5 epimerization of glucuronic acid to form iduronic acid residues, 2-O-sulfation of iduronic and glucuronic acid residues, as well as 6-O-sulfation and 3-O-sulfation of glucosamine residue. The enzymes that are responsible for the biosynthesis of HS have been cloned and characterized (Esko and Lindahl, 2001Go).

HS sulfotransferases, except for HS 2-O-sulfotransferase, are present in multiple isoforms (Aikawa et al., 2001Go; Habuchi et al., 2000Go, 2003Go; Liu and Rosenberg, 2002Go). The expression levels of these isoforms could play roles in regulating the biosynthesis of the HS with defined saccharide sequences (Liu et al., 1999bGo). It is now known that the isoforms are expressed at different levels in different tissues. Grobe and Esko (2002)Go reported that 5'-untranslated regions of N-deacetylase/N-sulfotransferase isoforms play an important role in determining the translational efficiency of the enzymes in different tissues. N-deacetylase/N- sulfotransferase isoforms and 3-O-sulfotransferase (3-OST) isoforms recognize the saccharide sequences around the modification sites to exhibit different substrate specificities (Aikawa et al., 2001Go; Liu et al., 1999bGo; Xia et al., 2002Go), whereas distinct substrate specificities among isoforms of 6-OST were not detected (Jemth et al., 2003Go; Smeds et al., 2003Go).

The 3-O-sulfation of glucosamine represents the last important modification step during the biosynthesis of biologically active HS. The 3-O-sulfated HSs are known to bind to antithrombin, herpes simplex virus (HSV) 1 glycoprotein D (gD) and growth factor receptor and fibroblast growth factor 7 (Liu et al., 1996Go; McKeehan et al., 1999Go; Shukla et al., 1999Go; Ye et al., 2001Go). The diversified biological functions of 3-O-sulfated HS are likely biosynthesized by different 3-OST isoforms. A total of six different 3-OST isoforms have been identified (Liu and Rosenberg, 2002Go; Xia et al., 2002Go). Recent reports have shown that the expression of 3-OST-2 in rat pineal is daylight sensitive and regulated by ß-adrenergic signaling pathway (Borjigin et al., 2003Go). In addition, a lower expression of 3-OST-2 was discovered in various human cancers, suggesting a potential role of 3-OST-2 in controlling the transformation of normal cells to cancer cells (Miyamoto et al., 2003Go).

The specific structures of the HSs that play roles in regulating anticoagulation and assisting HSV infection are known. It was reported that the enzymes for biosynthesizing antithrombin (AT)-binding HS and gD-binding HS belong to two 3-OST isoforms (Liu et al., 1996Go; Shukla et al., 1999Go). The 3-OST-1-modified HS binds to AT, exhibiting anticoagulant activity, whereas the 3-OST-3-modified HS binds to gD, serving as an entry receptor for HSV-1. The results from structural analysis indicated that the saccharide structures of AT-binding sites and gD- binding sites are different (Atha et al., 1985Go; Liu et al., 2002Go). We have recently reported that 3-OST-5 generates both an AT-binding site and an entry receptor for HSV-suggesting that 3-OST-5 has unique substrate specificity (Xia et al., 2002Go). In this article, we describe expression and purification of 3-OST-5 from insect cells using baculovirus expression approach. Using the purified enzyme, we uncovered a novel substrate specificity of 3-OST-5 that has broader substrate specificity than those of either 3-OST-1 or 3-OST-3. It is possible that 3-OST-5 may contribute to the biosynthesis of HS with unique biological functions.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Purification of recombinant 3-OST-5
Infection of SF9 cells with recombinant 3-OST-5 baculovirus elevated the level of HS sulfotransferase activity by 20-fold in the media, suggesting that the 3-OST-5 enzyme was successfully expressed. We employed both heparin-Sepharose and 3',5'-ADP chromatographies to purify 3-OST-5 because both columns were previously used to purify 3-OST-1, 3-OST-3A, and other HS sulfotransferases (Habuchi et al., 1995Go; Kobayashi et al., 1996Go; Liu et al., 1996Go, 1999aGo; Wlad et al., 1994Go). From 1450 ml of media, we obtained 560 µg protein with 27.0-fold purification and 31.4% recovery yield, as shown in Table I. The purity of the purified 3-OST-5 was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel (Figure 1). The purified protein was predominantly migrated in three bands at 34–40 kDa, labeled B1, B2, and B3 in Figure 1. The molecular weights of those observed bands are close to the calculated molecular weight of 37 kDa for 3-OST-5. The bands were cut from the gel and subjected to in-gel trypsin digestion. The resultant peptides were analyzed by mass spectrometry. Our results confirmed that all three bands contain the amino acid sequences of 3-OST-5, suggesting that the protein is pure. The observed multiple bands for purified 3-OST-5 are likely to be the products of the incomplete glycosylation or partial proteolysis during the expression in SF9 cells.


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Table I. Summary of the purification of 3-OST-5 from SF9 cells media

 


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Fig. 1. Analysis of purified 3-OST-5 by SDS–PAGE. Purified 3-OST-5 (1 µg) was analyzed by 13% SDS–PAGE and stained with Coomassie blue.

