A quantitative method to detect non-antithrombin-binding 3-O-sulfated units in heparan sulfate
2020; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1074/jbc.ra120.015864
ISSN1083-351X
AutoresHideo Mochizuki, Hideyuki Futatsumori, Eriko Suzuki, Koji Kimata,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoHeparan sulfate is synthesized by most animal cells and interacts with numerous proteins via specific sulfation motifs to regulate various physiological processes. Various 3-O-sulfated motifs are considered to be key in controlling the binding specificities to the functional proteins. One such motif synthesized by 3-O-sulfotransferase-1 (3OST-1) serves as a binding site for antithrombin (AT) and has been thoroughly studied because of its pharmacological importance. However, the physiological roles of 3-O-sulfates produced by other 3OST isoforms, which do not bind AT, remain obscure, in part due to the lack of a standard method to analyze this rare modification. This study aims to establish a method for quantifying 3-O-sulfated components of heparan sulfate, focusing on non-AT-binding units. We previously examined the reaction products of human 3OST isoforms and identified five 3-O-sulfated components, including three non-AT-binding disaccharides and two AT-binding tetrasaccharides, as digestion products of heparin lyases. In this study, we prepared these five components as a standard saccharide for HPLC analysis. Together with eight non-3-O-sulfated disaccharides, a standard mixture of 13 units was prepared. Using reverse-phase ion-pair HPLC with a postcolumn fluorescent labeling system, the separation conditions were optimized to quantify the 13 units. Finally, we analyzed the compositional changes of 3-O-sulfated units in heparan sulfate from P19 cells before and after neuronal differentiation. We successfully detected the 3-O-sulfated units specifically expressed in the differentiated neurons. This is the first report that shows the quantification of three non-AT-binding 3-O-sulfated units and establishes a new approach to explore the physiological functions of 3-O-sulfate. Heparan sulfate is synthesized by most animal cells and interacts with numerous proteins via specific sulfation motifs to regulate various physiological processes. Various 3-O-sulfated motifs are considered to be key in controlling the binding specificities to the functional proteins. One such motif synthesized by 3-O-sulfotransferase-1 (3OST-1) serves as a binding site for antithrombin (AT) and has been thoroughly studied because of its pharmacological importance. However, the physiological roles of 3-O-sulfates produced by other 3OST isoforms, which do not bind AT, remain obscure, in part due to the lack of a standard method to analyze this rare modification. This study aims to establish a method for quantifying 3-O-sulfated components of heparan sulfate, focusing on non-AT-binding units. We previously examined the reaction products of human 3OST isoforms and identified five 3-O-sulfated components, including three non-AT-binding disaccharides and two AT-binding tetrasaccharides, as digestion products of heparin lyases. In this study, we prepared these five components as a standard saccharide for HPLC analysis. Together with eight non-3-O-sulfated disaccharides, a standard mixture of 13 units was prepared. Using reverse-phase ion-pair HPLC with a postcolumn fluorescent labeling system, the separation conditions were optimized to quantify the 13 units. Finally, we analyzed the compositional changes of 3-O-sulfated units in heparan sulfate from P19 cells before and after neuronal differentiation. We successfully detected the 3-O-sulfated units specifically expressed in the differentiated neurons. This is the first report that shows the quantification of three non-AT-binding 3-O-sulfated units and establishes a new approach to explore the physiological functions of 3-O-sulfate. Heparan sulfate is synthesized by most animal cells as a component of proteoglycans and interacts with numerous proteins such as growth factors, morphogens, receptors, and extracellular matrix proteins to regulate various physiological processes (1Sarrazin S. Lamanna W.C. Esko J.D. Heparan sulfate proteoglycans.Cold Spring Harb. Perspect. Biol. 2011; 