title: Hierarchy of post-translational modifications involved in the circulatory longevity of glycoproteins. Demonstration of concerted contributions of glycan sialylation and subunit assembly to the pharmacokinetic behavior of bovine acetylcholinesterase.
abstract: The tetrameric form of native serum-derived bovine acetylcholinesterase is retained in the circulation for much longer periods (mean residence time, MRT = 1390 min) than recombinant bovine acetylcholinesterase (rBoAChE) produced in the HEK-293 cell system (MRT = 57 min). Extensive matrix-assisted laser desorption ionization-time of flight analyses established that the basic structures of the N-glycans associated with the native and recombinant enzymes are similar (the major species (50-60%) are of the biantennary fucosylated type and 20-30% are of the triantennary type), yet the glycan termini of the native enzyme are mostly capped with sialic acid (82%) and alpha-galactose (12%), whereas glycans of the recombinant enzyme exhibit a high level of exposed beta-galactose residues (50%) and a lack of alpha-galactose. Glycan termini of both fetal bovine serum and rBoAChE were altered in vitro using exoglycosidases and sialyltransferase or in vivo by a HEK-293 cell line developed specifically to allow efficient sialic acid capping of beta-galactose-exposed termini. In addition, the dimeric and monomeric forms of rBoAChE were quantitatively converted to tetramers by complexation with a synthetic peptide representing the human ColQ-derived proline-rich attachment domain. Thus by controlling both the level and nature of N-glycan capping and subunit assembly, we generated and characterized 9 distinct bovine AChE glycoforms displaying a 400-fold difference in their circulatory lifetimes (MRT = 3.5-1390 min). This revealed some general rules and a hierarchy of post-translation factors determining the circulatory profile of glycoproteins. Accordingly, an rBoAChE was generated that displayed a circulatory profile indistinguishable from the native form.
authors.affiliation: Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel.
authors.forename: C
authors.initials: C
authors.lastname: Kronman
authors.forename: T
authors.initials: T
authors.lastname: Chitlaru
authors.forename: E
authors.initials: E
authors.lastname: Elhanany
authors.forename: B
authors.initials: B
authors.lastname: Velan
authors.forename: A
authors.initials: A
authors.lastname: Shafferman
mesh_terms: D000110:Acetylcholinesterase / Q000097:blood* / Q000235:genetics / Q000493:pharmacokinetics; D000818:Animals; D002417:Cattle; D002460:Cell Line; D019281:Dimerization; D006023:Glycoproteins / Q000097:blood*; D006801:Humans; D019158:N-Acetylneuraminic Acid; D011499:Protein Processing, Post-Translational
chemical_list: D006023:Glycoproteins; D000110:Acetylcholinesterase; D019158:N-Acetylneuraminic Acid