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

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

doi:10.1042/bj20051606

Cited by 17 publications

(19 citation statements)

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“…The N-terminal 214 amino acids that are responsible for Golgi localization are not required for the enzymatic function of XT-I, because we could show that the deletion of amino acids 1-266 of XT-I did not influence the enzymatic activity (45). Similar results have been reported for GnT-V, a Golgi-resident protein whose stem region is not required for its enzymatic activity but is responsible for its intracellular localization (41).…”

Section: Discussionsupporting

confidence: 85%

Cloning and Recombinant Expression of Active Full-length Xylosyltransferase I (XT-I) and Characterization of Subcellular Localization of XT-I and XT-II

Schön

Prante

Bahr

et al. 2006

Journal of Biological Chemistry

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Xylosyltransferase I (XT-I) catalyzes the transfer of xylose from UDP-xylose to serine residues in proteoglycan core proteins. This is the first and apparently rate-limiting step in the biosynthesis of the tetrasaccharide linkage region in glycosaminoglycan-containing proteoglycans. The XYLT-II gene codes for a highly homologous protein, but its physiological function is not yet known. Here we present for the first time the construction of a vector encoding the full-length GFP-tagged human XT-I and the recombinant expression of the active enzyme in mammalian cells. We expressed XT-I-GFP and various GFP-tagged XT-I and XT-II mutants with C-terminal truncations and deletions in HEK-293 and SaOS-2 cells in order to investigate the intracellular localization of XT-I and XT-II. Immunofluorescence analysis showed a distinct perinuclear pattern of XT-I-GFP and XT-II-GFP similar to that of ␣-mannosidase II, which is a known enzyme of the Golgi cisternae. Furthermore, a co-localization of native human XT-I and ␣-mannosidase II could also be demonstrated in untransfected cells. Using brefeldin A, we could also show that both xylosyltransferases are resident in the early cisternae of the Golgi apparatus. For its complete Golgi retention, XT-I requires the N-terminal 214 amino acids. Unlike XT-I, for XT-II, the first 45 amino acids are sufficient to target and retain the GFP reporter in the Golgi compartment. Here we show evidence that the stem regions were indispensable for Golgi localization of XT-I and XT-II.

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Section: Discussionsupporting

confidence: 85%

Cloning and Recombinant Expression of Active Full-length Xylosyltransferase I (XT-I) and Characterization of Subcellular Localization of XT-I and XT-II

Schön

Prante

Bahr

et al. 2006

Journal of Biological Chemistry

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“…In addition, human XT-II was also found to be a Golgi-resident enzyme present in the early compartments [19]. The amino acid sequences of the XT-I and XT-II stem regions are rather divergent [22], with less than 15% identical amino acids (Fig. 2).…”

Section: Intracellular Localization Of the Human Xylosyltransferasesmentioning

confidence: 97%

“…The corresponding murine genes are located on chromosomes 7 and 11, respectively, and share an exon-intron structure similar to the human xylosyltransferase genes. All higher organisms investigated so far have two xylosyltransferase genes encoding putative functional xylosyltransferases [18,20,22,23,25,29], while only a single xylosyltransferase gene is present in the fly and the worm genomes [26,27]. Yeast and bacteria possess no xylosyltransferase orthologs.…”

Section: Purification and Cloning Of The Xylosyltransferasesmentioning

confidence: 99%

“…Furthermore, 260 amino acids at the aminoterminal end of human XT-I can be deleted without any loss of enzyme activity [22]. However, deletion of 272 amino acids results in an inactive protein, indicating a crucial role for the 12-amino acid motif Gly 261 -Lys-Glu-AlaIso-Ser-Ala-Leu-Ser-Arg-Ala-Lys 272 for proper folding, which is supported by the observation that a selected deletion of this motif leads to a complete loss of activity [22].…”

