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Journal of Lipid Research logoLink to Journal of Lipid Research
. 2012 Apr;53(4):653–663. doi: 10.1194/jlr.M021204

Defective lipid remodeling of GPI anchors in peroxisomal disorders, Zellweger syndrome, and rhizomelic chondrodysplasia punctata

Noriyuki Kanzawa *, Nobuyuki Shimozawa †, Ronald J A Wanders §, Kazutaka Ikeda **, Yoshiko Murakami *, Hans R Waterham §, Satoru Mukai ††, Morihisa Fujita *, Yusuke Maeda *, Ryo Taguchi **, Yukio Fujiki ††, Taroh Kinoshita *,§§,1
PMCID: PMC3307642  PMID: 22253471

Abstract

Many cell surface proteins in mammalian cells are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The predominant form of mammalian GPI contains 1-alkyl-2-acyl phosphatidylinositol (PI), which is generated by lipid remodeling from diacyl PI. The conversion of diacyl PI to 1-alkyl-2-acyl PI occurs in the ER at the third intermediate in the GPI biosynthetic pathway. This lipid remodeling requires the alkyl-phospholipid biosynthetic pathway in peroxisome. Indeed, cells defective in dihydroxyacetone phosphate acyltransferase (DHAP-AT) or alkyl-DHAP synthase express only the diacyl form of GPI-anchored proteins. A defect in the alkyl-phospholipid biosynthetic pathway causes a peroxisomal disorder, rhizomelic chondrodysplasia punctata (RCDP), and defective biogenesis of peroxisomes causes Zellweger syndrome, both of which are lethal genetic diseases with multiple clinical phenotypes such as psychomotor defects, mental retardation, and skeletal abnormalities. Here, we report that GPI lipid remodeling is defective in cells from patients with Zellweger syndrome having mutations in the peroxisomal biogenesis factors PEX5, PEX16, and PEX19 and in cells from patients with RCDP types 1, 2, and 3 caused by mutations in PEX7, DHAP-AT, and alkyl-DHAP synthase, respectively. Absence of the 1-alkyl-2-acyl form of GPI-anchored proteins might account for some of the complex phenotypes of these two major peroxisomal disorders.

Keywords: glycosylphosphatidylinositol, plasmalogens, peroxisomal disorder, rhizomelic chondrodysplasia punctata

The peroxisome, a single membrane-bounded organelle, contains over 50 different enzymes catalyzing various metabolic pathways important for human physiology (1–3). Biosynthesis of plasmalogens and β-oxidation of very long chain fatty acids are among its essential functions. Genetic defects affecting peroxisome biogenesis or one of the enzymes localized in peroxisomes cause peroxisomal disorders (1, 3–6).

Zellweger syndrome (ZS), the most severe peroxisomal disorder, is characterized by very early lethality, neonatal neurodegeneration, mental retardation, seizures, enlarged liver, and characteristic facial features (3, 7, 8). In ZS, either biogenesis of the organelle membrane or import of the major group of enzymes bearing a type 1 peroxisome targeting signal (PTS1) into the organelle is defective. The former defect is caused by mutation in PEX3, PEX16, or PEX19, which are involved in protein incorporation into the peroxisomal membrane (9). The latter defect is caused by mutations in other PEX genes, such as PEX1, -5, -6, -12, and -26. Among them, PEX5 is a receptor for PTS1 (Fig. 1).

Fig. 1.

Fig. 1.

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Peroxisome-dependent lipid remodeling of GPI and biosynthesis of plasmalogens. Plasmalogens are synthesized from DHAP through multiple reaction steps. The first two steps, mediated by DHAP-AT and alkyl-DHAP synthase, occur in the peroxisome; further reactions occur in the ER. Lipid remodeling of GPI, namely conversion of diacyl GPI into 1-alkyl-2-acyl GPI, occurs in the ER, requiring a putative alkyl donor lipid that is derived from 1-alkyl-DHAP. Import of DHAP-AT and alkyl-DHAP synthase into the peroxisome is dependent upon PEX5 and PEX7, respectively. Incorporation of membrane proteins into the peroxisomal membrane is dependent upon PEX3, PEX16, and PEX19. Deficiencies of PEX3, PEX5, PEX16, or PEX19 cause ZS, whereas those of PEX7, DHAP-AT, and alkyl-DHAP synthase cause RCDP types 1, 2, and 3, respectively.

