Chondroitin sulfate synthase-2 is necessary for chain extension of chondroitin sulfate but not critical for skeletal development

Chondroitin sulfate (CS) is a linear polysaccharide consisting of repeating disaccharide units of N-acetyl-D-galactosamine and D-glucuronic acid residues, modified with sulfated residues at various positions. Based on its structural diversity in chain length and sulfation patterns, CS provides specific biological functions in cell adhesion, morphogenesis, neural network formation, and cell division. To date, six glycosyltransferases are known to be involved in the biosynthesis of chondroitin saccharide chains, and a hetero-oligomer complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is known to have the strongest polymerizing activity. Here, we generated and analyzed CSS2(-/-) mice. Although they were viable and fertile, exhibiting no overt morphological abnormalities or osteoarthritis, their cartilage contained CS chains with a shorter length and at a similar number to wild type. Further analysis using CSS2(-/-) chondrocyte culture systems, together with siRNA of CSS1, revealed the presence of two CS chain species in length, suggesting two steps of CS chain polymerization; i.e., elongation from the linkage region up to Mr ～10,000, and further extension. There, CSS2 mainly participated in the extension, whereas CSS1 participated in both the extension and the initiation. Our study demonstrates the distinct function of CSS1 and CSS2, providing a clue in the elucidation of the mechanism of CS biosynthesis.     

Structural analysis of the linkage region oligosaccharides and unsaturated disaccharides from chondroitin sulfate using CarboPac PA1.

Swarm rat chondrosarcoma cell cultures were metabolically labeled with [35S]sulfate, [3H]glucose, or [3H]glucosamine. Chondroitin sulfate chains were isolated from purified aggrecan using alkaline borohydride treatment and Superose 6 chromatography. Various linkage region oligosaccharide alditols were derived from these chains using sequential chondroitinase digestions (ABC lyase followed by ACII lyase). They were then further processed by mercuric acetate treatment, which removed the 4,5-unsaturated uronosyl residue from the nonreducing end of the linkage, and then beta-galactosidase digestion which liberated the 2 galactose residues from the xylitol reducing terminus. Alkaline phosphatase digestions were performed to verify the presence of phosphate esters. All linkage region structures were isolated and identified using a combination of Progel-TSK G2500 and CarboPac PA1 chromatography steps in conjunction with monosaccharide analyses. This study revealed that chondroitin sulfate chains from aggrecan synthesized by rat chondrosarcoma cells in vitro have the following properties: 1) three out of every four of their linkage regions carry a phosphate ester on xylose, 2) nearly three out of every five chains begin the repeating disaccharide region with an unsulfated first disaccharide unit, 3) nearly twice as many nonphosphorylated chains have a sulfated first disaccharide than their phosphorylated counterparts, and 4) the vast majority of these chains do not contain sulfated galactose in their linkage regions. This report also describes a borohydride reduction procedure to confer alkali stability to the 3-substituted, unsaturated disaccharides derived from chondroitinase digests of chondroitin sulfate. Furthermore, a CarboPac PA1 method is demonstrated that separates these reduced disaccharides with exceptional resolution.     

Structural characterization of heparan sulfate and chondroitin sulfate of syndecan-1 purified from normal murine mammary gland epithelial cells. Common phosphorylation of xylose and differential sulfation of galactose in the protein linkage region tetrasaccharide sequence

