Search

A partial-length human cDNA with a predicted amino acid sequence homologous to a previously described heparan sulfate iduronyl 2-sulfotransferase (Kobayashi, M., Habuchi, H., Yoneda, M., Habuchi, O., and Kimata, K. (1997) J. Biol. Chem. 272, 13980-13985) was obtained by searching the expressed sequence-tagged data bank. Northern blot analysis was performed using this homologous cDNA as a probe, which demonstrated ubiquitous expression of messages of 5.1 and 2.0 kilobases in a number of human tissues and in several human cancer cell lines. Since the human lymphoma Raji cell line had the highest level of expression, it was used to isolate a full-length cDNA clone. The full-length cDNA was found to contain an open reading frame that predicted a type II transmembrane protein composed of 406 amino acid residues. The cDNA in a baculovirus expression vector was expressed in Sf9 insect cells, and cell extracts were then incubated together with 3'-phosphoadenosine 5'-phospho[35S]sulfate and potential glycosaminoglycan acceptors. This demonstrated substantial sulfotransferase activity with dermatan sulfate, a small degree of activity with chondroitin sulfate, but no sulfotransferase activity with desulfated N-resulfated heparin. Analysis of [35S]sulfate-labeled disaccharide products of chondroitin ABC, chondroitin AC, and chondroitin B lyase treatment demonstrated that the enzyme only transferred sulfate to the 2-position of uronyl residues, which were preponderantly iduronyl residues in dermatan sulfate, but some lesser transfer to glucuronyl residues of chondroitin sulfate.

Microsomal preparations from chondroitin 6-sulfate-producing chick embryo epiphyseal cartilage, and from chondroitin 4-sulfate-producing mouse mastocytoma cells, were incubated with UDP-[14C]glucuronic acid and UDP-N-acetylgalactosamine to form non-sulfated proteo[14C]chondroitin. Aliquots of the incubations were then incubated with 3'-phosphoadenylylphosphosulfate (PAPS) in the presence or absence of various detergents. In the absence of detergents, there was good sulfation of this endogenous proteo[14C]chondroitin by the original microsomes from both sources. Detergents, with the exception of Triton X-100, markedly inhibited sulfation in the mast cell system but not in the chick cartilage system. These results indicate that sulfation and polymerization are closely linked on cell membranes and that in some cases this organization can be disrupted by detergents. When aliquots of the original incubation were heat inactivated, and then reincubated with new microsomes from chick cartilage and/or mouse mastocytoma cells plus PAPS, there was no significant sulfation of this exogenous proteo[14C] chondroitin with either system unless Triton X-100 was added. Sulfation of exogenous chondroitin and chondroitin hexasaccharide was compared with sulfation of endogenous and exogenous proteo[14C]chondroitin. Sulfate incorporation into hexasaccharide and chondroitin decreased as their concentrations (based on uronic acid) approached that of the proteo[14C]chondroitin. At the same time, the degree of sulfation in percent of substituted hexosamine increased. However, the degree of sulfation did not reach that of the endogenous proteo[14C]chondroitin. Hexasaccharide and chondroitin sulfation were stimulated by the presence of Triton X-100. However, in contrast to the exogenous proteo[14C]chondroitin, there was some sulfation of hexasaccharide and chondroitin in the absence of this detergent. These results indicate that the intact microsomal system was not accessible to the larger substrates, and that even with detergents exogenous substrates were not sulfated as effectively as newly formed proteo[14C]chondroitin in an intact microsomal system. When the proteo[14C]chondroitin formed by the chick cartilage microsomal system was incubated together with the mast cell microsomal system and PAPS, sulfation only occurred at the 4-position. When the proteo[14C]chondroitin formed by the mouse mast cell microsomal system was incubated together with the chick cartilage microsomal system and PAPS, sulfation only occurred at the 6-position.(ABSTRACT TRUNCATED AT 400 WORDS)

A soluble sulfotransferase that could 6-sulfate both chondroitin sulfate and corneal keratan sulfate was purified 27,500-fold using a sequence of affinity chromatographic steps with heparin-Sepharose, wheat germ agglutinin-agarose, and 3',5'-ADP-agarose. The essentially pure enzyme had a specific activity 40 times greater than the most purified chondroitin 6-sulfotransferase previously reported and exhibited a single sharp Coomassie Blue-stained and a heavy silver-stained protein band of 75 kDa on SDS-polyacrylamide gel electrophoresis. Chromatography of the purified enzyme on Sephacryl demonstrated a size of 150 kDa, which indicated that the native enzyme exists as a dimer. In addition to 6-sulfation of nonsulfated GalNAc, the purified serum enzyme had the ability to sulfate GalNAc 4-sulfate residues to give GalNAc 4,6-disulfate residues. The purified enzyme exhibited a Km of 40 microM for adenosine 3'-phosphate 5'-phosphosulfate when either chondroitin sulfate or corneal keratan sulfate were used as the acceptors. Use of both chondroitin sulfate and keratan sulfate in the same experiment demonstrated mutual competition, establishing that the sulfation of these substrates is by the same enzyme. Photoaffinity labeling of the purified enzyme with 2-azidoadenosine 3',5'-di[5'-32P]phosphate occurred only with the 75-kDa protein, confirming that this is the chondroitin 6-sulfotransferase/keratan sulfotransferase.

