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Glycobiology Advance Access published online on October 9, 2003
Glycobiology, doi:10.1093/glycob/cwh012
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Submitted on August 15, 2003
Revised on September 24, 2003
Accepted on September 24, 2003

© 2003 Oxford University Press

ORIGINAL ARTICLES

Structural and topological studies on the lipid-mediated assembly of a membrane-associated lipomannan in Micrococcus luteus

Leroy S. Pakkiri 1, Beata A. Wolucka 2, Eric J. Lubert 1, and Charles J. Waechter 1*

1 Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536
2 Department of Molecular Microbiology, Flanders Interuniversity Institute for Biotechnology (VIB), K.U. Leuven, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Belgium

* To whom correspondence should be addressed. E-mail: waechte{at}pop.uky.edu.

Abstract

The biosynthesis of three mannolipids and the presence of a membrane-associated lipomannan in Micrococcus luteus (formerly Micrococcus lysodeikticus) were documented over 30 years ago. Structural and topological studies have been conducted to learn more about the possible role of the mannolipids in the assembly of the lipomannan. The major mannolipid has been purified and characterized as {alpha}-D-mannosyl-(1->3)-{alpha}-D-mannosyl-(1->3)-diacylglycerol (Man2-DAG) by negative-ion electrospray-ionization multistage mass spectrometry (ESI-MSn). Analysis of the fragmentation patterns indicates that the sn-1-position is predominantly acylated with a 12-methyltetradecanoyl group and the sn-2 position is acylated with a myristoyl group. The lipomannan is shown to be located on the exterior face of the cytoplasmic membrane, and not exposed on the surface of intact cells, by staining of intact protoplasts with fluorescein isothiocyanate (FITC)-linked concanavalin A (Con A). When cell homogenates of M. luteus are incubated with GDP-[3H]mannose (GDP-Man), [3H]mannosyl units are incorporated into Man1-2-DAG, mannosylphosphorylundecaprenol (Man-P-Undec) and the membrane-associated lipomannan. The addition of amphomycin, an inhibitor of Man-P-Undec synthesis, had no effect on the synthesis of Man1-2-DAG, but blocked the incorporation of [3H]mannose into Man-P-Undec, and consequently the lipomannan. These results strongly indicate that GDP-Man is the direct mannosyl donor for the synthesis of Man1-2-DAG, and that the majority of the 50 mannosyl units in the lipomannan are derived from Man-P-Undec. Protease-sensitivity studies with intact and lysed protoplasts indicate that the active sites of the mannosyltransferases catalyzing the formation of Man1-2-DAG and Man-P-Undec are exposed on the inner face, and the Man-P-Undec-mediated reactions occur on the outer surface of the cytoplasmic membrane. Based on all of these results a topological model is proposed for the lipid-mediated assembly of the membrane-bound lipomannan.


lipomannan, mannosylphosphorylundecaprenol, mannosyldiacylglycerols, flippases
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