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      Engineering Novel Complement Activity into a Pulmonary Surfactant Protein*

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          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.

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          Most cited references29

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          A novel serum protein similar to C1q, produced exclusively in adipocytes.

          We describe a novel 30-kDa secretory protein, Acrp30 (adipocyte complement-related protein of 30 kDa), that is made exclusively in adipocytes and whose mRNA is induced over 100-fold during adipocyte differentiation. Acrp30 is structurally similar to complement factor C1q and to a hibernation-specific protein isolated from the plasma of Siberian chipmunks; it forms large homo-oligomers that undergo a series of post-translational modifications. Like adipsin, secretion of Acrp30 is enhanced by insulin, and Acrp30 is an abundant serum protein. Acrp30 may be a factor that participates in the delicately balanced system of energy homeostasis involving food intake and carbohydrate and lipid catabolism. Our experiments also further corroborate the existence of an insulin-regulated secretory pathway in adipocytes.
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            Paths reunited: Initiation of the classical and lectin pathways of complement activation.

            Understanding the structural organisation and mode of action of the initiating complex of the classical pathway of complement activation (C1) has been a central goal in complement biology since its isolation almost 50 years ago. Nevertheless, knowledge is still incomplete, especially with regard to the interactions between its subcomponents C1q, C1r and C1s that trigger activation upon binding to a microbial target. Recent studies have provided new insights into these interactions, and have revealed unexpected parallels with initiating complexes of the lectin pathway of complement: MBL-MASP and ficolin-MASP. Here, we develop and expand these concepts and delineate their implications towards the key aspects of complement activation via the classical and lectin pathways.
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              Structural basis of sequence-specific collagen recognition by SPARC.

              Protein interactions with the collagen triple helix play a critical role in collagen fibril formation, cell adhesion, and signaling. However, structural insight into sequence-specific collagen recognition is limited to an integrin-peptide complex. A GVMGFO motif in fibrillar collagens (O denotes 4-hydroxyproline) binds 3 unrelated proteins: von Willebrand factor (VWF), discoidin domain receptor 2 (DDR2), and the extracellular matrix protein SPARC/osteonectin/BM-40. We report the crystal structure at 3.2 A resolution of human SPARC bound to a triple-helical 33-residue peptide harboring the promiscuous GVMGFO motif. SPARC recognizes the GVMGFO motifs of the middle and trailing collagen chains, burying a total of 720 A(2) of solvent-accessible collagen surface. SPARC binding does not distort the canonical triple helix of the collagen peptide. In contrast, a critical loop in SPARC is substantially remodelled upon collagen binding, creating a deep pocket that accommodates the phenylalanine residue of the trailing collagen chain ("Phe pocket"). This highly restrictive specificity pocket is shared with the collagen-binding integrin I-domains but differs strikingly from the shallow collagen-binding grooves of the platelet receptor glycoprotein VI and microbial adhesins. We speculate that binding of the GVMGFO motif to VWF and DDR2 also results in structural changes and the formation of a Phe pocket.
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                Author and article information

                Journal
                J Biol Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                0021-9258
                1083-351X
                2 April 2010
                29 January 2010
                29 January 2010
                : 285
                : 14
                : 10546-10552
                Affiliations
                From the Departments of []Infection, Immunity, and Inflammation and
                []Biochemistry, University of Leicester, Leicester LE1 9HN and
                the [§ ]Clinical Sciences Research Institute, University of Warwick, Coventry CV4 7AL, United Kingdom
                Author notes
                [4 ] Research Council United Kingdom Academic Fellow. To whom correspondence should be addressed: Dept. of Infection, Immunity, and Inflammation, Maurice Shock Bldg., University of Leicester. P. O. Box 138, Leicester LE1 9HN, United Kingdom. Tel.: 44 (0)116 252 5089; Fax: 44 (0)116 252 5030; E-mail: rw73@ 123456le.ac.uk .
                [1]

                Both authors contributed equally to this work.

                [2]

                Research Council United Kingdom Academic Fellow.

                [3]

                Wellcome Trust Value in People Award Fellow.

                Article
                M109.097493
                10.1074/jbc.M109.097493
                2856262
                20118239
                b91c5bc3-b08e-4fea-812e-991fb17cf1f2
                © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version full access.

                Creative Commons Attribution Non-Commercial License applies to Author Choice Articles

                History
                : 21 December 2009
                : 21 January 2010
                Categories
                Protein Structure and Folding

                Biochemistry
                collagen,lectin,complement,immunology,immunochemistry,proteolytic enzymes,protein engineering,protein-protein interactions

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