Hofinger, Edith S. A. and Spickenreither, Martin and Oschmann, Jan and Bernhardt, Günther and Rudolph, Rainer and Buschauer, Armin (2007) Recombinant human hyaluronidase Hyal-1: insect cells versus E. coli as expression system and identification of low molecular weight inhibitors. Glycobiology 17 (4), pp. 444-453.
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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 IC(50) of 50 +/- 4 microM comparable with glycyrrhizic acid.
|Institutions:||Chemistry and Pharmacy > Institute of Pharmacy > Pharmaceutical/Medicinal Chemistry II (Prof. Buschauer)|
|Projects:||GRK 760, Graduiertenkolleg Medizinische Chemie|
|Subjects:||500 Science > 570 Life sciences|
600 Technology > 610 Medical sciences Medicine
|Refereed:||Yes, this version has been refereed|
|Created at the University of Regensburg:||Yes|
|Owner:||Prof. Armin Buschauer|
|Deposited On:||21 Feb 2008 08:12|
|Last Modified:||05 Aug 2009 13:42|