The N-Terminal Domain and Glycosomal Localization of Leishmania Initial Acyltransferase LmDAT Are Important for Lipophosphoglycan Synthesis

Ether glycerolipids of Leishmania major are important membrane components as well as building blocks of various virulence factors. In L. major, the first enzyme of the ether glycerolipid biosynthetic pathway, LmDAT, is an unusual, glycosomal dihydroxyacetonephosphate acyltransferase important for parasite's growth and survival during the stationary phase, synthesis of ether lipids, and virulence. The present work extends our knowledge of this important biosynthetic enzyme in parasite biology. Site-directed mutagenesis of LmDAT demonstrated that an active enzyme was critical for normal growth and survival during the stationary phase. Deletion analyses showed that the large N-terminal extension of this initial acyltransferase may be important for its stability or activity. Further, abrogation of the C-terminal glycosomal targeting signal sequence of LmDAT led to extraglycosomal localization, did not impair its enzymatic activity but affected synthesis of the ether glycerolipid-based virulence factor lipophosphoglycan. In addition, expression of this recombinant form of LmDAT in a null mutant of LmDAT did not restore normal growth and survival during the stationary phase. These results emphasize the importance of this enzyme's compartmentalization in the glycosome for the generation of lipophosphoglycan and parasite's biology

The Trypanosoma brucei dihydroxyacetonephosphate acyltransferase TbDAT is dispensable for normal growth but important for synthesis of ether glycerophospholipids.

Glycerophospholipids are the most abundant constituents of biological membranes in Trypanosoma brucei, which causes sleeping sickness in humans and nagana in cattle. They are essential cellular components that fulfill various important functions beyond their structural role in biological membranes such as in signal transduction, regulation of membrane trafficking or control of cell cycle progression. Our previous studies have established that the glycerol-3-phosphate acyltransferase TbGAT is dispensable for growth, viability, and ester lipid biosynthesis suggesting the existence of another initial acyltransferase(s). This work presents the characterization of the alternative, dihydroxyacetonephosphate acyltransferase TbDAT, which acylates primarily dihydroxyacetonephosphate and prefers palmitoyl-CoA as an acyl-CoA donor. TbDAT restores the viability of a yeast double null mutant that lacks glycerol-3-phosphate and dihydroxyacetonephosphate acyltransferase activities. A conditional null mutant of TbDAT in T. brucei procyclic form was created and characterized. TbDAT was important for survival during stationary phase and synthesis of ether lipids. In contrast, TbDAT was dispensable for normal growth. Our results show that in T. brucei procyclic forms i) TbDAT but not TbGAT is the physiologically relevant initial acyltransferase and ii) ether lipid precursors are primarily made by TbDAT

HV-<i>Lm</i>DAT-ΔC₃ does not localize in the glycosomes.

<p>Wild type expressing recombinant HV-<i>Lm</i>DAT-ΔC₃ was analyzed by phase contrast (panel 1) or immunofluorescence microscopy using anti-V5 antibody (panel 2) or polyclonal antiserum specific to hypoxanthine guanine phosphoribosyltransferase (panel 3). Panel 4 shows the merge of panels 2 and 3.</p

Characterization of mutant forms of <i>Lm</i>DAT.

<p>(A) Schematic representation of human DHAPAT (<i>h</i>DHAPAT) and mutant forms of <i>Lm</i>DAT. The grey rectangle, the black rectangle and the hatched area depict the HV tag, the conserved domain, and the C-terminal glycosomal targeting tripeptide, respectively, and the asterisk depicts the point mutation. B) DHAPAT activity was quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027802#s2" target="_blank">Materials and Methods</a>. Equivalent of 0.5 mg protein extracts were applied for the assay. Null mutant alone or expressing HV-tagged wild-type and mutant forms of <i>Lm</i>DAT were used as a source of protein extracts. Activity is expressed as percentage of the positive control, the wild type (WT). The assay was performed twice in duplicate, and the graph depicts one representative experiment. Standard deviations are shown. (C) Western blot analyses in the presence of V5-specific (upper; V5) and hypoxanthine guanine phosphoribosyltransferase specific (lower; HGPRT; loading control) antibodies. Equivalent of 5×10⁷ cells were loaded in each lane. The apparent molecular weight is shown on the left. (D) Western blot analysis in the presence of WIC79.3 antibody to detect LPG. Equivalent of 10⁶ cells were loaded in each lane. (B, C, D): 1, <i>Δlmdat/Δlmdat</i>; 2, <i>Δlmdat/Δlmdat [HV-LmDAT NEO]</i>; 3, <i>Δlmdat/Δlmdat [HV-ΔN₅₄₆-LmDAT NEO]</i>; 4, <i>Δlmdat/Δlmdat [HV-ΔN₆₈₆-LmDAT NEO]</i>; 5, <i>Δlmdat/Δlmdat [HV-LmDAT-ΔC₇₃₃ NEO]</i>; 6, <i>Δlmdat/Δlmdat [HV-LmDAT-ΔC₃ NEO]</i>; 7, <i>Δlmdat/Δlmdat [HV-LmDAT^K852L NEO]</i>; WT, wild type.</p

Growth curves.