 
Characterization of 3-OST-5 modified HS
3-OST-5-modified HS was degraded by nitrous acid at pH 1.5, and the resultant disaccharides were resolved by reverse phase ion-pairing–high-performance liquid chromatography (RPIP-HPLC) (Figure 2). As expected, we observed three 3-O-[35S]sulfated disaccharides, IdoUA2S-[35S]2,5-anhydromannitol (AnMan) 3S, GlcUA-[3-35S] AnMan3S6S, and IdoUA2S-[3-35S]AnMan3S6S (labeled disaccharides 3, 5, and 6, respectively). Those disaccharides were reported in the 3-OST-5-modified HS, where the enzyme was prepared from COS-7 cells, in a prior publication (Xia et al., 2002Go). The results suggested that the recombinant enzyme from insect cells has similar substrate specificity to that of the enzyme expressed in COS-7 cells. It should be noted that disaccharide 4 was also observed in our previous study (Xia et al., 2002Go) and designated as unknown. However, we found that disaccharides 1, 2, and 4 are all 3-O-[35S]sulfated disaccharides in this study, as will be described. It should be noted that we did not investigate whether 3-OST-5 (prepared from COS-7 cells) generates monosulfated disaccharides in the prior publication. The reason was that the levels of monsulfated disaccharides were low, which made it difficult to identify those disaccharides in the presence of large amount of [35S]sulfate (Xia et al., 2002Go).



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Fig. 2. RPIP-HPLC chromatogram of the disaccharide analysis of 3-OST-5-modified HS. Purified 3-OST-5 enzyme was incubated with unlabeled HS and [35S]PAPS to prepare the 3-O-[35S]sulfated HS. The [35S]HS was degraded by nitrous acid at pH 1.5, followed by sodium borohydride reduction. The resultant 35S-labeled disaccharides were resolved by RPIP-HPLC. The elution positions of the disaccharide standards are indicated by arrows, where arrow 1 represents GlcUA-AnMan3S; arrow 2 represents IdoUA-AnMan3S; arrow 3 represents IdoUA2S-AnMan3S; arrow 4 represents IdoUA-AnMan3S6S; arrow 5 represents GlcUA-AnMan3S6S; and arrow 6 represents IdoUA2S-AnMan3S6S. The results for proving the structures of IdoUA-AnMan3S and IdoUA-AnMan3S6S are shown in Figure 3.

 


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Fig. 3. PAMN-HPLC chromatograms of disaccharides 2 and 4 before and after the digestions of {alpha}-iduronidase. (A) Elution profile of {alpha}-iduronidase-digested disaccharide 2. (B) Elution profile of {alpha}-iduronidase-digested disaccharide 4. The elution positions of undigested disaccharides 2 and 4 on PAMN-HPLC were indicated by arrows in A and B, respectively. The actions of {alpha}-iduronidase on disaccharides 2 and 4 are illustrated on the sides of A and B, respectively.

 
We tested the specificity of 3-OST-5 toward various glycosaminoglycans and various desulfated heparins (Table II). Both heparins and HSs serve as good receptors for 3-O-[35S]sulfation, whereas hyaluronic acid, chondroitin sulfate A, and chondroitin sulfate B are not good substrates for 3-OST-5. The result suggests that 3-OST-5 is specific for heparin and HS. We also examined the susceptibility of chemically desulfated heparins to 3-OST-5 modification. Both 2-O-desulfated heparin and 6-O-desulfated heparin serve as substrates for the enzyme. However, completely O-desulfated heparin is not a substrate for 3-OST-5. The results suggest that the enzyme requires either 2-O- or 6-O-sulfation in heparin to be modified by the 3-OST-5 enzyme. N-desulfated heparin is not a substrate, suggesting that N-sulfation is required for 3-OST-5 modification. It should be noted that heparin is not a substrate for 3-OST-3 (as opposed to HS) (Liu et al., 1999aGo) but is a substrate for 3-OST-1 (unpublished data). We noted that a higher amount of 35S-counts were observed using hyaluronic acid, chondroitin sulfate A, and chondroitin sulfate B as substrates than those of using completely O-desulfated heparin and N- desulfated heparin as substrates. A possible reason is that those glycosaminoglycans were contaminated by heparin or HS.


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Table II. Sulfation of glycosaminoglycans and desulfated heparins by 3-OST-5

 
We determined the bindings of 3-OST-5-modified HS to HSV-1 gD and AT. The 3-OST-5-modified HS was prepared by incubating [3H]HS, which was isolated from Chinese hamster ovary (CHO) cells grown in a medium containing [3H]glucosamine,and purified 3-OST-5 enzyme. We also prepared 3-OST-1-modified HS and 3-OST-3-modified HS as positive controls for the binding to AT and gD, respectively. As shown in Table III, 8.3% of 3-OST-5 modified HS bound to gD. About 19.8% 3-OST-5-modified HS bound to AT. Thus, using purified enzyme, we confirmed our previous conclusion that &!QJ;3-OST-5 generates both an AT-binding site and a &!QJ;gD-binding site (Xia et al., 2002Go).


