Kinetics of Hyal-1 and PH-20 hyaluronidases: Comparison of minimal substrates and analysis of the transglycosylation reaction

Abstract

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 (> or = 500 microM). 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.

Hyaluronidase Activity of Human Hyal1 Requires Active Site Acidic and Tyrosine Residues

“…As an alternative, we expressed wild-type and mutant human Hyal1 with a FLAG epitope tag and immunoaffinity purified the resulting protein from the conditioned media of either human prostate carcinoma cells or embryonic kidney cells. Michaelis-Menten kinetic values were not previously determined for Hyal1 but specific activity reported at a given concentration (22,24) is within an order of magnitude of the values we report here. Using the octasaccharide substrate, saturation was essentially observed within the same range we utilized (22), adjusted for the 10-fold difference in molar mass of the 2-kDa octasaccharide versus the 20-kDa polymeric HA substrate.…”

“…Michaelis-Menten kinetic values were not previously determined for Hyal1 but specific activity reported at a given concentration (22,24) is within an order of magnitude of the values we report here. Using the octasaccharide substrate, saturation was essentially observed within the same range we utilized (22), adjusted for the 10-fold difference in molar mass of the 2-kDa octasaccharide versus the 20-kDa polymeric HA substrate. The somewhat lower activity of Hyal1 purified from insect cells relative to the enzyme we have prepared from human cell culture may be due either to differences in utilization of these substrate sizes or in glycosylation.…”

“…Members of this family are endoglycosidases, with homology to chitinases and lysozyme (18 -21). Several of them, including Hyal1 (22)(23)(24), exhibit an acidic activity profile, consistent with their lysosomal function. Scheme 1 illustrates the proposed reaction mechanism, indicating specific acidic amino acid residues directly implicated in catalysis by sequence conservation and homology to the experimentally characterized sperm acrosomal hyaluronidase, PH-20 (25).…”

“…fied from insect cells demonstrated its minimum octasaccharide substrate requirement (22). Several naturally occurring inhibitors have been reported, but rational design of high affinity and/or mechanism-based inhibitors to control disease progression requires detailed characterization of Hyal1 chemistry.…”

“…To establish a system for detailed kinetic analysis, it was first necessary to generate large scale expression and purification schemes. Active Hyal1 had been produced previously using insect cell expression systems (22)(23)(24), but the sugar content of glycosyl moieties is often not accurately reproduced by insects relative to the human sequences. Because both PH-20 and Hyal1 hyaluronidases have shown sensitivity to glycosylation, the nature of these modifications has the potential to impact enzymatic function.…”