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.
Kask
Lena
L
The Wallenberg Laboratory, Department of Clinical Chemistry, Lund University, University Hospital, SE-205 02 Malmö, Sweden.
Hillarp
Andreas
A
Ramesh
Bala
B
Dahlbäck
Björn
B
Blom
Anna M
AM
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