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

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 DeltaUA2S-GlcNS3S and a tetrasaccharide with a structure of DeltaUA2S-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.     

Disaccharide compositional analysis of heparin and heparan sulfate using capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) was used to separate eight commercial disaccharide standards of the structure delta UA2X(1----4)-D-GlcNY6X (where delta UA is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is 2-deoxy-2-aminoglucopyranose, S is sulfate, Ac is acetate, X may be S, and Y is S or Ac). These eight disaccharides had been prepared from heparin, heparan sulfate, and derivatized heparins. A similar CZE method was recently reported for the analysis of eight chondroitin and dermatan sulfate disaccharides (A. Al-Hakim and R.J. Linhardt, Anal. Biochem. 195, 68-73, 1991). Two of the standard heparin/heparan sulfate disaccharides, having an identical charge of -2, delta UA2S(1----4)-D-GlcNAc and delta UA(1----4)-D-GlcNS, were not fully resolved using standard sodium borate/boric acid buffer. This buffer had proven effective in separating chondroitin/dermatan sulfate disaccharides of identical charge. Resolution of these two heparin/heparan sulfate disaccharides could be improved by extending the capillary length, preparing the buffer in 2H2O, or eliminating boric acid. Baseline resolution was achieved in sodium dodecyl sulfate in the absence of buffer. The structure and purity of each of the eight new commercial heparin/heparan sulfate disaccharide standards were confirmed using fast-atom-bombardment mass spectrometry and high-field 1H-NMR spectroscopy. Heparin and heparan sulfate were then depolymerized using heparinase (EC 4.2.2.7), heparin lyase II (EC 4.2.2.-), heparinitase (EC 4.2.2.8), and a combination of all three enzymes. CZE analysis of the products formed provided a disaccharide composition of each glycosaminoglycan. As little as 50 fmol of disaccharide could be detected by ultraviolet absorbance.     

The disaccharide repeating-units of heparan sulfate

Five disaccharides have been isolated after degradation of heparan sulfate by heparinase (heparin lyase) and heparitinase (heparan sulfate lyase) and are suggested to represent the repeating units of the polysaccharide. They all contain a 4,5-unsaturated uronic acid residue and are: (a) A trisulfated disaccharide that is apparently identical to a disaccharide repeating-unit of heparin; (b) a disulfated disaccharide that seems unique for heparan sulfate and contains 2-deoxy-2-sulfamidoglucose and uronic acid sulfate residues; (c) a nonsulfated disaccharide containing a 2-acetamido-2-deoxyglucose residue; (d) a monosulfated disaccharide containing a 2-acetamido-2-deoxyglucose sulfate residue; and (e) a monosulfated disaccharide containing a 2-deoxy-2-sulfamidoglucose residue. Yields of these disaccharides from different heparan sulfate fractions are discussed in relation to possible arrangements of these units in the intact polymer.     

Organ-specific sulfation patterns of heparan sulfate generated by extracellular sulfatases Sulf1 and Sulf2 in mice

Heparan sulfate endosulfatases Sulf1 and Sulf2 hydrolyze 6-O-sulfate in heparan sulfate, thereby regulating cellular signaling. Previous studies have revealed that Sulfs act predominantly on UA2S-GlcNS6S disaccharides and weakly on UA-GlcNS6S disaccharides. However, the specificity of Sulfs and their role in sulfation patterning of heparan sulfate in vivo remained unknown. Here, we performed disaccharide analysis of heparan sulfate in Sulf1 and Sulf2 knock-out mice. Significant increases in ΔUA2S-GlcNS6S were observed in the brain, small intestine, lung, spleen, testis, and skeletal muscle of adult Sulf1(-/-) mice and in the brain, liver, kidney, spleen, and testis of adult Sulf2(-/-) mice. In addition, increases in ΔUA-GlcNS6S were seen in the Sulf1(-/-) lung and small intestine. In contrast, the disaccharide compositions of chondroitin sulfate were not primarily altered, indicating specificity of Sulfs for heparan sulfate. For Sulf1, but not for Sulf2, mRNA expression levels in eight organs of wild-type mice were highly correlated with increases in ΔUA2S-GlcNS6S in the corresponding organs of knock-out mice. Moreover, overall changes in heparan sulfate compositions were greater in Sulf1(-/-) mice than in Sulf2(-/-) mice despite lower levels of Sulf1 mRNA expression, suggesting predominant roles of Sulf1 in heparan sulfate desulfation and distinct regulation of Sulf activities in vivo. Sulf1 and Sulf2 mRNAs were differentially expressed in restricted types of cells in organs, and consequently, the sulfation patterns of heparan sulfate were locally and distinctly altered in Sulf1 and Sulf2 knock-out mice. These findings indicate that Sulf1 and Sulf2 differentially contribute to the generation of organ-specific sulfation patterns of heparan sulfate.     

