Solvent accessibility of native and hydrolyzed human complement protein 3 analyzed by hydrogen/deuterium exchange and mass spectrometry

J Immunol. 2005 Mar 15;174(6):3469-74. doi: 10.4049/jimmunol.174.6.3469.


Complement protein C3 is a 187-kDa (1641-aa) protein that plays a key role in complement activation and immune responses. Its hydrolyzed form, C3(H2O), is responsible for the initiation of the activation of alternative complement pathway. Previous analyses using mAbs, anilinonaphthalenesulfonate dyes, and functional studies have suggested that C3 is conformationally different from C3(H2O). We have used amide hydrogen/deuterium exchange and MALDI-TOF mass spectrometry to identify and localize structural differences between native C3 and C3(H2O). Both proteins were incubated in D2O for varying amounts of time, digested with pepsin, and then subjected to mass-spectrometric analysis. Of 111 C3 peptides identified in the MALDI-TOF analysis, 31 had well-resolved isotopic mass envelopes in both C3 and C3(H2O) spectra. Following the conversion of native C3 to C3(H2O), 17 of these 31 peptides exhibited a change in deuterium incorporation, suggesting a conformational change in these regions. Among the identified peptides, hydrogen/deuterium exchange data were obtained for peptides 944-967, 1211-1228, 1211-1231, 1259-1270, 1259-1273, 1295-1318, and 1319-1330, which span the factor H binding site on C3d and factor I cleavage sites, and peptides 1034-1048, 1049-1058, 1069-1080, 1130-1143, 1130-1145, 1211-1228, 1211-1231, 1259-1270, and 1259-1273, spanning 30% of the C3d region of C3. Our results suggest that hydrolysis may produce a looser (more open) structure in the C3d region, in which some of the changes affect the conversion of helical segments into coil segments facilitating interactions with factors I and H. This study represents the first detailed study mapping the regions of C3 involved in conformational transition when hydrolyzed to C3(H2O).

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Complement C3 / chemistry*
  • Complement C3 / genetics
  • Complement C3 / metabolism
  • Complement Factor H / metabolism
  • Deuterium / chemistry
  • Fibrinogen / metabolism
  • Humans
  • Hydrogen / chemistry
  • Hydrolysis
  • In Vitro Techniques
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Conformation
  • Protein Subunits
  • Solvents
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization


  • CFH protein, human
  • Complement C3
  • Peptide Fragments
  • Protein Subunits
  • Solvents
  • Hydrogen
  • Complement Factor H
  • Fibrinogen
  • Deuterium