Overexpression of heparan sulfate 6-O-sulfotransferases in human embryonic kidney 293 cells results in increased N-acetylglucosaminyl 6-O-sulfation

Heparan sulfate (HS) interacts with a variety of proteins and thus mediates numerous complex biological processes. These interactions critically depend on the patterns of O-sulfate groups within the HS chains that determine binding sites for proteins. In particular the distribution of 6-O-sulfated glucosamine residues influences binding and activity of HS-dependent signaling molecules. The protein binding domains of HS show large structural variability, potentially because of differential expression patterns of HS biosynthetic enzymes along with differences in substrate specificity. To investigate whether different isoforms of HS glucosaminyl 6-O-sulfotransferase (6-OST) give rise to differently sulfated domains, we have introduced mouse 6-OST1, 6-OST2, and 6-OST3 in human embryonic kidney 293 cells and compared the effects of overexpression on HS structure. High expression of any one of the 6-OST enzymes resulted in appreciably increased 6-O-sulfation of N-sulfated as well as N-acetylated glucosamine units. The increased 6-O-sulfation was accompanied by a decrease in nonsulfated as well as in iduronic acid 2-O-sulfated disaccharide structures. Furthermore, overexpression led to an altered HS domain structure, the most striking effect was the formation of extended 6-O-sulfated predominantly N-acetylated HS domains. Although the effect was most noticeable in 6-OST3-expressing cells, these results were largely independent of the particular 6-OST isoform expressed and mainly influenced by the level of overexpression.     

Biosynthesis of heparin. O-sulfation of D-glucuronic acid units

Incubation of a microsomal fraction from murine mastocytoma, with UDP-[1-3H]GlcA, UDP-GlcNAc, and adenosine 3'-phosphate 5'-phosphosulfate (PAPS), yielded labeled, N-sulfated polysaccharides, in which most of the incorporated O-sulfate groups were located at C2 of L-iduronic acid and at C6 of D-glucosamine units. Analysis by anion-exchange high pressure liquid chromatography of disaccharides, generated by deaminative cleavage of these polysaccharides, revealed that, in addition, an appreciable portion of the -GlcNSO3-HexA-GlcNSO3- sequences in the intact polymers contained O-sulfated (at C2 or C3) D-glucuronic acid units. Calculations based on such compositional analysis of the N- and O-sulfated biosynthetic product, isolated by chromatography on DEAE-cellulose, showed that glucuronosyl 2/3-O-sulfate accounted for approximately 12% of the total incorporated O-sulfate groups. With [35S]PAPS (at a low total PAPS concentration) as an alternative source of label, the sulfated glucuronic acid residues were again detectable, albeit in much smaller amounts (1.8% of the total O-sulfate groups). Incorporation of label from UDP-[5-3H]GlcA was retained by the O-sulfated glucuronic acid units, thus demonstrating that these components had in fact been formed by sulfation of glucuronic acid residues and not by "back epimerization" of sulfated iduronic acid units. Structural analysis of polysaccharide intermediates at various stages of biosynthetic polymer modification, separated by ion-exchange chromatography, showed O-sulfation of glucuronic and iduronic acid units to appear simultaneously and before the 6-O-sulfation of glucosamine residues.     

Selective effects of sodium chlorate treatment on the sulfation of heparan sulfate

We have analyzed the effect of sodium chlorate treatment of Madin-Darby canine kidney cells on the structure of heparan sulfate (HS), to assess how the various sulfation reactions during HS biosynthesis are affected by decreased availability of the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate. Metabolically [(3)H]glucosamine-labeled HS was isolated from chlorate-treated and untreated Madin-Darby canine kidney cells and subjected to low pH nitrous acid cleavage. Saccharides representing (i) the N-sulfated domains, (ii) the domains of alternating N-acetylated and N-sulfated disaccharide units, and (iii) the N-acetylated domains were recovered and subjected to compositional disaccharide analysis. Upon treatment with 50 mM chlorate, overall O-sulfation of HS was inhibited by approximately 70%, whereas N-sulfation remained essentially unchanged. Low chlorate concentrations (5 or 20 mM) selectively reduced the 6-O-sulfation of HS, whereas treatment with 50 mM chlorate reduced both 2-O- and 6-O-sulfation. Analysis of saccharides representing the different domain types indicated that 6-O-sulfation was preferentially inhibited in the alternating domains. These data suggest that reduced 3'-phosphoadenosine 5'-phosphosulfate availability has distinct effects on the N- and O-sulfation of HS and that O-sulfation is affected in a domain-specific fashion.     