In rhizomelic chondrodysplasia punctata (RCDP) type 1, which is characterized by lethality between 2–3 years of age, a typical facial appearance, cataracts, skeletal dysplasia, microcephaly, and severe psychomotor defects, biosynthesis of alkyl-phospholipids and plasmalogens, and degradation of phytanic acid derived from phytol in chlorophyl are defective, whereas peroxisome membrane biogenesis per se and incorporation of PTS1-bearing proteins are not severely affected. RCDP type 1 is caused by mutation in PEX7 that is essential for transferring the minor group of enzymes bearing a PTS2, such as 3-ketoacyl-CoA thiolase of fatty-acid β-oxidation pathway, alkyl-dihydroxyacetone phosphate synthase (alkyl-DHAP synthase), which is required for synthesis of alkyl-phospholipids, and phytanoyl-CoA 2-hydroxylase, which is required for degradation of phytanic acid (3). Specific defects in DHAP-acyltransferase (DHAP-AT) and alkyl-DHAP synthase (also called alkylglycerone phosphate synthase), the first two enzymes in the alkyl-phospholipid biosynthetic pathway, cause disorders similar to RCDP type 1, termed RCDP type 2 and type 3, respectively (10–12), indicating that the major symptoms of RCDP are due to defective biosynthesis of plasmalogens and/or other alkyl-phospholipids, such as platelet activating factor.

More than 150 different cell surface proteins in mammalian cells are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). GPI-anchored proteins (GPI-APs) include receptors (such as folate receptor and urokinase-type plasminogen activator receptor), adhesion molecules (such as neural cell adhesion molecule), enzymes (such as alkaline phosphatase and 5′-nucleotidase), and complement regulatory proteins (CD55 and CD59), playing roles in cell-cell and cell-environment interactions (13–15). GPI, consisting of phosphatidylinositol (PI), glucosamine (GlcN), three mannoses (Man), and three ethanolamine phosphates, is synthesized from PI in the ER, and preassembled GPI is transferred to the protein's carboxyl terminus (14–16). In mammalian cells, the majority of protein-linked GPI contains 1-alkyl-2-acyl PI, whereas most free PIs from which GPIs are generated are in the diacyl form. The first and second intermediates of GPI precursors, N-acetyl glucosaminyl (GlcNAc)-PI and GlcN-PI, are mostly diacyl forms, whereas about 60% of the third intermediate, GlcN-(inositol-acylated)-PI (GlcN-acyl-PI), contain 1-alkyl-2-acyl PI, indicating that the 1-alkyl chain is generated after the third biosynthetic step in the ER (17). We reported that generation of the 1-alkyl-2-acyl form of GPI is dependent upon the peroxisomal alkyl-phospholipid biosynthetic pathway (18): Two different Chinese hamster ovary (CHO) mutant cell lines, NRel-4 and NZel-1 cells, which were defective in DHAP-AT or alkyl-DHAP synthase, respectively (19, 20), did not produce a detectable amount of the 1-alkyl-2-acyl form of GPI (18). It is conceivable that a putative alkyl-containing lipid donor is generated in the ER from alkyl-DHAP and is used in the remodeling reaction that converts the diacyl form GlcN-acyl-PI to the 1-alkyl-2-acyl form (Fig. 1).

These results suggested that remodeling of the diacyl form of GPI to the 1-alkyl-2-acyl form would not occur in cells from patients with RCDP types 2 and 3. Cells from patients with RCDP type 1 might also be defective in this remodeling. It is also likely that cells from patients with ZS are defective in generating 1-alkyl-2-acyl GPI. In this study, we investigated these hypotheses using CHO cells defective in PEX7, PEX5, or PEX19 and fibroblasts from patients with RCDP types 1, 2, or 3, or ZS (21–23). Our results indicate that these cells generate only the diacyl form of GPI.

