O-GLYCOSYLATION AND N-GLYCOSYLATION OF THE LEISHMANIA-MEXICANA SECRETED ACID-PHOSPHATASE - CHARACTERIZATION OF A NEW CLASS OF PHOSPHOSERINE-LINKED GLYCANS

The protozoan parasite Leishmania mexicana secretes a heavily glycosyl ated 100-kDa acid phosphatase (sAP) which is associated with one or mo re polydisperse proteophosphoglycans. Most of the glycans in this comp lex were released using mild acid hydrolysis conditions that preferent ially cleave phosphodiester linkages. The released saccharides were sh own to consist of monomeric mannose and a series of neutral and phosph orylated glycans by Dionex high performance liquid chromatography, met hylation analysis, exoglycosidase digestions, and one-dimensional H-1 NMR spectroscopy. The neutral species comprised a linear series of oli gosaccharides with the structures [Man alpha 1-2](1-5)Man. The phospho rylated oligosaccharides were characterized as PO4-6Gal beta 1-4Man an d PO4-6[Glc beta 1-3]Gal beta 4Man. The attachment of these glycans to the polypeptide backbone via the linkage, Man alpha 1-PO4-Ser, is sug gested by: 1) the finding that more than 60% of the serine residues in the polypeptide are phosphorylated and 2) the resistance of the phosp hoserine residues to alkaline phosphatase digestion unless the sAP was first treated with either mild acid (to release all glycans) or jack bean alpha-mannosidase (to release neutral mannose glycans). Analysis of the partially resolved components of the complex indicated that the most of the O-linked glycans on the 100-kDa phosphoglycoprotein compr ised mannose and the mannoseoligosaccharides. In contrast the major O- linked glycans on the proteophosphoglycan were short phosphoglycan cha ins, containing on average two repeat units per chain. In addition to the O-linked glycans, both components in the sAP complex contained N-l inked glycans. The N-glycanase F-released glycans were characterized b y Bio-Gel P4 chromatography and exoglycosidase digestions to be the bi antennary oligomannose type with the structures Glc(1)Man(6)GlcNAc(2) and Man(6)GlcNAc(2). The O-linked glycans of the sAP complex are simil ar to those found in the phosphoglycan chains of the abundant surface lipophosphoglycan, but differ in having much shorter phosphoglycan cha ins and a more diverse series of mannose cap oligosaccharides. These d ata suggest that there are marked differences in the ability of differ ent glycosyltransferases to utilize peptide-linked versus glycolipid-l inked accepters.