Irreversible glucuronyl C5-epimerization in the biosynthesis of heparan sulfate

Glucuronyl C5-epimerase catalyzes the conversion of d-glucuronic acid to l-iduronic acid units in heparan sulfate biosynthesis. Substrate recognition depends on the N-substituent pattern of the heparan sulfate precursor polysaccharide and requires the adjacent glucosamine residue toward the non-reducing end to be N-sulfated. Epimerization of an appropriately N-sulfated substrate is freely reversible in a soluble system, with equilibrium favoring retention of d-gluco configuration (Hagner-McWhirter, A., Lindahl, U., and Li, J.-P. (2000) Biochem. J. 347, 69-75). We studied the reversibility of the epimerase reaction in a cellular system, by incubating human embryonic kidney 293 cells with d-[5-(3)H]galactose. The label was incorporated with glucuronic acid units into the heparan sulfate precursor polysaccharide and was lost upon subsequent C5-epimerization to iduronic acid. However, analysis of oligosaccharides obtained by deaminative cleavage of the mature heparan sulfate chains indicated that all glucuronic acid units retained their C5-(3)H label, irrespective of whether they had occurred in sequences susceptible or resistant to the epimerase. All (3)H-labels of the final products resisted incubation with epimerase in a soluble system, apparently due to blocking O-sulfate groups. These results indicate that glucuronic acid C5-epimerization is effectively irreversible in vivo and argue for a stringent organization of the biosynthetic machinery.     

Structural diversity of N-sulfated heparan sulfate domains: distinct modes of glucuronyl C5 epimerization, iduronic acid 2-O-sulfation, and glucosamine 6-O-sulfation

The N-sulfated regions (NS domains) represent the modified sequences of heparan sulfate chains and mediate interactions of the polysaccharide with proteins. We have investigated the relationship between the type/extent of polymer modification and the length of NS domains in heparan sulfate species from human aorta, bovine kidney, and cultured NMuMG and MDCK cells. C5 epimerization of D-glucuronic acid to L-iduronic acid was found to be extensive and essentially similar in all heparan sulfate species studied, regardless of domain size, whereas the subsequent 2-O-sulfation of the formed iduronic acid residues varies appreciably. In aorta heparan sulfate, up to 90% of the formed iduronate residues were 2-O-sulfated, whereas in kidney heparan sulfate 2-O-sulfation occurred only in 相似文献   

The structural motif in chondroitin sulfate for adhesion of Plasmodium falciparum-infected erythrocytes comprises disaccharide units of 4-O-sulfated and non-sulfated N-acetylgalactosamine linked to glucuronic acid

An important characteristic of malaria parasite Plasmodium falciparum-infected red blood cells (IRBCs) is their ability to adhere to host endothelial cells and accumulate in various organs. Sequestration of IRBCs in the placenta, associated with excess perinatal and maternal mortality, is mediated in part by adhesion of parasites to the glycosaminoglycan chondroitin sulfate A (CSA) present on syncytiotrophoblasts lining the placental blood spaces. To define key structural features for parasite interactions, we isolated from CSA oligosaccharide fractions and established by electrospray mass spectrometry and high performance liquid chromatography disaccharide composition analysis their differing chain length, sulfate content, and sulfation pattern. Testing these defined oligosaccharide fragments for their ability to inhibit IRBC adhesion to immobilized CSA revealed the importance of non-sulfated disaccharide units in combination with 4-O-sulfated disaccharides for interaction with IRBCs. Selective removal of 6-O-sulfates from oligo- and polysaccharides to increase the proportion of non-sulfated disaccharides enhanced activity, indicating that 6-O-sulfation interferes with the interaction of CSA with IRBCs. Dodecasaccharides with four or five 4-O-sulfated and two or one non-sulfated disaccharide units, respectively, comprise the minimum chain length for effective interaction with IRBCs. Comparison of the activities of CSA and CSB oligo- and polysaccharides with a similar sulfation pattern and content achieved from partial desulfation demonstrated that glucuronic acid rather than iduronic acid residues are important for IRBC binding.     

