Effect of glycosylation on the stability of alpha1-antitrypsin toward urea denaturation and thermal deactivation.

The effects of glycosylation on the stability of human alpha1-antitrypsin were investigated. The transition midpoints in urea-induced equilibrium unfolding of a non-glycosylated recombinant, a yeast version of glycosylated, and human plasma alpha1-antitrypsin were 1.8 M, 2.2 M, and 2.5 M at 25 degrees C, respectively. Kinetic analyses of unfolding and refolding revealed that glycosylation retarded the unfolding without affecting the refolding rate significantly, suggesting that the stability increase is due to the stabilization of the native state as opposed to the destabilization of the unfolded state. In thermal deactivation, which is a heat-induced aggregation process, the unglycosylated recombinant alpha1-antitrypsin was deactivated most easily, which was followed in order by the yeast, and the plasma form. The results indicate that glycosylation confers the increase in stability of alpha1-antitrypsin, and that the oligomannose sugars present on the yeast form produce a less stable molecule than the complex type sugars on the plasma form. It appears that the effect of glycosylation on the enhancement of thermal resistance is exerted through the increase in conformational stability. However, a stable recombinant variant (Phe 51 --> Cys) that showed the same conformational stability as the plasma form was less resistant to thermal denaturation than the plasma alpha1-antitrypsin. The results suggest that the existence of carbohydrate moiety per se as well as the conformational stability contribute to the kinetic stability of alpha1-antitrypsin toward aggregation.