Hierarchy of Post-translational Modifications Involved in the Circulatory Longevity of Glycoproteins

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 ␣-galactose (12%), whereas glycans of the recombinant enzyme exhibit a high level of exposed ␤-galactose residues (50%) and a lack of ␣-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 ␤-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.

Controlled Concealment of Exposed Clearance and Immunogenic Domains by Site-specific Polyethylene Glycol Attachment to Acetylcholinesterase Hypolysine Mutants

“…Cell Culture Techniques-HEK-293 cell lines stably expressing high levels of ⌬C-rHuAChE and various hypolysine mutant rHuAChEs were generated as described previously (8,9). AChEs produced and secreted by these cells are consistently sialylated in a partial manner (ϳ60% sialylation) (8 -10).…”

“…Enzyme Activity, Specific Activity, and Thermostability Assays-AChE activities of purified AChE preparations were measured according to Ellman et al (19) as described previously (8). The various hypolysine AChE protein products were quantified by ELISA, and the specific activity of each mutant product was calculated by dividing the enzymatic activity to protein quantity.…”

“…AChEs produced and secreted by these cells are consistently sialylated in a partial manner (ϳ60% sialylation) (8 -10). The generation of mutant AChEs displaying highly sialylated N-glycans (ϳ90% sialylation) was achieved by transfecting the recombinant sialyltransferase expressor HEK-293ST-2D6 cell line (8) with the vectors coding for various hypolysine AChEs, followed by G418 selection to form stable producer cells.…”

“…The clearance patterns of the various enzyme preparations were usually biphasic and fitted to a bi-exponential elimination pharmacokinetic model (C t ϭ Ae Ϫk␣t ϩ Be Ϫk␤t ) as described previously (8,9). The pharmacokinetic parameter MRT (which reflects the average length of time the administered molecules are retained in the organism) was independently obtained by analyzing the clearance data according to a noncompartmental pharmacokinetic model using the WinNonlin computer program (20).…”

“…Desialylation of purified AChEs was performed as described previously (18). Release, recovery, purification, labeling, and analysis by MALDI-TOF of N-glycans were all described before (8).…”