title: Effects of single N-glycosylation site knockout on folding and defibrinogenating activities of acutobin recombinants from HEK293T.
abstract: Acutobin, the α-fibrinogenase from Deinagkistrodon acutus venom, contains four N-glycosylation sites with disialylated complex-typed glycans. Here, we explore the functional roles of each of the N-glycan by site-directed mutagenesis. The wild-type (ATB-wt) and single glycan-knockout mutants of recombinant acutobin were prepared from HEK293T, demonstrating that mutations at Asn(77), Asn(81) and Asn(100) impaired the folding while the S79A mutant and various Asn(229)-deglycosylated mutants were correctly folded. Based on homology modeling of acutobin and multiple sequence alignment with various venom thrombin-like enzymes, the importance of a hydrophilic environment at each glycosylation site to the enzyme folding could be rationalized. Remarkably, all the mutants showed similar catalytic activities for the chromogenic substrate and similar thermal stabilities as ATB-wt, suggesting that the glycan knockout did not affect the gross conformation and stability of the active sites. Although SDS-PAGE analyses revealed that ATB-wt and the D229-mutant degraded all human fibrinogen subunits faster but less specifically in vitro as compared with other mutants that cleaved only the α-subunit, ATB-wt and D229-mutant were not able to release fibrinogen-peptide A and thus coagulated human plasma slower than the other mutants did. In the mice model, the defibrinogenating effect of ATB-wt was stronger and lasting-longer than those of all the mutants. Taken together, all the glycans contribute to the pharmacokinetics of acutobin and ATB-wt in vivo, and the microenvironment around the Asn(229)-glycan appears to regulate the fibrinogen-chain specificity of acutobin while the N-glycans at positions 77, 81 and 100 are crucial for its folding.
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan; Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan. Electronic address: bc201@gate.sinica.edu.tw.
authors.forename: Inn-Ho
authors.initials: IH
authors.lastname: Tsai
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Ying-Ming
authors.initials: YM
authors.lastname: Wang
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Kai-Fa
authors.initials: KF
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry* / Q000494:pharmacology; D001777:Blood Coagulation / Q000187:drug effects; D003435:Crotalid Venoms / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D004795:Enzyme Stability; D005340:Fibrinogen / Q000737:chemistry; D055786:Gene Knockout Techniques; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013917:Thrombin / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology
chemical_list: D000925:Anticoagulants; D003435:Crotalid Venoms; D011993:Recombinant Fusion Proteins; D005340:Fibrinogen; D013917:Thrombin; C508893:acutobin, Deinagkistrodon acutus
keywords: Anticoagulant agent; Fibrinogenase chain specificity; HEK 293T recombinant; Mutagenesis of N-Glycosylation sites; Snake venom thrombin-like enzyme