Graduate Studies


First Advisor

Melanie A. Simpson

Date of this Version



A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Biochemistry, Under the Supervision of Professor Melanie A. Simpson. Lincoln, Nebraska : August 2018

Copyright (c) 2018 Eryn Cerelle Lee


Hyaluronidases (Hyal) are a class of enzymes that degrade hyaluronan (HA), and other abundant extracellular and cell surface polysaccharide glycans. There are five human hyaluronidases. Of these, Hyal1 has been extensively characterized for its role in hyaluronan turnover and signaling, and has been shown to promote prostate cancer progression. Hyal2 has been implicated in breast cancer progression, but its activity as a hyaluronidase is poorly understood. Structural similarity and conserved active site catalytic residues among members of the hyaluronidase family suggest that each of these isozymes would have catalytic activity. A better understanding of their structural and functional relationships has the potential to improve knowledge of disease progression mechanisms and serve as therapeutic drug targets. Though Hyal1 activity, cellular localization, trafficking and substrate specificity have been well characterized, previous reports suggest Hyal2 may be a cell surface receptor that may have little to no hyaluronidase activity. To address structural features that may underlie functional differences, we initially focused on characterizing recombinantly expressed Hyal2 in a solubly secreted GPI-truncated form compared to the full-length, GPI-linked and membrane-associated form. Point mutations were made in both the soluble and the full-length Hyal2 context. Upon successful production of our point mutants and purification of the soluble Hyal2, we quantitatively measured enzymatic activity by employing a modified Morgan-Elson assay and qualitatively screened for other glycan substrates by agarose gel electrophoresis. No significant HA or other glycan degradation by soluble Hyal2 was found. The following step of this analysis included the production of Hyal1 mutants that were predicted through in silico mutagenesis to potentially impact binding and degradation of HA in the active site. Point mutants were expressed in a similar fashion to Hyal2 mutants and were assayed for altered catalytic activity. We confirmed Arg134 to be essential in catalysis through HA positioning and Asp292 hinders HA binding but is necessary to maintain robust activity. These results provide useful information to further understand mechanisms involved in Hyal1 activity and our approach appears ideal if Hyal2 studies are revisited.

Advisor: Melanie A. Simpson