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 oligosaccharide chain of CVF is a symmetric fucosylated biantennary complex-type N-linked chain containing an alpha-galactosylated Le(x) antigenic epitope, 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-Gal 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-Gal Ab on CVF function as well as the effect of oligosaccharide modification or removal on CVF activity and immunoreactivity with the anti-alpha-Gal Ab. The immunoreactivity of CVF with the anti-alpha-Gal Ab is abolished upon de-alpha-galactosylation or complete deglycosylation. De-alpha-galactosylated CVF and deglycosylated CVF were found to be equally active in forming a stable C3/C5 convertase with human factor B and in decomplementing human serum indicating that the oligosaccharide chains of CVF are not required for its C-activating function. Consistent with the unaltered functional activity, deglycosylation of the molecule caused only minor changes in secondary structure as judged by far UV circular dichroism analysis. However, the oligosaccharide chains appear to contribute to the thermal stability of CVF, because deglycosylation caused the molecule to be more sensitive to temperature. Inasmuch as rCVF produced in mammalian cells can be expected to contain sialylated oligosaccharide chains, we also prepared sialylated CVF by enzymatic sialylation of de-alpha-galactosylated and de-alpha-fucosylated CVF with 2,6-sialyltransferase. It was found that the secondary structure and the activity of sialylated CVF were unchanged compared to native CVF.
Gowda
D C
DC
Department of Biochemistry and Molecular Biology, Georgetown University, Washington, DC 20007.
Petrella
E C
EC
Raj
T T
TT
Bredehorst
R
R
Vogel
C W
CW
D000818:Animals; D002240:Carbohydrate Sequence; D004546:Elapid Venoms / Q000737:chemistry / Q000276:immunology*; D006031:Glycosylation; D006168:Guinea Pigs; D006801:Humans; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D009844:Oligosaccharides / Q000276:immunology*; D017433:Protein Structure, Secondary; D012794:Sialic Acids; D013329:Structure-Activity Relationship
D004546:Elapid Venoms; D009844:Oligosaccharides; D012794:Sialic Acids; C016310:cobra venom factor; D019158:N-Acetylneuraminic Acid