title: Correlation between the glycan variations and defibrinogenating activities of acutobin and its recombinant glycoforms.
abstract: Acutobin isolated from Deinagkistrodon acutus venom has been used to prevent or treat stroke in patients. This defibrinogenating serine protease is a 39 kDa glycoprotein containing terminal disialyl-capped N-glycans. After sialidase treatment, the enzyme showed similar catalytic activities toward chromogenic substrate, and cleaved the Aα chain of fibrinogen as efficiently as the native acutobin did. However, the level of fibrinogen degradation products in mice after i.p.-injection of desialylated-acutobin was significantly lower than the level after acutobin injection, suggesting that the disialyl moieties may improve or prolong the half-life of acutobin. Two recombinant enzymes with identical protein structures and similar amidolytic activities to those of native acutobin were expressed from HEK293T and SW1353 cells and designated as HKATB and SWATB, respectively. Mass spectrometric profiling showed that their glycans differed from those of acutobin. In contrast to acutobin, HKATB cleaved not only the Aα chain but also the Bβ and γ chains of human fibrinogens, while SWATB showed a reduced α-fibrinogenase activity. Non-denaturing deglycosylation of these proteases by peptide N-glycosidase F significantly reduced their fibrinogenolytic activities and thermal stabilities. The in vivo defibrinogenating effect of HKATB was inferior to that of acutobin in mice. Taken together, our results suggest that the conjugated glycans of acutobin are involved in its interaction with fibrinogen, and that the selection of cells optimally expressing efficient glycoforms and further glycosylation engineering are desirable before a recombinant product can replace the native enzyme for clinical use.
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
authors.forename: Ying-Ming
authors.initials: YM
authors.lastname: Wang
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan.
authors.forename: Inn-Ho
authors.initials: IH
authors.lastname: Tsai
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
authors.forename: Jin-Mei
authors.initials: JM
authors.lastname: Chen
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
authors.forename: An-Chun
authors.initials: AC
authors.lastname: Cheng
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan.
authors.forename: Kay-Hooi
authors.initials: KH
authors.lastname: Khoo
mesh_terms: D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003435:Crotalid Venoms / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D004795:Enzyme Stability / Q000187:drug effects; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects; D005343:Fibrinolytic Agents / Q000494:pharmacology*; D006031:Glycosylation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D010449:Peptide Mapping; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000494:pharmacology; D011817:Rabbits; D011994:Recombinant Proteins / Q000494:pharmacology*; D012712:Serum Globulins / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects; D013917:Thrombin / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*
chemical_list: D003435:Crotalid Venoms; D005338:Fibrin Fibrinogen Degradation Products; D005343:Fibrinolytic Agents; D010455:Peptides; D011134:Polysaccharides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D012712:Serum Globulins; D013917:Thrombin; C508893:acutobin, Deinagkistrodon acutus; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase