Localization and characterization of the mannose-binding lectin (MBL)-associated-serine protease-2 binding site in rat ficolin-A: equivalent binding sites within the collagenous domains of MBLs and ficolins

J Immunol. 2007 Jul 1;179(1):455-62. doi: 10.4049/jimmunol.179.1.455.

Abstract

Ficolins and mannose-binding lectins (MBLs) are the first components of the lectin branch of the complement system. They comprise N-terminal collagen-like domains and C-terminal pathogen-recognition domains (fibrinogen-like domains in ficolins and C-type carbohydrate-recognition domains in MBLs), which target surface-exposed N-acetyl groups or mannose-like sugars on microbial cell walls. Binding leads to activation of MBL-associated serine protease-2 (MASP-2) to initiate complement activation and pathogen neutralization. Recent studies have shown that MASP-2 binds to a short segment of the collagen-like domain of MBL. However, the interaction between ficolins and MASP-2 is relatively poorly understood. In this study, we show that the MASP-2 binding site on rat ficolin-A is also located within the collagen-like domain and encompasses a conserved motif that is present in both MBLs and ficolins. Characterization of this motif using site-directed mutagenesis reveals that a lysine residue in the X position of the Gly-X-Y collagen repeat, Lys(56) in ficolin-A, which is present in all ficolins and MBLs known to activate complement, is essential for MASP-2 binding. Adjacent residues also make important contributions to binding as well as to MASP activation probably by stabilizing the local collagen helix. Equivalent binding sites and comparable activation kinetics of MASP-2 suggest that complement activation by ficolins and MBLs proceeds by analogous mechanisms.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • CHO Cells
  • Collagen / chemistry
  • Collagen / metabolism*
  • Complement Activation
  • Conserved Sequence
  • Cricetinae
  • Cricetulus
  • Kinetics
  • Lectins / biosynthesis
  • Lectins / blood
  • Lectins / chemistry*
  • Lectins / metabolism*
  • Lysine / chemistry
  • Lysine / metabolism
  • Mannose-Binding Lectins / chemistry
  • Mannose-Binding Lectins / metabolism
  • Mannose-Binding Protein-Associated Serine Proteases / chemistry*
  • Mannose-Binding Protein-Associated Serine Proteases / isolation & purification
  • Mannose-Binding Protein-Associated Serine Proteases / metabolism*
  • Molecular Sequence Data
  • Protein Binding
  • Protein Structure, Tertiary
  • Rats
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Substrate Specificity / immunology

Substances

  • Lectins
  • Mannose-Binding Lectins
  • Recombinant Proteins
  • ficolin
  • Collagen
  • Mannose-Binding Protein-Associated Serine Proteases
  • Masp2 protein, rat
  • Lysine