MATERIALS AND METHODS

Cells and materials

Fibroblast cell lines defective in PEX7, termed Gifu R01 and R03, were derived from two patients with RCDP type 1; those defective in PEX5, termed 2-07, and those defective in PEX16, termed D-01, were from patients with ZS; those defective in DHAP-AT, termed NL-#2 and #3, and those defective in alkyl-DHAP synthase, termed NL-#5 and #6, were from patients with RCDP type 2 and patients with RCDP type 3, respectively (24, 54). This study was approved by internal review committees in the Research Institute for Microbial Diseases of Osaka University, Gifu University School of Medicine, and University of Amsterdam. Written informed consent for specific research use was not obtained because all patients died within a few years after birth. The research use of their fibroblast cells that were taken for diagnostic purposes and stored frozen was approved by the internal review committees because these are rare lethal diseases for which investigation of the disease mechanism is important for the wellness of patients with ZS and RCDP and their families. To generate immortalized cell lines, we introduced cDNAs of human telomerase reverse transcriptase and SV40-T into fibroblast cell lines using a retrovirus vector (55–58). The immortalized cells were cultured in MEM medium with 10% FBS supplemented with 100 μg/ml of G418 and 0.5 μg/ml of puromycin to maintain plasmids. To complement RCDP fibroblasts, human PEX7 and DHAP-AT cDNAs were transduced using a retrovirus vector with a blasticidin resistance gene, and the cells were maintained in 10 μg/ml of blasticidin (InvivoGen, San Diego, CA). The PEX mutant and complemented CHO cells have been reported previously (21, 22, 34). ZPG207, ZP139, and ZP119 are defective in PEX7, PEX5, and PEX19, respectively. We used ZPG207 complemented with human PEX7 cDNA, termed ZPG207P7 (22), and ZP119 complemented with human PEX19, termed 119/P19 (34). ZPG207 complemented with FLAG-tagged hamster Pex7, termed ZPG207/FLAG-ClPEX7, and ZP139 complemented with HA-tagged hamster Pex5L, termed 139/5L-HA, were kind gifts from Drs. Yasuhiro Miyauchi and Kanji Okumoto, Kyushu University, respectively. For mass analysis of GPI, we generated ZPG207 cells stably expressing His-FLAG-GST-FLAG-tagged CD59 (HFGF-CD59). The CHO mutant cells NRel-4 and NZel-1 defective in DHAP-AT and alkyl-DHAP synthase, respectively, are gifts from R. A. Zoeller (Boston University, MA) (19, 20). pcDNA3.1/Zeo(+)-DHAP-AT plasmid was electroporated into NRel-4 and NZel-1 cells under the conditions of 1000 μF/260V. After 3 days, the cells were stained by mouse IgG1 monoclonal antibody clone 5D6 against hamster uPAR (40) and phycoerythrin-conjugated goat anti-mouse IgG (BD Biosciences, Franklin Lakes, NJ), and analyzed by FACS Calibur (BD Biosciences). In Western blotting, antibodies for Calnexin and Caveolin-1 (BD Biosciences), α-Tubulin (Sigma-Aldrich, St. Louis, MO), and glyceraldehydephosphate dehydrogenase (GAPDH, Life Technologies, Carlsbad, CA) were used.

Mass spectrometric analysis of PI from GPI-AP and HFGF-CD59

We used approximately 1 nmol (50 μg) of HFGF-CD59 for mass analysis of the PI moiety. The PI portion was isolated from affinity-purified HFGF-CD59 by deamination with sodium nitrite as described previously (18). PI derived from HFGF-CD59 was stored at –80°C until MS analysis. In CHO cells, data are cited from reference 18, and the method was described in same reference. For analysis of PI in ZPG207 cells, Nano ESI-MS/MS analysis was performed in negative ion mode using a 4000Q TRAP (AB Sciex, Foster City, CA) with a chip-based ionization source, TriVersa NanoMate (Advion BioSystems, Ithaca, NY). The ion spray voltage was set at –1.2 kV, the gas pressure at 0.3 psi, and the flow rate at 200 nl/min. The parameter settings were m/z 400–1,200 for scan range, –100 V for declustering potential, –50 to –60 volts for collision energy, and Q1/Q3 “unit” resolution. Samples were dissolved in C/M (1:2) containing 5 mM ammonium formate for injection into the mass spectrometer. The molecular species of PI that were liberated from HFGF-CD59 were directly subjected to flow injection and selectively analyzed by precursor ion scanning of the phosphoryl inositol part (59). The structure of each PI molecular species was confirmed by MS/MS analysis of the precursor ion.