Syndecan-1, present on the surfaces of normal murine mammary gland epithelial cells, is a transmembrane hybrid proteoglycan, which bears glycosaminoglycan (GAG) side chains of heparan sulfate (HS) and chondroitin sulfate (CS). Purified syndecan-1 ectodomains were analyzed for disaccharide composition and the GAG-protein linkage region after digestion with bacterial lyases. The HS chains contained predominantly a nonsulfated unit with smaller proportions of two monosulfated, two disulfated, and a trisulfated unit, whereas CS chains were demonstrated for the first time to bear GlcUA-GalNAc(4-O-sulfate) as a major component as well as GlcUA-GalNAc, GlcUA-GalNAc(6-O-sulfate), and an E disaccharide unit GlcUA-GalNAc(4,6-O-disulfate) as minor yet appreciable components. Two kinds of linkage region tetrasaccharides, GlcUA-Gal-Gal-Xyl and GlcUA-Gal-Gal-Xyl(2-O-phosphate), were found for the HS chains in a molar ratio of 55:45. In marked contrast, an additional sulfated tetrasaccharide, GlcUA-Gal(4-O-sulfate)-Gal-Xyl, was demonstrated only for the CS chains, and the unmodified phosphorylated and sulfated components were present at a molar ratio of 55:26:19. The present study thus provided conclusive evidence for the hypothesis that 4-O-sulfation of Gal is peculiar to CS chains in contrast to the phosphorylation of Xyl, which is common to both HS and CS chains. These modifications may be required for biosynthetic maturation of the linkage region tetrasaccharide sequence, which is a prerequisite for creating the repeating disaccharide region of GAG chains and/or biosynthetic selective chain assembly of CS and HS chains.     

Chondroitin sulfate N-acetylgalactosaminyltransferase 1 is necessary for normal endochondral ossification and aggrecan metabolism

Chondroitin sulfate (CS) is a glycosaminoglycan, consisting of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid residues, and plays important roles in development and homeostasis of organs and tissues. Here, we generated and analyzed mice lacking chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGalNAcT-1). Csgalnact1(-/-) mice were viable and fertile but exhibited slight dwarfism. Biochemically, the level of CS in Csgalnact1(-/-) cartilage was reduced to ～50% that of wild-type cartilage, whereas its chain length was similar to wild-type mice, indicating that CSGalNAcT-1 participates in the CS chain initiation as suggested in the previous study (Sakai, K., Kimata, K., Sato, T., Gotoh, M., Narimatsu, H., Shinomiya, K., and Watanabe, H. (2007) J. Biol. Chem. 282, 4152-4161). Histologically, the growth plate of Csgalnact1(-/-) mice contained shorter and slightly disorganized chondrocyte columns with a reduced volume of the extracellular matrix principally in the proliferative layer. Immunohistochemical analysis revealed that the level of both aggrecan and link protein 1 were decreased in Csgalnact1(-/-) cartilage. Western blot analysis demonstrated an increase in processed forms of aggrecan core protein. These results suggest that CSGalNAcT-1 is required for normal levels of CS biosynthesis in cartilage. Our observations suggest that CSGalNAcT-1 is necessary for normal levels of endochondral ossification, and the decrease in CS amount in the growth plate by its absence causes a rapid catabolism of aggrecan.     

Sulfation of glucuronic acid in the linkage tetrasaccharide by HNK-1 sulfotransferase is an inhibitory signal for the expression of a chondroitin sulfate chain on thrombomodulin

HNK-1 (human natural killer-1) carbohydrate epitope (HSO₃-3GlcAβ1-3Galβ1-4GlcNAc-) recognized by a HNK-1 monoclonal antibody is highly expressed in the nervous system and biosynthesized by a glucuronyltransferase (GlcAT-P or GlcAT-S), and sulfotransferase (HNK-1ST). A similar oligosaccharide (HSO₃-3GlcAβ1-3Galβ1-3Galβ1-4Xyl) also recognized by the HNK-1 antibody had been found in a glycosaminoglycan (GAG)-protein linkage region of α-thrombomodulin (TM) from human urine. However, which sulfotransferase is involved in sulfation of the terminal GlcA in the GAG-protein linkage region remains unclear. In this study, using CHO-K1 cells in which neither GlcAT-P nor GlcAT-S is endogenously expressed, we found that HNK-1ST has the ability to produce HNK-1 immunoreactivity on α-TM. We also demonstrated that HNK-1ST caused the suppression of chondroitin sulfate (CS) synthesis on TM and a reduction of its anti-coagulant activity. Moreover, using an in vitro enzyme assay system, the HNK-1-positive TM was found not to be utilized as a substrate for CS-polymerizing enzymes (chondroitin synthase (ChSy) and chondroitin polymerizing factor (ChPF)). These results suggest that HNK-1ST is involved in 3-O-sulfation of the terminal GlcA of the linkage tetrasaccharide which acts as an inhibitory signal for the initiation of CS biosynthesis on TM.     