Multi-faceted substrate specificity of heparanase

Heparan sulfate is a highly sulfated polysaccharide abundantly present in the extracellular matrix. Heparan sulfate consists of a disaccharide repeating unit of glucosamine and glucuronic and iduronic acid residues. The functions of heparan sulfate are largely dictated by its size as well as the sulfation patterns. Heparanase is an enzyme that cleaves heparan sulfate polysaccharide into smaller fragments, regulating the functions of heparan sulfate. Understanding the substrate specificity plays a critical role in dissecting the biological functions of heparanase and heparan sulfate. The prevailing view is that heparanase recognizes specific sulfation patterns in heparan sulfate. However, emerging evidence suggests that heparanase is capable of varying its substrate specificities depending on the saccharide structures around the cleavage site. The plastic substrate specificity suggests a complex role of heparanase in regulating the structures of heparan sulfate in matrix biology.     

A synthetic heparan sulfate oligosaccharide library reveals the novel enzymatic action of D-glucosaminyl 3-O-sulfotransferase-3a

Heparan sulfate (HS) glucosaminyl 3-O-sulfotranferases sulfate the C3-hydroxyl group of certain glucosamine residues on heparan sulfate. Six different 3-OST isoforms exist, each of which can sulfate very distinct glucosamine residues within the HS chain. Among these isoforms, 3-OST1 has been shown to play a role in generating ATIII-binding HS anticoagulants whereas 3-OST2, 3-OST3, 3-OST4 and 3OST-6 have been shown to play a vital role in generating gD-binding HS chains that permit the entry of herpes simplex virus type 1 into cells. 3-OST5 has been found to generate both ATIII- and gD-binding HS motifs. Previous studies have examined the substrate specificities of all the 3-OST isoforms using HS polysaccharides. However, very few studies have examined the contribution of the epimer configuration of neighboring uronic acid residues next to the target site to 3-OST action. In this study, we utilized a well-defined synthetic oligosaccharide library to examine the substrate specificity of 3-OST3a and compared it to 3-OST1. We found that both 3-OST1 and 3-OST3a preferentially sulfate the 6-O-sulfated, N-sulfoglucosamine when an adjacent iduronyl residue is located to its reducing side. On the other hand, 2-O-sulfation of this uronyl residue can inhibit the action of 3-OST3a on the target residue. The results reveal novel substrate sites for the enzyme actions of 3-OST3a. It is also evident that both these enzymes have promiscuous and overlapping actions that are differentially regulated by iduronyl 2-O-sulfation.     

Demonstration of a novel sulfotransferase in fetal bovine serum, which transfers sulfate to the C6 position of the GalNAc residue in the sequence iduronic acid alpha1-3GalNAc beta1-4iduronic acid in dermatan sulfate.

A novel sulfotransferase activity was discovered in fetal bovine serum using pig skin dermatan sulfate as an acceptor and [35S]3'-phosphoadenosine 5'-phosphosulfate as a sulfate donor. The enzyme was separated from chondroitin:GalNAc 6-O-sulfotransferase by chromatographic techniques. Enzymatic analysis of the reaction products demonstrated that the enzyme transferred sulfate to the C6 position of the GalNAc residue in the sequence -iduronic acid alpha1-3GalNAc beta1-4iduronic acid-. Thus, the enzyme has been identified as a hitherto unreported dermatan sulfate:GalNAc 6-O-sulfotransferase. The finding is in sharp contrast to the current concept that in dermatan sulfate biosynthesis GalNAc 4-O-sulfation is a prerequisite for iduronic acid formation by C5 epimerase.     

