STRUCTURE-FUNCTION ANALYSIS OF LEISHMANIA LIPOPHOSPHOGLYCAN - DISTINCT DOMAINS THAT MEDIATE BINDING AND INHIBITION OF ENDOTHELIAL-CELL FUNCTION

We have shown that Leishmania lipophosphoglycan (LPG) inhibits IL-1 be ta gene expression in human monocytes. Here, we show that LPC can bind in a time-dependent manner and suppress endothelial cell activation, possibly via specific LPG domains. Endotoxin (10 ng/ml, 4 h) consisten tly caused endothelium to increase monocyte adhesion (similar to 20-fo ld). LPC pretreatment (2 mu M, 2 h) completely blocked endotoxin-media ted monocyte adhesion. LPG did not grossly suppress endothelial functi ons because TNF-alpha- and IL-1 beta-mediated adhesion toward monocyte s were not affected. Using four highly purified LPC fragments (namely, repeating phosphodisaccharide (PGM), phosphoglycan, phosphosaccharide core-lyso-alkyl-phosphatidylinositol (core-PI), and lyso-alkyl-phosph atidylinositol (lyse-PI)), we examined whether these fragments can ind ependently inhibit endothelial adhesion. In contrast to that of intact LPG, neither the four LPG fragments (2 mu M, 2 h) independently nor t he co-addition of phosphoglycan and core-PI fragments blocked the endo toxin-mediated adhesion to monocytes. To determine whether the fragmen ts can reverse the effect of intact LPC, endothelial cells were first pretreated with the LPC fragments (10 mu M, 15 min), followed by the a ddition of LPG (2 mu M). All four LPC fragments fully reversed the eff ect of LPG. Simultaneous addition of LPC fragments and intact LPC caus ed only partial suppression (similar to 45%), while the addition of LP C fragments 15 min later had no reversal effect. Flow cytometry reveal ed that only core-PI and lyse-PI competitively inhibited (similar to 3 0%) LPC binding. Conversely, LPC competed with the binding of [H-3]lys o-PJ (similar to 30%). Furthermore, mAb against the PGM reversed (simi lar to 70%) the effect of LPC. Thus, the lyso-PI domain on LPC mediate s binding to endothelial cells, whereas the PGM domain mediates the ce ll inhibitory effect.