Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
PURPOSE: Plasmalogens (Plgs) are highly abundant lipids in the retina, and their deficiency leads to severe abnormalities during eye development. The first acylation step in the synthesis of Plgs is catalyzed by the enzyme glyceronephosphate O-acyltransferase (GNPAT), which is also known as dihydroxyacetone phosphate-acyltransferase (EC 2.3.1.42). GNPAT deficiency produces rhizomelic chondrodysplasia punctata type 2, a genetic disorder associated with developmental ocular defects. Despite the relevance of retinal Plgs, our knowledge of the mechanisms that regulate their synthesis, and the role of GNPAT during eye development is limited.
METHODS: Using the Xenopus laevis model organism, we characterized by in situ hybridization the expression pattern of gnpat and compared it to glycerol 3-phosphate acyltransferase mitochondrial (gpam or gpat1) during eye neurogenesis, lamination, and morphogenesis. The Xenopus Gnpat was biochemically characterized in a heterologous expression system in yeast.
RESULTS: During development, gnpat is expressed in proliferative cells of the retina and lens, and post-embryogenesis in proliferative cells of the ciliary marginal zone and lensepithelium. In contrast, gpam expression is mainly restricted to photoreceptors. Xenopus Gnpat expressed in yeast is present in both soluble and membrane fractions, but only the membrane-bound enzyme displays activity. The amino terminal of Gnpat, conserved in humans, shows lipid binding capacity that is enhanced by phosphatidic acid.
CONCLUSIONS: Enzymes involved in the Plgs and glycerophospholipid biosynthetic pathways are differentially expressed during eye morphogenesis. The gnpat expression pattern and the molecular determinants regulating Gnpat activity advance our knowledge of this enzyme, contributing to our understanding of the retinal pathophysiology associated with GNPAT deficiency.
Figure 1. De novo synthesis of alkyl and acyl phospholipids by GNPAT and GPAM. GNPAT and GPAM enzymes catalyze the initial transfer of an acyl chain from acyl-CoA to the sn-1 position of DHAP or G3P, respectively. In peroxisomes, GNPAT generates 1-acyl DHAP, which is channeled to the next enzyme in the pathway, acyl-DHAP-synthase (ADHAPS), for the formation of the first ether lipid intermediate, 1-alkyl-DHAP. Both 1-alkyl DHAP and 1-acyl DHAP are substrates of a reductase (ADHAPR) that produces their G3P counterparts. 1-Alkyl-G3P is then consumed in the ER, where the final enzymatic reactions leading to the synthesis of plasmalogens occur, introducing the characteristic vinyl-ether bond (red) in the sn-1 position of the glycerol backbone. GPAM, localized to the outer mitochondrial membrane, produces 1-acyl-glycerol-3-phosphate (1-acyl-G3P; also referred to as lysophosphatidic acid), which is converted to PA by a second acylation step. PA is the precursor of all acyl GPLs, which are the main structural components of cellular membranes. A-DHAPR, acyl/alkyl-DHAP reductase. FAR, fatty acyl-CoA reductase.
Figure 2. Expression of gnpat and gpam in the eye during development. gnpat: ISH in transverse sections with gnpat-specific antisense riboprobe on slides obtained from X. laevis tadpoles at the indicated embryonic stages. Expression becomes restricted to the CMZ during neuronal differentiation and lamination of the eye (compare stage 31 vs. stages 33/34 and 35/36). At stages 35 and 37, when most retinal cells have exited the cell cycle,29 expression recedes from the central retina (*). At stages 37/38, gnpat mRNA is restricted to the CMZ and PLFCs. At stage 39 and older, after lens formation, gnpat is expressed in the LE. gpam: ISH in transverse sections with gpam antisense riboprobe. Arrows at stages 33/34 potentially indicate newly born photoreceptors (PRs) that have not yet reached the outer retina. At stage 35, the arrow points to photoreceptors located in the dorsal part of the ONL. Note the dorsal–ventral gradient of gpam-expressing cells at stages 35 and 37, whereas expression occurs over the whole ONL by stages 39 and 42. The melanin pigment of the RPE precludes visualization of the ISH signal and is also detected with sense probes (Supplementary Fig. S1). Scale bar: 50 µM.