Glycosaminoglycans of the porcine central nervous system

Glycosaminoglycans (GAGs) are known to participate in central nervous system processes such as development, cell migration, and neurite outgrowth. In this paper, we report an initial glycomics study of GAGs from the porcine central nervous system. GAGs of the porcine central nervous system, brain and spinal cord were isolated and purified by defatting, proteolysis, anion-exchange chromatography, and methanol precipitation. The isolated GAG content in brain was 5 times higher than in spinal cord (0.35 mg/g of dry sample, compared to 0.07 mg/g of dry sample). In both tissues, chondroitin sulfate (CS) and heparan sulfate (HS) were the major and the minor GAG, respectively. The average molecular masses of CS from brain and spinal cord were 35.5 and 47.1 kDa, respectively, and those for HS from brain and spinal cord were 56.9 and 34 kDa, respectively. The disaccharide analysis showed that the compositions of CS from brain and spinal cords are similar, with uronic acid (1→3) 4-O-sulfo-N-acetylgalactosamine residue corresponding to the major disaccharide unit (CS type A) along with five minor disaccharide units. The major disaccharides of both brain and spinal cord HS were uronic acid (1→4) N-acetylglucosamine and uronic acid (1→4) 6-O-sulfo-N-sulfoglucosamine, but their composition of minor disaccharides differed. Analysis by (1)H and two-dimensional NMR spectroscopy confirmed these disaccharide analyses and provided the glucuronic/iduronic acid ratio. Finally, both purified CS and HS were biotinylated and immobilized on BIAcore SA biochips. Interactions between these GAGs and fibroblast growth factors (FGF1 and FGF2) and sonic hedgehog (Shh) were investigated by surface plasmon resonance.     

Glycosaminoglycans from bovine eye vitreous humour and interaction with collagen type II

Glycosaminoglycans (GAGs) play an important role in stabilizing the gel state of eye vitreous humour. In this study, the composition of GAGs present in bovine eye vitreous was characterized through disaccharide analysis by liquid chromatography-mass spectrometry. The interaction of GAGs with collagen type II was assessed using surface plasmon resonance (SPR). The percentage of hyaluronic acid (HA), chondroitin sulfate (CS) and heparan sulfate (HS), of total GAG, were 96.2%, 3.5% and 0.3%, respectively. The disaccharide composition of CS consisted of 4S (49%), 0S (38%) 6S (12%), 2S6S (1.5%) and 2S4S (0.3%). The disaccharide composition of HS consisted of 0S (80%), NS2S (7%), NS (7%), 6S (4%), NS6S (2%), and TriS, 2S and 4S6S (each at 0.1%). The average molecular weights of CS and HS were 148 kDa and 204 kDa, respectively. SPR reveals that collagen type II binds to heparin (primarily composed of TriS) with a binding affinity (K _D) of 755 nM and interacts with other GAGs, including CSB and CSE. Both bovine vitreous CS and HS interact with collagen type II, with vitreous HS showing a higher binding affinity.     