<p>Cells were inoculated at a cell density of 5×10⁵/ml and were enumerated with a hemacytometer as a function of time. The assay was performed twice and the graphs represent a typical experiment. Standard deviations are shown. (A) Black circles, wild type; grey circles, complemented line <i>Δlmdat</i>/<i>Δlmdat [HV-LmDAT NEO]</i>; white circles, <i>Δlmdat</i>/<i>Δlmdat</i>; white triangles, <i>Δlmdat/Δlmdat [HV-ΔN₅₄₆-LmDAT NEO]</i>; grey triangles, <i>Δlmdat/Δlmdat [HV-ΔN₆₈₆-LmDAT NEO]</i>; black triangles, <i>Δlmdat/Δlmdat [HV-LmDAT-ΔC₇₃₃ NEO]</i>. (B) Black circles, wild type; grey circles, complemented line <i>Δlmdat</i>/<i>Δlmdat [HV-LmDAT NEO]</i>; white circles, <i>Δlmdat</i>/<i>Δlmdat</i>; white triangles, <i>Δlmdat</i>/<i>Δlmdat [HV-LmDAT-ΔC₃ NEO]</i>; grey triangles, <i>Δlmdat</i>/<i>Δlmdat [HV-LmDAT^K852L NEO]</i>.</p

Digitonin fractionation followed by Western blot analysis.

<p>FV1 <i>[HV-LmDAT NEO]</i> and FV1 <i>[HV-LmDAT-ΔC₃ NEO]</i> were fractionated in the presence of digitonin as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027802#s2" target="_blank">Materials and Methods</a>. Cell supernatants were then subjected to Western blot analysis in the presence of monoclonal anti-V5 antibodies (V5), and of polyclonal immunoglobulins specific to arginase (ARG) and phosphomannomutase (PMM). Equivalent of 10⁷ cell supernants were loaded in each lane. The apparent molecular weight markers are shown.</p

Glycerolipid biosynthetic pathways in <i>Leishmania</i>.

<p>AGAT, 1-acyl-glycerol-3-phosphate acyltransferase; ADR, alkyl/acyl-DHAP reductase; <i>Lm</i>ADS, alkyl-DHAP synthase; DHAP, dihydroxyacetonephosphate; <i>Lm</i>DAT, DHAP acyltransferase; <i>Lm</i>FAR, fatty acyl-CoA reductase; G3P, glycerol-3-phosphate; <i>Lm</i>GAT, G3P acyltransferase; PA, phosphatidic acid. Genes encoding ADR and AGAT in <i>Leishmania</i> are unknown.</p

Tumor M2-PK: A novel urine marker of bladder cancer.

PurposeBladder cancer is a "Warburg-like" tumor characterized by a reliance on aerobic glycolysis and expression of pyruvate kinase M2 (PKM2). PKM2 oscillates between an active tetramer and an inactive dimer. We aim to further characterize PKM2, in particular PKM2 dimer, as a urinary biomarker of bladder cancer and a potential target for treatment.MethodsHTB-9, HTB-5, and UM-UC3 bladder cancer cells were assessed for proliferation under differential glucose levels using the hexosaminidase assay. Western blot and Blue-native analysis was performed for protein expression of PKM2. Shikonin, an herb that is known to bind and inhibit PKM2, was utilized to determine if PKM2 has a role in glucose usage and cellular proliferation in bladder cancer cells by caspase activity assay. Institutional review board approval was obtained to collect healthy control and bladder cancer patient urine samples. The ScheBo M2-PK EDTA Plasma Test was performed on urine samples to assess urine Tumor M2-PK values.ResultsThe three bladder cancer cell lines tested all demonstrate statistically significant increases in proliferation when exposed to higher level of glucose (200mg/dL). Similarly, low doses of glucose (25mg/dL) result in reduced proliferation. Increased cell growth in higher glucose concentration correlated with up-regulation of PKM2 protein expression. Shikonin, a PKM2 inhibitor, reduced cell proliferation and switched PKM2 isoforms from the dimer to tetramer. Lastly, dimer PKM2 (Tumor-M2PK) levels were assessed in the urine samples from bladder cancer (Bca) patients and healthy controls. Tumor M2-PK significantly correlated with the presence of BCa in our subjects.ConclusionsOur studies demonstrate the potential of PKM2, specifically the dimer (Tumor-M2PK) as a target of drug therapy and as a urinary marker for bladder cancer

Glycerophospholipid quantification.

<p>Glycerophospholipid quantification.</p

Contribution of <i>Tb</i>GAT and <i>Tb</i>DAT to glycerophospholipid biosynthesis.

<p><i>Tb</i>DAT acylates preferentially DHAP and is involved in the biosynthesis of ether and of ester glycerophospholipids (dotted arrow). In contrast, <i>Tb</i>GAT acylates both DHAP and G3P but is implicated mainly in ester glycerophospholipid biosynthesis (solid arrow) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181432#pone.0181432.ref031" target="_blank">31</a>].</p