Protozoan parasites of the genus Leishmania secrete a number of glycoproteins and mucin-like proteoglycans that appear to be important parasite virulence factors. We have previously proposed that the polypeptide backbones of these molecules are extensively modified with a complex array of phosphoglycan chains that are linked to Ser/Thr-rich domains via a common Man␣1-PO 4 -Ser linkage (Ilg, T., Overath, P., Ferguson, M. A. J., Rutherford, T., Campbell, D. G., and McConville, M. J. (1994) J. Biol. Chem. 269, 24073-24081). In this study, we show that Leishmania mexicana promastigotes contain a peptide-specific mannose-1-phosphotransferase (pep-MPT) activity that adds Man␣1-P to serine residues in a range of defined peptides. The presence and location of the Man␣1-PO 4 -Ser linkage in these peptides were determined by electrospray ionization mass spectrometry and chemical and enzymatic treatments. The pep-MPT activity was solubilized in non-ionic detergents, was dependent on Mn 2؉ , utilized GDP-Man as the mannose donor, and was expressed in all developmental stages of the parasite. The pep-MPT activity was maximal against peptides containing Ser/Thr-rich domains of the endogenous acceptors and, based on competition assays with oligosaccharide acceptors, was distinct from other leishmanial MPTs involved in the initiation and elongation of lipid-linked phosphoglycan chains. In subcellular fractionation experiments, pep-MPT was resolved from the endoplasmic reticulum marker BiP, but had an overlapping distribution with the cis-Golgi marker Rab1. Although Man-PO 4 residues in the mature secreted glycoproteins are extensively modified with mannose oligosaccharides and phosphoglycan chains, similar modifications were not added to peptide-linked Man-PO 4 residues in the in vitro assays. Similarly, Man-PO 4 residues on endogenous polypeptide acceptors were also poorly extended, although the elongating enzymes were still active, suggesting that the pep-MPT activity and elongating enzymes may be present in separate subcellular compartments.Parasitic protozoa of the genus Leishmania cause a spectrum of human and animal diseases that are transmitted by a sand fly vector. During their development in the digestive tract of the sand fly, these parasites differentiate from non-infective procyclic promastigotes to infective metacyclic promastigotes that target mammalian macrophages when introduced into the host during the insect's blood meal. Following their internalization into the macrophage phagolysosome, metacyclic promastigotes differentiate into a replicative amastigote stage and eventually rupture the host cell and perpetuate disease by infecting other host cells. A number of cell-surface and secreted virulence factors are thought to be crucial for the survival of these different developmental stages in their respective host environments. These include an abundant glycosylphosphatidylinositol-anchored lipophosphoglycan (LPG) 1 (1-3) and a number of secreted glycoproteins and proteophosphoglycans (PPGs) (reviewed in Refs. 4 ...
