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Solvent Accessibility of Native and Hydrolyzed Human Complement Protein 3 Analyzed by Hydrogen/Deuterium Exchange and Mass Spectrometry ¹

Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104

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).

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				N. Nishida, T. Walz, and T. A. Springer Structural transitions of complement component C3 and its activation products PNAS, December 26, 2006; 103(52): 19737 - 19742. [Abstract] [Full Text] [PDF]