title: The disulfide bonding pattern in ficolin multimers.
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.
authors.affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Tomoo
authors.initials: T
authors.lastname: Ohashi
authors.forename: Harold P
authors.initials: HP
authors.lastname: Erickson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry*; D002846:Chromatography, Affinity; D006224:Cricetinae; D017077:Culture Media, Conditioned / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D005990:Glycerol / Q000737:chemistry; D037102:Lectins; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000494:pharmacology; D000097604:Ficolins
chemical_list: D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D003545:Cysteine; D004220:Disulfides; D005990:Glycerol; D037102:Lectins; D010100:Oxygen; D011994:Recombinant Proteins; D014357:Trypsin; D000097604:Ficolins