Glycobiology Advance Access originally published online on June 29, 2007
Glycobiology 2007 17(9):963-971; doi:10.1093/glycob/cwm070
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Kinetics of Hyal-1 and PH-20 hyaluronidases: Comparison of minimal substrates and analysis of the transglycosylation reaction
Lehrstuhl für Pharmazeutische und Medizinische Chemie II, Institut für Pharmazie, Universität Regensburg, D-93040 Regensburg, 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 April 19, 2007; revised on June 21, 2007; accepted on June 24, 2007
The availability of recombinant expression systems for the production of purified human hyaluronidases PH-20 and Hyal-1 facilitated the first detailed analysis of the enzymatic reaction products. The human recombinant enzymes, both expressed by Drosophila Schneider-2 (DS-2) cells, were compared to bovine testicular hyaluronidase (BTH), a commercially available hyaluronidase preparation, which has long been considered a prototype of mammalian hyaluronidases. The conversion of low molecular weight hyaluronic acid (HA) fragments was detected by a capillary zone electrophoresis (CZE) method. Surprisingly, the HA hexasaccharide, which is generally accepted to be the minimum substrate of BTH, was not a substrate of recombinant human PH-20 and Hyal-1. However, HA octasaccharide was converted efficiently by both enzymes, thus representing the minimum substrate for human PH-20 and Hyal-1. Additionally, BTH was shown to catabolize the HA hexasaccharide at pH 4.0 mainly by hydrolysis, while at pH 6.0 transglycosylation prevailed. Human PH-20 was found to catalyze both hydrolysis and transglycosylation of the HA octasaccharide. On the contrary, human Hyal-1 converted the HA octasaccharide mainly by hydrolysis with transglycosylation products occurring only at high substrate concentrations (
500 µM). The differences between the hyaluronidase subtypes and isoenzymes were much more prominent than expected. Obviously, the different hyaluronidase subtypes have evolved into very specialized enzymes with respect to their catalytic mechanism of action.
Key words: capillary electrophoresis / Drosophila Schneider-2 cells / Hyal-1 / PH-20 / transglycosylation
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