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Amino acid transport in schistosomes: Characterization of the permeaseheavy chain SPRM1hc.
Krautz-Peterson G
,
Camargo S
,
Huggel K
,
Verrey F
,
Shoemaker CB
,
Skelly PJ
.
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Schistosomes are human parasitic flatworms that constitute an important public health problem globally. Adult parasites live in the bloodstream where they import nutrients such as amino acids across their body surface (the tegument). One amino acid transporter, Schistosome Permease 1 light chain, SPRM1lc, a member of the glycoprotein-associated family of transporters (gpaAT), has been characterized in schistosomes. Only a single member of the SLC3 family of glycoproteins that associate with gpaATs is found following extensive searching of the genomes of Schistosoma mansoni and S. japonicum. In this report, we characterize this schistosome permease heavy chain (SPRM1hc) gene and protein. The 72-kDa gene product is predicted to possess a single transmembrane domain, a (betaalpha)(8) (TIM barrel) conformation and a catalytic triad. Xenopus oocytes functionally expressing SPRM1hc with SPRM1lc import phenylalanine, arginine, lysine, alanine, glutamine, histidine, tryptophan, and leucine. Biochemical characterization demonstrates that in Xenopus extracts and in schistosome extracts SPRM1hc is associated into a high molecular weight complex with SPRM1lc that is disrupted by reducing agents. Quantitative real-time PCR and Western analysis demonstrate that SPRM1hc is expressed in each schistosome life stage examined (eggs, cercariae, schistosomula, adult males and females). SPRM1hc is widely distributed throughout adult male and female worms as determined by immunolocalization. Consistent with the hypothesis that SPRM1hc functions to facilitate nutrient uptake from host blood, immunogold electron microscopy confirms that the protein is distributed on the host-interactive tegumental membranes. We propose that surface-exposed, host-interactive, nutrient-transporting proteins like the SPRM1 heterodimer are promising vaccine candidates.
FIGURE 1. The SPRM1hc gene and predicted protein. A, diagrammatic representation of the SPRM1hc gene. Open rectangles, numbered 1–10, represent exons. The position of a poly(A) addition site is indicated (right). K represents kilobase pairs. B, alignment of SPRM1hc predicted amino acid sequence with homologs. GenBankTM accession numbers of these proteins are: SPRM1hc, EF204542; SjPRMhc, EF204543; rBAT (human), AAH93626; h4F2hc (human), NP 001013269; Ce BAT (or ATG1, C. elegans) CAB02316. Identical residues are indicated by shading. TM indicates the predicted transmembrane domain. 1– 8 and 1– 8 indicate domains comprising the TIM barrel. The arrowhead indicates a conserved cysteine that is postulated to be involved in SPRM1hc cross-linking to its light chain partner (SPRM1lc). Arrows at position Asp278, Glu332, and Asp408 highlight residues that may comprise a catalytic triad. The C-terminal peptide indicated in bold (615IDQPVGSQRVYLKSDGQPM633) was synthesized and used to generate anti-SPRM1hc antibodies.
FIGURE 2. Membrane topology model of the SPRM1hc/SPRM1lc het- erodimer. The gray barrel represents the single predicted transmembrane domain of SPRM1hc. Potential N-glycosylation sites are indicated by forks. The putative transmembrane domains of SPRM1lc are numbered 1–12. The putative cysteine residues involved in disulfide linkage are circled. OUT indi- cates the exterior of the cell; IN interior.
FIGURE 3. Functional characterization of SPRM1hc expressed in X. laevis oocytes. A, comparison of amino acid transport selectivity of SPRM1hc/SPRM1lc versus SPRM1hc/h4F2hc. L-Amino acid transport (mean S.E.) was determined in the presence ( ) or absence ( ) of Na . *, p 0.05; and ***, p 0.001. B, concentration dependence of L-Arg transport in oocytes expressing SPRM1hc/SPRM1lc versus SPRM1hc/h4F2hc. Bars repre- sent the mean S.E. from three independent experiments (n 24 oocytes). C, immunoprecipitation of SPRM1hc/SPRM1lc. Oocytes were injected with cRNA (as indicated) and labeled with [35S]methionine. Immu- noprecipitates, obtained using anti-SPRM1lc antibodies, were resolved by SDS-PAGE in the presence ( ) or absence ( ) of reducing agent and subjected to autoradiography. Arrowheads indicate the positions of SPRM1hc (hc) or SPRM1lc (lc). The arrow indicates the SPRM1hc/SPRM1lc (hc/lc) heterodimer.
FIGURE 4. Developmental expression of SPRM1hc. A, SPRM1hc protein expression determined by Western analysis. Membrane preparations from different parasite life stages (E, egg; C, cercariae; S, 24 h cultured schistoso- mula; , adult female (7-week-old); , adult male (7-week-old) were resolved by SDS-PAGE under reducing conditions; Adult, mixed populations of both males and females, prepared in the presence ( ) or absence ( ) of reducing agent were resolved by SDS-PAGE. The left panel shows a Coomassie Blue- stained gel, and the right panel shows a Western blot of an equivalent gel probed with anti-SPRM1hc antibodies. The arrow indicates the position of SPRM1hc, the arrowhead indicates the position of a higher molecular weight complex (likely SPRM1hc/SPRM1lc) seen when reducing agent is omitted. M, molecular mass markers, numbers represent kDa. B, SPRM1hc gene expres- sion determined by qRT-PCR. The following developmental stages were examined: egg, cercariae, schistosomula (15-day cultured), adult males (7-week-old), and adult females (7-week-old). The relative expression level in adult males was arbitrarily set at 100.
FIGURE 5. Immunolocalization of SPRM1hc in adult parasites. A, cross section through a male/female couple showing widespread staining with anti-SPRM1hc antibodies. B, higher magnification image of the peripheral tissue of an adult male (t, tegument; m, muscle). The arrow indicates the outer tegumental membrane. C, electron micrograph of the adult tegu- ment showing immunogold labeling of SPRM1hc. Arrowheads indicate gold particles at the host/parasite interface. Numbers above scale bars represent microns.
