UDP-D-xylose:proteoglycan core protein beta-D-xylosyltransferase (EC2.4.2.26) is the initial enzyme in the biosynthesis of chondroitin sulfate and dermatan sulfate proteoglycans in fibroblasts and chondrocytes. Secretion of xylosyltransferase into the extracellular space was determined in cultured human dermal fibroblasts. A more than 6-fold accumulation of xylosyltransferase activity in cell culture supernatant was observed (day 1, 0.6 microU per 106 cells; day 9, 4.1 microU per 106 cells); however, intracellular xylosyltransferase activity remained at a constant level (0.4 microU per 106 cells). Exposure of human chondrocytes to colchicine led to a 3-fold decreased level of xylosyltransferase and chondroitin-6-sulfate concentration in cell culture. Specific xylosyltransferase activity and chondroitin-6-sulfate concentration decreased in a concentration-dependent manner and in parallel in culture medium and accumulated 5-fold in cell lysates indicating that xylosyltransferase is secreted simultaneously into the extracellular space with chondroitin sulfate proteoglycans. Xylosyltransferase activities were determined in serum samples of 30 patients with systemic sclerosis. Xylosyltransferase activities in female (mean value 1.28 mU per liter, 90% range 1.10-1.55 mU per liter) and male patients (mean 1.39 mU per liter, 90% range 1.16-1. 57 mU per liter) with systemic sclerosis were significantly increased in comparison with blood donors of a corresponding age. Furthermore, xylosyltransferase activity was correlated with the clinical classification of systemic sclerosis. Female patients with diffuse cutaneous systemic sclerosis showed higher serum xylosyltransferase activities than patients with limited systemic sclerosis. These results confirm that the increase of proteoglycan biosynthesis in sclerotic processes of scleroderma is closely related to an elevated xylosyltransferase activity in blood and demonstrate the validity of xylosyltransferase as an additional diagnostic marker for determination of sclerotic activity in systemic sclerosis.

The formation of chondroitin sulfate is initiated by xylosyltransferase (XT) transferring xylose from UDPxylose to consensus serine residues of proteoglycan core proteins. Our alignment of 51 amino acid sequences of chondroitin sulfate attachment sites in 19 different proteins resulted in a consensus sequence for the recognition signal of XT. The complete recognition sequence is composed of the amino acids a-a-a-a-G-S-G-a-b-a, with a ‫؍‬ E or D and b ‫؍‬ G, E, or D. This sequence was confirmed by determination of the Michaelis-Menten constants for in vitro xylosylation of different synthetic proteins and peptides using an enriched XT preparation from conditioned cell culture supernatant of human chondrocytes. The highest acceptor activity was determined by the sequence Q-E-E-E-E-G-S-G-G-G-Q, which was found in the single chondroitin sulfate attachment site of bikunin, the inhibitory active component of the human inter-␣-trypsin inhibitor.We determined the Michaelis-Menten constant (K m ) of xylosylation of the synthetic bikunin analogous peptide Q-E-E-E-G-S-G-G-G-Q-K to be 22 M, which was 9-fold decreased in comparison to deglycosylated core protein from bovine cartilage (188 M), which was previously used as acceptor for the XT activity assay. The best XT acceptors were nonglycosylated recombinant wildtype bikunin (K m ‫؍‬ 0. These results imply that the primary structure of the acceptor is not the only determinant for recognition by xylosyltransferase. Thus, protein conformation is also a main factor in determining xylosylation.

Xylosyltransferase (XT) is the chain-initiating enzyme of the biosynthesis of chondroitin sulfate. So far, XT activity has been detected by incorporation of [14C]xylose in chemically deglycosylated cartilage proteoglycan or silk fibroin. However, these acceptors allow no reliable determination in blood. We found that recombinant bikunin is an excellent acceptor for XT. The Michaelis–Menten constants for the xylosylation of silk, deglycosylated cartilage proteoglycans, and bikunin were 545, 155, and 0.9 μmol/L, respectively. With recombinant bikunin as acceptor, we developed a sensitive assay that allows a precise determination of XT activity in serum. We measured the serum XT activities of 500 blood donors and observed a considerable sex and age dependence. XT activities in men (0.77–1.50 mU/L) were ∼30% higher than in women (0.58–1.20 mU/L) and reached a maximum in donors of ∼40 years of age. During the menstrual cycle, serum XT activity showed a significant coincidence with the β-estradiol concentration, and in the first trimester of pregnancy we observed a strong increase in serum XT activity.

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