New insights on the specificity of heparin and heparan sulfate lyases from Flavobacterium heparinum revealed by the use of synthetic derivatives of K5 polysaccharide from E. coli. and 2-O-desulfated heparin

The capsular polysaccharide from E. Coli, strain K5 composed of ...-->4)beta-D-GlcA(1-->4)alpha-D-GlcNAc(1-->4)beta-D-GlcA (1-->..., chemically modified K5 polysaccharides, bearing sulfates at C-2 and C-6 of the hexosamine moiety and at the C-2 of the glucuronic acid residues as well as 2-O desulfated heparin were used as substrates to study the specificity of heparitinases I and II and heparinase from Flavobacterium heparinum. The natural K5 polysaccharide was susceptible only to heparitinase I forming deltaU-GlcNAc. N-deacetylated, N-sulfated K5 became susceptible to both heparitinases I and II producing deltaU-GlcNS. The K5 polysaccharides containing sulfate at the C-2 and C-6 positions of the hexosamine moiety and C-2 position of the glucuronic acid residues were susceptible only to heparitinase II producing deltaU-GlcNS,6S and deltaU,2S-GlcNS,6S respectively. These combined results led to the conclusion that the sulfate at C-6 position of the glucosamine is impeditive for the action of heparitinase I and that heparitinase II requires at least a C-2 or a C-6 sulfate in the glucosamine residues of the substrate for its activity. Iduronic acid-2-O-desulfated heparin was susceptible only to heparitinase II producing deltaU-GlcNS,6S. All the modified K5 polysaccharides as well as the desulfated heparin were not substrates for heparinase. This led to the conclusion that heparitinase II acts upon linkages containing non-sulfated iduronic acid residues and that heparinase requires C-2 sulfated iduronic acid residues for its activity.     

Formation of dermatan sulfate by cultured human skin fibroblasts. Effects of sulfate concentration on proportions of dermatan/chondroitin

[3H,35S]Dermatan/chondroitin sulfate glycosaminoglycans produced during culture of fibroblasts in medium containing varying concentrations of sulfate were tested for their susceptibility to chondroitin ABC lyase and chondroitin AC lyase. Chondroitin ABC lyase completely degraded [3H]hexosamine-labeled and [35S] sulfate-labeled dermatan/chondroitin sulfate to disaccharides. Chondroitin AC lyase treatment of the labeled glycosaminoglycans produced different results. With this enzyme, dermatan/chondroitin sulfate formed at high concentrations of sulfate yielded small glycosaminoglycans and larger oligosaccharides but almost no disaccharide. This indicated that the dermatan/chondroitin sulfate co-polymer contained mostly iduronic acid with only an occasional glucuronic acid. As the medium sulfate concentration was progressively lowered, there was a concomitant increase in the susceptibility to degradation by chondroitin AC lyase. Thus, the labeled glycosaminoglycans formed at the lowest concentration of sulfate yielded small oligosaccharides including substantial amounts of disaccharide. The smaller chondroitin AC lyase-resistant [3H,35S]dermatan/chondroitin sulfate oligosaccharides were analyzed by gel filtration. Results indicated that, in general, the iduronic acid-containing disaccharide residues present in the undersulfated [3H,35S]glycosaminoglycan were sulfated, whereas the glucuronic acid-containing disaccharide residues were non-sulfated. This work confirms earlier reports that there is a relationship between epimerization and sulfation. Moreover, it demonstrates that medium sulfate concentration is critical in determining the proportions of dermatan to chondroitin (iduronic/glucuronic acid) produced by cultured cells.     

