In vitro expression of the beta subunit of human complement component C8.
Human C8 is one of five components of the cytolytic C5b-9 complex of complement. It is an oligomeric protein composed of three subunits (alpha, beta, gamma) encoded in separate genes. These are arranged as a disulfide-linked alpha-gamma dimer and a noncovalently associated beta chain. Biosynthesis studies and analyses of humans with hereditary C8 deficiencies suggest that C8 alpha-gamma synthesis and secretion can occur independently of C8 beta, but that serum levels of C8 beta are dependent on C8 alpha-gamma. One aim of the present study was to determine if functional human C8 beta could be synthesized in the absence of C8 alpha-gamma. Human C8 beta expression constructs were prepared and used to produce recombinant C8 beta (rC8 beta) in insect and COS-7 cells. Both cell types secreted rC8 beta that was similar in size to human C8 beta and exhibited similar ability to associate with human C8 alpha-gamma and form functional C8. A mutant form of C8 beta in which N-glycosylation sites were eliminated was also expressed and found to be functionally similar to rC8 beta and human C8 beta. These results indicate that C8 alpha-gamma is not required for intracellular processing and secretion of C8 beta. Furthermore, N-linked carbohydrate on C8 beta is not necessary for association with C8 alpha-gamma or for C8 activity.
Letson
C S
CS
Department of Chemistry and Biochemistry, University of South Carolina, Columbia 29208, USA.
Kaufman
K M
KM
Sodetz
J M
JM
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D003185:Complement C8 / Q000096:biosynthesis* / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000096:biosynthesis; D006031:Glycosylation; D006461:Hemolysis; D006801:Humans; D007313:Insecta / Q000166:cytology / Q000235:genetics; D010446:Peptide Fragments / Q000096:biosynthesis / Q000097:blood / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism
D003185:Complement C8; D010446:Peptide Fragments; D011994:Recombinant Proteins