ZG16p, an animal homolog of β-prism fold plant lectins, interacts with heparan sulfate proteoglycans in pancreatic zymogen granules

ZG16p is a soluble 16?kDa pancreatic protein having structural similarities with plant β-prism fold lectins such as the banana lectin BanLec and the jackfruit lectin jacalin. ZG16p is postulated to be involved in the formation of zymogen granules by interacting with proteoglycans (PGs) localized in pancreatic exocrine granule membranes, but direct evidence was lacking. We characterized the structural properties of rat pancreatic zymogen granule PGs and examined their interaction with ZG16p. Structural analysis of the glycosaminoglycans (GAGs) showed that rat pancreatic zymogen granule PGs have heparan sulfate chains with a unique property, a high degree of sulfation (ΔUA-GlcNAc:ΔUA-GlcNS:ΔUA-GlcNAc6S:ΔUA-GlcNS6S:ΔUA2S-GlcNS:ΔUA2S-GlcNS6S, 27.9:16.6:5.7:22.5:6.2:21.1). After heparin lyase II digestion, the core proteins derived from the PGs were detected at molecular weights of 66,000 and 35,000-40,000. An overlay binding assay revealed that ZG16p binds specifically to heparan sulfate PGs by recognizing their GAG chains. Affinity chromatography demonstrated that ZG16p binds most strongly to heparin among the zymogen granule proteins. Site-directed mutational analysis revealed that the basic amino acid residues located in two putative carbohydrate-binding sites (CBSs) of ZG16p, which were found in association with the crystal structure of BanLec, are responsible for the recognition of heparin. These observations suggest that ZG16p is the primary binding partner of the granule heparan sulfate PGs. ZG16p may cross-link the granule heparan sulfate chains via two CBSs and facilitate the formation of a submembranous matrix, a sorting platform for enzyme proteins on the luminal side of the zymogen granule membrane.     

Interaction of thrombospondin-1 and heparan sulfate from endothelial cells. Structural requirements of heparan sulfate

Cell surface-associated heparan sulfate proteoglycans, predominantly perlecan, are involved in the process of binding and endocytosis of thrombospondin-1 (TSP-1) by vascular endothelial cells. To investigate the structural properties of heparan sulfate (HS) side chains that mediate this interaction, the proteoglycans were isolated from porcine endothelial cells and HS chains obtained thereof by beta-elimination. To characterize the structural composition of the HS chains and to identify the TSP-1-binding sequences, HS was disintegrated by specific chemical and enzymatic treatments. Cell layer-derived HS chains revealed the typical structural heterogeneity with domains of non-contiguously arranged highly sulfated disaccharides separated by extended sequences containing predominantly N-acetylated sequences of low sulfation. Affinity chromatography on immobilized TSP-1 demonstrated that nearly all intact HS chains possessed binding affinity, whereas after heparinase III treatment only a small proportion of oligosaccharides were bound with similar affinity to the column. Size fractioning of the bound and unbound oligosaccharides revealed that only a specific portion of deca- to tetradecasaccharides possessed TSP-1-binding affinity. The binding fraction contained over 40% di- and trisulfated disaccharide units and was enriched in the content of the trisulfated 2-O-sulfated L-iduronic acid-N-sulfated-6-O-sulfated glucosamine disaccharide unit. Comparison with the disaccharide composition of the intact HS chains and competition experiments with modified heparin species indicated the specific importance of N- and 6-O-sulfated glucosamine residues for binding. Further depolymerization of the binding oligosaccharides revealed that the glucosamine residues within the TSP-1-binding sequences are not continuously N-sulfated. The present findings implicate specific structural properties for the HS domain involved in TSP-1 binding and indicate that they are distinct from the binding sequence described for basic fibroblast growth factor, another HS ligand and a potential antagonist of TSP-1.     