3a004952Crossref PubMed Scopus (740) Google Scholar). The heparan sulfate molecule is composed of densely sulfated regions connected by mostly nonsulfated regions (2Turnbull J.E. Gallagher J.T. Distribution of iduronate 2-sulphate residues in heparan sulphate. Evidence for an ordered polymeric structure.Biochem. J. 1991; 273: 553-559Crossref PubMed Scopus (121) Google Scholar). Most of the interactions between heparan sulfate and functional proteins are thought to occur at the sulfated regions having specific arrangements of sulfation motifs (3Gallagher J.T. Heparan sulfate: growth control with a restricted sequence menu.J. Clin. Invest. 2001; 108: 357-361Crossref PubMed Scopus (278) Google Scholar). The biosynthesis of heparan sulfate is initiated by the polymerization of alternating D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc) residues to form the repeating disaccharide structure -GlcA-β1,4-GlcNAc-α1,4-. This polymer is then partially N-deacetylated/N-sulfated and subsequently undergoes 5-epimerization of GlcA to L-iduronic acid (IdoA), 2-O-sulfation of hexuronic acid (HexA) residues and 6-O-sulfation of glucosamine residues. Then, a rare but functionally important modification, 3-O-sulfation of the glucosamine residues, also occurs. These modification processes are generally not uniform and result in a variety of disaccharide units (4Esko J.D. Lindahl U. Molecular diversity of heparan sulfate.J. Clin. Invest. 2001; 108: 169-173Crossref PubMed Scopus (751) Google Scholar). There are 12 sulfation patterns of disaccharide units (Fig. 1), and these units have isoforms of HexA, i.e., GlcA or IdoA. Combinations of these units enable the formation of disaccharide sequences specific for individual ligand proteins. Although the 3-O-sulfated units are usually less than 1% of total disaccharides (5Mochizuki H. Yoshida K. Shibata Y. Kimata K. Tetrasulfated disaccharide unit in heparan sulfate. Enzymatic formation and tissue distribution.J. Biol. Chem. 2008; 283: 31237-31245Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), there are seven isoform genes of heparan sulfate 3-O-sulfotransferase (3OST) in our genome. This rare modification was first described as an essential component of the antithrombin (AT)-binding site (6Lindahl U. Bäckström G. Thunberg L. Leder I.G. Evidence for a 3-O-sulfated D-glucosamine residue in the antithrombin-binding sequence of heparin.Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 6551-6555Crossref PubMed Scopus (398) Google Scholar). The 3OST-1, a specific enzyme for this modification, was purified and cloned as the first isoform of the gene family (7Liu J. Shworak N.W. Fritze L.M.S. Edelberg J.M. Rosenberg R.D. Purification of heparan sulfate D-glucosaminyl 3-O-sulfotransferase.J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 8Shworak N.W. Liu J. Fritze L.M.S. Schwartz J.J. Zhang L. Logeart D. Rosenberg R.D. Molecular cloning and expression of mouse and human cDNAs encoding heparan sulfate D-glucosaminyl 3-O-sulfotransferase.J. Biol. Chem. 1997; 272: 28008-28019Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). A number of reports related to the AT-binding 3-O-sulfate have been published to date because of its pharmacological importance. Unlike the 3OST-1, six other isoforms were searched out from the DNA database as a 3OST-1 homologue (9Shworak N.W. Liu J. Petros L.M. Zhang L. Kobayashi M. Copeland N.G. Jenkins N.A. Rosenberg R.D. Multiple isoforms of heparan sulfate D-glucosaminyl 3-O-sulfotransferase. Isolation, characterization, and expression of human cDNAs and identification of distinct genomic loci.J. Biol. Chem. 1999; 274: 5170-5184Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 10Xia G. Chen J. Tiwari V. Ju W. Li J.