Methylamine reaction and denaturation-dependent fragmentation of complement component 3. Comparison with...

Area of Science:

• Biochemistry
• Protein Chemistry
• Molecular Biology

Background:

• Complement protein C-3 plays a crucial role in the immune system.
• Understanding the reactive sites and fragmentation patterns of complement proteins is essential for elucidating their function.

Purpose of the Study:

• To investigate the covalent incorporation of methylamine into complement protein C-3.
• To identify the reactive site and characterize the fragmentation of C-3 under denaturation conditions.
• To compare the reactive site and fragmentation behavior of C-3 with alpha2-macroglobulin.

Main Methods:

• Covalent incorporation of [14C]methylamine into C-3.
• Identification of incorporated methylamine derivative using high-performance liquid chromatography and mass spectroscopy.
• Denaturation-dependent fragmentation of C-3 in sodium dodecyl sulfate at 90°C.
• Assessment of methylamine incorporation's effect on C-3 fragmentation.

Main Results:

• Complement protein C-3 incorporates [14C]methylamine stoichiometrically (0.85 mol/mol) into its Mr = 135,000 subunit.
• The incorporated methylamine forms a gamma-glutamylmethylamide derivative, identified via HPLC and mass spectrometry.
• C-3 undergoes denaturation-dependent fragmentation, converting the Mr = 135,000 subunit into Mr = 84,000 and 53,000 fragments.
• Pre-incubation of C-3 with methylamine completely prevents this fragmentation.

Conclusions:

• Complement protein C-3 possesses a reactive site, likely a glutamyl residue, susceptible to methylamine incorporation.
• This reactive site is involved in a denaturation-dependent fragmentation process.
• The findings suggest a common reactive site and mechanism between complement protein C-3 and alpha2-macroglobulin.