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149- IDENTIFICATION AND FUNCTIONAL , CHARACTERIZATION OF A NEW GENE ENCODING IXE MOUSE TERMINAL COMPLEMENT INHIBITOR CDS9

Qian,YMi,Qin,X’, Miwa, Ti,Sun, X ‘, Halperin JA’ and Song,WC’ Center for Experimental Therapeutics and Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; bepartment of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA

CD59 is a 18 to 20-kda, GPI-anchored membrane protein that functions as a key regulator of the terminal step of the complement activation cascade. It restricts binding of C8 and C9 to the C5b-8 complex, thereby preventing the formation of the membrane attack complex (CSb-9 of complement). A single human CD59 gene has been identified and corresponding genetic homologues from rat, mouse and pig have been characterized in previous studies. Here, we report the discovery and functional characterization of a separate cd59 gene in the mouse which we designate as mouse cd596 (to distinguish it from the previously characterized mouse cd59 gene which is designated as cd59a). Mouse cd596 is 85% and 63% identical to cd59a at the nucleotide and amino acid level, respectively, and has a higher sequence homology with human CD59 than does cd59a (44% vs. 41% sequence identity at the encoded protein level). Stable transfection of Chinese Hamster Ovary cells with cd59b cDNA significantly reduced the sensitivity of these cells to complement-mediated lysis, suggesting that the encoded protein functions as an inhibitorofthemembre attack complex formation. Northern blot analysis revealed that cd59b is expressed selectively in the mouse testis. In contrast, cd590 was shown to be expressed at high levels in the heart, kidney, liver and lung but only minimally in the testis. These results revealed the existence of two distinct cd59 genes in the mouse which are differentiallv reuulated and which mav have non-overlapping physiological tit&o& in viva.

150- DERIVATIZATION OF SOLUBLE HUMAN CD59 WI1 A MYRISTOYLATED PEPTIDE CREATES A POTENT MEMBRANE-BOUND INHIBITOR OF COMPLEMENT- MEDIATED LYSIS G.P. Smith. I. Dodd, A. Davies’, BP. Morgan’. P.J. Lachmann’ and R.A.G. Smith AdProTech plc. Unit 3, 2 Orchard Rd., Royston. SG8 5HD, UK ‘Centre for Veterinary Science, University of Cambridge, UK ‘Dept. Med. Biochem., IWCM, Heath Park, Cardiff CF4 4XN, UK.

CD59 is a membrane protein that protects cells from complement-mediated lysis by blocking the formation of the membrane attack complex. CD59 is attached to the cell surface through a glycosylphosphatidylinositol (GPI) anchor and association with the membrane is critial for its cytoprotective activity. Soluble forms of CD59 which lack a GPI anchor have been produced from human urine or by recombinant means but these proteins have low specific activity. To elucidate the therapeutic potential of CD59. it would be beneficial to convert material generated without a GPI anchor into a water-soluble, membrane-binding form. We found previously that membrane binding could be conferred on a soluble fragment of human CR1 by derivatization with a synthetic myristoyl-electrostatic switch peptide. A similar approach has now been applied to CD59. Free sulthydryl groups were introduced into soluble human CD59 by chemical modification and the product then linked covalently to a myristoylated peptide by disulftde exchange. Conditions were identified in which CD59 was modified by a single myristoylated peptide and which gave a product with good aqueous solubility. A reactive lysis assay demonstrated that this material had identical activity to GPI- anchored CD59 in protecting guinea pig erythrocytes from complement-mediated lysis by human serum. This approach provides a route to bulk production of a soluble CD59 analogue for assessing the activity of membrane-bound CD59 in models of human disease.

151- KINETICS AND PROPOSED MECHANISM OF BIN- DING OF THE COMPLEMENT INHIBITOR COMPSTATIN Arviid Sahu’, Athena Souliia*, Diitrios Morikifl,

complement activation in whole blood in in-vitro models for

152- PHARMACOLOGY OF SB 290157, A SELECTIVE SMALL MOLECULE ANTAGONIST OF THE C3a RECEPTOR RS. Ames, D. Lee., J.J. Foley, MA. Tome@ W. Bautsch*, B. Settmacher*, A. Klos*, A.C. Sulpizio, J.P. Hieble, G. McCafferty, and H.M. Sarau. SmithKline Beecham Pharmaceuticals, King of Prussia, PA and *Inst. of Medical Microbiology, Medical School Hatmover, Germany

The in vitro pharmacological profile of SB 290157, 2,2- diphenyl&oxyacetylarginine, a C3a receptor (C3aR) antagonist, is described. SB 290157 is a competitive antagonist of ’ ‘I-C3a mdioligand biding to RBL cells expressing the human C3aR (RBL-C3aR), with a K, = 34 nM. To assess cellular Iitncticsml activity of antagonists, inhibition of C3a- induced Ca*‘mobilization was used. SB 290157 inhibited 1 t&l C3a induced Ca*’ mobilization in RBL-C3aR cells and human neutrophils, with I& of 27 nM and 30 nM, respectively. Additionaly, C3a-mediated chemotaxis of HMC-1 was markedly inhibited by 5 uM SB 290157. Selectivity of SB 290157 was demonsttated in that thae was no inhibition of i”I- C5a binding or CSa-induced calcium mobilization in RBL-CSaR cells or PMNs, nor did it inhibit the Ca2’ responses induced by 6 other G-protein coupled receptors. Functional antagonism was not solely limited to the human C3aR, SB 290157 also inhibited C3a induced Ca” mobilization in RBL cells expressing the mouse and guinea pig C3a receptors, with P&, =I0 nM and 27 r&l, respectively. It potently inhibited C3a- mediated ATP release from guinea pig platelets (ICs,, = 30 nM) and inhibited C3a induced potentiation of the contractile response to field stimulation of perfused rat caudal artery, showing complete inhibition at 100 t&f. These data suggest that SB 290157 may be rt u&id tool compound to detine the physiological and pathophysiological roles of the C3aR