Section: Enzyme Structurementioning

confidence: 99%

“…Heparin binding sites are composed of either short stretches of positively charged amino acids, a combination of amino acids which are brought into close proximity due to protein conformation, or follow the CardinWeintraub rules, which identified a b-Arg/Lys-Arg/ Lys-b-Arg/Lys-b or b-Arg/Lys-Arg/Lys-Arg/Lys-b-bArg/Lys-b (with b representing nonpolar or hydrophobic amino acids) consensus sequence for heparin binding [115 -117]. Heparin binding of XT-I is independent of protein conformation, as binding is not abolished by protein denaturation [22]. Furthermore, a sequence motif similar to the Cardin-Weintraub consensus sequences was located in human XT-I.…”

Section: Interactions Of Human Xylosyltransferases and Heparinmentioning

confidence: 99%

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Human xylosyltransferases in health and disease

Götting

Kühn

Kleesiek

2007

Cell. Mol. Life Sci.

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The xylosyltransferases I and II (XT-I, XT-II, EC 2.4.2.26) catalyze the transfer of xylose from UDP-xylose to selected serine residues in the proteoglycan core protein, which is the initial and ratelimiting step in glycosaminoglycan biosynthesis. Both xylosyltransferases are Golgi-resident enzymes and transfer xylose to similar core proteins acceptors. XT-I and XT-II are differentially expressed in cell types and tissues, although the reason for the existence of two xylosyltransferase isoforms in all higher organisms remains elusive. Serum xylosyltransferase activity was found to be a biochemical marker for the assessment of disease activity in systemic sclerosis and for the diagnosis of fibrotic remodeling processes. Furthermore, sequence variations in the XT-I and XT-II coding genes were identified as risk factors for diabetic nephropathy, osteoarthritis or pseudoxanthoma elasticum. These findings point to the important role of the xylosyltransferases as disease modifiers in pathologies which are characterized by an altered proteoglycan metabolism. The present review discusses recent advances in mammalian xylosyltransferases and the impact of xylosyltransferases in proteoglycan-associated diseases.

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Endoplasmic reticulum retention of xylosyltransferase 1 (XYLT1) mutants underlying Desbuquois dysplasia type II

Al-Jezawi

Ali

2017

American J of Med Genetics Pt A

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Desbuquois syndrome is a heterogeneous rare type of skeletal dysplasia with a prevalence of less than 1 in 1,000,000 individuals. It is characterized by short-limbed dwarfism, dysmorphic facial features, and severe joint laxity. Two types have been recognized depending on the presence of distinctive carpal and phalangeal features. Mutations in the calcium activated nucleotidase 1 (CANT1) have been found to be responsible for type I and lately, for the Kim type of Desbuquois dysplasia. In addition, a number of Desbuquois dysplasia type II patients have been attributed to mutations in xylosyltransferase 1, encoded by the XYLT1 gene, an enzyme that catalyzes the transfer of UDP-xylose (a marker of cartilage destruction) to serine residues of an acceptor protein, essential for the biosynthesis of proteoglycans. We report here a patient with features consistent with Desbuquois dysplasia II including short long bones, flat face, mild monkey wrench appearance of the femoral heads. Whole exome sequencing revealed a novel homozygous duplication of a single nucleotide in XYLT1 gene (c.2169dupA). This variant is predicted to result in a frame-shift and stop codon p.(Val724Serfs*10) within the xylosyltransferase catalytic domain. Immunoflourescence staining of HeLa cells transfected with mutated XYLT1 plasmids constructs of the current as well as the previously reported missense mutations (c.1441C>T, p.(Arg481Trp) and c.1792C>T, p.(Arg598Cys)), revealed aberrant subcellular localization of the enzyme compared to wild-type, suggesting endoplasmic reticulum retention of these mutants as the likely mechanism of disease.

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

Cited by 17 publications

References 22 publications

Cloning and Recombinant Expression of Active Full-length Xylosyltransferase I (XT-I) and Characterization of Subcellular Localization of XT-I and XT-II

Cloning and Recombinant Expression of Active Full-length Xylosyltransferase I (XT-I) and Characterization of Subcellular Localization of XT-I and XT-II

Human xylosyltransferases in health and disease

Endoplasmic reticulum retention of xylosyltransferase 1 (XYLT1) mutants underlying Desbuquois dysplasia type II

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