Molecular dissection of placental malaria protein VAR2CSA interaction with a chemo-enzymatically synthesized chondroitin sulfate library

Placental malaria, a serious infection caused by the parasite Plasmodium falciparum, is characterized by the selective accumulation of infected erythrocytes (IEs) in the placentas of the pregnant women. Placental adherence is mediated by the malarial VAR2CSA protein, which interacts with chondroitin sulfate (CS) proteoglycans present in the placental tissue. CS is a linear acidic polysaccharide composed of repeating disaccharide units of d-glucuronic acid and N-acetyl-d-galactosamine that are modified by sulfate groups at different positions. Previous reports have shown that placental-adhering IEs were associated with an unusually low sulfated form of chondroitin sulfate A (CSA) and that a partially sulfated dodecasaccharide is the minimal motif for the interaction. However, the fine molecular structure of this CS chain remains unclear. In this study, we have characterized the CS chain that interacts with a recombinant minimal CS-binding region of VAR2CSA (rVAR2) using a CS library of various defined lengths and sulfate compositions. The CS library was chemo-enzymatically synthesized with bacterial chondroitin polymerase and recombinant CS sulfotransferases. We found that C-4 sulfation of the N-acetyl-d-galactosamine residue is critical for supporting rVAR2 binding, whereas no other sulfate modifications showed effects. Interaction of rVAR2 with CS is highly correlated with the degree of C-4 sulfation and CS chain length. We confirmed that the minimum structure binding to rVAR2 is a tri-sulfated CSA dodecasaccharide, and found that a highly sulfated CSA eicosasaccharide is a more potent inhibitor of rVAR2 binding than the dodecasaccharides. These results suggest that CSA derivatives may potentially serve as targets in therapeutic strategies against placental malaria.     

The low sulfated chondroitin sulfate proteoglycans of human placenta have sulfate group-clustered domains that can efficiently bind Plasmodium falciparum-infected erythrocytes

Plasmodium falciparum infection in pregnant women results in the chondroitin 4-sulfate-mediated adherence of the parasite-infected red blood cells (IRBCs) in the placenta, adversely affecting the health of the fetus and mother. We have previously shown that unusually low sulfated chondroitin sulfate proteoglycans (CSPGs) in the intervillous spaces of the placenta are the receptors for IRBC adhesion, which involves a chondroitin 4-sulfate motif consisting of six disaccharide moieties with approximately 30% 4-sulfated residues. However, it was puzzling how the placental CSPGs, which have only approximately 8% of the disaccharide 4-sulfated, could efficiently bind IRBCs. Thus, we undertook to determine the precise structural features of the CS chains of placental CSPGs that interact with IRBCs. We show that the placental CSPGs are a mixture of two major populations, which are similar by all criteria except differing in their sulfate contents; 2-3% and 9-14% of the disaccharide units of the CS chains are 4-sulfated, and the remainder are nonsulfated. The majority of the sulfate groups in the CSPGs are clustered in CS chain domains consisting of 6-14 repeating disaccharide units. While the sulfate-rich regions of the CS chains contain 20-28% 4-sulfated disaccharides, the other regions have little or no sulfate. Further, we find that the placental CSPGs are able to efficiently bind IRBCs due to the presence of 4-sulfated disaccharide clusters. The oligosaccharides corresponding to the sulfate-rich domains of the CS chains efficiently inhibited IRBC adhesion. Thus, our data demonstrate, for the first time, the unique distribution of sulfate groups in the CS chains of placental CSPGs and that these sulfate-clustered domains have the necessary structural elements for the efficient adhesion of IRBCs, although the CS chains have an overall low degree of sulfation.     

