Characterization of recombinant bovine sperm hyaluronidase and identification of an important Asn-X-Ser/Thr motif for its activity

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Title
Characterization of recombinant bovine sperm hyaluronidase and identification of an important Asn-X-Ser/Thr motif for its activity
Author(s)
C Park; Young-Hyun Kim; Sang-Rae Lee; S Park; Y Jung; Youngjeon LeeJi-Su Kim; T Eom; J S Kim; D M Lee; Bong-Seok SongBo Woong SimSun-Uk Kim; Kyu Tae Chang; E Kim
Bibliographic Citation
Journal of Microbiology and Biotechnology, vol. 28, no. 9, pp. 1547-1553
Publication Year
2018
Abstract
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.
Keyword
Cumulus oocyte complexFertilizationHyaluronic acidHyaluronidase
ISSN
1017-7825
Publisher
Korea Soc-Assoc-Inst
DOI
http://dx.doi.org/10.4014/jmb.1804.04016
Type
Article
Appears in Collections:
Ochang Branch Institute > Division of Bioinfrastructure > National Primate Research Center > 1. Journal Articles
Jeonbuk Branch Institute > Primate Resources Center > 1. Journal Articles
Ochang Branch Institute > Division of Bioinfrastructure > Futuristic Animal Resource & Research Center > 1. Journal Articles
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