Publications [#113165] of Harold P. Erickson

Papers Published

1. T Ohashi, HP Erickson, The disulfide bonding pattern in ficolin multimers., The Journal of biological chemistry, vol. 279 no. 8 (February, 2004), pp. 6534-9, ISSN 0021-9258 [doi]
   (last updated on 2013/05/16)

   Abstract:
   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.

   Keywords:
   Amino Acid Sequence • Animals • Blotting, Western • CHO Cells • Carrier Proteins • Chromatography, Affinity • Cricetinae • Culture Media, Conditioned • Cysteine • Dimerization • Disulfides • Electrophoresis, Polyacrylamide Gel • Glycerol • Lectins* • Microscopy, Electron • Models, Molecular • Molecular Sequence Data • Mutation • Oxygen • Protein Conformation • Protein Structure, Tertiary • Recombinant Proteins • Sequence Homology, Amino Acid • Trypsin • chemistry • chemistry* • metabolism • pharmacology