Structural requirements for the intracellular subunit polymerization of the complement inhibitor C4b-binding protein.
Författare
Summary, in English
C4b-binding protein (C4BP), an important inhibitor of complement activation, has a unique spider-like shape. It is composed of six to seven identical alpha-chains with or without a single beta-chain, the chains being linked by disulfide bridges in their C-terminal parts. To elucidate the structural requirements for the assembly of the alpha-chains, recombinant C4BP was expressed in HEK 293 cells. The expressed C4BP was found to contain six disulfide-linked alpha-chains. Pulse-chase analysis demonstrated that the recombinant C4BP was rapidly synthesized in the cells and the polymerized C4BP appeared in the medium after 40 min. The alpha-chains were polymerized in the endoplasmic reticulum (ER) already after 5 min chase. The polymerization process was unaffected by blockage of the transport from the ER to the Golgi mediated by brefeldin A or low temperature (10 degrees C). The C-terminal part of the alpha-chain (57 amino acids), containing 2 cysteine residues and an amphiphatic alpha-helix region, was required for the polymerization. We constructed and expressed several mutants of C4BP that lacked the cysteine residues and/or were truncated at various positions in the C-terminal region. Gel filtration analysis of these variants demonstrated the whole alpha-helix region to be required for the formation of stable polymers of C4BP, which were further stabilized by the formation of disulfide bonds.
Publiceringsår
2002
Språk
Engelska
Sidor
9349-9357
Publikation/Tidskrift/Serie
Biochemistry
Volym
41
Issue
30
Länkar
Dokumenttyp
Artikel i tidskrift
Förlag
The American Chemical Society (ACS)
Ämne
- Biochemistry and Molecular Biology
Nyckelord
- Hydrolysis
- Protein Conformation
- Receptors
- Complement : chemistry
- Complement : genetics
- Complement : isolation & purification
- Recombinant Proteins : chemistry
- Recombinant Proteins : genetics
- Recombinant Proteins : isolation & purification
- Spectroscopy
- Fourier Transform Infrared
- Human
- DNA Primers
- Chymotrypsin : metabolism
- Cell Line
- Biopolymers : chemistry
- Base Sequence
Status
Published
Forskningsgrupp
- Clinical Chemistry, Malmö
ISBN/ISSN/Övrigt
- ISSN: 0006-2960