C3b deposition on human erythrocytes induces the formation of a membrane skeleton-linked protein complex.
Decay-accelerating factor (DAF, also known as CD55), a glycosylphosphatidylinositol-linked (GPI-linked) plasma membrane protein, protects autologous cells from complement-mediated damage by inhibiting complement component 3 (C3) activation. An important physical property of GPI-anchored complement regulatory proteins such as DAF is their ability to translate laterally in the plasma membrane. Here, we used single-particle tracking and tether-pulling experiments to measure DAF lateral diffusion, lateral confinement, and membrane skeletal associations in human erythrocyte membranes. In native membranes, most DAF molecules exhibited Brownian lateral diffusion. Fluid-phase complement activation caused deposition of C3b, one of the products of C3 cleavage, onto erythrocyte glycophorin A (GPA). We then determined that DAF, C3b, GPA, and band 3 molecules were laterally immobilized in the membranes of complement-treated cells, and GPA was physically associated with the membrane skeleton. Mass spectrometry analysis further showed that band 3, alpha-spectrin, beta-spectrin, and ankyrin were present in a complex with C3b and GPA in complement-treated cells. C3b deposition was also associated with a substantial increase in erythrocyte membrane stiffness and/or viscosity. We therefore suggest that complement activation stimulates the formation of a membrane skeleton-linked DAF-C3b-GPA-band 3 complex on the erythrocyte surface. This complex may promote the removal of senescent erythrocytes from the circulation.
Karnchanaphanurach
Pallop
P
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02115, USA.
Mirchev
Rossen
R
Ghiran
Ionita
I
Asara
John M
JM
Papahadjopoulos-Sternberg
Brigitte
B
Nicholson-Weller
Anne
A
Golan
David E
DE
D018958:CD55 Antigens / Q000097:blood; D003167:Complement Activation; D003179:Complement C3b / Q000378:metabolism*; D004905:Erythrocyte Aging; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005614:Freeze Fracturing; D006021:Glycophorins / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000097:blood; D006801:Humans; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000097:blood; D052898:Optical Tweezers; D053719:Tandem Mass Spectrometry
D018958:CD55 Antigens; D006021:Glycophorins; D017261:Glycosylphosphatidylinositols; D046912:Multiprotein Complexes; D003179:Complement C3b