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Recombinant soluble CD59 inhibits reactive haemolysis with complement
1Molecular Medicine Research Laboratories, Yamanouchi Pharmaceutical Co. Ltd, Tsukuba, Japan.
Abstract:
Three soluble forms of membrane attack complex inhibitory factor (MACIF or CD59) were prepared using recombinant baculovirus-infected insect cells. They consisted of 70, 77 and 86 amino acids, starting from the amino terminus of naturally occurring CD59, and were designated recombinant (r) CD59 70, 77 and 86, respectively. All three rCD59 lacked a glycosyl-phosphatidylinositol (GPI) anchor, unlike membrane CD59 which has a GPI anchor at the anchor at the carboxyl terminus (77th amino acid). Their activities in inhibiting complement activation were assayed with C5b-7 intermediate cells and C8 and C9 components. The inhibitory activity of rCD59 70 was as high as that of rCD59 77 and twice that of rCD59 86. In addition, it was one-fourth and one-hundredth lower than the activities of urine and erythrocyte CD59, respectively. However, when assayed in the presence of human serum at a final concentration of 50% (v/v), the activities of both urine and erythrocyte CD59 were greatly decreased to to one-tenth of that of rCD59 70. Purified rCD59 70 molecules were all glycosylated, but rCD59 77 and 86 were mixtures of glycosylated and non-glycosylated molecules. The inhibitory activities of rCD59 77 and 86 were the same for the glycosylated and non-glycosylated forms. These results suggest that the soluble rCD59 provide a means for elucidating the biological roles of CD59.
Insights
Soluble recombinant CD59 (membrane attack complex inhibitory factor) variants were created to study complement inhibition. Recombinant CD59 70 demonstrated potent inhibitory activity, even in human serum, aiding CD59 function research.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Membrane attack complex inhibitory factor (CD59) regulates complement system activation.
- Understanding CD59's role requires soluble, functional variants for research.
Purpose of the Study:
- To produce and characterize soluble recombinant CD59 (rCD59) forms.
- To evaluate the complement inhibitory activities of these rCD59 variants.
Main Methods:
- Three soluble rCD59 forms (70, 77, 86 amino acids) were generated using recombinant baculovirus expression.
- Complement inhibition assays were performed using C5b-7 intermediate cells, C8, and C9.
- Glycosylation status and activity in human serum were assessed.
Main Results:
- rCD59 70 exhibited the highest inhibitory activity, comparable to rCD59 77 and twice that of rCD59 86.
- rCD59 70 retained significant activity in 50% human serum, unlike native CD59 forms.
- Glycosylation did not affect the inhibitory activity of rCD59 77 and 86.
Conclusions:
- Soluble rCD59 variants, particularly rCD59 70, are valuable tools for investigating CD59's biological functions.
- The stability of rCD59 70 in serum suggests potential for therapeutic applications or further in vivo studies.