Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Characterization of Recombinant Bovine Sperm Hyaluronidase and Identification of an Important Asn-X-Ser/Thr Motif for Its Activity

  • Park, Chaeri (College of Pharmacy, Catholic University of Daegu) ;
  • Kim, Young-Hyun (National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Lee, Sang-Rae (National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Park, Soojin (Graduate School of Medicine, Osaka University) ;
  • Jung, Yena (College of Pharmacy, Catholic University of Daegu) ;
  • Lee, Youngjeon (National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Kim, Ji-Su (National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Eom, Taekil (College of Applied Life Sciences, the Research Institute for Subtropical Agriculture and Biotechnology, Jeju National University) ;
  • Kim, Ju-Sung (College of Applied Life Sciences, the Research Institute for Subtropical Agriculture and Biotechnology, Jeju National University) ;
  • Lee, Dong-Mok (Daegyeong Regional Division, Technology Convergence R&BD Group, Korea Institute of Industrial Technology) ;
  • Song, Bong-Suk (National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Sim, Bo-Woong (National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Kim, Sun-Uk (National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Chang, Kyu-Tae (National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Kim, Ekyune (College of Pharmacy, Catholic University of Daegu)
  • Received : 2018.04.16
  • Accepted : 2018.07.19
  • Published : 2018.09.28
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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.

Keywords

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