Expression and characterization of recombinant subunits of human complement component C8: further analysis of the function of C8 alpha and C8 gamma.
Human C8 is composed of three nonidentical subunits (C8 alpha, C8 beta, and C8 gamma) that are encoded in separate genes. In C8 isolated from serum, these are arranged as a disulfide-linked C8 alpha-gamma dimer that is noncovalently associated with C8 beta. In this study, a recombinant form of C8 alpha-gamma was expressed independently of C8 beta in insect cells and COS-7 cells and was shown to be equivalent to serum-derived C8 alpha-gamma with respect to its ability to combine with C8 beta and form functional C8. Also expressed separately were mutant (mut) forms of C8 alpha and C8 gamma in which the single interchain disulfide bond was eliminated. MutC8 alpha exhibited the ability to combine with C8 beta and express hemolytic activity, although at a lower level than human C8. Addition of purified mutC8 gamma increased this activity, presumably by binding to mutC8 alpha. A possible role for C8 gamma as a retinol binding protein was also investigated. Absorbance spectroscopy and fluorescence emission and quenching revealed no specific binding of retinol to mutC8 gamma. Together, these results indicate that 1) the biosynthesis and secretion of C8 alpha-gamma is not dependent on C8 beta, which is consistent with in vivo observations in C8 beta-deficient humans; 2) C8 alpha can be synthesized independently of C8 gamma; therefore, protection of C8 alpha from premature membrane interactions during biosynthetic processing is not a likely function of C8 gamma; 3) C8 gamma enhances but is not required for expression of C8 activity; and 4) C8 gamma does not bind retinol; therefore, it cannot function as a retinol transport protein.
Schreck
S F
SF
Department of Chemistry and Biochemistry, School of Medicine, University of South Carolina, Columbia 29208, USA.
Plumb
M E
ME
Platteborze
P L
PL
Kaufman
K M
KM
Michelotti
G A
GA
Letson
C S
CS
Sodetz
J M
JM
D019943:Amino Acid Substitution / Q000235:genetics / Q000276:immunology; D000818:Animals; D019556:COS Cells; D003185:Complement C8 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D019281:Dimerization; D006801:Humans; D009036:Moths; D016297:Mutagenesis, Site-Directed / Q000276:immunology; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D012177:Retinol-Binding Proteins / Q000032:analysis
D003185:Complement C8; D011994:Recombinant Proteins; D012177:Retinol-Binding Proteins