Sulfation of sialyl N-acetyllactosamine oligosaccharides and fetuin oligosaccharides by keratan sulfate Gal-6-sulfotransferase

We have previously cloned keratan sulfate Gal-6-sulfotransferase (KSGal6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of Gal residue of keratan sulfate. In this study, we examined whether KSGal6ST could transfer sulfate to sialyl N -acetyllactosamine oligosaccharides or fetuin oligo-saccharides. KSGal6ST expressed in COS-7 cells catalyzed transfer of sulfate to NeuAcalpha2-3Galbeta1-4GlcNAc (3'SLN), NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Gl cNAc (SL1L1), NeuAcalpha2-3Galbeta1-4(6-sulfo)GlcNAcbeta1-3(6-sulfo) Galbeta1-4(6-su lfo)GlcNAc (SL2L4), and their desialylated derivatives except for Galbeta1-4GlcNAc, but not to NeuAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc (SLex). When the sulfated product formed from 3'SLN was degraded with neuraminidase and reduced with NaBH(4), the resulting sulfated disaccharide alditol showed the same retention time in SAX-HPLC as that of [(3)H]Gal(6SO(4))beta1-4GlcNAc-ol. KSGal6ST also catalyzed sulfation of fetuin. When the sulfated oligosaccharides released from the sulfated fetuin after sequential digestion with proteinase and neuraminidase were subjected to a reaction sequence of hydrazin-olysis, deaminative cleavage and NaBH(4)reduction, the major product was co-eluted with [(3)H]Gal(6SO(4))beta1-4anhydromannitol in SAX-HPLC. These observations show that KSGal6ST is able to sulfate position 6 of Gal residue of 3'SLN and fetuin oligosaccharides. The relative rates of the sulfation of SL2L4 was much higher than the rate of the sulfation of keratan sulfate. These results suggest that KSGal6ST may function in the sulfation of sialyl N -acetyllactosamine oligosaccharide chains attached to glycoproteins.     

Appican, the proteoglycan form of the amyloid precursor protein, contains chondroitin sulfate E in the repeating disaccharide region and 4-O-sulfated galactose in the linkage region

Chondroitin sulfate (CS)-D and CS-E, which are characterized by oversulfated disaccharide units, have been shown to regulate neuronal adhesion, cell migration, and neurite outgrowth. CS proteoglycans (CSPGs) consist of a core protein to which one or more CS chains are attached via a serine residue. Although several brain CSPGs, including mouse DSD-1-PG/phosphacan, have been found to contain the oversulfated D disaccharide motif, no brain CSPG has been reported to contain the oversulfated E motif. Here we analyzed the CS chain of appican, the CSPG form of the Alzheimer's amyloid precursor protein. Appican is expressed almost exclusively by astrocytes and has been reported to have brain- and astrocyte-specific functions including stimulation of both neural cell adhesion and neurite outgrowth. The present findings show that the CS chain of appican has a molecular mass of 25-50 kDa. This chain contains a significant fraction (14.3%) of the oversulfated E motif GlcUA beta 1-3GalNAc(4,6-O-disulfate). The rest of the chain consists of GlcUA beta 1-3GalNAc(4-O-sulfate) (81.2%) and minor fractions of GlcUA beta 1-3GalNAc and GlcUA beta 1-3GalNAc(6-O-sulfate). We also show that the CS chain of appican contains in its linkage region the 4-O-sulfated Gal structure. Thus, appican is the first example of a specific brain CSPG that contains the E disaccharide unit in its sugar backbone and the 4-O-sulfated Gal residue in its linkage region. The presence of the E unit is consistent with and may explain the neurotrophic activities of appican.     

