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

Abstract

Leishmania express lipophosphoglycans and proteophosphoglycans that contain Galbeta1-4Manalpha1-P phosphosaccharide repeat structures assembled by the sequential addition of Manalpha1-P and betaGal. The synthetic acceptor substrate Galbeta1-4Manalpha1-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 Galbeta1-4Manalpha1-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 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 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 was determined using 24 synthetic analogues of Galbeta1-4Manalpha1-P-decenyl. The three species gave similar results: the negative charge of the phosphodiester and the C-6 hydroxyl of the alphaMan residue are essential for substrate recognition, the latter most likely acting as a hydrogen bond acceptor. The C-6' hydroxyl of the betaGal residue is required for substrate recognition as well as for catalysis. The rate of Manalpha1-P transfer declines with increasing acceptor substrate chain length. The presence of a monosaccharide substituent at the C-3 position of the terminal betaGal 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.

Recent Progress in the Synthesis of Glycosyl Phosphate Derivatives

“…These compounds were originally developed as nucleic acid analogs with improved lipophilicity and stability to nucleases (49). Glycosyl boranophosphates have also been synthesized from glycosyl H-phosphonates (50)(51)(52) and by nucleophilic substitution of a glycosyl bromide with dimethyl boranophosphate (53). However, the glycosyl boranophosphates have only been used as probes to investigate Leishmania mannosyl phosphate transferase (52).…”

“…Glycosyl boranophosphates have also been synthesized from glycosyl H-phosphonates (50)(51)(52) and by nucleophilic substitution of a glycosyl bromide with dimethyl boranophosphate (53). However, the glycosyl boranophosphates have only been used as probes to investigate Leishmania mannosyl phosphate transferase (52). The properties of glycosyl boranophosphates are not well-known, but they have been reported to be more stable than the glycosyl phosphate diester counterparts under acidic conditions (51).…”

“…Such a compound would be useful as a probe to study enzymatic reactions. It is noteworthy that the equivalent compound containing a C=C bond, which would be reduced by boronating agents, cannot be synthesized using the previous method of silylation and boronation of a glycosyl H-phosphonate intermediate (50)(51)(52).…”

Probing Elongating and Branching β-d-Galactosyltransferase Activities in Leishmania Parasites by Making Use of Synthetic Phosphoglycans

“…27,28 Cell lysate aliquots were thawed on ice. The membranes were washed with storage buffer (see above) and pelleted in a Beckman JS13-1 rotor (7700 × g) for 10…”

“…Compounds 3 and 4 (fragments of the LPG with one phosphodiester group) showed 15% inhibition. Compound 8, the C-phosphonate analogue with two phosphonic esters in the chain, inhibited the enzyme for about 19%.We also surveyed a range of synthetic compounds 12-21 ( Figure 5B), 31-36 which were successful substrates for the eMPT from L. donovani, 27,28,34,36 for their capacity to inhibit the eGalT from the same parasite at an equimolar concentration (200 μM) to the acceptor substrate 6. All of the compounds contained a β-D-galactose residue at the non-reducing termini of the chain that abrogated their ability to work as substrates for the elongating galactosyltransferase.…”