IMMUNOREACTIVITY AND FUNCTION OF OLIGOSACCHARIDES IN COBRA VENOM FACTOR

Cobra venom factor (CVF) is the nontoxic C-activating glycoprotein in cobra venom. It is a structural and functional analogue of complement component C3. Previous work has established that the major oligosaccha ride chain of CVF is a symmetric fucosylated biantennary complex-type N-linked chain containing an alpha-galactosylated Le(x) antigenic epit ope, Gal alpha 1-3Gal beta 1-4 (Fuc alpha 1-3) GlcNAc beta 1, a novel carbohydrate structure. We show that naturally occurring anti-alpha-Ga l Ab present in normal human serum binds to CVF. In view of a possible human application of CVF, we studied the effect of this anti-alpha-Ga l Ab on CVF function as well as the effect of oligosaccharide modifica tion or removal on CVF activity and immunoreactivity with the anti-alp ha-Gal Ab. The immunoreactivity of CVF with the anti-alpha-Gal Ab is a bolished upon de-alpha-galactosylation or complete deglycosylation. De -alpha-galactosylated CVF and deglycosylated CVF were found to be equa lly active in forming a stable C3/C5 convertase with human factor B an d in decomplementing human serum indicating that the oligosaccharide c hains of CVF are not required for its C-activating function. Consisten t with the unaltered functional activity, deglycosylation of the molec ule caused only minor changes in secondary structure as judged by far UV circular dichroism analysis. However, the oligosaccharide chains ap pear to contribute to the thermal stability of CVF, because deglycosyl ation caused the molecule to be more sensitive to temperature. Inasmuc h as rCVF produced in mammalian cells can be expected to contain sialy lated oligosaccharide chains, we also prepared sialylated CVF by enzym atic sialylation of de-alpha-galactosylated and de-alpha-fucosylated C VF with 2,6-sialyltransferase. It was found that the secondary structu re and the activity of sialylated CVF were unchanged compared to nativ e CVF.