Characterization of the Elongating α-d-Mannosyl Phosphate Transferase from Three Species of Leishmania Using Synthetic Acceptor Substrate Analogues

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Abstract
Leishmania express lipophosphoglycans and proteophosphoglycans that contain Galβ1-4Manα1-P phosphosaccharide repeat structures assembled by the sequential addition of Manα1-P and βGal. The synthetic acceptor substrate Galβ1-4Manα1-P-decenyl and a series of analogues were used to probe Leishmania α-d-mannosyl phosphate transferase activity. We show that the activity detected with Galβ1-4Manα1-P-decenyl is the elongating α-d-mannosyl phosphate transferase associated with lipophosphoglycan biosynthesis (eMPTLPG). Differences in the apparent Km values for the donor and acceptor substrates were found using L. major, L. mexicana, and L. donovani promastigote membranes, but total activity correlated with the number of lipophosphoglycan repeats. Further comparisons showed that lesion-derived L. mexicana amastigotes, that do not express lipophosphoglycan, lack eMPTLPG and that nondividing L. major metacyclic promastigotes contain 5-fold less eMPTLPG activity than dividing procyclic promastigotes. The fine specificity of promastigote eMPTLPG activity was determined using 24 synthetic analogues of Galβ1-4Manα1-P-decenyl. The three species gave similar results: the negative charge of the phosphodiester and the C-6 hydroxyl of the αMan residue are essential for substrate recognition, the latter most likely acting as a hydrogen bond acceptor. The C-6‘ hydroxyl of the βGal residue is required for substrate recognition as well as for catalysis. The rate of Manα1-P transfer declines with increasing acceptor substrate chain length. The presence of a monosaccharide substituent at the C-3 position of the terminal βGal residue abrogates Man-P transfer, showing that chain elongation must precede side chain modification during lipophosphoglycan biosynthesis. In contrast, substitution of the penultimate phosphosaccharide repeat does not abrogate transfer but is slightly stimulatory in L. mexicana and inhibitory in L. major.

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