Figure 3. Determination of retinal cells expressing gnpat and gpam. (A) ISH with gnpat-specific riboprobes was followed by immunohistochemistry against calbindin (red; cones) and Islet1 (green; retinal ganglion cells and amacrine cells) on transverse sections of stage 42 eye. Nuclei were revealed by DAPI staining. Higher magnification of the indicated region and merged pictures are shown in the bottom panels. Note that gnpat mRNA in the CMZ is absent from the aadjacent neuronal marker Isl1-expressing cells. In the lens, gnpat expression overlaps with cell nuclei (DAPI staining) of the LE, but it is not expressed in cells that have lost their nuclei (no DAPI staining). Scale bar: 50 µm. (B) ISH against gpam followed by immunohistochemistry against calbindin (red; cones) and rhodopsin (green; rods) on transverse sections of a stage 39 embryo. Nuclei were revealed by DAPI staining. Enlarged magnifications are shown at the bottom. Note that gpam is expressed in the ONL and is co-expressed by both cones (calbindin positive, filled arrow) and rods (rhodopsin positive, open arrows). Scale bar: 50 µm.
Figure 4. Gnpat or Gpam general overexpression induces severe defects in early embryonic development. (A) Schematic of stages 1 and 2 (two cells) indicating the microinjection site to produce widespread overexpression. (B) Representative examples showing the developmental defects induced by general overexpression with the indicated plasmids (stages 35/36). (C) Quantitation of normal embryos or those with gastrulation (Gastr.) or embryonic malformation (Malform.) defects after microinjection. (D) Quantitation of embryonic survival of embryos microinjected with Xl-gpam or catalytically inactive Xl-GpamR317A constructs. The total number of embryos analyzed (n) is indicated at the top of each bar (N = 4).
Figure 5. Retinal lamination appears normal with eye-targeted overexpression or eye electroporation of Gnpat or Gpam. (A) Schematic of stage 3 (four cells; animal view) and stages 27/28 embryos indicating the microinjection site and the electroporation technique used to produce eye-targeted overexpression, respectively. Eye structure and lamination were analyzed at stage 39. The yellow line indicates the approximate location of sections. (B) Quantitation of normal embryos or those with gastrulation (Gastr.) or developmental malformation (Malform.) defects after microinjection of stage 3 embryos targeting the eye. The total number of embryos analyzed (n) is indicated at the top of each bar (N = 4). (C) Representative pictures of GFP expression in lateral views of the head of eye-targeted stage 39 embryos microinjected at the four-cell stage with the indicated plasmid DNA together with pCS2–GFP. E, eye; B, brain; L, lens; S, skin. (D, E) Immunohistochemistry of central retinal sections of stage 39 eyes electroporated at stages 27/28 either for GFP (D) or against GFP and activated caspase 3 (red cells; white arrows) (E). The nuclei were stained with DAPI. GCL, ganglion cell layer; INL, inner nuclear layer; L, lens; PR, photoreceptor. Scale bar: 50 µm.
Figure 6.
X
.
laevis GNPAT and GPAM are functional in yeast. (A) Conditional lethal yeast strains lacking endogenous yeast GPATs Sct1 and Gpt2 (sct1Δ gpt2Δ) were successfully generated by introducing the sequences coding for the indicated acyltransferases in URA3-based plasmids under the galactose inducible promoter. Shift of these strains to glucose represses expression from the plasmid resulting in the lethal phenotype. (B) Expression of Gnpat-V5/His6 and Gpam-V5/His6 analyzed by western blotting using anti-V5 antibodies in lysates and soluble and membrane preparations obtained from subcellular fractionation. (C) Acyltransferase activity assay. Lysates were obtained from a conditional lethal sct1Δ gpt2Δ strain expressing Gnpat-V5/His6 under the GAL promoter. Activity was expressed relative to that obtained with Gnpat lysate at pH 7.5 with DHAP and palmitoyl-CoA as substrates (activity lysate at pH 7.5 = 3 pmol/min/mg protein). ***P < 0.001 ANOVA followed by Bonferroni's test. (D) Gnpat lacks activity when soluble. The western blot and acyltransferase activity assays are shown in C and D, respectively. Note that, for the soluble fraction, the signal of the band corresponding to Gnpat was five times higher than the one corresponding to the membrane fraction in the western blot (activity membrane protein = 1.4 pmol/min/mg protein). Shown are representative results of one of two independent experiments.