Biosynthesis of heparin. Availability of glucosaminyl 3-O-sulfation sites

Heparin preparations isolated from pig intestinal mucosa and from bovine lung were fractionated with regard to affinity for antithrombin. The resulting fractions, with high (HA) or low (LA) affinity for the proteinase inhibitor, were analyzed by 13C NMR or by identification of di- and tetrasaccharides obtained through deaminative cleavage with nitrous acid. Structural differences between corresponding HA and LA fractions were essentially restricted to minor constituents, in particular 3-O-sulfated glucosamine units that occurred (1 or 2 residues/chain) in all HA preparations but were scarce or absent in LA heparin. The HA fractions also consistently showed higher contents of nonsulfated iduronic acid and, to a lesser extent, N-acetylated glucosamine units than the LA fractions. The two tetrasaccharide sequences, -IdoA-GlcNAc(6-OSO3)-GlcA-GlcNSO3- and -IdoA-GlcNAc(6-OSO3)-GlcA-GlcNSO3(6-OSO3)- , recently implicated as part of the acceptor site for glucosaminyl 3-O-sulfate groups (Kusche, M., B?ckstr?m, G., Riesenfeld, J., Petitou, M., Choay, J., and Lindahl, U. (1988) J. Biol. Chem. 263, 15474-15484), were identified in mucosal LA heparin; it was calculated that the preparation contained approximately one potential acceptor site/polysaccharide chain. Yet this material did not yield any labeled HA components on incubation with adenosine 3'-phosphate 5'-phospho-[35S]sulfate in the presence of glucosaminyl 3-O-sulfotransferase, solubilized from a mouse mastocytoma microsomal fraction. The failure to incorporate any 3-O-sulfate groups could conceivably be explained by the occurrence of a D-glucuronic rather than L-iduronic acid unit linked at the reducing ends of the above tetrasaccharide sequences. Alternatively, 3-O-sulfation may be restricted by other, as yet unidentified, inhibitory structural elements that are preferentially expressed in polysaccharide sequences selected for the generation of LA heparin.     

Biosynthesis of dermatan sulfate: chondroitin-glucuronate C5-epimerase is identical to SART2

We identified the gene encoding chondroitin-glucuronate C5-epimerase (EC 5.1.3.19) that converts D-glucuronic acid to L-iduronic acid residues in dermatan sulfate biosynthesis. The enzyme was solubilized from bovine spleen, and an approximately 43,000-fold purified preparation containing a major 89-kDa candidate component was subjected to mass spectrometry analysis of tryptic peptides. SART2 (squamous cell carcinoma antigen recognized by T cell 2), a protein with unknown function highly expressed in cancer cells and tissues, was identified by 18 peptides covering 26% of the sequence. Transient expression of cDNA resulted in a 22-fold increase in epimerase activity in 293HEK cell lysate. Moreover, overexpressing cells produced dermatan sulfate chains with 20% of iduronic acid-containing disaccharide units, as compared with 5% for mock-transfected cells. The iduronic acid residues were preferentially clustered in blocks, as in naturally occurring dermatan sulfate. Given the discovered identity, we propose to rename SART2 (Nakao, M., Shichijo, S., Imaizumi, T., Inoue, Y., Matsunaga, K., Yamada, A., Kikuchi, M., Tsuda, N., Ohta, K., Takamori, S., Yamana, H., Fujita, H., and Itoh, K. (2000) J. Immunol. 164, 2565-2574) with a functional designation, chondroitin-glucuronate C5-epimerase (or DS epimerase). DS epimerase activity is ubiquitously present in normal tissues, although with marked quantitative differences. It is highly homologous to part of the NCAG1 protein, encoded by the C18orf4 gene, genetically linked to bipolar disorder. NCAG1 also contains a putative chondroitin sulfate sulfotransferase domain and thus may be involved in dermatan sulfate biosynthesis. The functional relation between dermatan sulfate and cancer is unknown but may involve known iduronic acid-dependent interactions with growth factors, selectins, cytokines, or coagulation inhibitors.     

Structure of a fucose-branched chondroitin sulfate from sea cucumber. Evidence for the presence of 3-O-sulfo-beta-D-glucuronosyl residues

The structure of a unique focose-branched chondroitin sulfate isolated from the body wall of a sea cucumber was examined in detail. This glycosaminoglycan contains side chain disaccharide units of sulfated fucopyranosyl units linked to approximately one-half of the glucuronic acid moieties through the O-3 position of the acid. The intact polysaccharide is totally resistant to chondroitinase degradation, whereas, after defucosylation, it is partially degraded by the enzyme. However, only after an additional step of desulfation, the chondroitin from sea cucumber is almost totally degraded by chondroitinase AC or ABC. This result, together with the methylation and NMR studies of the native and chemically modified polysaccharide, suggest that besides the fucose branches, the sea cucumber chondroitin sulfate contains sulfate esters at position O-3 of the beta-D-glucuronic acid units. Furthermore, the proteoglycan from the sea cucumber chondroitin sulfate is recognized by anti-Leu-7 monoclonal antibody, which specifically recognizes 3-sulfoglucuronic acid residues. In analogy with the fucose branched units, the 3-O-sulfo-beta-D-glucuronosyl residues are resistant to chondroitinase degradation. Regarding the position of the glycosidic linkage and site of sulfation in the fucose branches, our results suggest high heterogeneity. Tentatively, it is possible to suggest the preponderance of disaccharide units formed by 3,4-di-O-sulfo-alpha-L-fucopyranosyl units glycosidically linked through position 1----2 to 4-O-sulfo-alpha-L-fucopyranose. Finally, the presence of unusual 4/6-disulfated disaccharide units, together with the common 6-sulfated and non-sulfated units, was detected in the chondroitin sulfate core of this polysaccharide.     

