Identification of a homologue of CD59 in a cyclostome: implications for the evolutionary development of the complement system

We have employed a COS cell expression cloning procedure to isolate a full length cDNA clone encoding a hagfish leukocyte-associated membrane protein (HLMP1). The protein. which is identified by a monoclonal antibody (JB3) ge nerated in our laboratory, is present on the majority of hagfish leukocytes and is also expressed on erythrocytes. The cDNA clone contained an open re ading frame encoding a 120 residue polypeptide which exhibits 33% amino aci d sequence identity with the precursor protein of human CD59, a leukocyte-a ssociated membrane protein which regulates the action of the complement mem brane attack complex on homologous cells. CD59 belongs to a family of struc turally related glycoproteins which includes the Ly-6 proteins expressed on mouse lymphocytes. In addition to significant overall sequence homology HL MP1 shows conservation of 8 key cysteine residues with members of the CD59/ Ly-6 family. Comparison of the hagfish sequence with that of the mature hum an CD59 protein suggested a processed protein consisting of 74 amino acids associated with the cell membrane via a GPI anchor. The latter was confirme d by immuno-flow cytometry following treatment of transfected COS cells wit h phospholipase. Phylogenetic analysis and tissue distribution of this prot ein in the hagfish are consistent with HLMP1 being a homologue of CD59. A t hree-dimensional model of HLMP1, constructed using the NMR-determined struc ture for human CD59 as a template, indicated conservation of a core structu re of five strands of B-sheet and a short helix stabilised by four disulfid e bonds. These findings, when taken together with our previous identificati on of C5a-like chemotactic activity in LPS-activated serum, provide indirec t evidence for the existence of the terminal lytic complement pathway (C5 t o C9) in these primitive vertebrates. (C) 1999 Published by Elsevier scienc e Ltd. All rights reserved.