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Table III. The bindings of 3-OST-5-modified HS to gD and AT

 
We also determined the Kms and Vmaxs of 3-OST-5 toward both HS and 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The Vmax and Km for HS are 4.7 pmoles of sulfate/min and 1.6 µM, respectively. The Vmax and Km for PAPS are 4.7 pmoles of sulfate/min and 4.0 µM, respectively.

Characterization of the substrate specificity of 3-OST-5
3-OST-5 sulfates the glucosamine residue that is linked to a nonsulfated iduronic acid residue at the nonreducing end
As described in Figure 2, three unidentified 35S-labeled components were detected in nitrous acid–degraded 3-OST-5-modified HS, designated disaccharide 1 (eluted at 18.5 min), disaccharide 2 (21.0 min), and disaccharide 4 (43.0 min). Although the signals were relatively low, we found that those components were present consistently. We decided to purify these components by RPIP-HPLC to determine if they represented 3-O-sulfated disaccharides. We noted that both disaccharide 1 and 2 were eluted on RPIP-HPLC in the region where monosulfated disaccharides were eluted. Disaccharide 4 was eluted on RPIP-HPLC in the region where disulfated disaccharides were eluted. Those observations suggested that disaccharide 1 and 2 are monosulfated disaccharides, whereas disaccharide 4 is a disulfated disaccharide.

We determined susceptibilities of disaccharides 1, 2, and 4 to ß-glucuronidase and {alpha}-iduronidase digestions. For disaccharide 1, we observed that the 35S-peak was shifted from 81 min to 8 min after ß-glucuronidase digestion on silica-based polyamine HPLC (PAMN-HPLC), suggesting that disaccharide 1 has a structure of GlcUA-AnMan3S (data not shown). For disaccharide 2, we observed that the 35S-peak was shifted from 86 min to 8 min on PAMN-HPLC after {alpha}-iduronidase digestion, suggesting that Disaccharide 2 has the structure of IdoUA-AnMan3S (Figure 3A). For disaccharide 4, we observed that the 35S-peak was shifted from 110 min to 87 min after {alpha}-iduronidase digestion on PAMN-HPLC (Figure 3B), and the resultant 35S-peak was coeluted with AnMan3S6S standard, suggesting that Disaccharide 4 has the structure of IdoUA-AnMan3S6S.

3-OST-5 sulfates N-sulfated glucosamine and N-unsubstituted glucosamine residues
It is known that 3-OST-1 and 3-OST-3 sulfate N-sulfated glucosamine and N-unsubstituted glucosamine residue, respectively (Liu et al., 1999aGo; Zhang et al., 1999Go). We attempted to determine which type of glucosamine residue is modified by 3-OST-5. To this end, we used a mixture of heparin lyases, including heparin lyase I, II, III and heparitinase IV, to degrade 3-OST-5-modified HS. About 50% of [35S]HS was degraded to disaccharides (or [35S]sulfate) and 40% of [35S]HS was degraded to tetrasaccharides (Figure 4A). It is very important to note that 3-O-sulfated HS, generated by 3-OST-1 and 3-OST-3, could not be degraded to disaccharides by a mixture of heparin lyases (Liu et al., 1999aGo; Zhang et al., 1999Go). To further prove that a 35S-labeled disaccharide was present in the heparin-lyase–digested 3-OST-5-modified HS, we resolved the disaccharide pool using RPIP-HPLC (Figure 4B). As shown in Figure 4B, we indeed observed one major [35S]disaccharide (disaccharide X) eluted in the region where trisulfated disaccharides are eluted, suggesting that disaccharide X is a trisulfated disaccharide. (A large amount of [35S]sulfate was also observed. We speculated that [35S]sulfate was the desulfated product from 3-O-[35S]sulfated HS during the digestion of heparin lyases. We also found numerous minor [35S]peaks around disaccharide X. Because the amount of those minor peaks was low, we were unable to determine their structures readily.)



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Fig. 4. Elution profile of heparin lyases–digested 3-OST-5-modified HS on BioGel P-6 and RPIP-HPLC chromatogram of the disaccharide portion from BioGel P-6. (A) Profile of heparin lyases–digested HS on BioGel P-6. The elution positions of the disaccharides and tetrasaccharides were determined by coeluting with 3H-labeled standards. (B) RPIP-HPLC chromatogram of the disaccharide fraction.

 
To rule out a remote possibility that disaccharide X is a tetrasulfated disaccharide, we determined its molecular mass by nanoelectrospray ionization mass spectrometry (nESI-MS). We observed a strong singly charged signal at m/z 677.4, which is identical to the calculated molecular mass for a trisulfated disaccharide monotriethylammonium salt (spectrum not shown). (To improve the nESI-MS spectrum, disaccharide X was desalted on a DEAE column, and it was eluted with 1 M triethylammonium bicarbonate [pH 8]. The resultant was then desalted on a BioGel P-2 column. Under these conditions, it is very possible that disaccharide X is present in the form of triethylammonium salt.) We noted that disaccharide X was eluted very close to a trisulfated disaccharide with a structure of {Delta}UA2S-GlcNS6S on RPIP-HPLC. Thus disaccharide X could potentially be contaminated by an unlabeled trisulfated disaccharide because both disaccharides have identical molecular mass. Given the fact that we did not observe a signal that represented a tetrasulfated disaccharide, we were able to conclude that disaccharide X is a trisulfated disaccharide.