Figure 7. Catalytically active forms of Xenopus Gpam and Gnpat support life of yeast devoid of endogenous GPATs. A double knock-out sct1Δ gpt2Δ strain expressing GPT2 under the GAL promoter in a LEU2 plasmid (previously introduced in Fig. 6A) was transformed with a URA3-based plasmid containing wild-type gpam or catalytically dead gpamR317A (A) or wild-type gnpat or catalytically dead gnpatH145A (C). Four independent transformants were grown in selective –ura/–leu plates containing galactose and then restreaked on galactose or glucose plates and grown for 3 days at 30°C (A, C). Expression of the catalytically dead enzymes GpamR317A (B) or GnpatH145A (D) confirmed by western blot of wild-type yeast transformed with empty vector or the same plasmids used in A and C. Membranes stained with Ponceau Red dye after transfer are shown for loading control. Primary antibodies against V5 epitope fused to Gpam (B) or GFP fused to Gnpat (D) detected heterologous expression of the proteins.
Figure 8. Comparative protein analysis between Xenopus and human GNPAT. (A–D) Hydrophobicity plot detected by analysis with TMHMM software (A), which is also indicated as a gray
shade in the protein alignment in B. The protein alignment (B) was performed between X.
laevis (Uniprot Q6GM34) and Homo sapiens GNPAT (Uniprot O15228-1). The HeliQuest analysis (C) shows putative AHs (light blue) associated with regions encoded by exons 2 (AH2) and 3 (AH3), which are also indicated in the protein alignment (B) and the schematic of the protein structure (D). HeliQuest analysis was carried out for the N-terminal region of both proteins encoded by exons 1 to 3, using a window of 18 amino acids. The N-terminus region encoded by exons 1, 2 (black underlined), and 3 (red underlined) is shown in B and D. The middle catalytic region contains the four motifs with signature conserved amino acid sequence (red box), and the peroxisomal targeting signal 1 (PTS1, yellow) is located in the carboxy-end region.
Figure 9. The amino end of Gnpat is necessary for a functional enzyme. (A) A double knock-out sct1Δ gpt2Δ strain expressing GPT2 under the GAL promoter in a LEU2 plasmid was transformed with a URA3-based plasmid containing wild-type gnpat or truncated Δ129N-gnpat. Four independent transformants were grown in selective –ura/–leu plates containing galactose and then re-streaked on galactose or glucose plates and grown for 3 days at 30°C. (B) Expression of Gnpat-GFP, ΔN-Gnpat-GFP, and GFP in wild-type cells analyzed by western blotting using anti-GFP antibodies in lysates and soluble and membrane preparations obtained from subcellular fractionation. Membranes stained with Ponceau Red dye after transfer are shown for the loading control.
Figure 10. The GNPAT amino-terminus has binding preference for phosphatidic acid. (A) The N-terminal end of Xl-Gnpat tagged with V5/His6 was expressed in E. coli and purified by Ni-NTA chromatography. Western blot analysis with anti-V5 primary antibodies is shown on the left. Expected molecular weight for the N-terminal end is 18.4 kDa. Total protein stained with TCE is shown (right). (B) Protein lipid overlay. Indicated lipid dots were incubated with proteins and detected as in A. GFP tagged with V5/His6 was used as a negative control. (C) Flow chart illustrating the steps of the flotation assay. (D) Dot-blot analysis of fractions obtained from the top to the bottom of the gradient (1–4) using anti-V5 primary antibody. Densitometry shown below the blots combined fractions 1 and 2 (top) and 3 and 4 (bottom), expressed as a percentage of the total signal from all combined fractions. POPC, palmitoyl oleoyl phosphatidylcholine.
Acar,
Plasmalogens in the retina: in situ hybridization of dihydroxyacetone phosphate acyltransferase (DHAP-AT)--the first enzyme involved in their biosynthesis--and comparative study of retinal and retinal pigment epithelial lipid composition.
2007, Pubmed
Acar,
Plasmalogens in the retina: in situ hybridization of dihydroxyacetone phosphate acyltransferase (DHAP-AT)--the first enzyme involved in their biosynthesis--and comparative study of retinal and retinal pigment epithelial lipid composition.
2007,
Pubmed
Alberti,
A suite of Gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae.
2007,
Pubmed
Atkinson-Leadbeater,
Dynamic expression of axon guidance cues required for optic tract development is controlled by fibroblast growth factor signaling.
