Glycobiology Advance Access originally published online on January 16, 2007
Glycobiology 2007 17(4):444-453; doi:10.1093/glycob/cwm003
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Recombinant human hyaluronidase Hyal-1: insect cells versus Escherichia coli as expression system and identification of low molecular weight inhibitors +
2 Lehrstuhl für Pharmazeutische und Medizinische Chemie II, Institut für Pharmazie, Universität Regensburg, D-93053 Regensburg, Germany
3 Institut für Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, D-06120 Halle (Saale), Germany
4 Novoplant GmbH, Am Schwabeplan 1b, D-06466 Gatersleben, Germany
1 To whom correspondence should be addressed; Tel: +49 941 9434827; Fax: +49 941 9434820; e-mail: armin.buschauer{at}chemie.uni-regensburg.de
Received on November 27, 2006; revised on January 9, 2007; accepted on January 9, 2007
The human hyaluronidase Hyal-1, one of six human hyaluronidase subtypes, preferentially degrades hyaluronic acid present in the extracellular matrix of somatic tissues. Modulations of Hyal-1 expression have been observed in a number of malignant tumors. However, its role in disease progression is discussed controversially due to limited information on enzyme properties as well as the lack of specific inhibitors. Therefore, we expressed human Hyal-1 in a prokaryotic and in an insect cell system to produce larger amounts of the purified enzyme. In Escherichia coli, Hyal-1 formed inclusion bodies and was refolded in vitro after purification by metal ion affinity chromatography. However, the enzyme was produced with extremely low folding yields (0.5%) and exhibited a low specific activity (0.1 U/mg). Alternatively, Hyal-1 was secreted into the medium of stably transfected Drosophila Schneider-2 (DS-2) cells. After several purification steps, highly pure enzyme with a specific activity of 8.6 U/mg (consistent with the reported activity of human Hyal-1 from plasma) was obtained. Both Hyal-1 enzymes showed pH profiles similar to the hyaluronidase of human plasma with an activity maximum at pH 3.5–4.0. Deglycosylation of Hyal-1, expressed in DS-2 cells, resulted in a decrease in the enzymatic activity determined by a colorimetric hyaluronidase activity assay. Purified Hyal-1 from DS-2 cells was used for the investigation of the inhibitory activity of new ascorbic acid derivatives. Within this series, L-ascorbic acid tridecanoate was identified as the most potent inhibitor with an IC50 of 50 ± 4 µM comparable with glycyrrhizic acid.
Key words: Drosophila Schneider-2 cells / Hyal-1 / hyaluronidase / inhibitors / protein folding
+ Dedicated to Prof. Dr. Gerhard Franz, Regensburg, on the occasion of his 70th birthday.
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