Purification and structural characterization of a filamentous, mucin-like proteophosphoglycan secreted by Leishmania parasites.
Parasitic protozoa of the genus Leishmania secrete a filamentous macromolecule that forms networks and appears to be associated with cell aggregation. We report here the purification of this parasite antigen from Leishmania major culture supernatant and its compositional (75.6% carbohydrate, 20% phosphate, 4.4% amino acids, w/w), structural, and ultrastructural characterization as a highly unusual proteophosphoglycan (PPG). Mild acid hydrolysis, which cleaves preferentially hexose 1-phosphate bonds, releases the PPG glycans. Their structures are Galbeta1-4Man, Manalpha1-2Man, Galbeta1-3Galbeta1-4Man, PO4-6(Galbeta1-3)0-2Galbeta1-4Man, and PO4-6(Arabeta1-2Galbeta1-3)Galbeta1-4Man. These glycans are also components of the parasite glycolipid lipophosphoglycan, but their relative abundance and structural organization in PPG are different. Some of them represent novel forms of protein glycosylation. 31P NMR on native PPG demonstrates that phosphate is exclusively in phosphodiester bonds and that the basic structure R-Manalpha1-PO4-6-Gal-R connects the glycans. A phosphodiester linkage to phosphoserine (most likely R-Manalpha1-PO4-Ser) anchors the PPG oligosaccharides to the polypeptide. PPG has a unique amino acid composition; glycosylated phosphoserine (>43 mol %), serine, alanine, and proline account for more than 87 mol % and appear to be clustered in large proteinase-resistant domains. Electron microscopy of purified PPG reveals cable-like, flexible, long (to 6 microm), and unbranched filaments. The overall structure of PPG shows many similarities to mammalian mucins. Potential functions of this novel mucin-like molecule for the parasites are discussed.
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Walter and Eliza Hall Institute of Medical Research, P. O. Royal Melbourne Hospital, Victoria 3050, Australia.
Stierhof
Y D
YD
Craik
D
D
Simpson
R
R
Handman
E
E
Bacic
A
A
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D008401:Gas Chromatography-Mass Spectrometry; D006031:Glycosylation; D018320:Leishmania major / Q000737:chemistry* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry*; D011509:Proteoglycans / Q000737:chemistry / Q000302:isolation & purification*; D013403:Sugar Phosphates / Q000737:chemistry / Q000302:isolation & purification*
D000596:Amino Acids; D009077:Mucins; D011509:Proteoglycans; D013403:Sugar Phosphates