In vivo labeling of cells with 3H-mannose and a test for the alkali resistance of GPI

Before labeling, cells (3 × 106 in a 60-mm dish) were cultured in medium containing 10 μM BE49385A/YW3548, a PIG-N inhibitor (a gift from Banyu Pharmaceutical) for 12–16 h (60, 61). When PIG-N, which transfers ethanolamine-phosphate side-branch to the first Man, is inhibited, Man-containing GPI intermediates lacking the side-branch are accumulated, and efficient radio-labeling is achieved (60). Cells were then incubated in 2.5 ml of glucose-free RPMI-1640 medium (Gibco/Invitrogen) containing 10 μg/ml tunicamycin (Wako Pure Chemical Industries, Ltd.), 10% dialyzed FBS, 20 mM HEPES, and 100 μg/ml D-glucose for 1 h. After tunicamycin treatment, 40 μCi/ml for CHO cells or 10 μCi/ml for fibroblasts of D-[2-3H(N)]mannose (American Radiolabeled Chemicals) was added, and incubation was continued for 1 h. Lipids were extracted from the cell pellet using two 300-μl aliquots of water-saturated butanol (BuOH). The extracted lipids were treated with 500 μl of 0.1 N KOH in methanol (MeOH) or MeOH only for 1 h at 37°C. To neutralize the solutions, 50 μl of 1 M acetic acid or water was added, and the samples were evaporated to dryness using a speed-vac. The lipids were extracted using two 300-μl aliquots of water-saturated BuOH and back-washed with 200 μl of BuOH-saturated water. After evaporation, the extracted lipids were used as samples for TLC on activated high-performance TLC silica gel 60 plates (Merck) with a developing solvent system of chloroform/MeOH/H2O (10:10:3).

Immunofluorescence microscopy

Cells were washed with PBS, fixed with PBS containing 4% paraformaldehyde for 20 min at room temperature, and permeabilized with PBS containing 1% BSA, 0.1% NaN3, and 0.1% Triton X-100. Cells were stained with combinations of rabbit anti-human catalase serum (1:3,000) and Alexa 594-conjugated goat secondary antibodies. For uPAR staining, cells were permeabilized with PBS containing 0.1% saponin instead of Triton X-100 and stained with 5 μg/ml anti-uPAR mAb (clone 5D6) and Alexa 488-conjugated goat secondary antibodies (Invitrogen). Images were acquired on a FluoView FV1000 (Olympus).

RESULTS

GPI lipid remodeling to generate 1-alkyl-2-acyl GPI is defective in PEX7 mutant CHO cells and fibroblasts from patients with RCDP type 1

PEX7 is required for transporting proteins bearing a PTS2, such as alkyl-DHAP synthase, into the peroxisome and is a causal gene for RCDP type 1 (24–27). We tested whether a defect in PEX7 affects the lipid remodeling of GPI. We first tested the PEX7-mutant CHO cell line ZPG207 and ZPG207 transfected with human PEX7 cDNA (22). Normalization of the PTS2-bearing protein distribution by human PEX7 cDNA was confirmed by the punctate pattern of PTS2-GFP staining that coincided with the staining pattern of catalase, an endogenous peroxisome enzyme (Fig. 2). We then analyzed production of the 1-alkyl-2-acyl form of GPI by testing alkali sensitivity (Fig. 3A). Due to the sensitivity of ester linkage to KOH treatment, diacyl form GPI is lost from the organic phase, whereas 1-alkyl-2-lyso GPI generated from 1-alkyl-2-acyl from GPI remained in the organic phase and can be detected by TLC analysis. In ZPG207 cells, all spots of GPI biosynthetic intermediates labeled with mannose disappeared after KOH treatment (Fig. 3B, lane 4), and expression of PEX7 restored the alkali-resistant 1-alkyl-2-lyso forms of GPI intermediates (Fig. 3B, lanes 7 and 8). To determine the structure of the PI moiety of protein-linked GPI, we generated ZPG207 cells stably expressing CD59, a human GPI-anchored protein, bearing tandem His-, FLAG-, glutathione S-transferase (GST)-, and FLAG-tags at the N-terminus (HFGF-CD59). We affinity-purified HFGF-CD59 with glutathione-Sepharose and released the PI moiety from approximately 1 nmol of protein by treatment with sodium nitrite (28, 29) and subjected it to nano ESI-MS analysis (Fig. 3C). All PIs in HFGF-CD59 from ZPG207 cells were the diacyl form (Fig. 3C, left bottom). MS/MS analysis of the major species of m/z = 865.80 demonstrated 18:0 fatty acids representing stearic acid, cyclic lyso phosphatidic acid bearing 18:0 chain (18:0 cPA), and inositol-phosphate (m/z = 241.0), indicating that it is di-stearoyl-PI (right bottom). Similarly, MS/MS analysis of a minor species of m/z = 837.70 demonstrated that it is 1-palmitoyl-2-stearoyl-PI (Fig. 3C, right top). In contrast, wild-type CHO-K1 cells contained mostly 1-alkyl-2-acyl PI (Fig. 3C, left top) (18). Therefore, PEX7 is required for the production of the 1-alkyl-2-acyl form of GPI anchors.