The positions of sulfate groups of disaccharide X were determined by examining its susceptibility to nitrous acid degradation and {Delta}4,5-glycuronate-2-sulfatase digestion (Figure 5). We found that the retention time of disaccharide X on RPIP-HPLC was shifted from 72 min to 62 min by treating with nitrous acid at pH 1.5 (Figure 5A). However, we did not observe a shift in retention time when the disaccharide was treated with nitrous acid at pH 4.5 (data not shown). The results suggest that disaccharide X contains an N-sulfated glucosamine residue based on the specificity of nitrous acid degradation at different pHs (Shively and Conrad, 1976Go). We also found that disaccharide X is susceptible to {Delta}4,5-glycuronate-2-sulfatase digestion. The retention time of disaccharide X was shifted from 150 min to 138 min on PAMN-HPLC after digestion by {Delta}4,5-glycuronate-2-sulfatase, suggesting it contains a {Delta}UA2S residue (Figure 5B) (McLean et al., 1984Go). Consistent with this conclusion, treatment of disaccharide X with HS glycuronidase showed no shift in retention time on PAMN-HPLC (data not shown). Taken together, our results suggest that disaccharide X has a structure of {Delta}UA2S-GlcNS3S.



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Fig. 5. HPLC chromatograms of nitrous acid-degraded and {Delta}4,5-glycuronidase-digested disaccharide X. (A) RPIP-HPLC chromatogram of nitrous acid (pH 1.5)–degraded disaccharide X. (B) PAMN-HPLC chromatogram of {Delta}4,5-glycuronate-2-sulfatase-digested disaccharide X. The arrows indicate the elution positions of undegraded disaccharide X. The reactions of nitrous acid degradation and {Delta}4,5-glycuronate-2-sulfatase digestions are illustrated on the side of A and B, respectively.

 
Whether 3-OST-5 sulfates N-unsubstituted glucosamine residue was investigated. Tetra-1 was isolated from the tetrasaccharide pool that was fractionated by BioGel P-6 using PAMN-HPLC (Figure 6A). We found that Tetra-1 coeluted with a previously published tetrasaccharide standard ({Delta}UA2S-GlcNS-IdoUA2S-GlcNH23S6S) on PMAN-HPLC, suggesting that Tetra-1 contains an N-unsubstituted glucosamine residue at the reducing end (data not shown) (Liu et al., 2002Go). To further prove that Tetra-1 indeed carries an N-unsubstituted glucosamine residue, we tested the susceptibility of Tetra-1 to the nitrous acid degradation at pH 4.5. It should be noted that the tetrasaccharide standard, {Delta}UA2S-GlcNS-IdoUA2S-GlcNH23S6S, reacts to nitrous acid degradation (pH 4.5) (Liu et al., 1999aGo). As expected, we observed that the retention time of Tetra-1 was shifted from 46 min to 36 min on PAMN-HPLC after nitrous acid degradation (Figure 6B). Thus our result was consistent with the conclusion that an N-unsubstituted glucosamine residue is present at the reducing end of Tetra-1.



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Fig. 6. PAMN-HPLC chromatograms of tetrasaccharide pool and nitrous acid (pH = 4.5)–degraded Tetra-1. (A) Chromatogram of tetrasaccharide pool which was obtained from heparin lyases-digested 3-OST-5-modified HS (Figure 4A). (B) Chromatogram of nitrous acid (pH 4.5)–degraded Tetra-1.

 
We noted that Tetra-1 only represents a small portion of total [35S]sulfate counts in the tetrasaccharide pool. As shown in Figure 6A, we observed numerous 35S-labeled peaks, including a large [35S]sulfate peak, in the tetrasaccharide pool. One possible reason for a large amount of [35S]sulfate in the tetrasaccharide pool is that the tetrasaccharides that were generated by 3-OST-5 were unstable, and the degradation of the tetrasaccharides yielded [35S]sulfate. Presently we could not determine the structures of those unstable tetrasaccharides. Regardless of the limitation in this experiment, we still can conclude that 3-OST-5 sulfates an N-unsubstituted glucosamine residue.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The 3-OST-5 enzyme was previously reported to generate both an AT-binding site and an entry receptor for HSV-1. The specificity of this enzyme was determined at the disaccharide level using crude cell extract from COS-7 cells transfected with the plasmid expressing 3-OST-5 (Xia et al., 2002Go). In this study, we expressed the enzyme in SF9 cells using a baculovirus approach at a high level and purified to apparent homogeneity. Using the purified enzyme, we investigated its substrate specificity in details and eliminated the potential effects of other HS sulfotransferases on the action of 3-OST-5. Our results demonstrated that the recombinant 3-OST-5 has very similar substrate specificity to the enzyme that was expressed in COS-7 cells as determined by the disaccharide analysis of 3-OST-5-modified HS. We also found that 3-OST-5-modified HS generated by the purified enzyme binds to AT and HSV-1 gD. Considered together, our results suggest that the recombinant 3-OST-5 enzyme from SF9 cells has the similar enzymatic activity to the preparation that is prepared by mammalian cells. This observation is consistent with those for previously reported 3-OST-1 and 3-OST-3A (Hernaiz et al., 2000Go; Liu et al., 1999aGo). However, whether the substrate specificity of 3-OST-5 observed in vitro truly represents its specificity in vivo remains to be investigated.

