Comp. Biochem. Physiol. Vol. l13B, No. 1, pp. 79-87, 1996 Copyright © 1996 Elsevier Science Inc. ELSEVIER ISSN 0305-0491/96/$15.00 SSDI 0305-0491 (96)02002-3 Structural Characterization of an Lyt-2/3 Homolog Expressed on Bufo regularis Lymphocytes Hoda I. Negm,* Mohamed H. Mansour, / Abdel Hakim Saadi and Raghad S. Abdel Halim~: * DEPARTMENT OF ZOOLOGY, FACULTY OF SCIENCE, MONOF1A UNIVERSITY; tDEPARTMENT OF ZOOLOGY, FACULTY OF SCIENCE, CAIRO UNIVERSITY; AND ~RESEARCH INSTITUTE OF OPHTHALMOLOGY, CAIRO, EGYPT ABSTRACT. Two alloantisera and a monoclonal antibody (mAb 53-6.7) of proven specificities to the murine Lyt-2/3 macromolecule labeled, in indirect immunoflourescent assays, a distinct lymphocyte population in the toad, Bufo regularis. Lyt-2/3 antigenic activities expressed by B. regularis lymphocytes have been solubilized and purified by mAb 53-6.7 affinity chromatography and found to be associated with a single 67 kDa macromole- cule in SDS-PAGE. Upon reduction, this macromolecule resolved into 38 kDa, 34 kDa and 28 kDa subunits corresponding to the c~, c~' and/3 subunits of the murine Lyt-2/3 complex. Comparisons based on the SAQ index of differences in amino acid compositions of HPLC-purified c~- and c~'-subunits of the amphibian Lyt-2/ 3 molecule indicated a significant structural relatedness to their murine counterpart as well as to the human CD8 polypeptide. Our observations point to an early phylogenetic emergence of Lyt-2/3 as an important component of the T cell cytolytic apparatus during vertebrate evolution. COMP mOCSEMPHYSIOL 1l 3B, 79-87, 1996. KEY WORDS. Bufo regularis, Lyt-2/3 complex, CDS, T lymphocytes, differentiation markers, lymphoid tissues, antigenic conservation, SAQ index INTRODUCTION The murine Lyt-2/3 molecular complex, and its counterpart in humans (CDS), are glycoproteins expressed on thymocytes and peripheral T cells and have classically been considered differentiation markers for the cytotoxic T cell subpopulation (Cantor and Boyse, 1977; Ledbetter et al., 1980; Reinherz and Schlossman, 1980). Antibodies to either molecules inhibit T cell cytotoxicity by blocking the recognition phase of the response (Spits et al., 1982; Swain, 1981; 1983), thus impli- cating both molecules in stabilizing T cell-target cell interac- tions, perhaps by binding non-polymorphic domains of class I major histocompatibility complex (MHC)-encoded polypep- tides (Macdonald et al., 1982; Meuer et al., 1982; Swain, 1983). Biochemical analyses of Lyt-2/3 and CD8 have re- vealed that both macromolecules are assembled into subunits that are disulfide-bonded into a variety of multimeric forms (Jay et al., 1982; Ledbetter and Seaman, 1982; Meuer et al., 1982). Genes encoding Lyt-2 and CD8 are closely linked to the variable gene locus of immunoglobulins in each species (Gottlieb, 1974; Sukhatme et al., 1985a), and as deduced from cDNA sequences, both molecules bear significant struc- tural homology to each other and to immunoglobulin variable Correspondence to: Dr. Mohamed H. Mansour, Department of Zoology, Fac- ulty of Science, Cairo University, Cairn 12613, Egypt Received 24 December 1994; revised 17 May 1995; accepted 22 May 1995. (IgV) domains (Littman et al., 1985; Nakauchi et al., 1985; Sukhatme et al., 1985b; Zamoyska et al., 1985). This struc- tural homology extends to other IF-like domains among mem- bers of the Ig superfamily (Williams, 1984; 1985; Mansour and Cooper, 1993) which are proposed to share a common evolutionary origin from an ancestral gene encoding a primor- dial domain-like structure. The structural and functional properties ascribed to Lyt-2/ 3 and CD8 suggest an evolutionary significance for these mole- cules in understanding how IF-related structures evolved to mediate specific lymphocyte functions. Their structural relat- edness to V domains of the Ig light chains in particular pro- voked speculation that they may have diverged from the pri- mordial domain prior to the phylogenetic emergence of Ig light chains (Hood et al., 1985). In support for this notion, a structural homolog for the murine Lyt-2/3 has been recently identified and characterized in tunicates, the protochordate group of invertebrates believed to be the direct ancestors of vertebrates (Negm et al., 1992). Nonetheless, no information is available regarding the possible occurrence of structurally- related molecules in vertebrate representatives other than mammals. In the present study we report serological and bio- chemical evidence for the presence of an Lyt-2/3 homolog in amphibians. Our findings suggest the conservation of Lyt-2/3 as an important component of the cellular cytolytic apparatus during early phases of vertebrate evolution.