Microsomal preparations from chondroitin 6-sulfate-producing chick embryo epiphyseal cartilage and from chondroitin 4-sulfate-producing mouse mastocytoma cells were incubated with varying concentrations of 3'-phosphoadenylylphospho[35S]sulfate and chondroitin hexasaccharide in the presence or absence of Triton X-100. [35S]Sulfate incorporation into hexasaccharide and into endogenous microsomal chondroitin 6-sulfate or endogenous microsomal chondroitin 4-sulfate was measured. With both microsomal systems, Triton X-100 increased the incorporation of [35S]sulfate into hexasaccharide but had much less effect on the incorporation into endogenous chondroitin sulfate. Higher concentrations of hexasaccharide inhibited the incorporation of [35S]sulfate into endogenous chondroitin sulfate. The apparent Km for 3'-phosphoadenylylphosphosulfate for both the 6-sulfotransferase and 4-sulfotransferase with hexasaccharide and with endogenous chondroitin sulfate in the presence or absence of Triton X-100 were all similar. However, the apparent Km for hexasaccharide was lower in the presence of Triton X-100 for both the microsomal sulfotransferases. This is consistent with solubilization of sulfotransferases, and indicates that hexasaccharide access to these enzymes had been limited in the particulate system. Examination of 35S-oligosaccharide products formed with each system demonstrated good 6-sulfation or 4-sulfation of penultimate GalNAc at the non-reducing end of the chondroitin hexasaccharide. However, no 6-sulfation of terminal GalNAc at the non-reducing end of a chondroitin pentasaccharide derived from hexasaccharide was observed, and there was only minimal 4-sulfation of this terminal GalNAc. Concurrent addition of GalNAc to hexasaccharide resulting in heptasaccharide did not appear to promote significant 6-sulfation or 4-sulfation of newly added non-reducing terminal GalNAc.

The potential relationship of an intact membrane organization for the synthesis of chondroitin and chondroitin 4-sulfate was examined after modification of a mouse mast cell microsomal system with the nonionic detergent, Triton X-100. The results indicated that Triton X-100 had no effect on the rate of polymerization but had a slight effect on the size of glycosaminoglycan chains. An “all or nothing” pattern of sulfation of newly formed chondroitin was obtained in both the presence and the absence of Triton X-100, and this pattern did not change whether sulfation was initiated concurrent with or subsequent to polymerization. Sulfation of exogenous [14C]chondroitin and exogenous proteo[3H]chondroitin by the microsomal system required Triton X-100 but still produced an all or nothing pattern rather than a random sulfation pattern. When a 100,000 x g supernatant fraction was utilized for sulfation of [14C]chondroitin or proteo[3H]chondroitin, Triton X-100 was not needed, and a partial sulfation pattern was obtained. However, it was similar to the all or nothing pattern in that it still produced two populations, with some chains nonsulfated and others approximately 50% sulfated. When chondroitin hexasaccharide was used with 3′-phosphoadenylylphospho[35S]sulfate, multiple GalNAc residues of the individual hexasaccharides were found to be sulfated. This was relatively independent of Triton X-100 or the concentration of the hexasaccharide acceptors. With soluble enzyme, sulfation of multiple GalNAc residues on the individual hexasaccharide molecules was even greater, so that trisulfated products were found. These results suggest that efficient sulfation of chondroitin is related to enzyme-substrate interaction more than to membrane organization.

A photoaffinity analogue, [beta-32P]5-azido-UDP-GlcA, was used to photolabel the enzymes that utilize UDP-GlcA in cartilage microsomes and rat liver microsomes. SDS-polyacrylamide gel electrophoresis analysis of photolabeled cartilage microsomes, which are specialized in chondroitin sulfate synthesis, showed a major radiolabeled band at 80 kDa and other minor radiolabeled bands near 40 and 60 kDa. Rat liver microsomes, which are enriched for enzymes of detoxification by glucuronidation, had a different pattern with multiple major labeled bands near 50-60 and 35 kDa. To determine that the photolabeled 80-kDa protein is the GlcA transferase II, we have purified the enzyme from cartilage microsomes. This membrane-bound enzyme, involved in the transfer of GlcA residues to non-reducing terminal GalNAc residues of the chondroitin polymer, has now been solubilized, stabilized, and then purified greater than 1350-fold by sequential chromatography on Q-Sepharose, heparin-Sepharose, and WGA-agarose. The purified enzyme exhibited a conspicuous silver-stained protein band on SDS-polyacrylamide gel electrophoresis that coincided with the major radiolabeled band of 80 kDa. SDS-polyacrylamide gel analysis of photoaffinity-labeled active fractions from the Q-Sepharose, heparin-Sepharose, and WGA-agarose also indicated only the single radiolabeled band at 80 kDa. Intensity of photolabeling in each of the fractions examined coincided with enzyme activity. The photolabeling of this 80-kDa protein was saturable with the photoprobe and could be inhibited by the addition of UDP-GlcA prior to the addition of the photoprobe. Thus, the photolabeling with [beta-32P]5-azido-UDP-GlcA has identified the GlcA transferase II as an 80-kDa protein. The purified enzyme was capable of transferring good amounts of GlcA residues to chondroitin-derived pentasaccharide with negligible transfer to pentasaccharides derived from hyaluronan or heparan.