Biosynthesis of a Leishmania virulence factor, lipophosphoglycan: Role of the Leishmania donovani LPG4A gene in repeat unit assembly

A critical cell-surface virulence factor of Leishmania promastigotes, Lipophosphoglycan (LPG) is a GPI-anchored polymer of Gal(β1,4)Man(α1-)PO ₄ repeat units. Repeat unit composition is a highly regulated aspect of the Leishmania infectious cycle. Polymer assembly involves the transfer of Man-PO₄ from GDP-Man by an elongation-specific mannosylphosphoryltransferase (eMPT) to existing repeat units.; The LPG4A gene was identified by the complementation of an lpg-L. donovani mutant, JEDI that is defective in the biosynthetic step catalyzed by the eMPT. LPG4A is a predicted type II membrane protein with some sequence homology to putative bacterial transferases. Using in vitro eMPT assays we demonstrate that LPG4A restores eMPT activity to JEDI, thus making it a likely candidate for the eMPT. To test whether LPG4A encodes the eMPT, we expressed the protein in mammalian and insect cells. However no activity was detected, suggesting that the gene might not encode the transferase or might require accessory proteins for activity. We were unable to reconstitute activity in heterologous system by addition of JEDI membranes. Appropriate controls were performed to ensure that the recombinant protein had not formed insoluble aggregates in the heterologous system. Preliminary cross-linking experiments to identify interacting proteins suggest that LPG4A could be part of a complex.; In addition to restoring LPG biosynthesis to JEDI, LPG4A restores phosphoglycosylation of other phosphoglycan-bearing molecules, secreted acid phosphatase (sAP) and proteophosphoglycan (PPG). The LPG4A gene has two potential start sites separated by 90 bp, encoding two in-frame ORFs, LPG4A₁ and LPG4A₂. During metacyclogenesis, as the procyclic promastigotes transform into the infective metacyclics, the number of repeat units doubles. When transfected into JEDI, LPG4A ₁ generates metacyclic LPG while LPG4A₂ generates procyclic LPG, suggesting the involvement of LPG4A in regulation of chain length. These differences are mirrored in the sizes of sAP and PPG, demonstrating broader involvement of LPG4A in repeat unit assembly. Overall our data indicate that LPG4A is unlikely to encode a functional transferase and is possibly an accessory factor or part of a larger complex required for eMPT activity. The involvement of LPG4A in the regulation of repeat unit composition highlights its possible role as a regulatory element.