Structural characterization of glycosaminoglycans from zebrafish in different ages

The zebrafish (Danio rerio) is a popular model organism for the study of developmental biology, disease mechanisms, and drug discovery. Glycosaminoglycans (GAGs), located on animal cell membranes and in the extracellular matrix, are important molecules in cellular communication during development, in normal physiology and pathophysiology. Vertebrates commonly contain a variety of GAGs including chondroitin/dermatan sulfates, heparin/heparan sulfate, hyaluronan and keratan sulfate. Zebrafish might represent an excellent experimental organism to study the biological roles of GAGs. A recent study showing the absence of heparan sulfate in adult zebrafish, suggested a more detailed evaluation of the GAGs present in this important model organism needed to be undertaken. This report aimed at examining the structural alterations of different GAGs at the molecular level at different developmental stages. GAGs were isolated and purified from zebrafish in different stages in development ranging from 0.5 days to adult. The content and disaccharide composition of chondroitin sulfate and heparan sulfate were determined using chemical assays, liquid chromotography and mass spectrometry. The presence of HS in adult fish was also confirmed using ¹H-NMR.     

Dromedary glycosaminoglycans: molecular characterization of camel lung and liver heparan sulfate

Glycosaminoglycans (GAGs) are the portion of a proteoglycan that determine its final shape and function. The molecular structure of predominant GAG species in camel liver and lung is reported for the first time. The one-humped camel survives in an extreme, arid habitat and, thus, offers a good model to study the role of glycomics on homeostasis. Heparan sulfate (HS) from the lung and liver of the one-humped camel were isolated. Characterization of these newly isolated glycosaminoglycans included (1)H NMR spectroscopy and disaccharide compositional analysis. The relative molecular weight of these GAGs was estimated by gradient polyacrylamide gel electrophoresis and their degree of sulfation was also assessed. Anticoagulant activity was determined using an anti-factor Xa assay and the HS from camel lung shows approximately 50% of heparin's activity. The structural differences of camel liver GAGs compared to human and porcine liver heparin and HS is discussed. Camel lung heparan sulfate resembles both heparin and HS in its structure and properties suggesting that it is either a highly sulfated form of HS, a mixture of heparin and HS or an undersulfated heparin.     

Exposure to common food additive carrageenan leads to reduced sulfatase activity and increase in sulfated glycosaminoglycans in human epithelial cells

The commonly used food additive carrageenan, including lambda (λ), kappa (κ) and iota (ι) forms, is composed of galactose disaccharides linked in alpha-1,3 and beta-1,4 glycosidic bonds with up to three sulfate groups per disaccharide residue. Carrageenan closely resembles the endogenous galactose or N-acetylgalactosamine-containing glycosaminoglycans (GAGs), chondroitin sulfate (CS), dermatan sulfate (DS), and keratan sulfate. However, these GAGs have beta-1,3 and beta-1,4 glycosidic bonds, in contrast to the unusual alpha-1,3 glycosidic bond in carrageenan. Since sulfatase activity is inhibited by sulfate, and carrageenan is so highly sulfated, we tested the effect of carrageenan exposure on sulfatase activity in human intestinal and mammary epithelial cell lines and found that carrageenan exposure significantly reduced the activity of sulfatases, including N-acetylgalactosamine-4-sulfatase, galactose-6-sulfatase, iduronate sulfatase, steroid sulfatase, arylsulfatase A, SULF-1,2, and heparan sulfamidase. Consistent with the inhibition of sulfatase activity, following exposure to carrageenan, GAG content increased significantly and showed marked differences in disaccharide composition. Specific changes in CS disaccharides included increases in di-sulfated disaccharide components of CSD (2S6S) and CS-E (4S6S), with declines in CS-A (4S) and CS-C (6S). Specific changes in heparin-heparan sulfate disaccharides included increases in 6S disaccharides, as well as increases in NS and 2S6S disaccharides. Study results suggest that carrageenan inhibition of sulfatase activity leads to re-distribution of the cellular GAG composition with increase in di-sulfated CS and with potential consequences for cell structure and function.     