A fingerprinting method for chondroitin/dermatan sulfate and hyaluronan oligosaccharides

A previously published method for the analysis of glycosaminoglycan disaccharides by high pH anion exchange chromatography (Midura,R.J., Salustri,A., Calabro,A., Yanagishita,M. and Hascall,V.C. (1994), Glycobiology,4, 333-342) has been modified and calibrated for chondroitin and dermatan sulfate oligosaccharides up to hexasaccharide in size and hyaluronan oligosaccharides up to hexadecasaccharide. For hyaluronan oligosaccharides chain length controls elution position; however, for chondroitin and dermatan sulfate oligosaccharides elution times primarily depend upon the level of sulfation, although chain length and hence charge density plays a role. The sulfation position of GalNAc residues within an oligosaccharide is also important in determining its elution position. Compared to 4-sulfation a reducing terminal 6-sulfate retards elution; however, when present on an internal GalNAc residue it is the 4-sulfate containing oligosaccharide which elutes later. These effects allow discrimination between oligosaccharides differing only in the position of GalNAc sulfation. Using this simple methodology, a Dionex CarboPac PA-1 column with NaOH/NaCl eluents and detection by absorbance at 232 nm, a quantitative analytical fingerprint of a chondroitin/dermatan sulfate chain may be obtained, allowing a determination of the abundance of chondroitin sulfate, dermatan sulfate, and hyaluronan along with an analysis of structural features with a linear response to approximately 0.1 nmol. The method may readily be calibrated using either commercial disaccharides or the di- and tetrasaccharide products of a limit digest of commercial chondroitin sulfate by chondroitin ABC endolyase. Commercially available and freshly prepared shark, whale, bovine, and human cartilage chondroitin sulfates have been examined by this methodology and we have confirmed that freshly isolated shark cartilage CS contains significant amounts of the biologically important GlcA2Sbeta(1-3)GalNAc6S structure.     

Adaptation of FACE methodology for microanalysis of total hyaluronan and chondroitin sulfate composition from cartilage

Protocols for analyzing the fine structure of hyaluronan and chondroitin sulfate using fluorophore-assisted carbohydrate electrophoresis of 2-aminoacridone-derivatized hyaluronidase/chondroitinase digestion products were adapted for direct analysis of previously characterized cartilage-derived samples. The chondroitin sulfate disaccharide compositions for fetal and 68 year human aggrecan from FACE analyses were DeltaDi4S (50%), DeltaDi6S (43%), and DeltaDi0S (7%); and DeltaDi4S (3%), DeltaDi6S (96%), and DeltaDi0S (1%), respectively. The nonreducing terminal structures included predominantly 4S-galNAc with minor amounts of 6S-galNAc and Di6S for the fetal aggrecan sample and, in addition, included 4,6S-galNAc in the 68 year aggrecan sample. FACE analysis of a proteinase K digest of rat chondrosarcoma tissue gave an internal disaccharide composition for its chondroitin sulfate chains of DeltaDi0S (7%) and DeltaDi4S (93%) with no DeltaDi6S and DeltaDi4, 6S detected, while DeltaDiHA from hyaluronan was 5% of the total. Analysis of nonreducing terminal structures indicated the presence of 4S-galNAc (51%), galNAc (27%), and Di4S (22%) with no 4,6S-galNAc or Di6S detected. Unexpectedly, FACE analysis detected putative linkage oligosaccharide structures from the chondroitin sulfate chains including both unsulfated (85%) and 4-sulfated (15%) linkage oligosaccharides. Finally, the number averaged chain length estimated from the ratio of the molar fluorescence of the Deltadisaccharides to that of the nonreducing termini or the linkage oligosaccharide structures was calculated as approximately 16 kDa. A tissue glucose concentration of 0.72 g/l was also measured. These results for both samples as determined by FACE analysis were similar to results previously reported, using more labor and time intensive procedures, validating the FACE protocols.     

