Demonstration by heterologous expression that the Leishmania SCA1 gene encodes an arabinopyranosyltransferase.
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 alpha1,2-D-arabinopyranose caps to beta1,3-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 alpha1,2-D-Arap capping. SCA1/2 exhibit canonical glycosyltransferase motifs, and overexpression of either gene leads to elevated microsomal alpha1,2-D-ArapT activity, resulting in arabinopyranosylation of beta1,3-Gal side chains in LPG (hereafter called side chain D-arabinopyranosyltransferase [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 guanidine 59-diphosphate (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.
Goswami
Mamta
M
Department of Biochemistry, University of Kentucky Medical Center, Lexington, KY 40536, USA.
Dobson
Deborah E
DE
Beverley
Stephen M
SM
Turco
Salvatore J
SJ
D000818:Animals; D001089:Arabinose / Q000378:metabolism*; D002236:Carbohydrate Conformation; D002460:Cell Line; D002522:Chlorocebus aethiops; D002851:Chromatography, High Pressure Liquid; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D006028:Glycosphingolipids / Q000737:chemistry; D007891:Leishmania / Q000201:enzymology* / Q000235:genetics*; D008861:Microsomes / Q000378:metabolism; D018411:Spodoptera; D014166:Transferases / Q000235:genetics* / Q000378:metabolism*
D006028:Glycosphingolipids; C008290:lipophosphonoglycan; D001089:Arabinose; D014166:Transferases