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Structural requirements for the intracellular subunit polymerization of the complement inhibitor C4b-binding protein.

Kask, Lena LU ; Hillarp, Andreas LU ; Ramesh, Bala; Dahlbäck, Björn LU and Blom, Anna LU (2002) In Biochemistry 41(30). p.9349-9357
Abstract
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... (More)
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. (Less)
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organization
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type
Contribution to journal
publication status
published
subject
keywords
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
in
Biochemistry
volume
41
issue
30
pages
9349 - 9357
publisher
The American Chemical Society
external identifiers
  • pmid:12135356
  • wos:000177111600010
  • scopus:0037199490
ISSN
0006-2960
DOI
10.1021/bi025980+
language
English
LU publication?
yes
id
5be1f4a6-d9f6-43e4-84be-f1c903a8b6fe (old id 109552)
alternative location
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12135356&dopt=Abstract
date added to LUP
2007-07-25 14:55:56
date last changed
2017-12-03 03:22:33
@article{5be1f4a6-d9f6-43e4-84be-f1c903a8b6fe,
  abstract     = {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.},
  author       = {Kask, Lena and Hillarp, Andreas and Ramesh, Bala and Dahlbäck, Björn and Blom, Anna},
  issn         = {0006-2960},
  keyword      = {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},
  language     = {eng},
  number       = {30},
  pages        = {9349--9357},
  publisher    = {The American Chemical Society},
  series       = {Biochemistry},
  title        = {Structural requirements for the intracellular subunit polymerization of the complement inhibitor C4b-binding protein.},
  url          = {http://dx.doi.org/10.1021/bi025980+},
  volume       = {41},
  year         = {2002},
}