Uncovering biphasic catalytic mode of C5-epimerase in heparan sulfate biosynthesis

Heparan sulfate (HS), a highly sulfated polysaccharide, is biosynthesized through a pathway involving several enzymes. C(5)-epimerase (C(5)-epi) is a key enzyme in this pathway. C(5)-epi is known for being a two-way catalytic enzyme, displaying a "reversible" catalytic mode by converting a glucuronic acid to an iduronic acid residue, and vice versa. Here, we discovered that C(5)-epi can also serve as a one-way catalyst to convert a glucuronic acid to an iduronic acid residue, displaying an "irreversible" catalytic mode. Our data indicated that the reversible or irreversible catalytic mode strictly depends on the saccharide substrate structures. The biphasic mode of C(5)-epi offers a novel mechanism to regulate the biosynthesis of HS with the desired biological functions.     

Biosynthesis of heparin

The formation of labeled heparin-precursor polysaccharide (N-acetylheparosan) from the nucleotide sugars, UDP-[¹⁴C]glucuronic acid and UDP-N-acetylglucosamine, in a mouse mastocytoma microsomal fraction was abolished by the addition of 1% Triton X-100. In contrast, the detergent-treated microsomal preparation retained the ability to convert such preformed polysaccharide into sulfated products during incubation with 3-phosphoadenylylsulfate (PAPS). However, as shown by ion-exchange chromatography of these products, the detegent treatment changed the kinetics of sulfation from the rapid, repetivive process characteristic of the unperturbed system to a slow, progressive sulfation, which involved all polysarccharide molecules simultaneously and yielded, ultimately, a more highly sulfated product. The detergent effect was attributed to solubilization of sulfotransferases from the microsomal membranes, along with other polymer-modifying enzymes and the polysaccharide substrate. The resulting product showed an apparently random distribution ofN-acetyl andN-sulfate groups, instead of the predominantly block-wise arrangement achieved through membrane-associated biosynthesis.O-Sulfation occurred mainly at C2 of the iduronic acid units in the membrane-bound polysaccharide but at C6 of the glucosamine residues in the presence of detergent.A capsular polysaccharide fromEscherichia coli K5, previously found to have a structure identical to that of the nonsulfated heparin-precursor polysaccharide, was sulfated in the solubilized system in a fashion similar to that of the endogenous substrate, but was not accessible to the membrane-bound enzymes.These findings suggest that the regulation of the polymer-modification process, and hence the structure of the final polysaccharide product, depends heavily on the organization of the enzymes and their proteoglycan substrate in the endoplasmic membranes of the cell.Abbreviations PAPS 3-phosphoadenylylsulfate - Hepes 4-(2-hydroxy-ethyl)piperazineethanesulfonic acid - GlcUA glucuronic acid This is Paper XIV of a series in which the preceeding reports are refs 10 and 12. A preliminary report has appeared [28].     

