Identification of a macrophage-binding determinant on lipophosphoglycan from Leishmania major promastigotes.

Abstract

Leishmania are obligatory intracellular parasites in mammalian macrophages that gain entry by receptormediated phagocytosis. Their major cell surface glycoconjugate, lipophosphoglycan (LPG), has been implicated in this process. A monoclonal antibody specific for Leishmania major LPG (WIC 79.3), which has been shown to block promastigote attachment to macrophages, was used to identify a macrophage-binding determinant of LPG. WIC 79.3 bound exclusively to the phosphorylated repeats of LPG and not to the saccharide core or lipid anchor. Furthermore, the epitope recognized by WIC 79.3 mapped to the phosphorylated oligosaccharide P5b, P04-6[Gal(g31-3)Gal(fi1-3)Gal(fl1-3)]Gal(131-4)Man(al-, which is unique to the LPG of promastigotes of L.major. Phosphorylated oligosaccharides P3, 3)JGal(f1l-4)Man(a1-, and P4b, LPGs form a polymorphic family of similar, but antigenically and structurally distinct, molecules present on all Leishmania species. The LPG molecule has a tripartite structure consisting of a series of phosphorylated oligosaccharide repeats (PORs) and a conserved phosphorylated saccharide core attached to a conserved, unusual lyso-alkyl phosphatidylinositol anchor ( Fig. 1; for review, see ref. 10). The PORs consist of a backbone of phosphodiester-linked disaccharide repeats [PO4-6Gal(31-4)Man(a1-], which is conserved in all species (9, 11). Variability between species involves side chain branches of varying complexity extending out from this common backbone (9, 11). Leishmania donovani displays the minimal structure with no branching, whereas L. major has the most complex, with the backbone sequence substituted with mono-, di-, tri-, and tetrasaccharide side chains consisting of combinations of galactose, arabinose, and glucose residues ( Fig. 1; ref. 9 tTo whom reprint requests should be addressed. 6The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Specific Recognition of Leishmania major Poly-β-galactosyl Epitopes by Galectin-9

“…It has been proposed that the difference between L. major and L. donovani in the induction of initial immune responses is accounted for partly by variation in the surface molecules of these Leishmania species, such as the phosphoglycan structure of LPG and by the interaction of such species-specific molecules with tissue macrophages or various cells present at the site of infection (13,38). Further, various reports suggest that the L. major-specific poly-␤-galactosyl epitopes are implicated in the progression of immune responses against Leishmania infections (13,39,40,42,54), although the host receptors/lectins have remained unclarified. As our data in this and the previous paper (16) suggest that galectin-3 and galectin-9 can distinguish L. major from L. donovani, galectin-3 and 9 are two such host factors, which can be implicated in the species-specific tropism of the disease.…”

“…1) (36 -38). Interestingly, the L. major-specific polygalactosyl epitope is implicated in two stages of the Leishmania life cycle: the host-pathogen interactions (39,40) and the attachment of Leishmania to its sand fly vector (41,42). It is likely that such roles of the polygalactosyl epitopes are mediated by ␤-galactoside-binding proteins such as galectins.…”

Monoclonal antibodies directed against Leishmania secreted acid phosphatase and lipophosphoglycan

“…This study is restricted to a comparison of the mAb reactivity with lipophosphoglycan of known structure and the use of chemical or enzymic degradation procedures and, therefore, provides only a first categorization of the epitope structure. A next step would be to use defined fragments derived from the natural antigens or by synthesis [5, 521. The evidence provided for the assignment of the epitopes for class AI, A111 mAbs and for WIC70.3 [52] and Klon 3.8 (group AIV mAbs) is relatively clear. mAb WIC 108.3 is a highly promiscuous antibody reacting with lipophosphoglycan isolated from at least three species (compare also Table 2); however, our experiments suggest that it has high preference for L. major lipophosphoglycan.…”

“…All three monoclonal antibodies showed mutual quantitative competition for binding sites on L. major lipophosphoglycan and recognized repetitive epitopes as could be shown by twosite-ELISA (data not shown). By analogy to WIC79.3, which has been shown to recognize repeats with terminating p1,3-linked Gal residues specific for this species [52], it is proposed that WIC108.3 and Klon3.8 prefentially react with these L. major-specific modifications. However, the crossreaction of WIC108.3 with L. mexicana and L. donovani lipophosphoglycan suggests that this mAb also recognizes pl,3Glc-substituted and unsubstituted repeats, albeit with decreasing affinity, as indicated by a much lower sensitivity of a two-site ELISA.…”

“…The epitope recognized by two mAbs (CA7AE and BF9CC) has been shown to include the disaccharidephosphate repeat (P04-6GalP1 ,4Man) common to lipophosphoglycan of at least three Leishmania species [ 5 ] ; in contrast, the L. majorspecific mAb WIC79.3 reacts with repeats substituted by ~1,3-galactosyl-residues (see [52] and Fig. 1).…”