C-reactive Protein and SAP-like Pentraxin are both Present in Limulus polyphemus Haemolymph: Crystal Structure of Limulus SAP Annette K. Shrive 1 , Alison M. Metcalfe 1,2 , Jamie R. Cartwright 1 and Trevor J. Greenhough 1 * 1 Structural Biology Research Group, School of Life Sciences Keele University, Keele Staffordshire, ST5 5BG, UK 2 North Staffordshire Hospital NHS Trust, City General Hospital, Newcastle Road Stoke-on-Trent, Staffordshire ST4 6QG, UK C-reactive protein and serum amyloid P component are members of the pentraxin family of oligomeric serum proteins which has been conserved through evolution. In humans both have pentameric structures and both play complex roles in the immune response, C-reactive protein being the classical acute-phase reactant produced in response to tissue damage and in¯ammation. An invertebrate SAP-like pentraxin has not previously been identi®ed and it has been postulated that C-reactive protein and serum amyloid P component are products of a gene duplication event within vertebrate evolution. We have isolated serum amyloid P com- ponent from the haemolymph of the phylogenetically ancient ``living fossil'', the horseshoe crab Limulus polyphemus and determined the three- dimensional structure by X-ray crystallography. The structure of the previously undiscovered Limulus serum amyloid P component, the ®rst invertebrate lectin structure to be determined, reveals the pentraxin fold and a novel doubly stacked octameric ring. The crystal structure and the discovery that both prototypic pentraxins are present in Limulus raises the possibility that both were present in the common ancestors of arthro- pods and chordates over 500 million years ago. The impact of the results on our understanding of the origins and evolution of pentraxins and innate immunity is discussed. # 1999 Academic Press Keywords: pentraxin; C-reactive protein; serum amyloid P component; lectin; octameric structure *Corresponding author Introduction The serum pentraxins C-reactive protein (CRP) and amyloid P component (SAP) are members of a conserved family of cyclic oligomeric proteins (Osmand et al., 1977; Pepys et al., 1978; Gewurz et al., 1995), homologues of which have been found in mammals, ®sh and amphibians (see, for example, Pepys et al., 1978, 1982; de Beer et al., 1982; Robey et al., 1983; Woo et al., 1985; Maudsley et al., 1987; Lin & Liu, 1993; Rubio et al., 1993; Jensen et al., 1997; Lund & Olafsen, 1998), and the phylogenetically ancient horseshoe crab Limulus polyphemus (Marchalonis & Edelman, 1968; Roche & Monsigny, 1974; Kaplan et al., 1977; Robey & Liu, 1981; Nguyen et al., 1986a,b; Myles et al., 1990; Tennent et al., 1993; Armstrong et al., 1996a,b). CRP and SAP are characterised by their high Ca-dependent binding af®nities for phosphocho- line (PC) and phosphoethanolamine (PE), respect- ively (Schwalbe et al., 1992), although CRP also binds PE. In humans, CRP plays a pivotal and complex role in the immune response and is the classical acute-phase reactant produced in response to tissue damage and in¯ammation (Gewurz et al., 1982, 1995; Volanakis, 1982). SAP-like pentraxin homologues are lectins whose ligands include PE and carbohydrate moieties (Hind et al., 1985), but not PC (Schwalbe et al., 1992). The known pentrax- in structures, human CRP (hCRP) (Shrive et al., 1996a), human SAP (hSAP) (Emsley et al., 1994) and rat CRP (T.J.G., M. Hopkins, D.A.A. Myles, E-mail address of the corresponding author: t.j.greenhough@keele.ac.uk Abbreviations used: CRP, C-reactive protein; SAP, serum amyloid P component; PE, phosphoethanolamine; PC, phosphocholine; hCRP, human C-reactive protein; hSAP, human serum amyloid P component; EDTA, ethylenediaminetetraacetic acid; ncs, non- crystallographic symmetry. Article No. jmbi.1999.2956 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 290, 997±1008 0022-2836/99/300997±12 $30.00/0 # 1999 Academic Press