Disaccharide compositional analysis of heparan sulfate and heparin polysaccharides using UV or high-sensitivity fluorescence (BODIPY) detection

One of the first steps in characterizing heparan sulfate (HS) and its close relative heparin is to conduct disaccharide composition analysis. This provides an overall picture of the structure of the polysaccharide in terms of its constituent disaccharides. This is of importance, for example, in the initial characterization of spatially and temporally regulated structures. Two protocols for conducting disaccharide analysis are presented here, both exploiting exhaustive digestion of the polysaccharide, yielding constituent disaccharides, by bacterial heparin lyases. The first method, suitable for microgram quantities of material, relies on the separation of the disaccharides by high-performance liquid chromatography (HPLC) coupled to ultraviolet absorbance detection and can be performed in 2 d. The second exploits reducing end-labeling with the fluorophore BODIPY hydrazide, separation by HPLC, and subsequent fluorescence detection and quantitation. The latter is a high-sensitivity method that requires nanograms of starting material and has a detection limit in the low fmol range, and is thus the most sensitive method for disaccharide compositional analysis of HS yet reported. Fluorescence detection can be routinely carried out in 3 d.     

Enzymatic preparation of heparin disaccharides as building blocks in glycosaminoglycan synthesis.

Pharmaceutical heparin and heparan sulfate, isolated from a side-stream of a commercial heparin manufacturing process, have been enzymatically depolymerzed with heparin lyases obtained from Flavobacterium heparinun. Heparin afforded a trisulfated disaccharide product that was recovered from the reaction mixture using gel permeation chromatography. Heparan sulfate afforded unsulfated disaccharide that was conveniently recovered from the product mixture by ion exchange chromatography. Both disaccharides were obtained in gram amounts at 90% or higher purity. Both enzymatically prepared disaccharides were chemically protected to prepare building blocks required for the future chemical synthesis of therapeutically valuable heparin oligosaccharides.     

Compositional profiling of heparin/heparan sulfate using mass spectrometry: assay for specificity of a novel extracellular human endosulfatase

An important class of carbohydrates studied within the field of glycobiology, heparin and heparan sulfate (HS) have been implicated in a diverse array of biological functions. Changes in their sulfation pattern and domain organization have been associated with different pathological situations such as viral infectivity, tumor growth, and metastasis. To obtain structural information about these biomolecules, and the modifications they may undergo during different stages of cell growth and development, a mass spectrometry-based method was developed and used to obtain unambiguous structural information on the glycosaminoglycans (GAGs) that comprise heparin/HS. The method was applied to assay for the heparin substrate specificity of a newly discovered human extracellular endosulfatase, HSulf-2, which has been implicated in tumorigenesis. This new protocol incorporates 12 known heparin disaccharides, including three sets of isomers. A unique response factor (R) is determined for each disaccharide, whereas a multiplexed and data processing method is incorporated for faster data acquisition and quantification purposes. Proof of principle was performed by using various heparin/HS samples isolated from bovine and porcine tissues.     

Examination of the substrate specificity of heparin and heparan sulfate lyases

We have examined the activities of different preparations of heparin and heparan sulfate lyases from Flavobacterium heparinum. The enzymes were incubated with oligosaccharides of known size and sequence and with complex polysaccharide substrates, and the resulting degradation products were analyzed by strong-anion-exchange high-performance liquid chromatography and by oligosaccharide mapping using gradient polyacrylamide gel electrophoresis. Heparinase (EC 4.2.2.7) purified in our laboratory and a so-called Heparinase I (Hep I) from a commercial source yielded similar oligosaccharide maps with heparin substrates and displayed specificity for di- or trisulfated disaccharides of the structure----4)-alpha-D-GlcNp2S(6R)(1----4)-alpha-L-IdoAp2S( 1----(where R = O-sulfo or OH). Oligosaccharide mapping with two different commercial preparations of heparan sulfate lyase [heparitinase (EC 4.2.2.8)] indicated close similarities in their depolymerization of heparan sulfate. Furthermore, these enzymes only degraded defined oligosaccharides at hexosaminidic linkages with glucuronic acid:----4)-alpha-D-GlcNpR(1----4)-beta-D-GlcAp(1----(where R = N-acetamido or N-sulfo). The enzymes showed activity against solitary glucuronate-containing disaccharides in otherwise highly sulfated domains including the saccharide sequence that contains the antithrombin binding region in heparin. A different commercial enzyme, Heparinase II (Hep II), displayed a broad spectrum of activity against polysaccharide and oligosaccharide substrates, but mapping data indicated that it was a separate enzyme rather than a mixture of heparinase and heparitinase/Hep III. When used in conjunction with the described separation procedures, these enzymes are powerful reagents for the structural/sequence analysis of heparin and heparan sulfate.     

