Leishmania braziliensis brazilensis and L. donovani chagasi

This study describes the identification of aqueous-soluble antigens in Leishmania promastigotes immunologically and biochemically closely related to the major surface antigen. Proteins from surface-iodinated L. braziliensis braziliensis and L. donovani chagasì promastigotes, extracted and separated by partitioning in the detergent Triton X-114, were analyzed. Immunoblotting of the extracted proteins, using homologous antisera, showed recognition of a 72-kDa labeled, amphiphilic antigen of L. b. braziliensis and a 65-kDa surface antigen of L. d. chagasi. The respective homologous sera also recognized non-labeled hydrophilic antigens, similar in their apparent molecular weights to the major surface antigens. The amphiphilic and hydrophilic antigens of each species were found to share common antigenic determinants, inasmuch as monospecific antibodies that recognized the amphiphilic protein reacted with the hydrophilic antigen. Structural homology was also obtained in the peptideidigestion profiles of the amphiphilic and the respective hydrophilic major antigens. Zymogram assay showed that both amphibilic and hydrophilic fractions displayed proteolytic activity that could be directly attributed to the major L. b. braziliensis and L. d. chagasi antigens. The hydrophilic antigens found in this study are probably not hydrolytic products of the surface antigens and occur in large quantities in the promastigote cytosol. Reprint requesis to: S. Kutner Present addresses: * Department of Cell Biology, Faculty of Sciences, Universidad Central de Venezuela, P.O. BOX 47069, Los Chaguaramos, Caracas 1041, Venezuela. ** Station de Technologie Alimentaire, INRA, 369 rue Jules Guesde, B.P. 39-59651 Villeneuve d'Ascq Cedex, France, *** Laboratoire de chimiotaxonomie des parasites, ORSTOM, 2051 AV. du Val de Montferrand, BP 5045, 34032 Montpellier Cedex, France Many studies on surface antigens of different Leishmania species from the Old and Nem7 World assert the presence of a major surface antigen with an apparent molecular weight of 63-65 kDa in promastigotes of L. tropica (Handman and Curtis 1982; Gardiner and Dwyer 1983), L. major (Bouvier et al. 1985), L. donovani (Lepay et al. 1983; Lemesre et al. 1985), L. mexicana (Chang et al. 1986) and L. brasiliensis (Misle et al. 1985) and that of a 72-kDa major surface antigen for the sub-species L. b. braziliensis (Legrand et al. 1987). Except for the latter, these glycoproteins exhibit cross-reactivity with heterologous sera and share a common primary structure in most Leishmania species tested (Etges et al. 1985; Colomer-Gould et al. 1985). Recently, proteolytic activity related to the major surface antigen in promastigotes was also reported (Etges et al. 1986; Bordier 1987; Bouvier et al. 1987; Chaudhuri and Chang 1988). Several methods have been used to purify the major surface antigen of Leishmania, including affinity chromatography with specific monoclonal antibodies (Chang and Chang 1986) or concanavalin A (Russell and Wilhelm 1986) or phase partitioning with the detergent Triton X-114 (Bouvier et al. 1985). Using the latter technique with L. major promastigotes, Bouvier and co-workers identi"fied a soluble form of the surface antigen generated in vitro during its purification, probably by the hydrolysis of a lipid-containing myristyl residue that anchored the protein in the membrane. In this work we report the identification of cytoplasmic soluble antigens that are biochemically and immunologically closely related to the major surface antigens of L. b. braziliensis and L. d. chagasi promastigotes. The results suggest that these soluble antigens are not generated in vitro during the Triton X-114 extraction, but rather are present