Involvement of a cysteine protease in the secretion process of human xylosyltransferase I.

Xylosylation of core proteins takes place in the Golgi-apparatus as the transfer of xylose from UDP-xylose to specific serine residues in proteoglycan core proteins. This initial and rate-limiting step in glycosaminoglycan biosynthesis is catalyzed by human xylosyltransferase I (XT-I). XT-I is proteolytically cleaved from the Golgi surface and shed in its active form into the extracellular space. The secreted, circulating glycosyltransferase represents a serum biomarker for various diseases with an altered proteoglycan metabolism, whereas a physiological function of secreted XT-I is still unknown. To shed light on the secretion process of XT-I and on its biological function, the cleavage site was examined and the group of proteases involved in the cleavage was identified in this study. The peptide mass fingerprint from partly purified secreted XT-I revealed the cleavage site to be localized in the aminoterminal 231 amino acids. The addition of a cysteine protease inhibitor cocktail to cells recombinantly expressing XT-I led to a concentration-dependent shift of enzyme activity towards the cell lysates attended by consistent total intracellular and extracellular XT-I activities. In conclusion, our findings provide a first insight into the XT-I secretion process regulated by a cysteine protease and may contribute to understanding the biological and pathological role of this process.

Xylosyltransferase gene variants and their role in essential hypertension.

BACKGROUND: An accumulation of extracellular matrix molecules, such as proteoglycans, is observed in the vascular wall of hypertensive patients. Xylosyltransferases I and II (XT-I and XT-II), the chain-initiating enzymes in the biosynthesis of proteoglycans, catalyze the transfer of D-xylose from UDP-D-xylose to specific serine residues of the core protein. Because associations between XYLT polymorphisms and an altered blood pressure have been observed, genetic variations in the XYLT genes might predispose to essential hypertension. The localization of the XYLT2 gene on chromosome 17q increases its attractiveness as this region has been reported to be a potential candidate locus for essential hypertension. METHODS: Genotyping of four polymorphisms in the genes XYLT1 and XYLT2 was performed in 150 unrelated essential hypertension patients and 150 age- and sex-matched normotensive controls using restriction fragment length polymorphism analysis. RESULTS: The allele and genotype frequencies of the XYLT variants investigated did not show any significant differences between patients and controls, among allele-carriers and nonallele-carriers and among recessive and nonrecessive allele-carriers comparing patients and controls. Systolic and diastolic blood pressures did not differ significantly between the genotypes concerning all XYLT variants analyzed. Two XYLT2 variants deviated from Hardy-Weinberg equilibrium (HWE) in the hypertensive group. CONCLUSIONS: No statistically significant association was found between four XYLT variants and hypertension or blood pressure, suggesting that they do not play a significant role in the development of essential hypertension. The deviation from HWE of two XYLT2 variants might be due to gene-phenotype associations which remain to be explored, as well as the possibility of gene-gene interactions.

Human xylosyltransferase II is involved in the biosynthesis of the uniform tetrasaccharide linkage region in chondroitin sulfate and heparan sulfate proteoglycans.

Human xylosyltransferase I (XT-I) initiates the biosynthesis of the glycosaminoglycan (GAG) linkage tetrasaccharide in proteoglycans. Xylosyltransferase II (XT-II) is a protein homologous to XT-I but with hitherto unknown activity or physiological function. Here, we report the enzymatic activity of XT-II and provide evidence that XT-II initiates the biosynthesis of both heparan sulfate and chondroitin sulfate GAGs. Transfection of the xylosyltransferase-deficient Chinese hamster ovary mutant pgsA-745 with XT-I or XT-II coding cDNA completely restored GAG biosynthesis. GAG disaccharide analysis revealed that XT-I- and XT-II-transfected pgsA-745 cells produced similar amounts of chondroitin sulfate and heparan sulfate. Furthermore, a high xylosyltransferase activity was measured after transfection with cDNAs encoding either isozyme. Analysis of the enzyme activity revealed that XT-II catalyzes the transfer of xylose to similar peptide acceptors as XT-I but with different efficiency. The optimal XT-II acceptor was observed using a bikunin-related peptide (K(m) 5.2 microM). Analysis of XT-I and XT-II mRNA expression in murine tissues showed a differential expression pattern for both enzymes. In particular, XT-II is highly expressed in liver tissue, where XT-I transcripts were not detected. This is the first report on the enzyme activity of XT-II and its involvement in chondroitin sulfate and heparan sulfate biosynthesis.