Purification, Photoaffinity Labeling, and Characterization of a Single Enzyme for 6-Sulfation of both Chondroitin Sulfate and Keratan Sulfate

Abstract

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 K m of 40 M 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[5-32 P]phosphate occurred only with the 75-kDa protein, confirming that this is the chondroitin 6-sulfotransferase/keratan sulfotransferase.Proteoglycans contain sulfated glycosaminoglycans that are covalently attached to a wide range of core protein families. In addition to the considerable variation and heterogeneity in the size and number of glycosaminoglycan chains that are linked to a core protein, there is heterogeneity in the position and degree of sulfation. The multiple functions of these molecules appear to be contributed, at least in part, by defined sulfate substitution of the glycosaminoglycan chains, so that the process of sulfation may have a direct effect on function.

Biosynthesis of Chondroitin/Dermatan Sulfate

“…The first chondroitin sulfotransferase to be purified, cloned, and expressed was a chicken 6-sulfotransferase (59) that catalyzes the sulfation of the Gal residues of keratan in addition to the GalNAc residues of chondroitin (60,61). Subsequently several chondroitin 6-sulfotransferases with varying degrees of homology to the avian chondroitin 6-sulfotransferase have also been cloned and expressed (62), but it remains to be established if these individual isoforms have different substrate specificities.…”

Purification of Heparan Sulfate D-Glucosaminyl 3-O-Sulfotransferase

“…The large batches of spent medium were then utilized to carry out an extensive purification procedure that included heparin-AF Toyopearl affinity chromatography, Mono Q-FPLC, TSK SW3000-HPLC, and 3Ј,5Ј-ADP-agarose affinity chromatography. The combination of heparin-Sepharose chromatography and 3Ј,5Ј-ADP-agarose affinity chromatography has previously permitted the isolation of heparan sulfate N-and 6-O-sulfotransferases, heparin N-and a mixture of O-sulfotransferases, and (33,34,41,(43)(44)(45). However, the isolation of HS act conversion activity required two additional chromatographic steps because of the need to achieve 10 -100-fold greater extent of purification as compared with other GAG biosynthetic enzymes.…”

Biosynthesis of Chondroitin Sulfate

“…While the only chondroitin sulfate-synthesizing enzymes as yet purified are the xylosyl transferase (10) and chondroitin 6-sulfotransferase (11,12), seven enzymes participating in synthesis of heparin have been purified (13)(14)(15)(16)(17)(18)(19). Moreover, some of these have been shown to be multifunctional, so that a single protein has been shown to contain two separate enzyme activities (15,18,20,21), thus facilitating multiple catalytic events along a single polymer.…”