Three additional 3-O-sulfated disaccharides were observed in nitrous acid–degraded (pH 1.5) 3-OST-5-modified HS, including GlcUA-AnMan3S, IdoUA-AnMan3S, and IdoUA-AnMan3S6S. We proved the structures by determining their susceptibilities to ß-glucuronidase and {alpha}-iduronidase, respectively. In a previous study, we observed the presence of IdoUA-AnMan3S6S but were unable to prove the structure because the amount of IdoUA-AnMan3S6S was low. Using the purified enzyme, we prepared a large amount of 3-OST-5-modified HS, which allowed us to obtain sufficient amount of the disaccharide to prove its structure. In addition, we purified and proved the presence of GlcUA-AnMan3S and IdoUA-AnMan3S. Together our results suggest that 3-OST-5 sulfates a glucosamine residue that is linked to an iduronic acid residue at the nonreducing end.

Both IdoUA-AnMan3S and IdoUA-AnMan3S6S are rare in the HS isolated from natural sources. The disaccharide of IdoUA-AnMan3S was observed in the HS isolated from glomerular basement membrane (Edge and Spiro, 1990Go, 2000Go), whereas the disaccharide of IdoUA-AnMan3S6S was observed in the clams Anomalocardia brasiliana and Tivela mactroides (Pejler et al., 1987Go). The biological functions of the HS containing IdoUA-AnMan3S6S is unknown, and it is not associated with antithrombin binding (Pejler et al., 1987Go). Edge and Spiro (2000)Go reported that decreased levels of IdoUA-AnMan3S and IdoUA2S-AnMan3S in HS isolated from the glomerular basement membrane were observed in diabetic patients. The authors suggested that the specific 3-O-sulfated HS might contribute to defects in the anionic filtration barrier in diabetic patients. It should be noted that the previously characterized 3-OST-1, 3-OST-3A, and 3-OST-2 do not generate either IdoUA-AnMan3S or IdoUA-AnMan3S6S (Liu et al., 1996Go, 1999aGo, 1999bGo). (Zhang and colleagues reported that 3-OST-1-modified HS can contain IdoUA-AnMan3S and IdoUA-AnMan3S6S [Zhang et al., 2001bGo]. However, the altered substrate specificity of 3-OST-1 was observed by using the HS that is absence of 2-O-sulfated iduronic acid or 2-O-sulfated glucuronic acid residues [Bai and Esko, 1996Go].) Given the fact that 3-OST-5 enzyme generates the saccharide sequences containing both IdoUA-AnMan3S and IdoUA2S-AnMan3S, it is possible that 3-OST-5-modified HS plays roles in regulating the functions of anionic filtrations, in addition to the activities in generating anticoagulant HS and an entry receptor for HSV-1.

3-OST-5 enzyme sulfates both N-sulfated glucosamine (GlcNS) and N-unsubstituted glucosamine (GlcNH2) residues. This conclusion was based on the structural analysis of the products of heparin lyases–digested 3-OST-5- modified HS. Given the facts that 3-OST-1 sulfates GlcNS and 3-OST-3 sulfates GlcNH2, our results suggest that 3-OST-5 has more relaxed substrate specificities. Currently we do not know whether 3-OST-5 sulfates N-acetylated glucosamine residue because we could not analyze the structures of all 3-O-[35S]sulfated oligosaccharides, which are in low abundance or unstable. To our surprise, we observed a 3-O-sulfated disaccharide with a structure of {Delta}UA2S-GlcNS3S from the heparin lyases–digested products. It has been reported that HS tetrasaccharides containing 3-O-sulfated glucosamine residues are resistant to the digestions by heparin lyases (Liu et al., 1999aGo; Sundaram et al., 2003Go; Yamada et al., 1995Go; Zhang et al., 1999Go). (Two disaccharides carrying a 3-O-sulfate group, with structures of {Delta}UA-GlcNS3S and {Delta}UA-GlcNS3S6S, were observed in the heparin lyases–digested 3-OST-1-modifed in the presence of heparin/heparan sulfate interacting protein [Zhang et al., 2001aGo]. It is still unclear why this protein alters the substrate specificities of heparin lyases to yield the 3-O-sulfated disaccharides.) Indeed, we only found tetra- and hexasaccharides in the products of heparin lyases–digested 3-OST-3-modified HS (data not shown). It remains to be investigated why 3-OST-5-modified HS can be digested into a disaccharide and 3-OST-1 and 3-OST-3-modified HS cannot. It is possible that the saccharide sequences around the 3-O-sulfation sites within the 3-OST-5-modified HS are unique, which allows the actions of heparin lyases to occur.

