Identification and sub-cellular localization of a NAD transporter in Leishmania braziliensis (Lb NDT1)

Abstract

Nicotinamide adenine dinucleotide (NAD) is one of the central molecules involved in energy homeostasis, cellular signaling and antioxidative defense systems. Consequently, its biosynthetic pathways and transport systems are of vital importance. The nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT), a key enzyme in the biosynthesis of NAD, is distributed in all domains of life and exhibits various isoforms in free-living organisms in contrast with intracellular parasites, which displays a single enzyme. In Leishmania braziliensis a unique cytosolic NMNAT has been reported to date and the mechanisms through which adequate levels of NAD are maintained among the different sub-cellular compartments of this parasite are unknown. Experimental evidences have related the transport of NAD to the Nucleotide Transporters (NTTs) family, whose members are located in the cytoplasmic membrane of parasitic life organisms. Additionally, the Mitochondrial Carrier Family (MCF), a group of proteins located in the membrane of internal organelles such as the mitochondria of free life organisms, has been implicated in NAD transport. Applying bioinformatics tools, the main characteristics of the MCF were found in a transporter candidate that we have designated as Nicotinamide Adenine Dinucleotide Transporter 1 of L. braziliensis (LbNDT1). The expression of LbNDT1 was tested both in axenic amastigotes and promastigotes of L. braziliensis, through immunodetection using polyclonal avian antibodies produced in this study. N-glycosylation of LbNDT1 was observed in both stages. Additionally, a possible partial mitochondrial distribution for LbNDT1 in amastigotes and a possible glycosomal location in promastigotes are proposed. Finally, the capability of LbNDT1 to transport NAD was confirmed by complementation assays in Saccharomyces cerevisiae. Our results demonstrate the existence of LbNDT1 in L. braziliensis becoming the first NAD transporter identified in protozoan parasites to date.

Keywords: Biochemistry; Bioinformatics; Biomolecules; Cell biology; Energetic metabolism; LbNDT1; Leishmania braziliensis; Molecular biology; NAD transport.

Figure 1

LbNDT1 displays typical characteristics of the MCF. A. The topology model predicts the three tandem sequences (Repeats I–III) that establish six trans-membrane α helices (H1 – H6), and the N and C terminal regions toward the outer face of the membrane. B-E. A predictive model was obtained from the ROBETTA server, whose validation was completed by Ramachandran plots analysis with MolProbity. B. Top visualization of the six trans-membrane α helices (H1 – H6). C. Front visualization of the short α helices (h12, h34 and h56), located toward the inner face of the membrane. D. The N and C terminal regions are positioned towards the outer face of the membrane. E. Location of the Px[DE]xx[KR] (METS) (green) and [YF][DE]xx[KR] motifs (red). Images generated with UCSF Chimera (v 1.10.2).

Figure 2

LbNDT1 shows conformational similarity with MCF members. A. Predicted tertiary structure for LbNDT1 (aa 1–362 red) by ROBETTA server. B. Tertiary structure of the ADP/ATP transporter of S. cerevisiae ADT2 (PDB 4C9G, cyan). C. Superposition between the LbNDT1 model and the ADT2 tertiary structure (RMSD between 143 pairs of atoms: 1.090 Å). Images generated by UCSF-Chimera v 1.10.2.

Figure 3

LbNDT1 partially restores the phenotype of the S. cerevisiae mutant ΔNDT1 strain, deficient in mitochondrial NAD transport. Growth on MS Ura⁻ medium was evaluated for transfectants ΔNDT1 + pYES2/LbNDT1 (▲), ΔNDT1 + pYES2/AtNDT2 (●), ΔNDT1 + empty pYES2 (■), untransfected ΔNDT1 (♦) and the wild strain + empty pYES2 (x). To monitor the growth in liquid medium, readings of OD₆₀₀ were made every 24 h for 5 days. The solid medium images were documented for 4 days with the Imager® Gel DocTMXR image analyzer and the BIORAD™ Quantity One Basic 4.6.3 software. A. Growth in liquid medium containing a fermentable carbon source (raffinose 2% (w/v). B. Growth in liquid medium containing a non-fermentable carbon source (ethanol 2% (v/v) C. Growth in a solid medium containing a non-fermentable carbon source (ethanol 2% (v/v). Three biological replicates were performed for each assay.

