title: C-mannosylation of human hyaluronidase 1: possible roles for secretion and enzymatic activity.
abstract: Protein glycosylation, one of the post-translational modifications, is important for many protein functions, such as protein stability, folding and secretion. In the protein glycosylation, C-mannosylation was first identified in ribonuclease 2, and some proteins have been reported to be C-mannosylated; however, effects of its modifications for target proteins remain unclear. Hyaluronidase 1 (HYAL1), degrading hyaluronic acid (HA), has two predicted C-mannosylation sites at Trp¹³⁰ and Trp³²¹. In this study, we examined whether HYAL1 is C-mannosylated or not, and the effect of C-mannosylation on HYAL1. Using mass spectrometry, we first demonstrated that intracellular HYAL1 is C-mannosylated at Trp¹³⁰ but not at Trp³²¹. Surprisingly, although HYAL1 was secreted into conditioned medium and it possessed enzymatic activity, secreted HYAL1 was not C-mannosylated. Computer simulation demonstrated that C-mannosylation of HYAL1 at Trp¹³⁰ changed conformation of the catalytic active site, and faced Glu¹³¹ in the opposite direction toward its substrate, HA, indicating that C-mannosylation will negatively regulate its secretion, and will attenuate its enzymatic activity. Taken together, this is the first report that demonstrates the presence of C-mannosylation among HYAL family proteins, and our results suggest possible roles of C-mannosylation for secretion and enzymatic activity.
authors.affiliation: Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Goto
authors.affiliation: Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Niwa
authors.affiliation: Global Research Cluster, RIKEN, Japan.
authors.forename: Takehiro
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Global Research Cluster, RIKEN, Japan.
authors.forename: Naoshi
authors.initials: N
authors.lastname: Dohmae
authors.affiliation: Department of Molecular Target Medicine Screening, Aichi Medical University School of Medicine, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Umezawa
authors.affiliation: Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Japan.
authors.forename: Siro
authors.initials: S
authors.lastname: Simizu
mesh_terms: D020134:Catalytic Domain; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned; D006031:Glycosylation; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism*; D006821:Hyaluronoglucosaminidase / Q000737:chemistry* / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism*; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary
chemical_list: D017077:Culture Media, Conditioned; D006820:Hyaluronic Acid; D006821:Hyaluronoglucosaminidase; D008358:Mannose