Structural identification and quantification of unreported sialylated N-glycans in bovine testicular hyaluronidase by LC-ESI-HCD-MS/MS

Bovine testicular hyaluronidase (BTH), which accelerates the absorption and dispersion of drugs by decomposing hyaluronan in subcutaneous tissues, has been used in medical applications, including local anesthesia, ophthalmology, and dermatosurgery. The requirement of N-glycans for the activity of human hyaluronidase has been reported, and BTH has greater activity than human hyaluronidase. However, the N-glycan characteristics of BTH are unclear. From a commercial BTH source containing additional proteins, purified BTH (pBTH) was obtained using size exclusion chromatography, and the structures and quantities of its N-glycans were analyzed using liquid chromatography (LC)-electrospray ionization-higher energy collisional dissociation (HCD)-tandem mass spectrometry (MS/MS). In pBTH, 32 N-glycans were identified, with 12 sialylations (39.0% of total N- glycan content), nine core-fucosylations (31.5%), six terminal galactosylations (14.6%), five highmannosylations (13.7%), and four bisecting N-acetylglucosamine structures (7.8%). The presence of sialylated glycopeptides in pBTH was confirmed by nano-LC-HCD-MS/MS analysis. The absolute quantity of all N-glycans was calculated as 1.4 pmol (0.6 pmol for sialylation) in pBTH (1.0 pmol). The sialylation level (related to halflife, thermal stability, resistance to proteolysis, and solubility) was 24.4 times higher than that of human hyaluronidase. The hyaluronan degradation activity of de-sialylated pBTH decreased to 41.2 +/- 4.2%, showing that sialylated N-glycans were required for pBTH activity as well. This is the first study to identify and quantify 32 N- glycans of pBTH and investigate their structural roles in its activity. The presence of larger amounts of sialylated N-glycans in pBTH than in human hyaluronidase suggests a greater utilization of pBTH.