title: Engineering novel complement activity into a pulmonary surfactant protein.
abstract: Complement neutralizes invading pathogens, stimulates inflammatory and adaptive immune responses, and targets non- or altered-self structures for clearance. In the classical and lectin activation pathways, it is initiated when complexes composed of separate recognition and activation subcomponents bind to a pathogen surface. Despite its apparent complexity, recognition-mediated activation has evolved independently in three separate protein families, C1q, mannose-binding lectins (MBLs), and serum ficolins. Although unrelated, all have bouquet-like architectures and associate with complement-specific serine proteases: MBLs and ficolins with MBL-associated serine protease-2 (MASP-2) and C1q with C1r and C1s. To examine the structural requirements for complement activation, we have created a number of novel recombinant rat MBLs in which the position and orientation of the MASP-binding sites have been changed. We have also engineered MASP binding into a pulmonary surfactant protein (SP-A), which has the same domain structure and architecture as MBL but lacks any intrinsic complement activity. The data reveal that complement activity is remarkably tolerant to changes in the size and orientation of the collagenous stalks of MBL, implying considerable rotational and conformational flexibility in unbound MBL. Furthermore, novel complement activity is introduced concurrently with MASP binding in SP-A but is uncontrolled and occurs even in the absence of a carbohydrate target. Thus, the active rather than the zymogen state is default in lectin.MASP complexes and must be inhibited through additional regions in circulating MBLs until triggered by pathogen recognition.
authors.affiliation: Department of Infection, Immunity, and Inflammation, University of Leicester, Leicester LE1 9HN, UK.
authors.forename: Umakhanth
authors.initials: U
authors.lastname: Venkatraman Girija
authors.forename: Christopher
authors.initials: C
authors.lastname: Furze
authors.forename: Julia
authors.initials: J
authors.lastname: Toth
authors.forename: Wilhelm J
authors.initials: WJ
authors.lastname: Schwaeble
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Mitchell
authors.forename: Anthony H
authors.initials: AH
authors.lastname: Keeble
authors.forename: Russell
authors.initials: R
authors.lastname: Wallis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003167:Complement Activation; D015922:Complement C1q / Q000235:genetics* / Q000378:metabolism; D037601:Mannose-Binding Lectin / Q000235:genetics / Q000378:metabolism*; D050606:Mannose-Binding Protein-Associated Serine Proteases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering; D037662:Pulmonary Surfactant-Associated Protein A / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
chemical_list: D037601:Mannose-Binding Lectin; D037662:Pulmonary Surfactant-Associated Protein A; D011994:Recombinant Proteins; C069858:mannose binding protein A; D015922:Complement C1q; D050606:Mannose-Binding Protein-Associated Serine Proteases; C491338:Masp2 protein, rat