2010,
Pubmed
,
Xenbase
Baker,
The outer segment serves as a default destination for the trafficking of membrane proteins in photoreceptors.
2008,
Pubmed
,
Xenbase
Bertolesi,
Identification and expression analysis of GPAT family genes during early development of Xenopus laevis.
2012,
Pubmed
,
Xenbase
Bertolesi,
Wiring the retinal circuits activated by light during early development.
2014,
Pubmed
,
Xenbase
Borchman,
Lipids and the ocular lens.
2010,
Pubmed
Bozelli,
Plasmalogen Replacement Therapy.
2021,
Pubmed
Bratschi,
Glycerol-3-phosphate acyltransferases gat1p and gat2p are microsomal phosphoproteins with differential contributions to polarized cell growth.
2009,
Pubmed
Braverman,
Mutation analysis of PEX7 in 60 probands with rhizomelic chondrodysplasia punctata and functional correlations of genotype with phenotype.
2002,
Pubmed
Chang,
Sequential genesis and determination of cone and rod photoreceptors in Xenopus.
1998,
Pubmed
,
Xenbase
Chiquet,
Characterization of calbindin-positive cones in primates.
2002,
Pubmed
Clayton,
Isolated dihydroxyacetonephosphate acyltransferase deficiency presenting with developmental delay.
1994,
Pubmed
Costello,
Identification and Ultrastructural Characterization of a Novel Nuclear Degradation Complex in Differentiating Lens Fiber Cells.
2016,
Pubmed
Das,
Dietary ether lipid incorporation into tissue plasmalogens of humans and rodents.
1992,
Pubmed
de Vet,
Characterization of recombinant guinea pig alkyl-dihydroxyacetonephosphate synthase expressed in Escherichia coli. Kinetics, chemical modification and mutagenesis.
1999,
Pubmed
Dircks,
A conserved seven amino acid stretch important for murine mitochondrial glycerol-3-phosphate acyltransferase activity. Significance of arginine 318 in catalysis.
1999,
Pubmed
Gautier,
HELIQUEST: a web server to screen sequences with specific alpha-helical properties.
2008,
Pubmed
Gimeno,
Thematic review series: glycerolipids. Mammalian glycerol-3-phosphate acyltransferases: new genes for an old activity.
2008,
Pubmed
Gonzalez-Baro,
Mitochondrial glycerol phosphate acyltransferase contains two transmembrane domains with the active site in the N-terminal domain facing the cytosol.
2001,
Pubmed
Gorgas,
The ether lipid-deficient mouse: tracking down plasmalogen functions.
2006,
Pubmed
Hajra,
Induction of the peroxisomal glycerolipid-synthesizing enzymes during differentiation of 3T3-L1 adipocytes. Role in triacylglycerol synthesis.
2000,
Pubmed
Hajra,
Dihydroxyacetone phosphate acyltransferase.
1997,
Pubmed
Hollyfield,
Photoreceptor outer segment development: light and dark regulate the rate of membrane addition and loss.
1979,
Pubmed
,
Xenbase
Hollyfield,
Differential growth of the neural retina in Xenopus laevis larvae.
1971,
Pubmed
,
Xenbase
Holt,
Cellular determination in the Xenopus retina is independent of lineage and birth date.
1988,
Pubmed
,
Xenbase
Honsho,
Distinct Functions of Acyl/Alkyl Dihydroxyacetonephosphate Reductase in Peroxisomes and Endoplasmic Reticulum.
2020,
Pubmed
Honsho,
Plasmalogen homeostasis - regulation of plasmalogen biosynthesis and its physiological consequence in mammals.
2017,
Pubmed
Hsu,
Light regulates the ciliary protein transport and outer segment disc renewal of mammalian photoreceptors.
2015,
Pubmed
Huang,
Blastomeres show differential fate changes in 8-cell Xenopus laevis embryos that are rotated 90 degrees before first cleavage.
1998,
Pubmed
,
Xenbase
Huang,
Human lens phospholipid changes with age and cataract.
2005,
Pubmed
Itzkovitz,
Functional characterization of novel mutations in GNPAT and AGPS, causing rhizomelic chondrodysplasia punctata (RCDP) types 2 and 3.
2012,
Pubmed
Jones,
Assay of dihydroxyacetone phosphate acyltransferase with 32P-labeled substrate.
