Purchase this article with an account.
Robert A. Burdi, Edward Wagner, Loyal Walker, Algis Grybauskas, Ryan D. McCarty, Jeffrey P. Mayer, Paul A. Knepper; Hemopexin: An Inhibitor for Hyaluronidase-2. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6616.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
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.
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 4oC. Untreated samples were stored at 4oC. 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.
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.
This PDF is available to Subscribers Only