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 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-Ara(p) capping. SCA1/2 exhibit canonical glycosyltransferase motifs, and overexpression of either gene leads to elevated microsomal alpha 1,2-d-Ara(p)T activity, resulting in arabinopyranosylation of beta 1,3-Gal side chains in LPG (hereafter called side chain d-arabinopyranosyltransferase [sc-d-Ara(p)T]). Heterologous expression in a null arabinose background was used to determine whether the SCA1 gene encodes the actual sc-d-Ara(p)T. 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-Ara(p)T activity was observed only in the latter. In in vitro assays incubated with guanidine 59-diphosphate (GDP)-d-[H-3]Ara(p) as the sugar donor and utilizing exogenous LPG as an acceptor, significant sc-d-Ara(p)T 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-Ara(p)T and provide the first example of heterologous expression of a d-Ara(p)T gene.