Structural characterization of human liver heparan sulfate

The isolation, purification and structural characterization of human liver heparan sulfate are described. 1H-NMR spectroscopy demonstrates the purity of this glycosaminoglycan (GAG) and two-dimensional 1H-NMR confirmed that it was heparan sulfate. Enzymatic depolymerization of the isolated heparan sulfate, followed by gradient polyacrylamide gel, confirmed its heparin lyase sensitivity. The concentration of resulting unsaturated disaccharides was determined using reverse phase ion-pairing (RPIP) HPLC with post column derivatization and fluorescence detection. The results of this analysis clearly demonstrate that the isolated GAG was heparan sulfate, not heparin. Human liver heparan sulfate was similar to heparin in that it has a reduced content of unsulfated disaccharide and an elevated average sulfation level. The antithrombin-mediated anti-factor Xa activity of human liver heparan sulfate, however, was much lower than porcine intestinal (pharmaceutical) heparin but was comparable to standard porcine intestinal heparan sulfate. Moreover, human liver heparan sulfate shows higher degree of sulfation than heparan sulfate isolated from porcine liver or from the human hepatoma Hep 2G cell line.     

Glycosaminoglycan composition of the large freshwater mollusc bivalve Anodonta anodonta

In this paper, glycosaminoglycans from the body of the large freshwater mollusc bivalve Anodonta anodonta were recovered at about 0.6 mg/g of dry tissue, composed of chondroitin sulfate (approximately 38%), nonsulfated chondroitin (about 21%), and heparin (41%). This last polysaccharide was found to consist of a large percentage (approximately 88%) of a fast-moving species possessing a lower molecular mass and sulfate group amount and about 12% of a more sulfated, slow-moving component having a greater molecular mass. The chondroitin sulfate was composed of approximately 28% of the 6-sulfated disaccharide, 46% of the 4-sulfated disaccharide, and about 26% of the nonsulfated disaccharide, with a charge density value of 0.74. Heparin was subjected to the oligosaccharide mapping after treatment with heparinase and then separation of the resulting unsaturated oligosaccharides by SAX-HPLC. A heparin sample from Anodonta anodonta showed a degree of sulfation similar to that of bovine mucosal heparin because of the presence of approximately the same mol % of the trisulfated disaccharide (DeltaUA2S(1-->4)-alpha-D-GlcN2S6S), a slight modification of the other oligosaccharides, and a significant increase of the disaccharide bearing the sulfate group in position 3 of the N-sulfoglucosamine 6-sulfate (-->4)-beta-D-GlcA(1-->4)-alpha-D-GlcN2S3S6S(1-->) part of the ATIII-binding region. However, the anticoagulant activity of mollusc heparin was quite similar to that of pharmaceutical grade heparin. The data obtained again emphasize the heterogeneity of GAGs from molluscs.     