It remains to be investigated the relative sulfation efficiency by 3-OST-5 to GlcNH2 and GlcNS. It is likely that the sulfated saccharide sequences around GlcNH2 or GlcNS contribute to the susceptibility to 3-OST-5 sulfation. To eliminate the contribution of the sulfate patterns around the modification sites, the study will require a series of structurally defined oligosaccharides substrates differed by only GlcNH2 versus GlcNS residue. Unfortunately, those oligosaccharides are still unavailable.

The potential role of 3-OST-5 in biosynthesizing anticoagulant HS was proposed by HajMohammadi and colleagues (2003)Go. Analysis of 3-OST-1-null mice has failed to detect any phenotype in hemostasis. The authors suggested that another isoform of 3-OST performs the function of the biosynthesis of anitcoagulant HS. Given the fact that 3-OST-5 generates anticoagulant HS, further studies of the role of 3-OST-5 in biosynthesizing anticoagulant HS in vivo, in conjunction with 3-OST-1, could help us to understand the regulating role of HS in coagulation at molecular levels.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
Human 3-OST-5 expression plasmid (pcDNA3.1-3OST5) was prepared as described previously (Xia et al., 2002Go). Recombinant human 3-OST-3A and mouse 3-OST-1 were expressed in Sf9 cells using a baculovirus expression system and purified by heparin-Sepharose CL-6B (Pharmacia, Uppsala, Sweden) and 3', 5'-ADP-agarose (Sigma, St. Louis, MO) chromatographies (Hernaiz et al., 2000Go; Liu et al., 1999aGo). [35S] PAPS was prepared by incubating 0.4–2 mCi/ml [35S] Na2SO4 (carrier free, ICN, Costa Mesa, CA) and 16 mM ATP with 5 mg/ml dialyzed yeast extract (Sigma) (Bame and Esko, 1989Go). HS (isolated from bovine kidney) was from ICN. 3H-labeled HS was purified from wild-type CHO cells, which were grown in media containing 3H-glucosamine (ICN) (Zhang et al., 1999Go). Human AT was from Cutter Biological (Berkely, CA). Desulfated heparins were obtained from Neoparin (San Leandro, CA). A truncated form of HSV-1 gD-1 (306t) and monoclonal anti-gD (DL6) were generous gifts of Drs. Cohen and Eisenberg of the University of Pennsylvania (Nicola et al., 1996Go). Heparintinase IV, {Delta}4,5-glycuronate-2-sulfatase and HS glycuronidase are gifts from Dr. Keiichi Yoshida (Tokyo Research Institute of Seikagaku). The 3H-labeled disaccharide standards, IdoUA-AnMan6S, IdoUA2S-AnMan, GlcUA-AnMan3S6S, and IdoUA2S-AnMan6S, were prepared from 3H-labeled heparin (Shworak et al., 1994Go). The 35S-labeled disaccharide standards, IdoUA2S-AnMan3S and IdoUA2S-AnMan3S6S, were purified from nitrous acid (pH 1.5)–degraded HS that was modified by purified 3-OST-3 enzyme as described by Liu et al. (1999a)Go. {alpha}- Iduronidase was from Glyko (Novato, CA). ß-Glucuronidase was from Sigma.

Preparation of recombinant 3-OST-5 enzyme
Preparation of 3-OST-5 baculovirus expression plasmid
The secreted form of 3-OST-5 was constructed by removing the 29 amino acid residues from N-terminus consisting of the proposed transmembrane domain (Xia et al., 2002Go). The construction of the expression plasmid was involved in two-step cloning. A partial 3-OST-5 cDNA (about 900 bp) was first cloned from pcDNA3.1-3OST5 into the baculovirus expression vector containing a honeybee melittin signal using EcoR I/Xba I sites (Liu et al., 1999aGo; Xia et al., 2002Go). The remaining 3-OST-5 sequence was amplified by a polymerase chain reaction using two specific primers and pcDNA3.1–3OST5 as a template. The sequences of the two specific primers are: 5'-primer AATTTGGATCCCCAGAGTTGGGAGCTTGGATAG with a BamH I site (underscored); 3'-primer AACAAAACTTATTACAAGTTTGAGA. The product (559 bp) was digested with BamH I and EcoR I to yield a fragment with 82 bp. The resultant 82-bp fragment was then cloned into the construct that contains part of 3-OST-5 sequence using BamH I/EcoR I sites. The reading frame was confirmed by sequencing analysis. It should be noted that the construct does not contain the (His)6 sequence.

Expression of 3-OST-5
3-OST-5 recombinant baculovirus was prepared from 3-OST-5 baculovirus expression plasmid using the Bac-to-Bac baculovirus system (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Briefly, Sf9 cells (1 x 106 cells/ml, Invitrogen) were grown in serum-free media in a spinner bottle and were infected by 3-OST-5 recombinant virus. The medium was harvested 72 h after infection. The harvested medium was centrifuged at 1000 x g for 15 min, and 3-[(3-cholamidopropyl)diethylammonio]-1-propane sulfonate (CHAPS) was added to a final concentration of 0.6%. This solution was frozen in liquid nitrogen and stored at -80°C for subsequent purification.

