Localization of classical and alternative pathway regulatory activity within the decay-accelerating factor

Area of Science:

• Immunology
• Biochemistry
• Cell Biology

Background:

• Decay-accelerating factor (DAF) is a cell-surface protein regulating complement system activation.
• DAF protects host cells from autologous complement attack by dissociating C3 convertases.
• DAF comprises four short consensus repeats (SCRs) and is anchored by a glycoinositol phospholipid (GPI) moiety.

Purpose of the Study:

• To investigate the functional roles of individual SCR domains within DAF in regulating complement pathways.
• To determine the specific SCRs responsible for classical and alternative pathway C3 convertase regulation.
• To elucidate the mechanism of DAF's regulatory function, distinguishing domain-specific interactions from spatial arrangement.

Main Methods:

• Preparation of recombinant GPI-anchored DAF proteins lacking individual SCR domains.
• Incorporation of variant DAF proteins into sheep erythrocyte hemolytic intermediates.
• Assays to assess the intrinsic regulatory abilities of variant DAF on classical and alternative complement pathway activation.
• Fluid phase C3 activation assays to compare functional differences of variant DAF proteins.

Main Results:

• Classical pathway C3 convertase regulation is mediated by SCR-2 and SCR-3.
• Alternative pathway C3 convertase regulation involves SCR-2, SCR-3, and SCR-4.
• Functional differences arise from domain-specific interactions rather than altered spatial arrangements.
• DAF variants with SCR-1, -2, and -3 selectively inhibit classical pathway activation, excluding SCR-4.

Conclusions:

• SCR-2 and SCR-3 are essential for DAF's classical pathway regulatory function.
• SCR-2, -3, and -4 collectively contribute to DAF's alternative pathway regulatory function.
• DAF's regulatory efficacy is dependent on specific domain interactions within its SCRs.