Mass spectrometric evidence of heparin disaccharides for the catalytic characterization of a novel endolytic heparinase

Heparin like glycosaminoglycans (HLGAGs) are struc-turally complex linear polysaccharides composed of re-peating disaccharide unit of uronic (α-L-iduronic or β-D-glucuronic) acid linked 1→4 to α-D-glucosamine, whichis a highly variable sulfation pattern and ascribes to eachglycosaminoglycan (GAG) chain a unique structuralsignature. This signature dictates specific the GAG-pro-tein interactions underlying critical biological processesrelated to cell and tissue functions [1]. Only in fe…     

Characterization of a heparan sulfate 3-O-sulfotransferase-5, an enzyme synthesizing a tetrasulfated disaccharide

Heparan sulfate d-glucosaminyl 3-O-sulfotransferases (3-OSTs) catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 3 of the glucosamine residue of heparan sulfate and heparin. A sixth member of the human 3-OST family, named 3-OST-5, was recently reported (Xia, G., Chen, J., Tiwari, V., Ju, W., Li, J.-P., Malmstrom, A., Shukla, D., and Liu, J. (2002) J. Biol. Chem. 277, 37912-37919). In the present study, we cloned putative catalytic domain of the human 3-OST-5 and expressed it in insect cells as a soluble enzyme. Recombinant 3-OST-5 only exhibited sulfotransferase activity toward heparan sulfate and heparin. When incubated heparan sulfate with [35S]PAPS, the highest incorporation of35S was observed, and digestion of the product with a mixture of heparin lyases yielded two major35S-labeled disaccharides, which were determined as DeltaHexA-GlcN(NS,3S,6S) and DeltaHexA(2S)-GlcN(NS,3S) by further digestion with 2-sulfatase and degradation with mercuric acetate. However, when used heparin as acceptor, we identified a highly sulfated disaccharide unit as a major product. This had a structure of DeltaHexA(2S)-GlcN(NS,3S,6S). Quantitative real-time PCR analysis revealed that 3-OST-5 was highly expressed in fetal brain, followed by adult brain and spinal cord, and at very low or undetectable levels in the other tissues. Finally, we detected a tetrasulfated disaccharide unit in bovine intestinal heparan sulfate. To our knowledge, this is the first report to describe not only the natural occurrence of tetrasulfated disaccharide unit but also the enzymatic formation of this novel structure.     

The keratan sulfate disaccharide Gal(6S03) beta1,4-GlcNAc(6S03) modulates interleukin 12 production by macrophages in murine Thy-1 type autoimmune disease

It has been reported that disaccharides of the glycosaminoglycans (GAGs), heparin, or heparan sulfate suppress the production of cytokines. Therefore, we examined the effects of GAGs (keratan sulfate, hyaluronan, chondroitin, chondroitin sulfate, and heparin sulfate) disaccharides on production of interleukin (IL)-12, a pivotal cytokine in the Th-1 type immune system. Among the GAG disaccharides, only a keratan sulfate disaccharide, Gal(6-SO(3))-GlcNAc(6-SO(3)) (L4), suppressed IL-12 production in macrophages stimulated with lipopolysaccharides and interferon-gamma. Neither keratan sulfate chains nor keratan sulfate tetrasaccharides elicited any change in the IL-12 production. N-Acetyl-lactosamine, Gal-GlcNAc (LacNAc), also did not change IL-12 production. These results indicated that a certain size, i.e. disaccharide and sulfate, are essential to suppress IL-12 production. L4 was then applied to MRL-lpr/lpr mice, a Th-1 type autoimmune disease model. The treatment of MRL-lpr/lpr mice with L4 1) decreased in serum IL-12, 2) induced apoptosis in T cells in lymph nodes thereby suppressing lymphoaccumulation, and 3) suppressed hypergammaglobulinemia and glomerulonephritis. We showed previously that IL-12 suppresses cell death of T cells, thereby enhancing the lymphoaccumulation in MRL-lpr/lpr mice. Moreover, it has been reported that IL-12 deficiency in MRL-lpr/lpr mice diminishes lymphoaccumulation and delays glomerulonephritis. The treatment with L4 suppressed phosphoprotein kinase C and phosphoinositide 3-kinase expression in macrophages, suggesting that L4 suppresses IL-12 production by inhibiting phosphoprotein kinase C and phosphoinositide 3-kinase pathways.