Analysis of the DXD Motifs in Human Xylosyltransferase I Required for Enzyme Activity

Götting, Christian; Müller, Sandra; Schöttler, Manuela; Schön, Sylvia; Prante, Christian; Brinkmann, Thomas; Kühn, Joachim; Kleesiek, Knut

doi:10.1074/jbc.m401340200

Cited by 30 publications

(33 citation statements)

References 52 publications

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“…XT-I Activity Assay-Using whole protein extracts from cardiac tissue or cell culture supernatant, the analysis of XT-I activity was carried out as described previously (21). The method is based on the incorporation of […”

Section: Methodsmentioning

confidence: 99%

Transforming Growth Factor β1-regulated Xylosyltransferase I Activity in Human Cardiac Fibroblasts and Its Impact for Myocardial Remodeling

Prante

Milting

Kassner

et al. 2007

Journal of Biological Chemistry

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In cardiac fibrosis remodeling of the failing myocardium is associated with a complex reorganization of the extracellular matrix (ECM). Xylosyltransferase I and Xylosyltransferase II (XT-I and XT-II) are the key enzymes in proteoglycan biosynthesis, which are an important fraction of the ECM. XT-I was shown to be a measure for the proteoglycan biosynthesis rate and a biochemical fibrosis marker. Here, we investigated the XT-I and XT-II expression in cardiac fibroblasts and in patients with dilated cardiomyopathy and compared our findings with nonfailing donor hearts. We analyzed XT-I and XT-II expression and the glycosaminoglycan (GAG) content in human cardiac fibroblasts incubated with transforming growth factor (TGF)-␤ 1 or exposed to cyclic mechanical stretch. In vitro and in vivo no significant changes in the XT-II expression were detected. For XT-I we found an increased expression in parallel with an elevated chondroitin sulfate-GAG content after incubation with TGF-␤ 1 and after mechanical stretch. XT-I expression and subsequently increased levels of GAGs could be reduced with neutralizing anti-TGF-␤ 1 antibodies or by specific inhibition of the activin receptor-like kinase 5 or the p38 mitogen-activated protein kinase pathway. Usage of XT-I small interfering RNA could specifically block the increased XT-I expression under mechanical stress and resulted in a significantly reduced chondroitin sulfate-GAG content. In the left and right ventricular samples of dilated cardiomyopathy patients, our data show increased amounts of XT-I mRNA compared with nonfailing controls. Patients had raised levels of XT-I enzyme activity and an elevated proteoglycan content. Myocardial remodeling is characterized by increased XT-I expression and enhanced proteoglycan deposition. TGF-␤ 1 and mechanical stress induce XT-I expression in cardiac fibroblasts and have impact for ECM remodeling in the dilated heart. Specific blocking of XT-I expression confirmed that XT-I catalyzes a rate-limiting step during fibrotic GAG biosynthesis.

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Section: Methodsmentioning

confidence: 99%

Transforming Growth Factor β1-regulated Xylosyltransferase I Activity in Human Cardiac Fibroblasts and Its Impact for Myocardial Remodeling

Prante

Milting

Kassner

et al. 2007

Journal of Biological Chemistry

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“…In GT-A fold glycosyltransferases, which include XXTs, the DxD motif (Asp-x-Asp) plays an important role in coordinating an essential divalent cation (Busch et al, 1998;Wiggins and Munro, 1998;Zhang et al, 2001;Boeggeman and Qasba, 2002;Götting et al, 2004;Cavalier and Keegstra, 2006;Bobovská et al, 2014). Structural characterization of a putative glycosyltransferase from the Paramecium bursaria chlorella virus-1 (PBCV-1), which is predicted to belong to the same GT34 gene family as the XXTs, showed that the two Asp residues in the DxD motif participate in the coordination of the divalent cation (Zhang et al, 2007).…”

Section: Xxt5 Activity Requires the Dxd Motifmentioning

confidence: 99%

“…The DxD motif functions to coordinate the divalent cation that forms ionic interactions with the negatively charged diphosphate moiety in UDP-Xyl and is critical for catalytic activity in numerous other glycosyltransferases (Busch et al, 1998;Wiggins and Munro, 1998;Li et al, 2001;Götting et al, 2004). To determine the functional role of XXT5 in vivo, we overexpressed the XXT5 D228A:D230A mutant in the xxt5 knockout mutant plant.…”

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confidence: 99%

Enzymatic Activity of Xyloglucan Xylosyltransferase 5

et al. 2016

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ORCID IDs: 0000-0002-5107-9009 (R.F.); 0000-0001-6938-6923 (O.A.Z.).Xyloglucan, the most abundant hemicellulosic component of the primary cell wall of flowering plants, is composed of a b-(1,4)-glucan backbone decorated with D-xylosyl residues. Three xyloglucan xylosyltransferases (XXTs) participate in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana). Two of these, XXT1 and XXT2, have been shown to be active in vitro, whereas the catalytic activity of XXT5 has yet to be demonstrated. By optimizing XXT2 expression in a prokaryotic system and in vitro activity assay conditions, we demonstrate that nonglycosylated XXT2 lacking its cytosolic amino-terminal and transmembrane domain displays high catalytic activity. Using this optimized procedure for the expression of XXT5, we report, to our knowledge for the first time, that recombinant XXT5 shows enzymatic activity in vitro, although at a significantly slower rate than XXT1 and XXT2. Kinetic analysis showed that XXT5 has a 7-fold higher K m and 9-fold lower k cat compared with XXT1 and XXT2. Activity assays using XXT5 in combination with XXT1 or XXT2 indicate that XXT5 is not specific for their products. In addition, mutagenesis experiments showed that the in vivo function and in vitro catalytic activity of XXT5 require the aspartate-serine-aspartate motif. These results demonstrate that XXT5 is a catalytically active xylosyltransferase involved in xylosylation of the xyloglucan backbone.

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“…Based on experimental studies on a3ManT [52], XylT [53], GM2 synthase [54] and fringe [55], the DxD motif has been implicated in binding phosphate group and the metal ion.…”

Section: Location Of Residues Whose Functional Importance Has Been Stmentioning

confidence: 99%

Fold-recognition and comparative modeling of human β3GalT I, II, IV, V and VI and β3GalNAcT I: Prediction of residues conferring acceptor substrate specificity

Patel

Balaji

2007

Journal of Molecular Graphics and Modelling

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Analysis of the DXD Motifs in Human Xylosyltransferase I Required for Enzyme Activity

Abstract: Human xylosyltransferase I (XT-I

Cited by 30 publications

References 52 publications

Transforming Growth Factor β1-regulated Xylosyltransferase I Activity in Human Cardiac Fibroblasts and Its Impact for Myocardial Remodeling

Transforming Growth Factor β1-regulated Xylosyltransferase I Activity in Human Cardiac Fibroblasts and Its Impact for Myocardial Remodeling

Enzymatic Activity of Xyloglucan Xylosyltransferase 5

Fold-recognition and comparative modeling of human β3GalT I, II, IV, V and VI and β3GalNAcT I: Prediction of residues conferring acceptor substrate specificity

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