Identification, quantification, and structural role of N-glycans in two highly purified isoforms of sheep testicular hyaluronidase

Abstract

Animal-derived hyaluronidase, which hydrolyzes the polysaccharide hyaluronic acid, has been used in medical applications despite its limited purity. Additionally, the N-glycan characterization of sheep testicular hyaluronidase (STH) and its structural role remain poorly understood. In this study, STH was purified from the commercially available STH preparation (containing at least 14 impurity proteins) using heparin-affinity chromatography followed by size exclusion chromatography. The structure and quantity of N-glycans of STH were investigated using liquid chromatography-electrospray ionization-high energy collision dissociation-tandem mass spectrometry. Two isoforms, H3S1 and H3S2, of STH were obtained (purity >98 %) with a yield of 3.4 % and 5.1 %, respectively. Fourteen N-glycans, including nine core-fucosylated N-glycans (important for the stability and function of glycoproteins), were identified in both H3S1 and H3S2, with similar quantities of each N-glycan. The amino acid sequences of the proteolytic peptides of H3S1 and H3S2 were compared with those reported in STH. The hyaluronic acid-degrading activity of deglycosylated H3S1 and H3S2 was reduced to 70.8 % and 71.1 % compared to that (100 %) of H3S1 and H3S2, respectively. This is the first report of N-glycan characterization of two highly purified isoforms of STH. These H3S1 and H3S2 will be useful for medical use without unwanted effects of partially purified STH.