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.
Highlights
Complement is a central part of the immune system that neutralizes pathogens via antibody-dependent and -independent mechanisms and stimulates a variety of protective responses including phagocytosis, inflammation, and adaptive immunity [1]
We find that MASP binding, comparable with that by mannose-binding lectin (MBL), can be engineered through just three amino acid substitutions to the collagenous domain of surfactant protein-A (SP-A)
To examine the tolerance of MBL to such changes, we made a series of modified MBLs in which the collagenous domain was modified
Summary
Protein Components—Recombinant rat MBL and modified forms of rat MASP-2 were produced in a Chinese hamster ovary cell expression system and purified as described previously by affinity chromatography on mannose-Sepharose and nickel-Sepharose columns, respectively [9, 19]. Recombinant wild-type rat MASP-2 is toxic to producing Chinese hamster ovary cells and autoactivates during biosynthesis. Mutant forms of MBL were created by PCR and expressed in the same way as the wild-type protein. MASP-2K was mixed with wild-type or mutant MBL or SP-A and added to a suspension of mannose- or fucose-Sepharose (5 l of a 1:1 v/v suspension in a total volume of 30 l) in 50 mM Tris-HCl (pH 7.5), containing 150 mM NaCl and 5 mM CaCl2, at 37 °C with mixing. Data are the mean Ϯ S.E. from at least two separate experiments using different protein preparations, unless otherwise stated
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