Ion exchange HPLC microanalysis of chondroitin sulfate: quantitative derivatization of chondroitin lyase digestion products with 2-aminopyridine

Sulfated glycosaminoglycans such as chondroitin sulfate arecomposed of three structural domains, a linkage oligosaccharide,connecting the chain to the core protein, a variably sulfateddisaccharide repeat structure within the chain and a nonreducingterminal, and these domains may confer specific functions onparticular chain populations. We report here a new and highlysensitive method for the detection and quantitation of all nonreducingterminal residues and internal disaccharides obtained by chondroltinaseABC or ACII digestion of aggrecan chondroitin sulfate. The procedureinvolves a quantitative reductive amination of the reducingends of sulfated mono- and disaccharide chondroitinase productswith 2-aminopyridine and boranedimethylamine. All derivatizedsaccharides can be separated and quantitated by fluorescencein a single chromatographic step on an AS4A anion exchange column,eluted with a gradient (0–500 mM) of sodium trifluoroacetate.The reproducibility and stability of the derivatisation, togetherwith the sensitivity of the chromatography system, allowed forroutine quantitation in the range of 3–500 pmol of reducinggroup (corresponding to about 1.5–250 ng of disaccharideor 0.75–125 ng of monosaccharide). Moreover, the fluorescenceyield (fluorescence area units per pmol of reducing group) wasvirtually identical for all saccharides analyzed. Applicationof this method to an analysis of aggrecan purified from calfepiphyseal cartilage and from rat chondrosarcoma chondrocytecultures allowed a precise identification and quantitation ofthe internal disaccharides and the nonreducing terminal structures,together with an estimation of the number average molecularweight of CS chains in these aggrecan preparations. aggrecan chondroitin sulfate glycosaminoglycans proteoglycans     

Involvement of human natural killer-1 (HNK-1) sulfotransferase in the biosynthesis of the GlcUA(3-O-sulfate)-Gal-Gal-Xyl tetrasaccharide found in α-thrombomodulin from human urine

Thrombomodulin (TM) is an integral membrane glycoprotein, which occurs as both a chondroitin sulfate (CS) proteoglycan (PG) form (β-TM) and a non-PG form without a CS chain (α-TM) and hence is a part-time PG. An α-TM preparation isolated from human urine contained the glycosaminoglycan linkage region tetrasaccharide GlcUAβ1-3Galβ1-3Galβ1-4xylose, and the nonreducing terminal GlcUA residue is 3-O-sulfated. Because the human natural killer-1 sulfotransferase (HNK-1ST) transfers a sulfate group from 3'-phosphoadenosine 5'-phosphosulfate to the C-3 position of the nonreducing terminal GlcUA residue in the HNK-1 antigen precursor trisaccharide, GlcUAβ1-3Galβ1-4GlcNAc, the sulfotransferase activity toward the linkage region was investigated. In fact, the activity of HNK-1ST toward the linkage region was much higher than that toward the glucuronylneolactotetraosylceramide, the precursor of the HNK-1 epitope. HNK-1ST may be responsible for regulating the sorting of α- and β-TM. Furthermore, HNK-1ST also transferred a sulfate group from 3'-phosphoadenosine 5'-phosphosulfate to the C-3 position of the nonreducing terminal GlcUA residue of a chondroitin chain. Intriguingly, the HNK-1 antibody recognized CS chains and the linkage region if they contained GlcUA(3-O-sulfate), suggesting that HNK-1ST not only synthesizes the HNK-1 epitope but may also be involved in the generation of part-time PGs.     

