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The disulfide bonding pattern in ficolin multimers.

Publication ,  Journal Article
Ohashi, T; Erickson, HP
Published in: J Biol Chem
February 20, 2004

Ficolin is a plasma lectin, consisting of a short N-terminal multimerization domain, a middle collagen domain, and a C-terminal fibrinogen-like domain. The collagen domains assemble the subunits into trimers, and the N-terminal domain assembles four trimers into 12-mers. Two cysteine residues in the N-terminal domain are thought to mediate multimerization by disulfide bonding. We have generated three mutants of ficolin alpha in which the N-terminal cysteines were substituted by serines (Cys4, Cys24, and Cys4/Cys24). The N-terminal cysteine mutants were produced in a mammalian cell expression system, purified by affinity chromatography, and analyzed under nondenaturing conditions to resolve the multimer structure of the native protein and under denaturing conditions to resolve the disulfide-linked structure. Glycerol gradient sedimentation and electron microscopy in nondenaturing conditions showed that plasma and recombinant wild-type protein formed 12-mers. The Cys4 mutant also formed 12-mers, but Cys24 and Cys4/Cys24 mutants formed only trimers. This means that protein interfaces containing Cys4 are stable as noncovalent protein-protein interactions and do not require disulfides, whereas those containing Cys24-Cys24 require the disulfides for stability. Proteins were also analyzed by nonreducing SDS-PAGE to show the covalent structure under denaturing conditions. Wild-type ficolin was covalently linked into 12-mers, whereas elimination of either Cys4 or Cys24 gave dimers and monomers. We present a model in which symmetric Cys24-Cys24 disulfide bonds between trimers are the basis for multimerization. The model may also be relevant to collectin multimers.

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Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

February 20, 2004

Volume

279

Issue

8

Start / End Page

6534 / 6539

Location

United States

Related Subject Headings

  • Trypsin
  • Sequence Homology, Amino Acid
  • Recombinant Proteins
  • Protein Structure, Tertiary
  • Protein Conformation
  • Oxygen
  • Mutation
  • Molecular Sequence Data
  • Models, Molecular
  • Microscopy, Electron
 

Citation

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Ohashi, T., & Erickson, H. P. (2004). The disulfide bonding pattern in ficolin multimers. J Biol Chem, 279(8), 6534–6539. https://doi.org/10.1074/jbc.M310555200
Ohashi, Tomoo, and Harold P. Erickson. “The disulfide bonding pattern in ficolin multimers.J Biol Chem 279, no. 8 (February 20, 2004): 6534–39. https://doi.org/10.1074/jbc.M310555200.
Ohashi T, Erickson HP. The disulfide bonding pattern in ficolin multimers. J Biol Chem. 2004 Feb 20;279(8):6534–9.
Ohashi, Tomoo, and Harold P. Erickson. “The disulfide bonding pattern in ficolin multimers.J Biol Chem, vol. 279, no. 8, Feb. 2004, pp. 6534–39. Pubmed, doi:10.1074/jbc.M310555200.
Ohashi T, Erickson HP. The disulfide bonding pattern in ficolin multimers. J Biol Chem. 2004 Feb 20;279(8):6534–6539.

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

February 20, 2004

Volume

279

Issue

8

Start / End Page

6534 / 6539

Location

United States

Related Subject Headings

  • Trypsin
  • Sequence Homology, Amino Acid
  • Recombinant Proteins
  • Protein Structure, Tertiary
  • Protein Conformation
  • Oxygen
  • Mutation
  • Molecular Sequence Data
  • Models, Molecular
  • Microscopy, Electron