In vitro expression of the beta subunit of human complement component C8

Area of Science:

• Immunology
• Molecular Biology
• Protein Biochemistry

Background:

• Human complement component 8 (C8) is a crucial part of the cytolytic C5b-9 complex.
• C8 is an oligomeric protein comprising alpha, beta, and gamma subunits, with alpha and gamma forming a disulfide-linked dimer, and beta associated noncovalently.
• Previous studies suggest C8 alpha-gamma synthesis is independent of C8 beta, but C8 beta serum levels depend on C8 alpha-gamma.

Purpose of the Study:

• To investigate whether functional human C8 beta can be synthesized and secreted without the presence of C8 alpha-gamma.
• To determine the role of N-linked glycosylation on C8 beta in its secretion, association with C8 alpha-gamma, and overall C8 complex activity.

Main Methods:

• Expression of human C8 beta and a mutant C8 beta lacking N-glycosylation sites in insect and COS-7 cells.
• Analysis of secreted recombinant C8 beta (rC8 beta) size and its ability to associate with human C8 alpha-gamma.
• Functional assessment of the reconstituted C8 complex.

Main Results:

• Recombinant C8 beta (rC8 beta) was successfully synthesized and secreted by both insect and COS-7 cells.
• Secreted rC8 beta was similar in size to native human C8 beta and could functionally associate with C8 alpha-gamma to form active C8.
• A mutant C8 beta lacking N-glycosylation sites was also functionally similar to wild-type rC8 beta and native C8 beta.

Conclusions:

• The C8 alpha-gamma subunit is not required for the intracellular processing and secretion of C8 beta.
• N-linked glycosylation on C8 beta is dispensable for its association with C8 alpha-gamma and for the formation of a functional C8 complex.