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Glycobiology Advance Access published online on November 4, 2005
Glycobiology, doi:10.1093/glycob/cwj054
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© The Author 2005. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org
Received September 30, 2005
Revised November 1, 2005
Accepted November 1, 2005

Article

Demonstration by Heterologous Expression that the Leishmania SCA1 Gene encodes an Arabinopyranosyltransferase

Mamta Goswami 1, Deborah E. Dobson 2, Stephen M. Beverley 2, and Salvatore J. Turco 1*

1 Department of Biochemistry, University of Kentucky Medical Center, Lexington, KY 40536
2 Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110

* To whom correspondence should be addressed.
Salvatore J. Turco, E-mail: turco{at}uky.edu


  Abstract

In part of the life-cycle within their sand fly vector, Leishmania major parasites first attach to the fly’s midgut through their main surface adhesin lipophosphoglycan (LPG) and later resynthesize a structurally distinct LPG that results in detachment and eventual transmission. One of these structural modifications requires the addition of {alpha}1,2-D-arabinopyranose caps to {beta}1,3-galactose side chains in the phosphoglycan repeat unit domain of LPG. We had previously identified two Side Chain Arabinose genes (SCA1/2) that were involved in the {alpha}1,2-D-Arap capping. SCA1/2 exhibit canonical glycosyltransferase motifs and overexpression of either gene leads to elevated microsomal {alpha}1,2-D-ArapT activity, resulting in arabinopyranosylation of {beta}1,3-Gal side chains in LPG (hereafter referred to as sc-D-ArapT). Heterologous expression in a null arabinose background was used to determine whether the SCA1 gene encodes the actual sc-D-ArapT. SCA1 expression constructs introduced into both mammalian COS-7 cells and the baculovirus-sf9 cell system exhibited considerable expression of the protein. However, functional sc-D-ArapT activity was observed only in the latter. In in vitro assays incubated with GDP-D-[3H]Arap as the sugar donor and utilizing exogenous LPG as an acceptor, significant sc-D-ArapT activity was observed when microsomes from the baculovirus-sf9 cells were incubated in presence of the LPG acceptor. No activity was observed in the absence of LPG. These results demonstrate that SCA1 encodes a sc-D-ArapT and provide the first example of heterologous expression of a D-ArapT gene.

Keywords: Leishmania major/arabinose/arabinosyltransferase/lipophosphoglycan/metacyclogenesis.
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