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Related Experiment Videos

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

C S Letson1, K M Kaufman, J M Sodetz

  • 1Department of Chemistry and Biochemistry, University of South Carolina, Columbia 29208, USA.

Molecular Immunology
|December 1, 1996
PubMed
Summary

Functional human complement component 8 beta (C8 beta) can be synthesized and secreted independently of the C8 alpha-gamma subunit. N-linked glycosylation on C8 beta is not essential for its function or association with C8 alpha-gamma.

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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.

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  • 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.