Characterization of a Novel GDP-mannose:Serine-protein Mannose-1-phosphotransferase from Leishmania mexicana

Abstract

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 ...

Glycosylphosphatidylinositol biosynthetic enzymes are localized to a stable tubular subcompartment of the endoplasmic reticulum in Leishmania mexicana

“…Although a small proportion of BiP also sedimented in lighter fractions (in some but not all experiments), the distribution of this fraction was always distinct from that of the DPMS/plasma membrane markers (Figure 3A). Maximum DPMS activity was also consistently displaced from that of the peptide‐specific mannophosphotransferase (pepMPT) which initiates protein O ‐phosphoglycosylation in the Golgi apparatus (Moss et al ., 1999) (Figure 3A). These results suggest that both endogenous DPMS as well as the DPMS–GFP chimera are localized to a distinct membrane subcompartment and supports the notion that the DPMS tubule is connected to the subpellicular cytoskeleton–plasma membrane complex.…”

Lipophosphoglycan is not required for infection of macrophages or mice by Leishmania mexicana

“…Deletion mutants for phosphoglycan‐modified SAP, for example, retain their infectivity to macrophages and mice (Wiese, 1998). To solve this question, it may be necessary to perform targeted deletions on multiple PPG genes or on genes of biosynthetic enzymes that are selectively involved in assembly of the PPG glycans, such as the GDP‐mannose:serine‐protein mannose‐1‐phosphotrans ferase that has been characterized recently in L.mexicana promastigotes (Moss et al ., 1999). Further studies will be required to define the role of the PPG family for Leishmania virulence in mammals.…”

Secretory Pathway of Trypanosomatid Parasites

“…Early immunochemical and biochemical analyses indicated that these heavily glycosylated proteins were modified with similar phosphoglycans to those found on the LPG of the same parasites (28,162). Subsequent studies showed that serine residues in the protein backbone of the SAP were modified with Man-1-PO 4 , which was extended with short linear chains of ␣1-2-linked mannose or longer chains of repeating Gal␤1-4Man␣1-PO 4 -phosphodisaccharides (157,185,224). The SAPs belong to a heterogeneous family of PPGs, which include the promastigote filamentous PPG, the GPI-anchored cell surface PPG, and the nonfilamentous amastigote PPG (153,155,156).…”

“…Although the latter processing steps do not occur to the same extent in T. cruzi or Leishmania spp. (see below), many of the proteins in the latter species are modified with complex O-linked glycans or phosphoglycans by enzymes that also appear to be compartmentalized in the Golgi (131,224). Some of these Golgi-specific processing steps can be inhibited with the ionophore monensin (20,28), although this drug does not affect the surface transport of GPI proteins such as VSG (20,85).…”

“…The fluorescent lipid BODIPY-ceramide has been used to label the Golgi in live T. brucei BF [105,602], while this dye is specifically sequestered within the tubular lysosome of Leishmania promastigotes (159), highlighting possible differences in the lipid composition of secretory pathway membranes in different trypanosomatids. The different cisternae of the trypanosomatid Golgi appear to be biochemically and functionally differentiated, based on the polarized distribution of some Golgi marker proteins (56), the separation of different Golgi fractions in subcellular fractionation studies (125,224), and differences in the cytochemical staining of some cisternae (94). Moreover, T. brucei BF glycoproteins receive complex N-linked glycan modifications that FIG.…”