Hemopexin: An Inhibitor for Hyaluronidase-2

Abstract

Purpose: : Primary open angle glaucoma (POAG) is a common age-related optic neuropathy. Recently, our laboratory reported that the innate immune receptor Toll-4 is activated by low molecular weight hyaluronic acid (LMW HA) triggering a unified signaling pathway that causes an inflammatory cascade resulting in POAG. LMW HA is generated by an endo-glycosidase, hyaluronidase-2 (hyal-2), which degrades high molecular weight HA (HMW HA) into LMW HA. Thus, an exogeneous hyal-2 inhibitor could have clinical importance to prevent the generation of LMW HA. On the basis of reverse zymography and 2-D mass spectrometry, hemopexin was identified as a putative hyal-2 inhibitor.

Methods: : Testing of hyal-2 activity in human serum (HS) and bovine aqueous (BA) was accomplished via a functional assay measuring the amount of N-acetyl-D-glucosamine produced by hyal-2 degradation of HMW HA. HS and BA samples treated with a Stabilizing Protein Cocktail (SPC;Thermo Scientific) were diluted 1:4 with SPC and stored at 4^oC. Untreated samples were stored at 4^oC. Hyal-2 baseline activity for HS and BA samples was determined immediately upon sample acquisition. Seven days after baseline testing, treated and untreated BA and HS samples were retested to assess hyal-2 activity. Enzyme inhibition was determined by comparing the amount of hyal-2 activity with and without the addition of 0.1 to 100uM Vcpal, 0.1 to 100mM ascorbic acid, and 0.1 to 100uM hemopexin to the functional assay.

Conclusions: : Based on Vmax, Km, and percent hyal-2 activity values for BA and HS, it can be concluded that SPC maintained hyal-2 activity in HS and BA over a 7 day period. The Vmax values for BA with and without the addition of hemopexin also show that hyal-2 activity in BA was decreased by nearly 40 fold, and IC50 values show that hemopexin is approximately a 3 fold greater inhibitor of hyal-2 than Vcpal. These results provide a method of maintaining hyal-2 activity for detailed functional studies of both human serum and aqueous in POAG, and also provide evidence for hemopexin as a hyaluronidase inhibitor.

Keywords: enzymes/enzyme inhibitors • aqueous • protective mechanisms