-P. Malmström A. Shukla D. Liu J. Heparan sulfate 3-O-sulfotransferase isoform 5 generates both an antithrombin-binding site and an entry receptor for herpes simplex virus, type 1.J. Biol. Chem. 2002; 277: 37912-37919Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 11Xu D. Tiwari V. Xia G. Clement C. Shukla D. Liu J. Characterization of heparan sulphate 3-O-sulphotransferase isoform 6 and its role in assisting the entry of herpes simplex virus type 1.Biochem. J. 2005; 385: 451-459Crossref PubMed Scopus (84) Google Scholar). It has been reported that the 3OST isoforms catalyze the formation of non-AT-binding 3-O-sulfates. Although accumulating evidence suggests the physiological importance of non-AT-binding 3-O-sulfates (12Thacker B.E. Xu D. Lawrence R. Esko J.D. Heparan sulfate 3-O-sulfation: a rare modification in search of a function.Matrix Biol. 2014; 35: 60-72Crossref PubMed Scopus (115) Google Scholar), the structure–function relationships of this modification remain obscure. The lack of a standard method to analyze this rare modification is hindering the research to clarify the biochemical functions of non-AT-binding 3-O-sulfates. Previously, we analyzed a reaction product of 3OST isoforms and identified five 3-O-sulfated components, including three disaccharides and two tetrasaccharides, as a digestion product of heparin lyases (5Mochizuki H. Yoshida K. Shibata Y. Kimata K. Tetrasulfated disaccharide unit in heparan sulfate. Enzymatic formation and tissue distribution.J. Biol. Chem. 2008; 283: 31237-31245Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 13Mochizuki H. Yoshida K. Gotoh M. Sugioka S. Kikuchi N. Kwon Y.-D. Tawada A. Maeyama K. Inaba N. Hiruma T. Kimata K. Narimatsu H. Characterization of a heparan sulfate 3-O-sulfotransferase-5, an enzyme synthesizing a tetrasulfated disaccharide.J. Biol. Chem. 2003; 278: 26780-26787Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The three disaccharides have been determined to be ΔHexA-GlcNS3S6S (Di-(N,3,6)S), ΔHexA2S-GlcNS3S (Di-(2,N,3)S), and ΔHexA2S-GlcNS3S6S (Di-tetraS) units. However, the two tetrasaccharides, named Tetra-1 and Tetra-2, have not yet been determined. Heparin lyases produced by Flavobacterium heparinum have been used for years to analyze heparan sulfate-related polysaccharides including heparin (14Hovingh P. Linker A. The enzymatic degradation of heparin and heparitin sulfate. III. Purification of a heparitinase and a heparinase from flavobacteria.J. Biol. Chem. 1970; 245: 6170-6175Abstract Full Text PDF PubMed Google Scholar, 15Silva M.E. Dietrich C.P. Isolation and partial characterization of three induced enzymes from Flavobacterium heparinum involved in the degradation of heparin and heparitin sulfates.Biochem. Biophys. Res. Commun. 1974; 56: 965-972Crossref PubMed Scopus (35) Google Scholar). Using a mixture of three lyases (heparinase, heparitinase I, and heparitinase II), heparan sulfate is almost completely digested into unsaturated disaccharides with the exception of the AT-binding structure. The glucosaminidic linkage adjacent to GlcA-GlcNS3S ± 6S units, a critical component of the AT-binding site, is resistant to digestion and results in unsaturated tetrasaccharides (16Yamada S. Yoshida K. Sugiura M. Sugahara K. Khoo K.-H. Morris H.R. Dell A. Structural studies on the bacterial lyase-resistant tetrasaccharides derived from the antithrombin III-binding site of porcine intestinal heparin.J. Biol. Chem. 1993; 268: 4780-4787Abstract Full Text PDF PubMed Google Scholar). However, non-AT-binding 3-O-sulfated structures are susceptible to the lyases and digested into unsaturated disaccharides (5Mochizuki H. Yoshida K. Shibata Y. Kimata K. Tetrasulfated disaccharide unit in heparan sulfate. Enzymatic formation and tissue distribution.J. Biol. Chem. 2008; 283: 31237-31245Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Although there are a number of reports demonstrating the compositional analysis of heparan sulfate and heparin using heparin lyases, apart from our previous study, analysis of the 3-O-sulfated disaccharides has not been reported. Ours was the first to demonstrate the Di-tetraS unit as the most sulfated component of heparan sulfate. We also performed quantitative analysis of the Di-tetraS unit in heparan sulfate from various rat tissues and found rare but ubiquitous distribution of this unique structure (5Mochizuki H. Yoshida K. Shibata Y. Kimata K. Tetrasulfated disaccharide unit in heparan sulfate. Enzymatic formation and tissue distribution.J. Biol. Chem. 2008; 283: 31237-31245Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 13Mochizuki H. Yoshida K. Gotoh M. Sugioka S. Kikuchi N. Kwon Y.