title: Demonstration by heterologous expression that the Leishmania SCA1 gene encodes an arabinopyranosyltransferase.
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 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.
authors.affiliation: Department of Biochemistry, University of Kentucky Medical Center, Lexington, KY 40536, USA.
authors.forename: Mamta
authors.initials: M
authors.lastname: Goswami
authors.forename: Deborah E
authors.initials: DE
authors.lastname: Dobson
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Beverley
authors.forename: Salvatore J
authors.initials: SJ
authors.lastname: Turco
mesh_terms: 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*
chemical_list: D006028:Glycosphingolipids; C008290:lipophosphonoglycan; D001089:Arabinose; D014166:Transferases