Isoenzyme-specific differences in the degradation of hyaluronic acid by mammalian-type hyaluronidases

Abstract

Bovine testicular hyaluronidase (BTH) has been used as a spreading factor for many years and was primarily characterized by its enzymatic activity. As recombinant human hyaluronidases are now available the bovine preparations can be replaced by the human enzymes. However, data on the pH-dependent activity of hyaluronidases reported in literature are inconsistent in part or even contradictory. Detection of the pH-dependent activity of PH-20 type hyaluronidases, i.e. recombinant human PH-20 (rhPH-20) and BTH, showed a shift of the pH optimum from acidic pH values in a colorimetric activity assay to higher pH values in a turbidimetric activity assay. Contrarily, recombinant human Hyal-1 (rhHyal-1) and bee venom hyaluronidase (BVH) exhibited nearly identical pH profiles in both commonly used types of activity assays. Analysis of the hyaluronic acid (HA) degradation products by capillary zone electrophoresis showed that hyaluronan was catabolized by rhHyal-1 continuously into HA oligosaccharides. BTH and, to a less extent, rhPH-20 exhibited a different mode of action: at acidic pH (pH 4.5) HA was degraded as described for rhHyal-1, while at elevated pH (pH 5.5) small oligosaccharides were produced in addition to HA fragments of medium molecular weight, thus explaining the pH-dependent discrepancies in the activity assays. Our results suggest a sub-classification of mammalian-type hyaluronidases into a PH-20/BTH and a Hyal-1/BVH subtype. As the biological effects of HA fragments are reported to depend on the size of the molecules it can be speculated that different pH values at the site of hyaluronan degradation may result in different biological responses.

Role of hyaluronan and hyaluronan-binding proteins in lung pathobiology

“…HYAL-1 was found to have maximal and complete HA-degrading activity at pH 3.5-4.0, which is consistent with its role as a HA-degrading enzyme within the lysosome. PH-20 continuously degrades HMW-HA to small oligosaccharides at pH 4.5; at pH 5.5, HA is degraded to larger fragments (54). Unfortunately, the other hyaluronidase proteins were not examined in this study, but the authors speculate that the pH dependence of these enzymes could lead to different biological responses based on the production of different-sized HA fragments at the site of action.…”

“…Six hyaluronidase genes encode HYAL-1, -2, -3, and -4, PHYAL1 (a pseudogene), and PH-20 (27, 130 -132). A recent study by Hofinger et al (54) revealed that HA degradation by hyaluronidase enzymes may be pH-dependent. HYAL-1 was found to have maximal and complete HA-degrading activity at pH 3.5-4.0, which is consistent with its role as a HA-degrading enzyme within the lysosome.…”

Enzymatic Depletion of Tumor Hyaluronan Induces Antitumor Responses in Preclinical Animal Models

“…PH20 is unique as a membrane-bound, neutral active enzyme with high specific activity toward HA in vitro and in vivo, which is capable of degrading matrix-associated megadalton HA polymers to small tetrasaccharide and hexasaccharide products (16)(17)(18)(19). Given the rapid endogenous turnover of HA, accumulation in tumors could involve disequilibrium between the enzymes that synthesize HA and the enzymes that catabolize HA or receptors for cellular uptake, which subsequently mediate intracellular metabolism (20)(21)(22)(23).…”

Pro- and anti-inflammatory cytokines increase hyaluronan production by rat synovial membrane in vitro

“…The content of HA in tissues is controlled in mammals by two classes of enzymes: HA synthases (HAS1, HAS2 and HAS3) (47) and hyaluronidases (48,49). Particular isoforms of HAS probably synthesize HA of different sizes (50).…”