Characterization of the Elongating #alpha#-D-Mannosyl Phosphate Transferase from Three Species of Leishmania Using Synthetic Acceptor Substrate Analogues

摘要

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 acceptor substrate Gal#beta#1-4Man#alpha#1-P-decenyl and a series of analogues were used to probe Leishmania #alpha#-D-mannosyl phosphate transferase activity. We show that the activity detected with Gal#beta#-4Man#alpha#1-P-decenyl is the elongating #alpha#-D-mannosyl phosphate transferase associated with lipophosphoglycan biosynthesis (eMPT~LPG). Differences in the apparent K_m values for the donor and aceptor substrtes were found using L. major, L. mexicana ,and L. donovani promastigote membranes, but total derived L. mexicana amastigotes, that do not express lipophosphoglycan, lack eMPT~LPG and that nondividing L. major metacyclic promastigotes contain 5-fold less eMPT~LPG activity than dividing procyclic promastigotes. The fine specificity of promastigote eMPT~LPG activity eas determined using 24 synthetic analogues of Gal#beta1-4Man#alpha#1-P-decenyl. The three species gave similar results: the negative charge of the phosphodiester 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. The C-6' hydroxyl of the #beta#Gal residue is required for substrate recognition as well as for catalysis .The rate of Man#alpha#1-P transfer declines with increasing acceptor substrate chain length. The presence of a monosaccharide substituent at the C-3 position of the terminal #beta#Gal residue abrogates Man-P transfer, showing that chain elongation must prcede side chain modification during lipophosphoglycan biosynthesis. In contrast, substitution of the penultimate phosphosaccharide repeat does not transfer but is slightly stimulatory in L. mexicana and inhibitory in L. major.