RESUMO
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.
Assuntos
Anticoagulantes/metabolismo , Heparina/metabolismo , Pentosiltransferases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Anticoagulantes/química , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Heparina/química , Humanos , Dados de Sequência Molecular , Pentosiltransferases/química , Pentosiltransferases/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência , Análise de Sequência de Proteína , UDP Xilose-Proteína XilosiltransferaseRESUMO
Human xylosyltransferases I and II (XylT-I, XylT-II) are key enzymes in glycosaminoglycan biosynthesis. Knowledge about the in vivo molecular weight, oligomeric state or turnover number are essential characteristics which have been addressed in this study. XylT-II was purified from Pichia pastoris by fractionated ammonium sulfate precipitation, heparin affinity and ion exchange chromatography. XylT-II was purified over 7000-fold with a final yield of 2.6%. By utilizing mass spectra analysis we can prove its first in-gel detection showing a migration pattern behavior that confirms its in silico molecular weight of 95.8 kDa. We could determine a turnover number of 2.18 min(-1) or one transferred xylose molecule per one XylT-II molecule each 27.5s. The k(cat)/K(M) ratio was 0.357 min(-1)microM(-1) for XylT-II using the bikunin-homologous acceptor Bio-QEEEGSGGGQKK-F. The comparison to XylT-I derived from the same organism revealed a 2.4-fold higher catalytic efficiency (0.870 min(-1)microM(-1)) for XylT-I.
Assuntos
Pentosiltransferases/biossíntese , Pentosiltransferases/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Sequência de Aminoácidos , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Géis/química , Humanos , Cinética , Espectrometria de Massas , Dados de Sequência Molecular , Pentosiltransferases/química , Pichia/genética , Proteínas Recombinantes/química , UDP Xilose-Proteína XilosiltransferaseRESUMO
Human xylosyltransferase II (EC 2.4.2.26, XT-II) represents an isoform of xylosyltransferase I (XT-I). Recently, we and others provided first evidence that XT-II is capable of initiating the biosynthesis of glycosaminoglycan chains in proteoglycans. Here, a soluble form of human XT-II was expressed in the yeast Pichia pastoris and the substrate specificity for various acceptors was investigated, pointing to a modified bikunin peptide to be the optimal XT-II acceptor (K(M)=1.9 microM). Furthermore, biochemical characterization of XT-II showed that this enzyme was strongly inhibited by nucleotides and glycosaminoglycans. Its temperature optimum, stability, and ion dependency were further examined, demonstrating necessity for Mg(2+) or Mn(2+) ions for its enzymatic activity. Our data show for the first time that XT-I and XT-II are xylosyltransferases with similar but not identical properties, pointing to their potential role in modulating the cellular proteoglycan pool.
Assuntos
Pentosiltransferases/química , Pentosiltransferases/metabolismo , Pichia/metabolismo , Engenharia de Proteínas/métodos , Transfecção/métodos , Ativação Enzimática , Estabilidade Enzimática , Humanos , Pentosiltransferases/genética , Pentosiltransferases/isolamento & purificação , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , UDP Xilose-Proteína XilosiltransferaseRESUMO
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.
