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

被引:59
作者
Kronman, C [1 ]
Chitlaru, T [1 ]
Elhanany, E [1 ]
Velan, B [1 ]
Shafferman, A [1 ]
机构
[1] Israel Inst Biol Res, Dept Biochem & Mol Genet, IL-74100 Ness Ziona, Israel
关键词
D O I
10.1074/jbc.M004298200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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收藏
页码:29488 / 29502
页数:15
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