Altered fine structures of corneal and skeletal keratan sulfate and chondroitin/dermatan sulfate in macular corneal dystrophy

The content and fine structure of keratan and chondroitin/dermatan sulfate in normal human corneas and corneas affected by macular corneal dystrophies (MCD) types I and II were examined by fluorophore-assisted carbohydrate electrophoresis. Normal tissues (n = 11) contained 15 microg of keratan sulfate and 8 microg of chondroitin/dermatan sulfate per mg dry weight. Keratan sulfates consisted of approximately 4% unsulfated, 42% monosulfated, and 54% disulfated disaccharides with number of average chain lengths of approximately 14 disaccharides. Chondroitin/dermatan sulfates were significantly longer, approximately 40 disaccharides per chain, and consisted of approximately 64% unsulfated, 28% 4-sulfated, and 8% 6-sulfated disaccharides. The fine structural parameters were altered in all diseased tissues. Keratan sulfate chain size was reduced to 3-4 disaccharides; chain sulfation was absent in MCD type I corneas and cartilages, and sulfation of both GlcNAc and Gal was significantly reduced in MCD type II. Chondroitin/dermatan sulfate chain sizes were also decreased in all diseased corneas to approximately 15 disaccharides, and the contents of 4- and 6-sulfated disaccharides were proportionally increased. Tissue concentrations (nanomole of chains per mg dry weight) of all glycosaminoglycan types were affected in the disease types. Keratan sulfate chain concentrations were reduced by approximately 24 and approximately 75% in type I corneas and cartilages, respectively, and by approximately 50% in type II corneas. Conversely, chondroitin/dermatan sulfate chain concentrations were increased by 60-70% in types I and II corneas. Such changes imply a modified tissue content of individual proteoglycans and/or an altered efficiency of chain substitution on the core proteins. Together with the finding that hyaluronan, not normally present in healthy adult corneas, was also detected in both disease subtypes, the data support the conclusion that a wide range of keratocyte-specific proteoglycan and glycosaminoglycan remodeling processes are activated during degeneration of the stromal matrix in the macular corneal dystrophies.     

Phosphorylation and sulfation of oligosaccharide substrates critically influence the activity of human beta1,4-galactosyltransferase 7 (GalT-I) and beta1,3-glucuronosyltransferase I (GlcAT-I) involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans

We determined whether the two major structural modifications, i.e. phosphorylation and sulfation of the glycosaminoglycan-protein linkage region (GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1), govern the specificity of the glycosyltransferases responsible for the biosynthesis of the tetrasaccharide primer. We analyzed the influence of C-2 phosphorylation of Xyl residue on human beta1,4-galactosyltransferase 7 (GalT-I), which catalyzes the transfer of Gal onto Xyl, and we evaluated the consequences of C-4/C-6 sulfation of Galbeta1-3Gal (Gal2-Gal1) on the activity and specificity of beta1,3-glucuronosyltransferase I (GlcAT-I) responsible for the completion of the glycosaminoglycan primer sequence. For this purpose, a series of phosphorylated xylosides and sulfated C-4 and C-6 analogs of Galbeta1-3Gal was synthesized and tested as potential substrates for the recombinant enzymes. Our results revealed that the phosphorylation of Xyl on the C-2 position prevents GalT-I activity, suggesting that this modification may occur once Gal is attached to the Xyl residue of the nascent oligosaccharide linkage. On the other hand, we showed that sulfation on C-6 position of Gal1 of the Galbeta1-3Gal analog markedly enhanced GlcAT-I catalytic efficiency and we demonstrated the importance of Trp243 and Lys317 residues of Gal1 binding site for enzyme activity. In contrast, we found that GlcAT-I was unable to use digalactosides as acceptor substrates when Gal1 was sulfated on C-4 position or when Gal2 was sulfated on both C-4 and C-6 positions. Altogether, we demonstrated that oligosaccharide modifications of the linkage region control the specificity of the glycosyltransferases, a process that may regulate maturation and processing of glycosaminoglycan chains.     

