BIOSYNTHESIS OF LIPOPHOSPHOGLYCAN FROM LEISHMANIA-MAJOR - CHARACTERIZATION OF (BETA-1-3)-GALACTOSYLTRANSFERASE(S)

Lipophosphoglycan (LPG) is the major cell surface molecule of promasti gotes of all Leishmania species. It is comprised of three domains: a c onserved GPI anchor linked to a repeating phosphorylated disaccharide (P2; PO4-6-Gal(beta 1-4)Man(alpha 1-) backbone variously substituted w ith galactose, glucose and arabinose residues in L. major and capped w ith a neutral oligosaccharide. Using a microsomal membrane preparation from L. major, we have been able to demonstrate that galactose from U DP-[C-14]galactose can be transferred to an endogenous acceptor, chara cterized as LPG, An in vitro assay was established, based on anion-exc hange HPLC, that concurrently identifies and quantitates the products of the galactosyltransferases. We show that the products formed are [C -14]galactose-labelled P3 (PO4-6-[Gal(beta 1-3)]Gal(beta 1-4)Man(alpha 1-), P4b (PO4-6-[Gal(beta 1-3)Gal(beta 1-3)]Gal(beta 1-4)Man(alpha 1- ) and P5b (PO4-6-[Gal(beta 1-3)Gal(beta 1-3)]Gal(beta 1-4)Man(alpha 1- ). These are major galactosylated repeating units of the backbone of L . major LPG, The same products are also formed when LPG from L. donova ni, which contains an unbranched backbone of P2 repeats, is used as an exogenous acceptor with L. major microsomal membranes and UDP-[C-14]g alactose. In addition, no formation of radioactive backbone repeats (P 2) was detected in membrane incubations containing UDP[C-14]galactose with or without added unlabelled GDP-mannose, indicating that the addi tion of the (beta 1-3)-linked galactose branches is independent of the synthesis of the repeating disaccharide (P2) backbone. Preliminary ki netic analyses suggest that the addition of multiple (beta 1-3)-linked galactose residues may be catalysed by more than one (beta 1-3) galac tosyltransferase. The (beta 1-3)galactosyltransferase(s) activity was not detected in microsomal membrane preparations from promastigotes of L. donovarti.