Figure 4

Production of polyclonal avian α–LbNDT1 antibodies. A. Expression and purification of the recombinant Trx-LbNDT1 protein from IB was monitored by SDS-PAGE, Coomassie R250 staining and western blot. Lanes: non-induced cells with pBAD202/LbNDT1 (1) and induced (2). Insoluble fraction of induced cells (3). IB's washing with buffer I (4) and buffer II (5). Solubilized IB (6). Soluble (7) and insoluble (8) post dialysis fractions. B. Purification of the Trx-LbNDT1 IB from preparative SDS-PAGE was monitored by SDS-PAGE, Coomassie R250 staining and western blot. Lanes: eluates from the upper (1), intermediate (2) and Lower (3) bands extracted from the preparative SDS-PAGE. C. The specificity of the immune α-LbNDT1 (IS), non-related (NRS, from a different individual inoculated with 1X PBS as a control) and pre-immune sera (PS, from the same immunized individual before the inoculations as a control) at dilutions of 1:5000 was analyzed by western blot. Lanes: 300 ng of BSA (1) and recombinant Trx-LbNDT1 protein (2). Commercial α-Trx antibody (1:6000) was used as a positive control (3). Membranes were developed with the alkaline phosphatase system. The right black arrows indicate the recombinant Trx-LbNDT1 protein. MW, Thermo Scientific™ Pierce™ Unstained Protein Molecular Weight Marker (kDa).

Figure 5

The endogenous N-Glycosylated LbNDT1 was detected in insoluble protein fraction from axenic amastigotes and promastigotes of L. braziliensis. A. Detection of endogenous LbNDT1 in promastigotes (lanes 1–3) and axenic amastigotes (lanes 4–6). SDS-PAGE and western blot: total proteins (1) & (4). Soluble (2) & (5) and insoluble protein fractions (3) & (6). Recombinant Trx-LbNDT1 protein was loaded as a positive control (7). B. The de-glycosylation process of endogenous LbNDT1 was follow by western blot. Total proteins (1). Soluble (2) and insoluble protein fractions (3). De-glycosylation of insoluble protein fraction by chemical treatment (4) or enzymatic treatment with PNGase F (5) Recombinant Trx-LbNDT1 protein was loaded as a positive control (6). C. Immunodetection of the endogenous de-glycosylated LbNDT1 (PNGase F treatment). Lanes: insoluble protein fraction of promastigotes (1) and axenic amastigotes (2). Recombinant Trx-LbNDT1 protein was loaded as a positive control (3). Membranes were developed with the alkaline phosphatase system. The right arrows indicate the endogenous LbNDT1 protein. D. Potential glycosylation sites for LbNDT1 according its predicted topology. Each circle shows the amino acid sequence; green squares: N-Glycosilation on asparagine residues; red circles: O-Glycosylation on threonine and blue circles: O-Glycosylation on serine residues. Numbers 1–6 show the possible transmembrane regions.

Figure 6

The endogenous LbNDT1 protein exhibits a differential subcellular location in axenic amastigotes and promastigotes of L. braziliensis. A.In vitro differentiation process of promastigotes (Objective 20X) to axenic amastigotes (Objective 40X) observed under light microscope. B. The pre-immune (PI) and immune sera (α-LbNDT1) were used in indirect immunofluorescence assay on fixed axenic amastigotes and promastigotes of L. braziliensis. Alexa Fluor 488: endogenous LbNDT1 localization (green). MitoTracker: mitochondria localization (red). DAPI: nuclei localization (blue). Scale bars, 10 μm. Images were acquired with Nikon Eclipse C1 Plus microscope and the EZ-C1 Software. Objective 60X. LbNDT1 and MitoTracker co-localization is evident in amastigotes.