Identification of chondroitin sulfate glucuronyltransferase as chondroitin synthase-3 involved in chondroitin polymerization: chondroitin polymerization is achieved by multiple enzyme complexes consisting of chondroitin synthase family members

Recently, we demonstrated that chondroitin polymerization is achieved by any two combinations of human chondroitin synthase-1 (ChSy-1), ChSy-2 (chondroitin sulfate synthase 3, CSS3), and chondroitin-polymerizing factor (ChPF). Although an additional ChSy family member, called chondroitin sulfate glucuronyltransferase (CSGlcA-T), has been identified, its involvement in chondroitin polymerization remains unclear because it possesses only glucuronyltransferase II activity responsible for the elongation of chondroitin sulfate (CS) chains. Herein, we report that CSGlcA-T exhibits polymerization activity on alpha-thrombomodulin bearing the truncated linkage region tetrasaccharide through its interaction with ChSy-1, ChSy-2 (CSS3), or ChPF, and the chain length of chondroitin formed by the co-expressed proteins in various combinations is different. In addition, ChSy family members co-expressed in various combinations exhibited distinct but overlapping acceptor substrate specificities toward the two synthetic acceptor substrates, GlcUAbeta1-3Galbeta1-O-naphthalenemethanol and GlcUAbeta1-3Galbeta1-O-C(2)H(4)NH-benzyloxycarbonyl, both of which share the disaccharide sequence with the glycosaminoglycan-protein linkage region tetrasaccharide. Moreover, overexpression of CSGlcA-T increased the amount of CS in HeLa cells, whereas the RNA interference of CSGlcA-T resulted in a reduction of the amount of CS in the cells. Furthermore, the analysis using the CSGlcA-T mutant that lacks any glycosyltransferase activity but interacts with other ChSy family members showed that the glycosyltransferase activity of CSGlcA-T plays an important role in chondroitin polymerization. Overall, these results suggest that chondroitin polymerization is achieved by multiple combinations of ChSy-1, ChSy-2, CSGlcA-T, and ChPF and that each combination may play a unique role in the biosynthesis of CS. Based on these results, we renamed CSGlcA-T chondroitin synthase-3 (ChSy-3).     

Two N-acetylgalactosaminyltransferase are involved in the biosynthesis of chondroitin sulfate

Two N-acetylgalactosaminyltransferases, designated I and II, have been purified from the microsomal fraction of calf arterial tissue and separated on Bio-Gel A. N-Acetylgalactosaminyltransferase I was purified 450-fold. It requires Mn2+ for maximal activity and transfers N-acetylgalactosamine residues from UDP-[1-3H]GalNAc in beta-glycosidic configuration to the non-reducing terminus of the acceptor substrates GlcA(beta 1-3)Gal(beta 1-3)Gal, GlcA(beta 1-3)Gal(beta 1-4)Glc and GlcA(beta 1-3)Gal. Even-numbered chondroitin oligosaccharides serve as acceptors for N-acetylgalactosaminyltransferase II, which transfers N-acetylgalactosamine from UDP-[1-3H]GalNAc to the non-reducing glucuronic acid residues of oligosaccharide acceptor substrates. Maximum transfer rates were obtained with a decasaccharide derived from chondroitin. Longer or shorter-chain chondroitin oligosaccharides are less effective acceptor substrates. All reaction products formed by N-acetylgalactosaminyltransferases I and II are substrates of beta-N-acetylhexosaminidase, which splits off the transferred [1-3H]GalNAc completely. In the microsomal fraction N-acetylgalactosaminyltransferase II had a 300-fold higher specific activity than N-acetylgalactosaminyltransferase I. In contrast to enzyme I, enzyme II loses much of its activity during the purification procedure and undergoes rapid thermodenaturation. GlcA-Gal-Gal is a characteristic sequence of the carbohydrate-protein linkage region of proteochondrioitin sulfate. The acceptor capacity of this trisaccharide suggests that N-acetylgalactosaminyltransferase I is involved in the synthesis of the carbohydrate-protein linkage region. Since N-acetylgalactosaminyltransferase II is highly specific for chondroitin oligosaccharides, we conclude that it participates in chain elongation during chondroitin sulfate synthesis.