-D. Tawada A. Maeyama K. Inaba N. Hiruma T. Kimata K. Narimatsu H. Characterization of a heparan sulfate 3-O-sulfotransferase-5, an enzyme synthesizing a tetrasulfated disaccharide.J. Biol. Chem. 2003; 278: 26780-26787Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In this study, expanding our earlier work, we aimed to establish a method by which 3-O-sulfated components of heparan sulfate, especially non-AT-binding 3-O-sulfated units, can be quantified. Because the 3-O-sulfated tetrasaccharides have not been characterized, we first performed a structural analysis of the tetrasaccharides using a biochemical technique. We then prepared the five components as a standard saccharide for HPLC analysis. Together with non-3-O-sulfated disaccharides, the 13 units of heparan sulfate were analyzed by reverse-phase ion-pair HPLC using the postcolumn fluorescent labeling system. To test the method established here, we finally analyzed the compositional changes of 3-O-sulfated units in heparan sulfate from P19 cells before and after neuronal differentiation. Since the Tetra-1 and Tetra-2 were expected to be derived from the AT-binding structure and have GlcA-GlcNS3S ± 6S units as a reducing disaccharide, we planned to characterize the tetrasaccharides by determining these units. We first prepared five 3-O-[35S]sulfated units in the same manner reported previously (13Mochizuki H. Yoshida K. Gotoh M. Sugioka S. Kikuchi N. Kwon Y.-D. Tawada A. Maeyama K. Inaba N. Hiruma T. Kimata K. Narimatsu H. Characterization of a heparan sulfate 3-O-sulfotransferase-5, an enzyme synthesizing a tetrasulfated disaccharide.J. Biol. Chem. 2003; 278: 26780-26787Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and described in "Experimental Procedures." 35S-labeled heparan sulfate prepared by incubating heparan sulfate with [35S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) and recombinant human 3OST-5 was digested with a mixture of heparin lyases and separated by HPLC on a CarboPac PA1 column. When monitored at 232 nm, six major disaccharide units of heparan sulfate were identified (Fig. 2A, upper panel). The elution profile of radioactivity (lower panel) is consistent with our previous report, and five peaks have been characterized as follows. Peaks 2, 4, and 5 are Di-(N,3,6)S, Di-(2,N,3)S, and Di-tetraS, respectively. Peaks 1 and 3 are Tetra-1 and Tetra-2, respectively. The peak fractions were pooled and desalted for further analysis. The 35S-labeled tetrasaccharides were digested with human heparanase-1 (Hpa-1) and analyzed by HPLC on a CarboPac PA1 column as described in "Experimental Procedures" (Fig. 2B). It has been reported that the Hpa-1 cleaves glucuronidic linkage of GlcA-GlcNS3S ± 6S units in the reducing end of heparan sulfate and releases GlcNS3S ± 6S monosaccharides (17Okada Y. Yamada S. Toyoshima M. Dong J. Nakajima M. Sugahara K. Structural recognition by recombinant human heparanase that plays critical roles in tumor metastasis. Hierarchical sulfate groups with differential effects and the essential target disulfated trisaccharide sequence.J. Biol. Chem. 2002; 277: 42488-42495Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Nonsulfated GlcA is an essential residue of the cleavage reaction. The radio-chromatogram 1 shows the analysis of untreated [35S]Tetra-1. With the enzyme digestion, the retention time of 35S-labeled product was shifted to an earlier position (chromatogram 2). Chromatogram 3 shows the analysis of [35S]GlcNS3S monosaccharide, prepared from [35S]Di-(2,N,3)S by treatment with mercuric acetate as described in "Experimental Procedures." To confirm the molecular size, the digested product was also analyzed by HPLC on a gel filtration column. The 35S-labeled product was eluted at the position of [35S]GlcNS3S (data not shown). These data show that Tetra-1 has the GlcA-GlcNS3S unit as a reducing disaccharide. [35S]Tetra-2 was analyzed in the same way as above. The 35S-labeled product produced by the reaction of Hpa-1 was confirmed to be [35S]GlcNS3S6S monosaccharide by HPLC on a CarboPac PA1 column (Fig. 2B, lower panel) and a gel filtration column (data not shown). The results indicate that Tetra-2 has the GlcA-GlcNS3S6S unit as a reducing disaccharide. In consequence, the tetrasaccharides were renamed with the reducing disaccharides, i.e., Di-(N,3)SLR and Di-(N,3,6)SLR, where LR is lyase-resistant (Fig. 2C). 