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Covalent binding and hemolytic activity of complement proteins
Insights
Hydroxylamine inactivates the third component of complement (C3), directly correlating hemolytic and binding activities. This supports a model where C3b uses a reactive thioester to bind surfaces.
Area of Science:
- Biochemistry
- Immunology
- Complement System
Background:
- The third component of complement (C3) plays a crucial role in the immune response.
- Understanding the mechanism of C3 activation and its interaction with surfaces is vital for immunology.
Purpose of the Study:
- To investigate the inactivation of C3 by hydroxylamine and methylamine.
- To elucidate the mechanism of covalent binding of C3b to receptive surfaces.
Main Methods:
- Chemical inactivation of C3 using hydroxylamine and [14C]methylamine.
- Assaying C3 hemolytic activity and covalent binding activity.
- Quantifying the labeling of C3 in the C3d domain.
Main Results:
- Hydroxylamine directly inactivates C3, with identical kinetics for hemolytic and covalent binding activities.
- C3 inactivation correlates quantitatively with [14C]methylamine labeling in the C3d domain.
- The proposed model of internal thioester reactivity in C3b binding also applies to C4 but not C5.
Conclusions:
- Covalent, surface-bound C3b is hemolytically active.
- An internal thioester in C3 becomes reactive upon activation to C3b, mediating covalent surface binding.
- This thioester mechanism is conserved in C4 but absent in C5.
Abstract:
We report the inactivation of the third component of complement (C3) by hydroxylamine. C3 hemolytic and covalent binding activities decline with identical kinetics, demonstrating a direct correlation between the two activities. We conclude that covalent, surface-bound C3b is hemolytically active. The inactivation of C3 is first order with respect to hydroxylamine. We also studied C3 inactivation with [14C]methylamine. The inactivation corresponds quantitatively with the labeling of C3 in the C3d domain. The data obtained support the following hypothesis: there is an internal thioester within C3 which becomes highly reactive on activation to C3b, and C3b binds to receptive surfaces by transfer of the acyl function of the thioester to a hydroxyl group on the receptive surface. This proposed model for the reaction of C3 with receptive surfaces also applies to C4, which binds to membrane surfaces covalently and is able to be inactivated by hydroxylamine and methylamine. C5, on the other hand, is not inactivated by treatment with the amines.