University of Toronto Biochemistry

	David E. Isenman Professor B.Sc., University of Toronto, 1972 Ph.D., Universityof Toronto, 1976 WeizmannInstitute of Science (Rehovot, Israel)1977 Research Institute of Scripps Clinic (La Jolla, California), 1978
	Medical Sciences Building, Room 5326B 416-978-2703 d.isenman@utoronto.ca

Structure-Function Relations in Proteins of the Complement System

Research Synopsis

The complement system refers to a group of some 30 proteins, two thirds of which circulate in blood plasma and the remainder being present on the surfaces of various blood cells and blood vessel walls. Through their interactions, this group of proteins mediate the clearance of antigen-antibody complexes and additionally provide innate immunity against microbial pathogens via antibody-independent mechanisms. Moreover, recent studies confirm that complement plays an important role in the generation of an antibody response against foreign antigen, thereby providing a crucial link between the innate and adaptive immune systems.

The research interests of my laboratory focus on the biochemical characterization of the complement system proteins comprising the C3/C4/C5 superfamily. As can be appreciated from the schematic, activated forms of C3 and C4 are on the one hand subunits of multicomponent proteases which are the key points of amplification and effector function initiation in the complement system and on the other hand, when deposited onto a pathogen they act as opsonins that facilitate "bridging" to several classes of complement receptors (CR) present on circulating blood phagocytes and B lymphocytes. Understanding the nature of some of these protein-protein interactions in biochemical detail may at some point facilitate the rational design of compounds which might alleviate the acute or chronic inflammatory conditions which are mediated by complement.

The Central Role of C3 in Complement Biology

For components C3 and C4, there are respectively at least 12 and 7 distinct complement family proteins with which these molecules interact. In order to localize binding sites within C3 and C4 for the various soluble and membrane-bound proteins with which they interact, we have been employing a protein engineering approach to test candidate binding sites suggested by the cumulative data in the literature. This involves either making site-directed mutants within the context of the intact molecule and assessing specific functions of the expressed mutant molecules, or expressing segments of the molecule and testing whether these segments possess a particular binding function. Additionally, we have crystallized C3d and C4d, and in collaboration with the labs of Drs. James Rini and David Rose, respectively, obtained the structure of those two major fragments of C3 and C4 (see figure). Consequently, for binding interactions that are localized in whole or in part to the C3d or C4d domains, the mutagenic approach can now be guided by their three dimensional structure. Additional structural projects are ongoing.

Selected Publications

Nagar, B., Jones, R.G., Diefenbach, R.J., Isenman, D.E. and Rini, J.M. (1998) X- ray crystal structure of C3d: A C3 fragment and ligand for complement receptor 2. Science 280, 1277-1281.

Oran, A.E., and Isenman, D.E. (1999) Identification of residues within the 727-767 segment of human complement component C3 important for its interaction with factor H and with complement receptor 1 (CR1, CD35). J. Biol. Chem. 274, 5120-5130.

Pan, Q., Ebanks, R.O., and Isenman, D.E. (2000) Two clusters of acidic amino acids near the NH2-terminus of complement component C4 a'-chain are important for C2 binding J. Immunol. 165, 2518-2527.

Clemenza, L. and Isenman, D.E. (2000) Structure-guided identification of C3d residues essential for its binding to complement receptor 2 (CD21). J. Immunol. 165, 3839-3848.

van den Elsen, J.M.H., Martin, A., Wong, V.B., Clemenza, L., Rose, D.R., and Isenman, D.E. (2002) X-ray crystal structure of the C4d fragment of human complement component C4. J. Mol Biol. 322, 1103-1115.

Clemenza, L. and Isenman, D.E. (2004) The C4A and C4B isotypic forms of human complement fragment C4b have the same intrinsic affinity for complement receptor 1 (CR1/CD35). J. Immunol. 172, 1670-1680.

Leung, E., Blom, A., Clemenza, L. and Isenman, D.E. (2006). The complement regulator C4b-binding protein (C4BP) interacts with both the C4c and C4dg subfragments of the parent C4b ligand: Evidence for synergy in C4BP subsite binding. Biochemistry 45 , 8378-8392.

Burman, J., Leung, E., Atkins, K.L., O'Seaghdha, M.N., Lango, L., Bernadó, P., Bagby, S., Svergun, D.I., Foster, T.J., Isenman, D.E. , and van den Elsen, J.M.H. (2008). Interaction of Human Complement with Sbi, a Staphylococcal Immunoglobulin-binding Protein: Indications of a Novel Mechanism of Complement Evasion by Staphylococcus aureus. Journal of Biological Chemistry 283 , 17579-17593.

Upadhyay, A., Burman, J.D., Clark , E.A., Leung, E., Isenman, D.E. , van den Elsen, J.M.H. and Bagby, S. (2008). Structure-function analysis of the C3-binding region of Staphylococcus aureus immune subversion protein Sbi. Journal of Biological Chemistry 283 , 22113-22120.

Isenman, D.E. , Leung, E., Mackay, J.D., Bagby, S., and van den Elsen (2010) Mutational analyses reveal that the staphylococcal immune evasion molecule Sbi and complement receptor 2 (CR2) share overlapping contact residues on C3d: Implications for the controversy regarding the CR2/C3d cocrystal structure. Journal of Immunology 184 , 1946-1955.

Kajander, T., Lehtinen, M.J., Hyvärinen, S., Bhattacharjee, A., Leung, E., Isenman, D.E. , Meri, S., Goldman, A.,, and Jokiranta, T.S. (2011) Dual interaction of factor H with C3d and glycosaminoglycans in host-nonhost driscrimination by complement. Proc. Natl. Acad. Sci. U.S.A. 108 , 2897-2902.

van den Elsen, J.M.H. and Isenman, D.E. (2011) A crystal structure of the complex between human complement receptor 2 and its ligand C3d. Science 332 , 608-611.

Department of Biochemistry
University of Toronto
Toronto, Canada

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