3OST-5 produces the five 3-O-sulfated units from heparan sulfate and heparin in different proportions (5Mochizuki H. Yoshida K. Shibata Y. Kimata K. Tetrasulfated disaccharide unit in heparan sulfate. Enzymatic formation and tissue distribution.J. Biol. Chem. 2008; 283: 31237-31245Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). To obtain sufficient amounts of all components, these substrates (12 mg of each) were sulfated with an excess amount of 3OST-5. The mixture of 3-O-sulfated products was then digested with a mixture of heparin lyases and separated by HPLC on a gel filtration column as described in "Experimental Procedures." Figure 3A shows the elution profile monitored at 232 nm. The 35S-labeled saccharides prepared in "Characterization of Tetrasaccharides" were analyzed under the same conditions to confirm the elution positions of 3-O-sulfated saccharides, indicated by arrow heads. Because of insufficient separation, two tetrasaccharides (peaks 1 and 2) were pooled as a single fraction in this step and separated by the CarboPac PA1 column as described below. Peak 3 is tetrasulfated disaccharide, i.e., Di-tetraS. Peak 4 is trisulfated disaccharides containing Di-(N,3,6)S, Di-(2,N,3)S, and Di-(2,N,6)S. Peaks 5 and 6 are disulfated disaccharides and monosulfated disaccharides, respectively. The peak fractions containing 3-O-sulfated units were then separated into single components by HPLC on a CarboPac PA1 semipreparative column. Upper and bottom panels of Figure 3B show the separation of tetrasaccharides (the mixture of peaks 1 and 2 in A) and trisulfated disaccharides (peak 4 in A), respectively, monitored at 232 nm. To confirm the elution positions, the 35S-labeled saccharides were also analyzed under the same conditions (middle panel). Peaks a (Di-(N,3)SLR), b (Di-(N,3,6)SLR), c (Di-(N,3,6)S), and e (Di-(2,N,3)S) were pooled, desalted by a gel filtration column, and freeze-dried. Peaks d and d' are Di-(2,N,6)S and its anomer. The tetrasulfated disaccharide (peak 3 in A) was also applied to the CarboPac PA1 semipreparative column under the same conditions. A single peak fraction, eluted at the position of [35S]Di-tetraS, was pooled, desalted, and freeze-dried (data not shown). The freeze-dried saccharides were dissolved in 20 mM ammonium acetate buffer, pH 6.0. The standard saccharides were quantitated by measurement of the absorbance at 232 nm (18Kariya Y. Yoshida K. Morikawa K. Tawada A. Miyazono H. Kikuchi H. Tokuyasu K. Preparation of unsaturated disaccharides by eliminative cleavage of heparin and heparan sulfate with heparitinases.Comp. Biochem. Physiol. 1992; 103B: 473-479Google Scholar). Five 3-O-sulfated units, prepared in this study, and eight non-3-O-sulfated disaccharide units, obtained from Seikagaku Bio, were mixed as the standard mixture of 13 units. In the previous study, we employed reverse-phase ion-pair HPLC with a postcolumn fluorescent labeling system (Fig. 4A), described by Toyoda et al. (19Toyoda H. Yamamoto H. Ogino N. Toida T. Imanari T. Rapid and sensitive analysis of disaccharide composition in heparin and heparan sulfate by reversed-phase ion-pair chromatography on a 2 μm porous silica gel column.J. Chromatogr. A. 1999; 830: 197-201Crossref Scopus (47) Google Scholar, 20Toyoda H. Kinoshita-Toyoda A. Selleck S.B. Structural analysis of glycosaminoglycans in Drosophila and Caenorhabditis elegans and demonstration that tout-velu, a Drosophila gene related to EXT tumor suppressors, affects heparan sulfate in vivo.J. Biol. Chem. 2000; 275: 2269-2275Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar), in the analysis of the Di-tetraS unit. When the standard mixture was analyzed by the previous method, elution positions of 3-O-sulfated units overlapped with the Di-(2,N,6)S unit as shown in Figure 4B. Additionally, anomer separation of 3-O-sulfated units complicated the quantitative analysis. Thus we optimized the method to ensure accurate quantitative analysis of the 13 units. We tested gradient formations of eluant B, proportions of eluant C and D, water-soluble organic solvents for eluant D, pH adjustments of eluant C, and separation columns. As a result, we obtained a baseline separation of all components as shown in Figure 4C. The improved method is detailed in "Experimental