Single-Site Mutational Engineering and Following MonoPEGylation of the Human Lectin Galectin-2: Effects on Ligand Binding, Functional Aspects, and Clearance from Serum
Abstract
The emerging insights into the physiological significance of endogenous lectins prompted us to characterize the effect of monosubstitution with poly(ethylene glycol) (PEG; 5 kDa) on a human lectin. As role model, we used a member of the galectin family, that is, galectin-2, the Cys57Met (single-site) mutant and its monoPEGylated derivative.
The activities of these three proteins were comparatively studied by biochemical, cell biological, and histochemical methods, using surface-immobilized glycoproteins, different types of cells presenting gangliosides or (glyco)proteins as counterreceptors in vitro and tissue sections. PEGylation led to decreases in affinity/signal intensity with context dependence.
The introduction of the mutation, too, can influence reactivity. Assays on haemagglutination and inhibition of cell proliferation underscored that mutational engineering and substitution can (but must not necessarily) affect this protein's activity.
Serum clearance in rats was markedly retarded by PEGylation. Overall, the bulky substitution, spatially comparable to N-glycans, can markedly reduce binding of the galectin to physiological binding sites.