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

• Published in: The Journal of biological chemistry 1999-03, Vol.274 (10), p.6678-6688
• Main Authors: Moss, J M, Reid, G E, Mullin, K A, Zawadzki, J L, Simpson, R J, McConville, M J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Chromatography, High Pressure Liquid; Leishmania mexicana; Leishmania mexicana - metabolism; Mass Spectrometry; Molecular Sequence Data; Phosphotransferases (Alcohol Group Acceptor) - genetics; Phosphotransferases (Alcohol Group Acceptor) - isolation & purification; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Transferases (Other Substituted Phosphate Groups) - genetics; Transferases (Other Substituted Phosphate Groups) - isolation & purification; Transferases (Other Substituted Phosphate Groups) - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.10.6678

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 Manα1-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 Manα1-P to serine residues in a range of defined peptides. The presence and location of the Manα1-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.