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J. Biol. Chem., Vol. 281, Issue 28, 19512-19526, July 14, 2006

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The Carboxy Terminus of EmbC from Mycobacterium smegmatis Mediates Chain Length Extension of the Arabinan in Lipoarabinomannan^*

Libin Shi, Stefan Berg, Arwen Lee^¶, John S. Spencer¹, Jian Zhang, Varalakshmi Vissa¹, Michael R. McNeil, Kay-Hooi Khoo^¶||2, and Delphi Chatterjee³

From the Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523, Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, Liaoning, China, 116027, ^¶Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan, and ^||Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan

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, -D-Araf(12)--D-Araf(15)--D-Araf(15)--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 Ara-(18-22) 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.

Received for publication, December 28, 2005 , and in revised form, May 10, 2006.

^* This work was supported in part by National Institutes of Health Grant AI-37139 (to D. C.) and AI-33706 (to M. R. M.) and Taiwan National Science Council Grant 94-2311-B-001-071 (to K. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table I.

¹ Supported by NIAID, National Institutes of Health Grants NO1 AI-25469 and AI-47197.

² To whom correspondence may be addressed. E-mail: kkhoo{at}gate.sinica.edu.tw.

³ To whom correspondence may be addressed. Tel.: 970-491-7495; Fax: 970-491-1815; E-mail: delphi{at}lamar.colostate.edu.

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