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.
Müller
Sandra
S
Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstrasse 11, 32545 Bad Oeynhausen, Germany.
Disse
Jennifer
J
Schöttler
Manuela
M
Schön
Sylvia
S
Prante
Christian
C
Brinkmann
Thomas
T
Kuhn
Joachim
J
Kleesiek
Knut
K
Götting
Christian
C
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006493:Heparin; D006801:Humans; D007313:Insecta; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010430:Pentosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D000097608:UDP Xylose-Protein Xylosyltransferase
D006493:Heparin; D010430:Pentosyltransferases; D000097608:UDP Xylose-Protein Xylosyltransferase