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.
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.
Kronman
C
C
Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel.
Chitlaru
T
T
Elhanany
E
E
Velan
B
B
Shafferman
A
A
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
D006023:Glycoproteins; D000110:Acetylcholinesterase; D019158:N-Acetylneuraminic Acid