Measurement of 3-OST-5 activity
The activity of 3-OST-5 was determined by measuring the amount of [35S]sulfate transferred to HS polysaccharide. A similar method was used to determine the activity of 3-OST-3 (Liu et al., 1999aGo). Briefly, a typical 50-µl reaction contained various amount of 3-OST-5 enzyme, 200 µg/ml HS, 1 x 107 cpm of [35S] PAPS, 50 mM 2-[N-morpholino]ethanesulfonic acid, pH 7.0, 10 mM MnCl2, 5 mM MgCl2, 100 mM NaCl, 120 µg/ml bovine serum albumin, and 1% Triton X- 100 (v/v). The reaction mixture was incubated at 37°C for 1 h. The [35S] HS was isolated by a DEAE-Sephacel column (Xia et al., 2002Go).

Purification of recombinant 3-OST-5
The entire purification was carried out at 4°C. The harvested medium (about 1.5 L) was mixed with 4-[N-morpholino] propanesulfonic acid (MOPS) to a final concentration of 20 mM, adjusted pH to 7.0 with 1 M NaOH. The preparation was centrifuged to remove insoluble particles. The supernatant was mixed with an equal volume of cold 20 mM MOPS, pH 7.0, and then loaded on a heparin-Sepharose CL-6B column (1 x 10 cm), which was equilibrated with 20 mM MOPS, 0.6% CHAPS, 2% glycerol, pH 7.0 (MCG buffer) and 150 mM NaCl at 4 ml/min. The column was then washed with MCG buffer containing 150 mM NaCl for 20 min and eluted with a linear gradient of NaCl from 150 mM to 1000 mM in 50 min. The fractions (48 ml) containing 3-OST-5 activity were pooled and dialyzed against 50 mM NaCl in MCG buffer. The solution was then loaded on a 3',5'-ADP-agarose column (0.5 x 8 cm) equilibrated with 150 mM NaCl in MCG buffer at 0.3 ml/min. The column was washed with MCG buffer containing 150 mM NaCl for 33 min and eluted with a linear gradient of NaCl from 150 mM to 1000 mM in 67 min followed by a 17-min wash of 1000 mM NaCl in MCG buffer. The fractions (8 ml) containing 3-OST-5 activity were pooled.

Protein identification by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)
Purified 3-OST-5 was in-gel digested with a Genomics Solutions ProGest robot using trypsin in 50 mM ammonium bicarbonate (pH 8) (Borchers et al., 2000Go). Extracted peptides were lyophilized and reconstituted in 5 µl 50% methanol (v/v), 0.1% formic acid. Peptides (0.5 µl) were mixed with 1 µl saturated {alpha}-cyanohydroxycinnamic acid in 50% acetonitrile/0.1% trifluoracetic acid and analyzed using both a Bruker Reflex III MALDI-TOF and the Applied Biosystems 4700 MALDI-TOF/TOF. For protein identification, Mascot software (www.matrixscience.com) was used for matching molecular mass of peptides against the NCBInr database (2/15/2003). The digest (band 1 in Figure 1) was also analyzed by liquid chromatography–mass spectrometry on a Waters Q/TOF API US coupled to a Waters CapLC using a 75-min gradient elution on a LCPackings 75 µm x 15 cm PepMap C18 reverse phase column.

Characterization of 3-OST-5-modified HS
Preparation of 3-OST-5-modified HS
Purified 3-OST-5 (70 ng) was mixed with 1 µg HS and 20 µM [35S] PAPS (1 x 107 cpm) in the enzyme reaction buffer as described. Twenty reactions were prepared to obtain enough 3-OST-5-modified HS for structure analysis. Alternatively, the HS was replaced by 3H-labeled HS (0.1 µg), which was prepared from [3H]glucosamine (ICN) metabolically labeled CHO cells, to prepare [3H, 3-O-35S]HS.

Determination of 3-OST-5 modified HS binding to AT and to gD
The binding of 3-OST-5-modified HS to AT was determined using an AT/concanavalin A–Sepharose (Sigma) approach (Liu et al., 1996Go). The assay for determining the binding of 3-OST-5-modified HS to gD was carried out by an immunoprecipitation procedure using anti-gD monoclonal antibody (Shukla et al., 1999Go).

Disaccharide analysis
The [35S] HS was degraded by nitrous acid (pH 1.5) followed by reduction with sodium borohydride (Shively and Conrad, 1976Go). The resultant disaccharides were desalted on a BioGel P-2 column (0.5 x 200 cm), which was equilibrated with 0.1 M ammonium bicarbonate at 4 ml/h. The disaccharides were then resolved by RPIP-HPLC to determine the identities of 35S-labeled disaccharides by coeluting with appropriate standards (Liu et al., 2002Go; Xia et al., 2002Go).

Degradations of 3-O-[35S]HS by enzymes
The conditions for digesting HS by a mixture of heparin lyases, including heparinase, heparitinase I, heparitinase II and heparitinase IV, were described elsewhere (Zhang et al., 1999Go). The conditions for the digestion with {alpha}-iduronidase and ß-glucuronidase were previously described (Liu et al., 1999aGo). The conditions for the digestions by {Delta}4,5-glycuronate-2-sulfatase and HS glycuronidase were described elsewhere (Liu et al., 2002Go).

