PURIFICATION AND STRUCTURAL CHARACTERIZATION OF A FILAMENTOUS, MUCIN-LIKE PROTEOPHOSPHOGLYCAN SECRETED BY LEISHMANIA PARASITES

Parasitic protozoa of the genus Leishmania secrete a filamentous macro molecule that forms networks and appears to be associated with cell ag gregation. We report here the purification of this parasite antigen fr om Leishmania major culture supernatant and its compositional (75.6% c arbohydrate, 20% phosphate, 4.4% amino acids, w/w), structural, and ul trastructural characterization as a highly unusual proteophosphoglycan (PPG). Mild acid hydrolysis, which cleaves preferentially hexose 1-ph osphate bonds, releases the PPG glycans. Their structures are Gal beta 1-4Man, Man alpha 1-2Man, Gal beta 1-3Gal beta 1-4Man, PO4-6(Gal beta 1-3)(0-2)Gal beta 1-4Man, and PO4-6(Ara beta 1-2Gal beta 1-3)Gal beta 1-4Man. These glycans are also components of the parasite glycolipid lipophosphoglycan, but their relative abundance and structural organiz ation in PPG are different. Some of them represent novel forms of prot ein glycosylation. P-31 NMR on native PPG demonstrates that phosphate is exclusively in phosphodiester bonds and that the basic structure R- Man alpha 1-PO4-6-Gal-R connects the glycans. A phosphodiester linkage to phosphoserine (most likely R-Man alpha 1-PO4-Ser) anchors the PPG oligosaccharides to the polypeptide. PPG has a unique amino acid compo sition; glycosylated phosphoserine (>43 mol %), serine, alanine, and p roline account for more than 87 mol % and appear to be clustered in la rge proteinase-resistant domains. Electron microscopy of purified PPG reveals cable-like, flexible, long (to 6 mu m), and unbranched filamen ts. The overall structure of PPG shows many similarities to mammalian mucins. Potential functions of this novel mucin-like molecule for the parasites are discussed.