1994,
Pubmed
Kawai,
Transformation of Saccharomyces cerevisiae and other fungi: methods and possible underlying mechanism.
2010,
Pubmed
Ladner,
Visible fluorescent detection of proteins in polyacrylamide gels without staining.
2004,
Pubmed
Lewin,
Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction.
1999,
Pubmed
Marr,
Controlling lipid fluxes at glycerol-3-phosphate acyltransferase step in yeast: unique contribution of Gat1p to oleic acid-induced lipid particle formation.
2012,
Pubmed
McFarlane,
The Xenopus retinal ganglion cell as a model neuron to study the establishment of neuronal connectivity.
2012,
Pubmed
,
Xenbase
Mercurio,
Ultrastructural localization of glycerolipid synthesis in rod cells of the isolated frog retina.
1982,
Pubmed
Mizuno,
Lipid composition and (Na+ + K+)-ATPase activity in rat lens during triparanol-induced cataract formation.
1981,
Pubmed
Mochizuki,
The lens equator: a platform for molecular machinery that regulates the switch from cell proliferation to differentiation in the vertebrate lens.
2014,
Pubmed
Moser,
Molecular genetics of peroxisomal disorders.
2000,
Pubmed
Naufer,
pH of endophagosomes controls association of their membranes with Vps34 and PtdIns(3)P levels.
2018,
Pubmed
Ofman,
Acyl-CoA:dihydroxyacetonephosphate acyltransferase: cloning of the human cDNA and resolution of the molecular basis in rhizomelic chondrodysplasia punctata type 2.
1998,
Pubmed
Perron,
The genetic sequence of retinal development in the ciliary margin of the Xenopus eye.
1998,
Pubmed
,
Xenbase
Piano,
Recombinant human dihydroxyacetonephosphate acyl-transferase characterization as an integral monotopic membrane protein.
2016,
Pubmed
Racenis,
The acyl dihydroxyacetone phosphate pathway enzymes for glycerolipid biosynthesis are present in the yeast Saccharomyces cerevisiae.
1992,
Pubmed
Rakshit,
Rhodopsin Forms Nanodomains in Rod Outer Segment Disc Membranes of the Cold-Blooded Xenopus laevis.
2015,
Pubmed
,
Xenbase
Rodemer,
Inactivation of ether lipid biosynthesis causes male infertility, defects in eye development and optic nerve hypoplasia in mice.
2003,
Pubmed
Saab,
Plasmalogens in the retina: from occurrence in retinal cell membranes to potential involvement in pathophysiology of retinal diseases.
2014,
Pubmed
Smart,
Phylogenetic analysis of glycerol 3-phosphate acyltransferases in opisthokonts reveals unexpected ancestral complexity and novel modern biosynthetic components.
2014,
Pubmed
Stiemke,
Cell birthdays in Xenopus laevis retina.
1995,
Pubmed
,
Xenbase
Stiemke,
Photoreceptor outer segment development in Xenopus laevis: influence of the pigment epithelium.
1994,
Pubmed
,
Xenbase
Takeuchi,
Biochemistry, physiology, and genetics of GPAT, AGPAT, and lipin enzymes in triglyceride synthesis.
2009,
Pubmed
Thai,
Synthesis of plasmalogens in eye lens epithelial cells.
1999,
Pubmed
van Leyen,
A function for lipoxygenase in programmed organelle degradation.
1998,
Pubmed
Viet,
Modeling ocular lens disease in Xenopus.
2020,
Pubmed
,
Xenbase
Wanders,
Human dihydroxyacetonephosphate acyltransferase deficiency: a new peroxisomal disorder.
1992,
Pubmed
Waterham,
Genetics and molecular basis of human peroxisome biogenesis disorders.
2012,
Pubmed
Zaremberg,
Differential partitioning of lipids metabolized by separate yeast glycerol-3-phosphate acyltransferases reveals that phospholipase D generation of phosphatidic acid mediates sensitivity to choline-containing lysolipids and drugs.
2002,
Pubmed
Zheng,
The initial step of the glycerolipid pathway: identification of glycerol 3-phosphate/dihydroxyacetone phosphate dual substrate acyltransferases in Saccharomyces cerevisiae.
2001,
Pubmed
Zhou,
Advances in the Biosynthetic Pathways and Application Potential of Plasmalogens in Medicine.
2020,
Pubmed
