Effect of human acetylcholinesterase subunit assembly on its circulatory residence

Sialylated recombinant human acetylcholinesterase (rHuAChE), produced by st ably transfected cells, is composed of a mixed population of monomers, dime rs and tetramers and manifests a time-dependent circulatory enrichment of t he higher-order oligomeric forms. To investigate this phenomenon further, h omogeneous preparations of rHuAChE differing in their oligomerization statu ses were generated: (1) monomers, represented by the oligomerization-impair ed C580A-rHuAChE mutant, (2) wild-type (WT) dimers and (3) tetramers of WT- rHuAChE generated in vitro by complexation with a synthetic ColQ-derived pr oline-rich attachment domain ('PRAD') peptide. Three different series of ea ch of these three oligoform preparations were produced: (1) partly sialylat ed, derived from HEK-293 cells; (2) fully sialylated, derived from engineer ed HEK-293 cells expressing high levels of sialyltransferase; and (3) desia lylated, after treatment with sialidase to remove sialic acid termini quant itatively. The oligosaccharides associated with each of the various prepara tions were extensively analysed by matrix-assisted laser desorption ionizat ion-time-of-flight MS. With the enzyme preparations comprising the fully si alylated series, a clear linear relationship between oligomerization and ci rculatory mean residence time (MRT) was observed. Thus monomers, dimers and tetramers exhibited MRTs of 110, 195 and 740 min respectively. As the leve l of sialylation decreased, this differential behaviour became less pronoun ced; eventually, after desialylation all oligoforms had the same MRT (5 min ). These observations suggest that multiple removal systems contribute to t he elimination of AChE from the circulation. Hen we also demonstrate that b y the combined modulation of sialylation and tetramerization it is possible to generate a rHuAChE displaying a circulatory residence exceeding that of all other known forms of native or recombinant human AChE.