title: Establishment of a fluorescence-based method to evaluate endocytosis of desialylated glycoproteins in vitro.
abstract: Insufficient sialylation can result in rapid clearance of therapeutic glycoproteins by intracellular degradation, which is mainly mediated by asialoglycoprotein receptors (ASGPRs) on hepatic cells. In contrast, for glycoproteins, a long half-life is often related to high level of terminal sialic acid. These could be extremely important for insufficient sialylated biomedicines in clinic, and development of therapeutic glycoproteins in laboratory. However, how the desialylated glycoproteins are removed and how to evaluate the ASGPRs mediated endocytosis in vitro needs further investigate. Herein we described an integrative characterization of ASGPRs in vitro to elucidate its endocytosis properties. The endocytosis was determined by a fluorescence-based quantization method. The results showed that the ASGPRs could bind to poorly sialylated glycoproteins including asialofetuin and low sialylated recombinant Factor VIIa with a relatively higher ASGPRs binding affinity, and induce a more rapid endocytosis in vitro. Moreover, the mechanism under the internalization of ASGPRs was also investigated, which was found to depend on clathrin and caveolin. Utilizing the relative fluorescence quantification can be suitable for measurement of insufficient sialylated glycoprotein endocytosis and quality control of therapeutic glycoproteins, which could be useful for the understanding of the development of therapeutic glycoproteins.
authors.affiliation: Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009 China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Luo
authors.affiliation: Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009 China.
authors.forename: Song
authors.initials: S
authors.lastname: Chen
authors.affiliation: Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009 China.
authors.forename: Na
authors.initials: N
authors.lastname: Xu
authors.affiliation: Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009 China.
authors.forename: Wen Bo
authors.initials: WB
authors.lastname: Sai
authors.affiliation: Jiangsu Chia Tai Tianqing Pharmaceutical Co., Ltd., Nanjing, 210023 China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhao
authors.affiliation: Jiangsu Chia Tai Tianqing Pharmaceutical Co., Ltd., Nanjing, 210023 China.
authors.forename: Ying Chun
authors.initials: YC
authors.lastname: Li
authors.affiliation: Jiangsu Chia Tai Tianqing Pharmaceutical Co., Ltd., Nanjing, 210023 China.
authors.forename: Xiao Jing
authors.initials: XJ
authors.lastname: Hu
authors.affiliation: Jiangsu Chia Tai Tianqing Pharmaceutical Co., Ltd., Nanjing, 210023 China.
authors.forename: Hong
authors.initials: H
authors.lastname: Tian
authors.affiliation: Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009 China. Electronic address: xdgao@cpu.edu.cn.
authors.forename: Xiang Dong
authors.initials: XD
authors.lastname: Gao
authors.affiliation: Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009 China. Electronic address: wbyao@cpu.edu.cn.
authors.forename: Wen Bing
authors.initials: WB
authors.lastname: Yao
mesh_terms: D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000378:metabolism; D016466:CHO Cells; D022461:Caveolins / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D034281:Dynamins / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D015942:Factor VIIa / Q000378:metabolism; D060748:Fetuins / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005453:Fluorescence; D005470:Fluorometry / Q000379:methods*; D006023:Glycoproteins / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D013997:Time Factors
chemical_list: D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D022461:Caveolins; D002966:Clathrin; D060748:Fetuins; D006023:Glycoproteins; D011994:Recombinant Proteins; C017029:asialofetuin; C103587:recombinant FVIIa; D015942:Factor VIIa; D034281:Dynamins; D019158:N-Acetylneuraminic Acid; D016650:Fluorescein-5-isothiocyanate
keywords: Asialoglycoprotein receptors; Endocytosis; Fluorescence quantification