High-level expression and purification of human xylosyltransferase I in High Five insect cells as biochemically active form

Kühn, Joachim; Müller, Sandra; Schnölzer, Martina; Kempf, Tore; Schön, Sylvia; Brinkmann, Thomas; Schöttler, Manuela; Götting, Christian; Kleesiek, Knut

doi:10.1016/j.bbrc.2003.10.157

Cited by 18 publications

(18 citation statements)

References 28 publications

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“…Since the XylT2 sequence is similar to the sequence of XylT1, which has been shown to have xylosyltransferase activity in heterologous expression systems (13,14,27), the simplest interpretation is that XylT2 also functions as a xylosyltransferase. However, this activity has yet to be demonstrated directly (13,14).…”

Section: Discussionmentioning

confidence: 99%

Biosynthesis of Chondroitin and Heparan Sulfate in Chinese Hamster Ovary Cells Depends on Xylosyltransferase II

Cuellar

Chuong

Hubbell

et al. 2007

Journal of Biological Chemistry

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Xylosyltransferase (XylT) catalyzes the initial enzymatic reaction in the glycosaminoglycan assembly pathway for proteoglycan biosynthesis. Its activity is thought to be rate-limiting. Two xylosyltransferases have been found using genomic analyses, and one of these, XylT1, has been shown to have xylosyltransferase activity. On the other hand, the less studied XylT2 in recombinant form lacks xylosyltransferase activity and has no known function. Wild-type Chinese hamster ovary cells express abundant Xylt2 mRNA levels and lack detectable Xylt1 mRNA levels. Analysis of a previously described Chinese hamster ovary cell xylosyltransferase mutant (psgA-745) shows that it harbors an Xylt2 nonsense mutation and fails to assemble glycosaminoglycans onto recombinant biglycan. Transfection of this cell line with a murine Xylt2 minigene results in the production of recombinant chondroitin sulfate-modified biglycan core protein and restoration of fibroblast growth factor binding to cell surface-associated heparan sulfate. Expression analyses on 10 different human transformed cell lines detect exclusive XYLT2 expression in two and co-expression of XYLT1 and XYLT2 in the others but at disparate ratios where XYLT2 expression is greater than XYLT1 in most cell lines. These results indicate that XylT2 has a significant role in proteoglycan biosynthesis and that cell type may control which family member is utilized.Xylosyltransferase (UDP-D-xylose:protein ␤-D-xylosyltransferase, EC 2.4.2.26) (XylT) 3 activity was first isolated and studied in the early 1960s. This enzyme was one of the first glycosyltransferases isolated (1). It catalyzes the initial enzymatic reaction in the assembly of glycosaminoglycans to core proteins. This addition of a xylose to a designated serine is hypothesized to be rate-limiting for proteoglycan (PG) biosynthesis (2, 3). PGs are found on the cell surface and in the extracellular matrix. PGs are fundamental to cellular processes including enhancement of receptor binding of cytokines and growth factors and augmentation of enzyme-substrate interactions important in coagulation and lipid catabolism (4 -9). Extracellular matrix PGs maintain basement membrane integrity, augment growth factor and cytokine sequestration, and create morphogen gradients (4, 10 -12). Genomic analyses have identified two xylosyltransferase family members in mammals. XYLT1 encodes for xylosyltransferase I (XylT1), shown to have xylosyltransferase activity (13). XYLT2 encodes for xylosyltransferase II (XylT2), which when expressed in vitro failed to have xylosyltransferase activity (13,14), and therefore, its function is unknown. A highly useful tool for the study of PGs has been the xylosyltransferase-deficient Chinese hamster ovary cell (CHO) line pgsA-745 (15, 16). This cell line was isolated from an ethylmethane sulfonate mutagenesis screen of CHO-K1 cells for sulfation incorporation mutants. This cell line produces neither chondroitin nor heparan-sulfated PGs (15, 16). In our long term investigation of this enzymatic pathway an...

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Section: Discussionmentioning

confidence: 99%

Biosynthesis of Chondroitin and Heparan Sulfate in Chinese Hamster Ovary Cells Depends on Xylosyltransferase II

Cuellar

Chuong

Hubbell

et al. 2007

Journal of Biological Chemistry

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“…Heparin-We have shown in previous studies that heparin is a potent inhibitor of human XT-I and that the enzyme strongly binds to the heparin matrix during heparin affinity chromatography (17,18,28). Here, we used the generated XT-I mutants to investigate whether alterations of the DXD motifs affect the inhibitory effects of heparin.…”

Section: Inhibition Of Xt-i Activity By Heparin and Binding Of Rxt-i mentioning

confidence: 99%

“…A stable High Five/pCG255-1 insect cell clone expressing rXT-I-(⌬1-148)-V5-His was generated as described in detail (28). Synthesis of Recombinant Bikunin-Recombinant bikunin was expressed in E. coli strain BL21(DE3) as described previously (29).…”

Section: Materials-highmentioning

confidence: 99%

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

Götting

Müller

Schöttler

et al. 2004

Journal of Biological Chemistry

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“…High Five/pCG255-1 insect cells, which produce cell culture supernatant containing recombinant XT-I (rXT-I-His), were cultured as described previously (17 ).…”

Section: Serum Samplesmentioning

confidence: 99%

Measurement of Fibrosis Marker Xylosyltransferase I Activity by HPLC Electrospray Ionization Tandem Mass Spectrometry

et al. 2006

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High-level expression and purification of human xylosyltransferase I in High Five insect cells as biochemically active form

Cited by 18 publications

References 28 publications

Biosynthesis of Chondroitin and Heparan Sulfate in Chinese Hamster Ovary Cells Depends on Xylosyltransferase II

Biosynthesis of Chondroitin and Heparan Sulfate in Chinese Hamster Ovary Cells Depends on Xylosyltransferase II

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

Measurement of Fibrosis Marker Xylosyltransferase I Activity by HPLC Electrospray Ionization Tandem Mass Spectrometry

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