The xylosyltransferase (XT) isciforrils XT-I and XT-II initiate the posttranslational glycosaminoglycan (GAG) synthesis Here. we determined the relative expression of both isoforms in 33 human cell lines. The majority of tested cell lines showed dominant XYLT2 gene expression. while only in 23132/87, JAR, NCI-H510A and THP-1 was the XT-I mRNA expression higher. Nearly equal expression levels were detected in six cell lines Additionally, to shed light on Putative differences in acceptor specificities the acceptor properties of potential acceptor sequences were determined Peptides were expressed as glutathione-S-transferase fusion proteins containing putative or known GAG attachment sites of in vivo proteoglycans Kinetic analysis showed that K-m and V-max Values for XT-I mediated xylosylation were slightly higher than chose for XT-II, and that XT-I showed a lesser stringency concerning the acceptor sequence Mutagenesis of the bikunin peptide sequence in the G-S-G attachment site and flanking regions generated potential acceptor molecules. Here. mutations oil the N-terminal side and the attachment site were found to be more Susceptible to a loss of acceptor function than Mutations in the C-terminus Altogether the known consensus sequence a-a-a-a-G-S-G-a-a/G-a ('a' representing Asp Or Glu) for XT-I mediated xylosylation Could be approved and additionally extended to apply to XT-II as well (C) 2009 Elsevier Inc All rights reserved