INVOLVEMENT OF OLIGOMERIZATION, N-GLYCOSYLATION AND SIALYLATION IN THE CLEARANCE OF CHOLINESTERASES FROM THE CIRCULATION

The possible role of post-translational modifications such as subunit oligomerization, protein glycosylation and oligosaccharide processing on the circulatory life-time of proteins was studied using recombinant human acetylcholinesterase (rHuAChE). Different preparations of rHuAC hE containing various amounts of tetramers, dimers and monomers are cl eared at similar rates from the circulation, suggesting that oligomeri zation does not play an important role in determining the rate of clea rance. An engineered rHuAChE mutant containing only one N-glycosylatio n site was cleared from the circulation more rapidly than the wild-typ e triglycosylated enzyme, On the other hand, hyperglycosylated mutants containing either four or five occupied M-glycosylation sites, analag ous to those present on the slowly cleared fetal bovine serum acetylch olinesterase (FBS-AChE), were also cleared more rapidly from the blood stream than th(: wild-type species. Furthermore, the two different tet raglycosylated mutants were cleared at different rates while the penta glycosylated mutant exhibited the most rapid clearance profile. These results imply that though the number of N-glycosylation sites plays a role in the circulatory life-time of the enzyme, the number of N-glyca n units in itself does not determine the rate of clearance. When satur ating amounts of asialofetuin were administered together with rHuAChE, the circulatory half-life of the enzyme was dramatically increased (f rom 80 min to 19 h) and was found to be similar to that displayed by p lasma-derived cholinesterases while desialylation of these enzymes cau sed a sharp decrease in the circulatory half-life to approximately 3-5 min. Determination of the average number of sialic acid residues per enzyme subunit of the five different N-glycosylation species generated , revealed that the rate of clearance is not a function of the absolut e number of appended sialic acid moieties but rather of the number of unoccupied sialic acid attachment sites per enzyme molecule. Specifica lly, we demonstrate an inverse-linear relationship between the number of vacant sialic acid attachment sites and the values of the enzyme re sidence time within the bloodstream.