Structural requirements for the intracellular subunit polymerization of the complement inhibitor C4b-binding protein.
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.
The Wallenberg Laboratory, Department of Clinical Chemistry, Lund University, University Hospital, SE-205 02 Malmö, Sweden.
Lena
L
Kask
Andreas
A
Hillarp
Bala
B
Ramesh
Björn
B
Dahlbäck
Anna M
AM
Blom
D001483:Base Sequence; D001704:Biopolymers / Q000737:chemistry*; D002460:Cell Line; D002918:Chymotrypsin / Q000378:metabolism; D003169:Complement Inactivator Proteins; D017931:DNA Primers; D006023:Glycoproteins; D006801:Humans; D006868:Hydrolysis; D011487:Protein Conformation; D011951:Receptors, Complement / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017550:Spectroscopy, Fourier Transform Infrared
D001704:Biopolymers; D003169:Complement Inactivator Proteins; D017931:DNA Primers; D006023:Glycoproteins; D011951:Receptors, Complement; D011994:Recombinant Proteins; D002918:Chymotrypsin