Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme.

Human XT-I (xylosyltransferase I; EC 2.4.2.26) initiates the biosynthesis of the glycosaminoglycan linkage region and is a diagnostic marker of an enhanced proteoglycan biosynthesis. In the present study, we have investigated mutant enzymes of human XT-I and assessed the impact of the N-terminal region on the enzymatic activity. Soluble mutant enzymes of human XT-I with deletions at the N-terminal domain were expressed in insect cells and analysed for catalytic activity. As many as 260 amino acids could be truncated at the N-terminal region of the enzyme without affecting its catalytic activity. However, truncation of 266, 272 and 273 amino acids resulted in a 70, 90 and >98% loss in catalytic activity. Interestingly, deletion of the single 12 amino acid motif G261KEAISALSRAK272 leads to a loss-of-function XT-I mutant. This is in agreement with our findings analysing the importance of the Cys residues where we have shown that C276A mutation resulted in a nearly inactive XT-I enzyme. Moreover, we investigated the location of the heparin-binding site of human XT-I using the truncated mutants. Heparin binding was observed to be slightly altered in mutants lacking 289 or 568 amino acids, but deletion of the potential heparin-binding motif P721KKVFKI727 did not lead to a loss of heparin binding capacity. The effect of heparin or UDP on the XT-I activity of all mutants was not significantly different from that of the wild-type. Our study demonstrates that over 80% of the nucleotide sequence of the XT-I-cDNA is necessary for expressing a recombinant enzyme with full catalytic activity.

Impact of polymorphisms in the genes encoding xylosyltransferase I and a homologue in type 1 diabetic patients with and without nephropathy.

BACKGROUND: Xylosyltransferase I (XT-I) is the chain-initiating enzyme in the biosynthesis of heparan sulfate proteoglycans (HSPGs). It catalyses the transfer of xylose to specific serine residues in the core protein. The XYLT-II gene codes for a protein highly homologous to the XT-I but its biologic function is not yet known. HSPGs are thought to play an important role in the permeability properties of the glomerular basement membrane (GBM) and thus the xylotransferase genes might be potential candidate genes predisposing to diabetic nephropathy in type 1 diabetic patients. METHODS: We screened all XYLT-I and XYLT-II exons and flanking intron regions in 96 Caucasians with type 1 diabetes (48 with and 48 without diabetic nephropathy) using denaturing high-performance liquid chromatography (DHPLC). We also studied a nondiabetic control group. RESULTS: Applying this technique we identified 13 variations in XYLT-I and 20 in XYLT-II. The variations IVS6-9T>C and IVS6-14_IVS6-13insG in XYLT-II were found with a frequency of 5.2% (5/96) in nondiabetic nephropathy patients, while all nephropathy patients were negative (P= 0.06). Nondiabetic controls also showed the single nucleotide polymorphisms (SNP) at a frequency of 1.1% (5/440). The investigation of the SNPs' influence on clinical characteristics revealed significant associations for c.1989T>C (XYLT-I) and c.2402C>G (XYLT-II) with patients' blood pressure. CONCLUSION: We detected in our cohort associations between DNA sequence variations of genes encoding xylosyltransferases and the occurrence of altered clinical characteristics.

Serum xylosyltransferase I activity, the new biochemical fibrosis marker, is not affected by renal insufficiency.

OBJECTIVES: Serum xylosyltransferase I (XT-I) is a marker for the determination of tissue remodeling in systemic sclerosis. Here, we investigated whether renal insufficiency affects XT-I levels in blood. METHODS: We measured serum XT-I activity in 236 patients with different serum creatinine levels. RESULTS: XT-I activities in cohorts with increased creatinine levels were not significantly altered compared to controls. CONCLUSIONS: Serum XT-I activity is applicable as a fibrosis marker independent from renal function.

Human xylosyltransferase I: functional and biochemical characterization of cysteine residues required for enzymic activity.