Procedures." The use of ethanol for eluant D and pH adjustment of eluant C to 8.0 are critical factors to reduce the anomer separation and result in a sharp symmetric peak. Under the optimized condition, the amounts of 3-O-sulfated units correlated with the integrated fluorescence intensity, as shown in Figure 4D. The standard curves of non-3-O-sulfated units were also obtained with linear correlations of R2 > 0.999 (data not shown). Using the method established here, we examined the compositional changes of heparan sulfate from P19 cells before and after neuronal differentiation based on the evidence described in "Discussion." P19 cells were differentiated into neurons by treatment with retinoic acid as described in "Experimental Procedures." Heparan sulfates were prepared from P19 cells and differentiated neurons and digested with a mixture of heparin lyases. The digested products were analyzed by the HPLC with postcolumn fluorescent labeling method. Figure 5A shows the typical photomicrographs of P19 cells and differentiated neurons (upper and lower panels, respectively). Figure 5B shows representative chromatograms of the digested products derived from P19 cells (blue line) and differentiated neurons (red line). The inset represents an enlarged vertical axis with the same horizontal scale. Significant peaks of Di-(2,N,3)S and Di-tetraS were detected in the chromatogram derived from the neurons, as indicated by arrows, but not from the undifferentiated P19 cells. The molar percent of 13 components is shown in Table 1. The detection limit of the method was estimated to be 2 pmol of saccharides, ensured by the minimum points of standard curves in Figure 4D.Table 1Compositional analysis of heparan sulfate from P19 cells and their differentiated neurons (molar percent)Di-0SaFor abbreviations, see Figures 1 and 2C.Di-NSDi-6SDi-2SDi-(N,6)SDi-(2,N)SDi-(2,6)SDi-(N,3,6)SDi-(2,N,3)SDi-(2,N,6)SDi-(N,3)SLRDi-tetraSDi-(N,3,6)SLRP19 cell56 ± 0.7bData represent means ± S.E. of three independent culture experiment.20 ± 0.56.0 ± 0.020.37 ± 0.134.2 ± 0.1310 ± 0.20.10 ± 0.019NDcND, not detected.0.10 ± 0.0083.4 ± 0.21NDNDNDNeuron47 ± 1.921 ± 0.98.5 ± 0.170.99 ± 0.036.1 ± 0.188.4 ± 0.350.15 ± 0.0090.07 ± 0.0030.29 ± 0.0077.0 ± 0.640.10 ± 0.0020.29 ± 0.0040.07 ± 0.002a For abbreviations, see Figures 1 and 2C.b Data represent means ± S.E. of three independent culture experiment.c ND, not detected. Open table in a new tab We established a method that quantifies 13 components of heparan sulfate, including three non-AT-binding and two AT-binding 3-O-sulfated units. The five 3-O-sulfated units have been identified as a reaction product of 3OST isoforms in our previous studies (5Mochizuki H. Yoshida K. Shibata Y. Kimata K. Tetrasulfated disaccharide unit in heparan sulfate. Enzymatic formation and tissue distribution.J. Biol. Chem. 2008; 283: 31237-31245Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 13Mochizuki H. Yoshida K. Gotoh M. Sugioka S. Kikuchi N. Kwon Y.-D. Tawada A. Maeyama K. Inaba N. Hiruma T. Kimata K. Narimatsu H. Characterization of a heparan sulfate 3-O-sulfotransferase-5, an enzyme synthesizing a tetrasulfated disaccharide.J. Biol. Chem. 2003; 278: 26780-26787Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). 3OST-1 produces Di-(N,3)SLR and Di-(N,3,6)SLR as a critical component of the AT-binding site. 3OST-2, 3OST-3, and 3OST-4 produce Di-(2,N,3)S and Di-tetraS as non-AT-binding structures. 3OST-5 has broad specificity and produces all five units, including Di-(N,3,6)S unit specific for this isoform. The lyase-susceptible Di-(N,3,6)S unit would be derived from IdoA-GlcNS3S6S and different from the reducing disaccharide of Di-(N,3,6)SLR. IdoA-aMan3S6S, where aMan represents 2,5-anhydromannose, and GlcA-aMan3S6S have been reported as the reaction products of 3OST-5 by nitrous acid degradation (21Chen J. Duncan M.B. Carrick K. Pope R.M. Liu J. Biosynthesis of 3-O-sulfated heparan sulfate: unique substrate specificity of heparan sulfate 3-O-sulfotransferase isoform 5.Glycobiology. 