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Papers In Press, published online ahead of print May 10, 2006
Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-16782
Corresponding Author: delphi{at}lamar.colostate.edu
D-arabinofurans, attached to either a galactofuran or a lipomannan, are the primary constituents of mycobacterial cell wall, forming the unique arabinogalactan (AG) and lipoarabinomannan (LAM), respectively. Emerging data indicate that the arabinans of AG and LAM are distinguished by virtue of the additional presence of linear termini in LAM which entails some unknown feature of the EmbC protein for proper synthesis. In common with the two paralogous EmbA and EmbB proteins functionally implicated for the arabinosylation of AG, EmbC is predicted to carry 13 transmembrane spanning helices in an integral N-terminal domain followed by a hydrophilic extracytoplasmic C-terminal domain. To delineate the function of this C-terminal domain, the embC knock-out mutant of Mycobacterium smegmatis was complemented with plasmids expressing truncated embC genes. The expression level of serially truncated EmbC protein thus induced, was examined by EmbC-specific peptide antibody and their functional implications were inferred from ensuing detailed structural analysis of the truncated LAM variants synthesized. Apart from critically showing that the smaller arabinans are mostly devoid of the linear terminal motif, ß-D-Araf(12)-a-D-Araf(15)-a-D-Araf(15)-a-D-Araf, our studies clearly implicate the C-terminal domain of EmbC in the chain extension of LAM. For the first time, a full range of arabinan chains, as large as 18-22 Araf residues and beyond could be released intact by the use of an endogenous endo-D-arabinanase from M. smegmatis, profiled and sequenced directly by tandem mass spectrometry. In conjunction with NMR studies, our results unequivocally show that the LAM-specific linear termini are an extension on a well-defined inner branched Ara18-22 core. This hitherto unrecognized feature not only allows a significant revision of the structural model of LAM-arabinan since its first description a decade ago, but also furnishes a probable molecular basis of selectivity in biosynthesis, as conferred by the EmbC protein.
J. Biol. Chem, 10.1074/jbc.M513846200
Submitted on December 28, 2005
Revised on May 10, 2006
Accepted on May 10, 2006
The carboxy terminus of EmbC from Mycobacterium smegmatis mediates chain length extension of the Arabinan in lipoarabinomannan
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