Analysis of xylosyltransferase II binding to the anticoagulant heparin.
The key enzymes in the biosynthetic pathway of glycosaminoglycan production are represented by the human xylosyltransferase I and its isoform II (XylT-I and XylT-II). The glycosaminoglycan heparin interacts with a variety of proteins, thereby regulating their activities, also those of xylosyltransferases. The identification of unknown amino acids responsible for heparin-binding of XylT-II was addressed in this study. Thus, six XylT-II fragments were designed as fusion proteins with MBP and we received soluble and purified MBP/XylT-II from Escherichia coli. Heparin-binding studies showed that all fragments bound with low affinity to heparin. Prolonging of XylT-II fragments did not account for a cooperative effect of multiple heparin-binding motifs and in turn for a stronger heparin-binding. Sequence alignment and surface polarity plot led to the identification of two highly positively charged Cardin-Weintraub motifs with surface accessibility, resulting in combination with short clusters of basic amino acids for strong heparin-binding of native xylosyltransferases.
Casanova
Javier Carrera
JC
Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany.
Ambrosius
Michael
M
Kuhn
Joachim
J
Kleesiek
Knut
K
Götting
Christian
C
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D000097608:UDP Xylose-Protein Xylosyltransferase
D000925:Anticoagulants; D006493:Heparin; D010430:Pentosyltransferases; D011993:Recombinant Fusion Proteins; D000097608:UDP Xylose-Protein Xylosyltransferase