title: Recombinant human hyaluronidase Hyal-1: insect cells versus Escherichia coli as expression system and identification of low molecular weight inhibitors.
abstract: 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.
authors.affiliation: Lehrstuhl für Pharmazeutische und Medizinische Chemie II, Institut für Pharmazie, Universität Regensburg, D-93053 Regensburg, Germany.
authors.forename: Edith S A
authors.initials: ES
authors.lastname: Hofinger
authors.forename: Martin
authors.initials: M
authors.lastname: Spickenreither
authors.forename: Jan
authors.initials: J
authors.lastname: Oschmann
authors.forename: Günther
authors.initials: G
authors.lastname: Bernhardt
authors.forename: Rainer
authors.initials: R
authors.lastname: Rudolph
authors.forename: Armin
authors.initials: A
authors.lastname: Buschauer
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D002460:Cell Line; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D005978:Glutathione / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D019476:Insect Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D007313:Insecta / Q000201:enzymology*; D007700:Kinetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D017931:DNA Primers; D029968:Escherichia coli Proteins; D019476:Insect Proteins; D011994:Recombinant Proteins; D006821:Hyaluronoglucosaminidase; D005978:Glutathione; D001205:Ascorbic Acid; D019803:Glutathione Disulfide