Dissociation and re-association studies on the interaction domains of mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, provide evidence for heterodimer formation

Mol Immunol. 2014 May;59(1):1-9. doi: 10.1016/j.molimm.2013.12.003. Epub 2014 Jan 11.

Abstract

Activation of the lectin pathway of complement begins with the activation of mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, which are bound to the recognition molecules, MBL and ficolins. MASPs are Ca(2+)-dependent dimers. Dimerization and Ca(2+)-dependent association with the recognition molecules occurs via the first 3 domains, the CUB1-EGF-CUB2 region. The CUB1-EGF-CUB2 (D1-3) regions of MASP-1 and MASP-2, and also their tagged versions, were expressed in E. coli, refolded and purified. The first three domains of MASP-1 are identical with the respective regions of MASP-3 and MAp44, which are also associated with MBL and ficolins. The functionality of the fragments was checked by inhibition of C3 deposition from human serum. Time-course of the dissociation and re-association was examined by size exclusion chromatography. Both refolded proteins are tight Ca(2+)-dependent dimers, as expected. In buffer containing EDTA MASP-1_D1-3 dissociated to monomers, however it took about 1h to reach an equilibrium. Upon re-calcification dimers were re-formed, but this process was even slower; only after overnight incubation was the dimerization completed. MASP-2_D1-3 showed a somewhat different behavior: dissociation by EDTA was even slower, less complete, and higher MW aggregates also appeared. Heterodimer formation was detected by native PAGE. As modeled by the D1-3 fragments, MASP-1 and MASP-2 can readily form heterodimers after dissociation and re-association, however, in the presence of Ca(2+) exchange of subunits is slow between the homodimers. MASP-1:MASP-3 heterodimer formation was modeled by the tagged and untagged D1-3 fragments, and data indicate that subunits of these proteins are readily exchanged even in the presence of Ca(2+). The existence of heterodimers influences the current view on the composition of lectin pathway complexes and their activation.

Keywords: Ca(2+)-dependent dimer; Complement; Dissociation; Heterodimer; Lectin pathway; MBL-associated serine proteases.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites / genetics
  • Calcium / chemistry
  • Calcium / metabolism
  • Complement C3 / metabolism
  • Complement Pathway, Mannose-Binding Lectin
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Ficolins
  • Humans
  • Kinetics
  • Lectins / chemistry*
  • Lectins / metabolism
  • Mannose-Binding Lectin / chemistry*
  • Mannose-Binding Lectin / metabolism
  • Mannose-Binding Protein-Associated Serine Proteases / chemistry*
  • Mannose-Binding Protein-Associated Serine Proteases / genetics
  • Mannose-Binding Protein-Associated Serine Proteases / metabolism
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Protein Binding
  • Protein Multimerization
  • Protein Refolding
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism

Substances

  • Complement C3
  • Lectins
  • Mannose-Binding Lectin
  • Peptide Fragments
  • Recombinant Proteins
  • MASP1 protein, human
  • MASP2 protein, human
  • Mannose-Binding Protein-Associated Serine Proteases
  • Calcium