Chimeric and truncated forms of human complement protein C8 alpha reveal binding sites for C8 beta and C8 gamma within the membrane attack complex/perforin region.
Human C8 is one of five components of the membrane attack complex of complement. It is an oligomeric protein composed of three subunits (C8 alpha, C8 beta, and C8 gamma) that are derived from different genes. C8 alpha and C8 beta are homologous and both contain a pair of tandemly arranged N-terminal modules [thrombospondin type 1 (TSP1) + low-density lipoprotein receptor class A (LDLRA)], an extended middle segment referred to as the membrane attack complex/perforin region (MACPF), and a pair of C-terminal modules [epidermal growth factor (EGF) + TSP1]. During biosynthetic processing, C8 alpha and C8 gamma associate to form a disulfide-linked dimer (C8 alpha-gamma) that binds to C8 beta through a site located on C8 alpha. In this study, the location of binding sites for C8 beta and C8 gamma and the importance of the modules in these interactions were investigated by use of chimeric and truncated forms of C8 alpha in which module pairs were either exchanged for those in C8 beta or deleted. Results show that exchange or deletion of one or both pairs of modules does not abrogate the ability of C8 alpha to form a disulfide-linked dimer when coexpressed with C8 gamma in COS cells. Furthermore, each chimeric and truncated form of C8 alpha-gamma retains the ability to bind C8 beta; however, only those containing the TSP1 + LDLRA modules from C8 alpha are hemolytically active. These results indicate that binding sites for C8 beta and C8 gamma reside within the MACPF region of C8 alpha and that interaction with either subunit is not dependent on the modules. They also suggest that the N-terminal modules in C8 alpha are important for C9 binding and/or expression of C8 activity.
Plumb
M E
ME
Department of Chemistry and Biochemistry, School of Medicine, University of South Carolina, Columbia 29208, USA.
Scibek
J J
JJ
Barber
T D
TD
Dunlap
R J
RJ
Platteborze
P L
PL
Sodetz
J M
JM
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D003185:Complement C8 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015941:Complement Hemolytic Activity Assay; D015938:Complement Membrane Attack Complex / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*
C087169:C8G protein, human; D003185:Complement C8; D015938:Complement Membrane Attack Complex; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D052899:Pore Forming Cytotoxic Proteins; D011993:Recombinant Fusion Proteins; D054353:Perforin