2003; 13: 785-794Crossref PubMed Scopus (37) Google Scholar). It should be noted that 3OST-6, the youngest member of the 3OST gene family, has been cloned and characterized after our previous report. Xu et al. (11Xu D. Tiwari V. Xia G. Clement C. Shukla D. Liu J. Characterization of heparan sulphate 3-O-sulphotransferase isoform 6 and its role in assisting the entry of herpes simplex virus type 1.Biochem. J. 2005; 385: 451-459Crossref PubMed Scopus (84) Google Scholar) reported that the 3OST-6 produces no AT-binding structures, and the reaction specificity of this isoform is similar to that of 3OST-3. Since our primary objective is non-AT-binding 3-O-sulfates, the preparation of tetrasaccharide standards was limited to two major components that account for 70% of the total reaction products of 3OST-1 (5Mochizuki H. Yoshida K. Shibata Y. Kimata K. Tetrasulfated disaccharide unit in heparan sulfate. Enzymatic formation and tissue distribution.J. Biol. Chem. 2008; 283: 31237-31245Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), although other minor tetrasaccharides have been reported (16Yamada S. Yoshida K. Sugiura M. Sugahara K. Khoo K.-H. Morris H.R. Dell A. Structural studies on the bacterial lyase-resistant tetrasaccharides derived from the antithrombin III-binding site of porcine intestinal heparin.J. Biol. Chem. 1993; 268: 4780-4787Abstract Full Text PDF PubMed Google Scholar, 22Chen Y. Lin L. Agyekum I. Zhang X. St. Ange K. Yu Y. Zhang F. Liu J. Amster I.J. Linhardt R.J. Structural analysis of heparin-derived 3-O-sulfated tetrasaccharides: antithrombin binding site variants.J. Pharm. Sci. 2017; 106: 973-981Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). N-unsubstituted glucosamine residues have also been reported as a minor component of the heparan sulfate (23Norgard-Sumnicht K. Varki A. Endothelial heparan sulfate proteoglycans that bind to L-selectin have glucosamine residues with unsubstituted amino groups.J. Biol. Chem. 1995; 270: 12012-12024Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 24Westling C. Lindahl U. Location of N-unsubstituted glucosamine residues in heparan sulfate.J. Biol. Chem. 2002; 277: 49247-49255Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Improvement of the method to detect these minor components is the next step in achieving a comprehensive analysis of heparan sulfate. We employed reverse-phase ion-pair HPLC as the separation method and aimed to determine the optimum conditions for efficient separation of the 13 units as described in "Results." Consequently, a baseline resolution of all components was achieved. The detection limit of the postcolumn fluorescence labeling method is similar to that of mass spectrometry with a low pmol level (25Li G. Yang B. Li L. Zhang F. Xue C. Linhardt R.J. Analysis of 3-O-sulfo group-containing heparin tetrasaccharides in heparin by liquid chromatography-mass spectrometry.Anal. Biochem. 2014; 455: 3-9Crossref PubMed Scopus (30) Google Scholar). Although mass spectrometry is a powerful method for identifying the target molecule, application of the method is restricted to the HPLC system using volatile eluant. Therefore, the optimized HPLC conditions are unsuitable for mass spectrometry as is. A method by which all 13 units can be separated using a volatile buffer has yet to be established. Huang et al. (26Huang Y. Mao Y. Zong C. Lin C. Boons G.-J. Zaia J. Discovery of a heparan sulfate 3-O-sulfation specific peeling reaction.Anal. Chem. 2015; 87: 592-600Crossref PubMed Scopus (24) Google Scholar) reported the peeling reaction that specifically degrades heparan sulfate oligosaccharides having 3-O-sulfated glucosamine residue at the reducing end. This terminal residue is susceptible to degradation under mildly basic conditions (pH 8 and over), and temperature increases accelerate the reaction. We found that when the standard saccharides prepared in this study were dissolved in ammonium acetate buffer, pH 6.0, they could be stably stored at −80 °C for at least 1 year. In the compositional analysis, heparan sulfates were digested with the heparin lyases in a buffer solution of pH 7.0, at 30 °C for 4 h. To ensure stability of the digested products having 3-O-sulfated glucosamine residues, the standard mixture of 13 units was incubated under the same conditions. No quantitative changes and no degradation products were observed in the HPLC analysis (data not shown). Furthermore, we stop the lyase r
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