Demonstration by heterologous expression that the ...

1 Glycobiology vol. 16 no. 3 pp. 230 236, 2006. Advance Access publication on November 4, 2005. Demonstration by heterologous expression that the Leishmania SCA1 gene encodes an arabinopyranosyltransferase Mamta Goswami2, Deborah E. Dobson3, the alimentary tract of their sand fly vectors. Following Stephen M. Beverley3, and Salvatore J. Turco1,2 their inoculation into the human host, Leishmania exist as 2. Department of Biochemistry, University of Kentucky Medical Center, intracellular amastigotes in macrophages.

2 Lipophosphoglycan Lexington, KY 40536; and 3 Department of Molecular Microbiology, (LPG) is an abundant molecule on the surface of Leishmania Washington University School of Medicine, St. Louis, MO 63110 promastigotes (Turco and Descoteaux, 1992). The glyco- Received on September 30, 2005; revised on November 1, 2005; accepted on conjugate consists of a small oligosaccharide that caps a November 1, 2005 backbone structure of repeating -6 Gal( 1,4)-Man( 1)-PO4. units (n = 15 30), which are linked by an oligosaccharide In part of the life cycle within their sand fly vector, Leishmania core to a phosphatidylinositide anchor.

3 Species-specific major parasites first attach to the fly's midgut through their polymorphisms in LPG structure occur in the structure of main surface adhesin lipophosphoglycan (LPG) and later the oligosaccharide cap and in the composition and the resynthesize a structurally distinct LPG that results in number of oligosaccharide side chains that branch off from detachment and eventual transmission. One of these struc- the repeat units (Thomas et al., 1992; Turco and Descoteaux, tural modifications requires the addition of 1,2-D- 1992; McConville et al.)

4 , 1995). arabinopyranose caps to 1,3-galactose side chains in the LPG has been shown to serve as an adhesion molecule phosphoglycan repeat unit domain of LPG. We had previ- that mediates the interaction of promastigotes within the ously identified two side chain arabinose genes (SCA1/2) that midgut epithelium of the sand fly (Sacks and Kamhawi, were involved in the 1,2-D-Arap capping. SCA1/2 exhibit 2001). To maintain infection in the fly, the promastigote canonical glycosyltransferase motifs, and overexpression of must attach to the epithelial cells during elimination of the either gene leads to elevated microsomal 1,2-D-ArapT activ- digested bloodmeal.

5 Subsequently, detachment facilitates ity, resulting in arabinopyranosylation of 1,3-Gal side movement of the parasite from the midgut to the mouth- chains in LPG (hereafter called side chain D-arabinopyrano- parts for eventual transmission. In Leishmania major Friedlin syltransferase [sc-D-ArapT]). heterologous expression in a V1 strain (LmFV1), the 1,3-Gal-terminated side chains of null arabinose background was used to determine whether the LPG are critical for the attachment process which involves SCA1 gene encodes the actual sc-D-ArapT.

6 SCA1 expression binding to a recently identified galectin (PpGalec) (Kamhawi constructs introduced into both mammalian COS-7 cells et al., 2004) in the fly's midgut epithelium. During the pro- and the baculovirus-sf9 cell system exhibited considerable cess of metacyclogenesis (Sacks, 1989), the differentiation expression of the protein. However, functional sc-D-ArapT of weakly infective procyclic promastigotes into highly activity was observed only in the latter. In in vitro assays infective metacyclic promastigotes, L.

7 Major detach from the incubated with guanidine 5 -diphosphate (GDP)-D-[3H]Arap midgut to facilitate transmission. Detachment is mediated as the sugar donor and utilizing exogenous LPG as an accep- by structural changes in LPG in which the procyclic LPG. tor, significant sc-D-ArapT activity was observed when that is involved in attachment is replaced by a metacyclic microsomes from the baculovirus-sf9 cells were incubated in LPG that cannot attach to the midgut (Figure 1). In one of presence of the LPG acceptor.

8 No activity was observed in the modifications, there is an increase in size of LPG because the absence of LPG. These results demonstrate that SCA1 of an approximate doubling in the number of repeat units. encodes a sc-D-ArapT and provide the first example of heterol- Second, there is a pronounced decrease in surface LPG with ogous expression of a D-ArapT gene. 1,3-Gal-terminated side chains and a corresponding increase in 1,3-Gal side chains terminating in 1,2-D-Arap Key words: arabinose/arabinosyltransferase/Leishman ia (McConville et al.)

9 , 1992). Importantly, this side chain arabi- major/lipophosphoglycan/metacyclogenesis nose (SCA) (sc-D-Arap) structural modification is not recog- nized by any midgut lectin, and the parasite disengages. Arabinosyl-containing glycoconjugates are plentiful in Introduction microbes (Brennan and Nikaido, 1995), plants (Fincher et al., 1983), and protozoan parasites (Previato et al., 1982;. Leishmania parasites are the causative agents of leishmania- Xavier Da Silveira et al., 1998; Guha-Niyogi et al., 2001), sis, a disease that afflicts millions of people throughout the but are absent in mammalian cells.

10 Unlike other monosac- tropics and subtropics. The parasites spend the extracellu- charides, arabinose can occur naturally in glycoconjugates lar phase of their life cycle as flagellated promastigotes in in pyranose or furanose conformations and in D- and L- stereoisomers. For example, in the mycobacterial lipoara- binogalactan and arabinogalactan (Brennan and Nikaido, 1. To whom correspondence should be addressed; e-mail: 1995), D-arabinofuranose is abundantly present, whereas in The Author 2005. Published by Oxford University Press.