Characterization of the elongating alpha-D-mannosyl phosphate transferase from three species of Leishmania using synthetic acceptor substrate analogues

Leishmania express lipophosphoglycans and proteophosphoglycans that contain Gal beta 1-4Man alpha 1-P phosphosaccharide repeat structures assembled by the sequential addition of Man alpha 1-P and beta Gal. The synthetic accep tor substrate Gal beta 1-4Man alpha 1-P-decenyl and a series of analogues w ere used to probe Leishmania alpha-D-mannosyl phosphate transferase activit y. We show that the activity detected with Gal beta 1-4Man alpha 1-P-deceny l is the elongating cx-D-mannosyl phosphate transferase associated with lip ophosphoglycan biosynthesis (eMPT(LPG)). Differences in the apparent K-m va lues for the donor and acceptor substrates were found using L, major, L. me xicana, and L. donovani promastigote membranes, but total activity correlat ed with the number of lipophosphoglycan repeats. Further comparisons showed that lesion-derived L mexicana amastigotes, that do not express lipophosph oglycan, lack eMPT(LPG) and that nondividing L. major metacyclic promastigo tes contain 5-fold less eMPT(LPG) activity than dividing procyclic promasti gotes. The fine specificity of promastigote eMPT(LPG) activity was determin ed using 24 synthetic analogues of Gal beta 1-4Man alpha 1-P-decenyl. The t hree species gave similar results: the negative charge of the phosphodieste r and the C-6 hydroxyl of the alpha Man residue are essential for substrate recognition, the latter most likely acting as a hydrogen bond acceptor. Th e C-6' hydroxyl of the beta Gal residue is required for substrate recogniti on as well as for catalysis, The rate of Man alpha 1-P transfer declines wi th increasing acceptor substrate chain length. The presence of a monosaccha ride substituent at the C-3 position of the terminal beta Gal residue abrog ates Man-P transfer, showing that chain elongation must precede side chain modification during lipophosphoglycan biosynthesis. In contrast, substituti on of the penultimate phosphosaccharide repeat does not abrogate transfer b ut is slightly stimulatory in L. mexicana and inhibitory in L. major.