XT-I (xylosyltransferase I) is the initial enzyme in the post-translational biosynthesis of glycosaminoglycan chains in proteoglycans. To gain insight into the structure-function relationship of the enzyme, a soluble active form of human XT-I was expressed in High Five insect cells with an apparent molecular mass of 90 kDa. Analysis of the electrophoretic mobility of the protein under non-reducing and reducing conditions indicated that soluble XT-I does not form homodimers through disulphide bridges. In addition, the role of the cysteine residues was investigated by site-directed mutagenesis combined with chemical modifications of XT-I by N-phenylmaleimide. Replacement of Cys471 or Cys574 with alanine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of soluble recombinant XT-I by forming disulphide bonds. On the other hand, N-phenylmaleimide treatment showed no effect on wild-type XT-I but strongly inactivated the cysteine mutants in a dose-dependant manner, indicating that seven intramolecular disulphide bridges are formed in wild-type XT-I. The inhibitory effect of UDP on the XT-I activity of C561A (Cys561-->Ala) mutant enzyme was significantly reduced compared with all other tested cysteine mutants. In addition, we tested for binding to UDP-agarose beads. The inactive mutants revealed no significantly different nucleotide-binding properties. Our study demonstrates that recombinant XT-I is organized as a monomer with no free thiol groups and strongly suggests that the catalytic activity does not depend on the presence of free thiol groups, furthermore, we identified five cysteine residues which are critical for enzyme activity.

Analysis of the DXD motifs in human xylosyltransferase I required for enzyme activity.

Human xylosyltransferase I (XT-I) is the initial enzyme involved in the biosynthesis of the glycosaminoglycan linker region in proteoglycans. Here, we tested the importance of the DXD motifs at positions 314-316 and 745-747 for enzyme activity and the nucleotide binding capacity of human XT-I. Mutations of the 314DED316 motif did not have any effect on enzyme activity, whereas alterations of the 745DWD747 motif resulted in reduced XT-I activity. Loss of function was observed after exchange of the highly conserved aspartic acid at position 745 with glycine. However, mutation of Asp745 to glutamic acid retained full enzyme activity, indicating the importance of an acidic amino acid at this position. Reduced substrate affinity was observed for mutants D747G (Km=6.9 microm) and D747E (Km=4.4 microm) in comparison with the wild-type enzyme (Km=0.9 microm). Changing the central tryptophan to a neutral, basic, or acidic amino acid resulted in a 6-fold lower Vmax, with Km values comparable with those of the wild-type enzyme. Despite the major effect of the DWD motif on XT-I activity, nucleotide binding was not abolished in the D745G and D747G mutants, as revealed by UDP-bead binding assays. Ki values for inhibition by UDP were determined to be 1.9-24.6 microm for the XT-I mutants. The properties of binding of XT-I to heparin-beads, the Ki constants for noncompetitive inhibition by heparin, and the activation by protamine were not altered by the generated mutations.

High-level expression and purification of human xylosyltransferase I in High Five insect cells as biochemically active form.

Human xylosyltransferase I (XT-I) catalyzes the transfer of xylose from UDP-xylose to consensus serine residues of proteoglycan core proteins. Expression of a soluble form of recombinant histidine-tagged XT-I (rXT-I-HIS) was accomplished at a high level with High Five/pCG255-1 insect cells in suspension culture. The recombinant protein was purified to homogeneity by a combination of heparin affinity chromatography and metal (Ni(2+)) chelate affinity chromatography. Using the modern technique of perfusion chromatography, a rapid procedure for purification of the rXT-I-HIS from insect cell culture supernatant was developed. The purified, biologically active enzyme was homogeneous on SDS-PAGE, was detected with anti-XT-I-antibodies, and had the expected tryptic fragment mass spectrum. N-terminal amino acid sequencing demonstrated that the N-terminal signal sequence of the expressed protein was quantitatively cleaved. The total yield of the enzyme after purification was 18% and resulted in a specific XT-I activity of 7.9mU/mg. The K(m) of the enzyme for recombinant [Val(36),Val(38)](delta1),[Gly(92),Ile(94)](delta2)bikunin was 0.8microM. About 5mg purified enzyme could be obtained from 1L cell culture supernatant. The availability of substantial quantities of active, homogeneous enzyme will be of help in future biochemical and biophysical characterization of XT-I and for the development of a immunological XT-I assay.