HPLC
We employed both RPIP-HPLC and anion-exchange HPLC to resolve 3-O-[35S]sulfated disaccharides and 3-O-[35S]tetrasaccharides. For the analysis of the mixture of disaccharides containing trisulfated disaccharides, we used a C18-reversed phase column (0.46 x 25 cm, Vydac) under the RPIP-HPLC condition (Liu et al., 1999aGo). The column was eluted with acetonitrile at 8% for 45 min, at 15% for 15 min, and at 19.5% for 30 min, in a solution containing 38 mM ammonium phosphate monobasic, 2 mM phosphoric acid, and 1 mM tetrabutylammonium phosphate monobasic (Fluka) at a flow rate of 0.5 ml/min.

For the analyses of {alpha}-iduronidase, glucuronidase, and {Delta}4,5-glycuronate-2-sulfatase-treated 3-O-[35S]sulfated disaccharides, we employed a PAMN-HPLC column (0.46 x 25 cm, Waters, Milford, CT). The column was eluted with 30 mM KH2PO4 for 70 min, followed by a linear gradient of KH2PO4 from 30 to 1000 mM in 130 min at a flow rate of 0.8 ml/min. To isolate 3-O-[35S]sulfated tetrasaccharides, the PAMN-HPLC was eluted with a linear gradient of KH2PO4 from 350 mM to 1000 mM in 60 min, followed by additional wash with 1000 mM KH2PO4 for 20 min at a flow rate of 0.8 ml/min.

nESI-MS
The experiment was carried out on a Micromass Quattro II with QhQ geometry, a Z-spray source, and pulled borosilicate glass nanovials. The conditions for the analysis of HS disaccharides were described elsewhere (Pope et al., 2001Go). An extensive desalting procedure was used to obtain the MS spectrum of disaccharide X. The disaccharide was desalted by a DEAE-Sephacel column and eluted with 1 M triethylammonium bicarbonate (pH 8). The resultant disaccharide was further desalted by a BioGel P-2 column, which was eluted with 0.1 M ammonium acetate at a flow rate of 4 ml/h.

Kinetic analysis
We determined the kinetic constants (Km and Vmax) of 3-OST-5 toward both HS and PAPS. We have observed that the rate of reaction remained constant up to 10 min. Thus we incubated the reactions for 5 min, and the reaction velocity represented the initial velocity.

To determine the Km and Vmax with respect to HS, various concentrations of HS (0.125–8 µM) were employed in a standard 50-µl reaction as described, containing 20 µM 35[S]PAPS (1 x 107 cpm). The molar concentrations of HS were calculated using the average molecular weight at 14,000 Da (provided by manufacturer). Reactions were quenched by adding 100 µl of a buffer containing 50 mM sodium acetate, 6 M Urea, 150 mM NaCl, 1 mM ethylenediamine tetra-acetic acid and 0.1% Triton X-100, pH 5.5. The samples were then subjected into a 200 µl DEAE-Sepharose column. The resultant [35S] HS were eluted from the column by 1000 mM NaCl (Liu et al., 1996Go).

To determine the Km and Vmax with respect to PAPS, various concentrations of unlabeled PAPS (from 1 to 20 µM) and 1 x 107 cpm [35S]PAPS were employed in a standard 50-µl reaction. The reactions were quenched after 5 min incubation. The corresponding initial velocities were plotted against the concentrations of HS or PAPS. The plot was fitted to the equation V = Vmax * S/(Km + S), where S represents either the concentration of HS or PAPS, to obtain Km and Vmax.


    Acknowledgements
 
This work is supported by a grant from the National Institutes of Health (AI50050) and a grant from North Carolina Biotechnology Center. M.D. is a recipient of predoctoral fellowship (2001-17095) from the David and Lucile Packard Foundation. K.C. is a recipient of a postdoctoral fellowship from the Kech Foundation administrated by the North Carolina Biotechnology Center. We thank Drs. Gary Cohen and Roselyn Eisenberg (University of Pennsylvania) for providing purified recombinant gD and anti-gD monoclonal antibody (DL6) and Dr. Keiichi Yoshida (Tokyo Research Institute of Seikagaku) for HS glycuronidase, {Delta}4,5-glycuronate-2-sulfatase and heparitinase IV. While this manuscript was under preparation, Mochizuki et al. (2003) reported the isolation of two trisulfated disaccharides from 3-OST-5-modified HS, and a tetrasulfated disaccharide from 3-OST-5-modified heparin. However, they did not investigate whether 3-OST-5 sulfates a glucosamine residue that is linked to a nonsulfated iduronic acid residue at the nonreducing end.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: jian_liu{at}unc.edu Back


    Abbreviations
 
AnMan, 2,5-anhydromannitol; AT, antithrombin; CHAPS, 3-[(3-cholamidopropyl)diethylammonio]-1-propane sulfonate; CHO, Chinese hamster ovary; gD, glycoprotein D, HS, heparan sulfate; HSV, herpes simplex virus; OST, O-sulfotransferase; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; MOPS, 4-[N-morpholino] propanesulfonic acid; nESI-MS, nanoelectrospray ionization mass spectrometry; PAMN, silica-based polyamine; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; RPIP-HPLC, reverse phase ion-pairing–high-performance liquid chromatography; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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