Mutational study of heparan sulfate 2-O-sulfotransferase and chondroitin sulfate 2-O-sulfotransferase

Heparan sulfate (HS) and chondroitin sulfate (CS) are highly sulfated polysaccharides with a wide range of biological functions. Heparan sulfate 2-O-sulfotransferase (HS-2OST) transfers the sulfo group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the 2-OH position of the hexauronic acid that is adjacent to N-sulfated glucosamine, whereas chondroitin sulfate 2-O-sulfotransferase (CS-2OST) transfers the sulfo group to the hexauronic acid that is adjacent to N-acetylated galactosamine. Here we report a systematic mutagenesis study of HS-2OST and CS-2OST based on their structural homology to estrogen sulfotransferase and HS 3-O-sulfotransferase isoform 3 (3-OST3), for which crystal structures exist. We have identified six residues possibly involved in binding to PAPS. HS-2OST carrying mutations of these residues lacks sulfotransferase activity and the ability to bind 3'-phosphoadenosine 5'-phosphate, a PAPS analogue, as determined by isothermal titration calorimetry. Similar residues involved in binding to PAPS were also identified in CS-2OST. Additional residues that participate in carbohydrate substrate binding were also identified in both enzymes. Mutations at these residues led to the loss of sulfotransferase activity but maintained the ability to bind to phosphoadenosine 5'-phosphate. The catalytic function of HS-2OST appears to involve two histidine residues (His140 and His142), whereas only one histidine (His168) of CS 2-OST is likely to be critical. This unique feature of HS 2-OST catalytic residues directed us to characterize the Drosophila heparan sulfate 2-O-sulfotransferase. The results from this study provide insight into the differences and similarities various residues play in the biological roles of the HS-2OST and CS-2OST enzymes.     

Effects of sulfate deprivation on the production of chondroitin/dermatan sulfate by cultures of skin fibroblasts from normal and diabetic individuals

Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with [3H]glucosamine in the presence of diminished concentrations of sulfate. Although total synthesis of [3H]chondroitin/dermatan glycosaminoglycans varied somewhat between cell lines, glycosaminoglycan production was not affected within any line when sulfate levels were decreased from 0.3 mM to 0.06 mM to 0.01 mM to 0 added sulfate. Lowering of sulfate concentrations resulted in diminished sulfation of chondroitin/dermatan in a progressive manner, so that overall sulfation dropped to as low as 19% for one of the lines. Sulfation of chondroitin to form chondroitin 4-sulfate and chondroitin 6-sulfate was progressively and equally affected by decreasing the sulfate concentration in the culture medium. However, sulfation to form dermatan sulfate was preserved to a greater degree, so that the relative proportion of dermatan sulfate to chondroitin sulfate increased. Essentially all the nonsulfated residues were susceptible to chondroitin AC lyase, indicating that little epimerization of glucuronic acid residues to iduronic acid had occurred in the absence of sulfation. These results confirm the previously described dependency of glucuronic/iduronic epimerization on sulfation, and indicate that sulfation of the iduronic acid-containing disaccharide residues of dermatan can take place with sulfate concentrations lower than those needed for 6-sulfation and 4-sulfation of the glucuronic acid-containing disaccharide residues of chondroitin. There were considerable differences among the six fibroblast lines in susceptibility to low sulfate medium and in the proportion of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate. However, there was no pattern of differences between normals and diabetics.     

1H-n.m.r. investigation of naturally occurring and chemically oversulphated dermatan sulphates. Identification of minor monosaccharide residues.

The 1H-n.m.r. spectra of various dermatan sulphate preparations present, besides the major signals of the basic disaccharide unit, several other minor signals. We have assigned most of them by n.m.r., using two-dimensional proton-proton double-quantum-correlation and nuclear-Overhauser-effect spectroscopy experiments. This allowed us to identify 2-O-sulphated L-iduronic acid and D-glucuronic acid residues as well as 6-sulphated N-acetylgalactosamine (presumably 4-O-sulphated as well). 2-O-Sulphated iduronic acid was present to similar extents (6-10% of total uronic acids) in pig skin dermatan sulphate and pig intestine dermatan sulphate, whereas glucuronic acid represented 17% of the uronic acid of pig skin dermatan sulphate and was virtually absent (1%) from the other preparation. 6-O-Sulphated N-acetylgalactosamine was present in minor amounts in pig intestine dermatan sulphate only. The influence of sulphation of iduronic acid units on their conformation was assessed by using chemically oversulphated pig intestine dermatan sulphate. Introduction of sulphate groups in this unit in dermatan sulphate tends to shift the conformational equilibrium towards the 1C4 conformer.