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KMID : 0545120180280091547
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Journal of Microbiology and Biotechnology
2018 Volume.28 No. 9 p.1547 ~ p.1553
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Characterization of Recombinant Bovine Sperm Hyaluronidase and Identification of an Important Asn-X-Ser/Thr Motif for Its Activity
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Park Chae-Ri
Kim Young-Hyun
Lee Sang-Rae
Park Soo-Jin
Jung Ye-Na
Lee Young-Jeon
Kim Ji-Su
Eom Tae-Kil
Kim Ju-Sung
Lee Dong-Mok
Song Bong-Suk
Sim Bo-Woong
Kim Sun-Uk
Chang Kyu-Tae
Kim E-Kyune
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Abstract
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Hyaluronidases are a family of enzymes that catalyse the breakdown of hyaluronic acid, which is abundant in the extracellular matrix and cumulus oocyte complex. To investigate the activity of recombinant bovine sperm hyaluronidase 1 (SPAM1) and determine the effect of the Asn-X-Ser/Thr motif on its activity, the bovine SPAM1 open reading frame was cloned into the mammalian expression vector pCXN2 and then transfected to the HEK293 cell line. Expression of recombinant bovine hyaluronidase was estimated using a hyaluronidase activity assay with gel electrophoresis. Recombinant hyaluronidase could resolve highly polymeric hyaluronic acid and also caused dispersal of the cumulus cell layer. Comparative analysis with respect to enzyme activity was carried out for the glycosylated and deglycosylated bovine sperm hyaluronidase by N-glycosidase F treatment. Finally, mutagenesis analysis revealed that among the five potential N-linked glycosylation sites, only three contributed to significant inhibition of hyaluronic activity. Recombinant bovine SPAM1 has hyaluronan degradation and cumulus oocyte complex dispersion ability, and the N-linked oligosaccharides are important for enzyme activity, providing a foundation for the commercialization of hyaluronidase.
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KEYWORD
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Hyaluronidase, hyaluronic acid, fertilization, cumulus oocyte complex
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