Fig. 2.

Fig. 2.

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Transfection of human PEX7 restored the localization of PTS2-bearing protein at the peroxisome in PEX7-defective CHO cells. PTS2-GFP is diffuse in PEX7-defective ZPG207 cells (top panels), whereas a punctate distribution of PTS2-GFP was restored by transfection with human PEX7 (bottom panels). Cells were observed for GFP (left-hand panels) and stained with anti-catalase antibody (middle panels). Merged profiles are shown in the right-hand panels. Scale bars, 20 μm.

Fig. 3.

Fig. 3.

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GPI lipid remodeling to generate 1-alkyl-2-acyl GPI is defective in RCDP type1 cells. A: Schematic showing the alkali-sensitive and -resistant characteristics of diacyl and 1-alkyl-2-acyl GPI intermediates, respectively. After KOH treatment, 1-alkyl-2-acyl GPI is converted to 1-alkyl-2-lyso GPI, which is retained in the lipid fraction. B: Alkali-sensitivity of GPI mannolipids derived from PEX7-defective ZPG207 cells. Cells were metabolically labeled with D-[2-3H] mannose in the presence of BE49385A to accumulate late GPI intermediates. The extracted lipids were treated with 0.1 N KOH in MeOH or with MeOH alone and analyzed by TLC. H3-H7’, mannose-containing GPI intermediates; H3*-H7’*, alkaline-resistant part of H3-H7’. Lanes 1 and 3, wild-type CHO-K1; lanes 2 and 4, ZPG207; lanes 5 and 7, ZPG207 complemented with human PEX7; lanes 6 and 8, ZPG207 complemented with hamster Pex7; lanes 1, 2, 5, and 6, MeOH-treated cells; lanes 3, 4, 7, and 8, KOH-treated cells. C: Mass analysis of PI moieties in HFGF-CD59 from the plasma membrane of wild-type CHO-K1 and PEX7-defective ZPG207 cells. In CHO-K1 cells, 1-alkyl-2-acyl species (16:0e-18:0, 18:1e-18:0, and 18:0e-18:0) were dominant (left top, data cited from reference 18), whereas only diacyl species were found in ZPG207 cells (left bottom). Fatty chain compositions of PI species are indicated as A:B-C:D, where A and C are the number of carbons, B and D are the number of unsaturated bonds, and A:B and C:D are sn1 and sn2 chain, respectively. e indicates 1-alkyl form. Fatty acid chain compositions of the major (m/z = 865.80) and minor (m/z = 837.70) species of PI in ZPG207 were determined by MS/MS analysis (right bottom and right top, respectively). FA, fatty acid; cPA, cyclic lyso phosphatidic acid; Ino-P, phosphoryl inositol. The 18:1e-18:0 species seen in CHO-K1 cells might contain a plasmalogen type. The exact structure is yet to be determined. D: Alkali sensitivity of GPI mannolipids derived from RCDP type 1 fibroblasts from two patients. Lanes 1 and 4, healthy controls; lanes 2 and 5, RCDP type1 cell line R01; lanes 3 and 6, another RCDP type1 cell line R03; lanes 7 and 9, R01 complemented with PEX7; lanes 8 and 10, R03 complemented with PEX7.

We next analyzed fibroblast cell lines previously established from patients with RCDP type 1. As in CHO cells, significant fractions of GPI intermediates in wild-type fibroblasts were resistant to alkali, indicating generation of 1-alkyl-2-acyl GPI (Fig. 3D, lanes 1 and 4). Alkali-resistant GPI was greatly decreased in patients’ cells (Fig. 3D, lanes 5 and 6). After complementation with human PEX7 cDNA, the alkali-resistant GPIs were generated at levels comparable to those in wild-type cells (Fig. 3D, lanes 9 and 10), establishing that generation of 1-alkyl-2-acyl GPI anchors is defective in RCDP type 1.

Fibroblasts from patients with RCDP types 2 and 3 are defective in 1-alkyl-2-acyl GPIs

We next assessed generation of alkali-resistant GPI in fibroblast cell lines from patients with RCDP types 2 and 3. As expected from our previous results with mutant CHO cells defective in DHAP-AT and alkyl-DHAP synthase, all spots of GPI-mannolipids generated in these cells from RCDP type 2 and type 3 patients disappeared after alkali treatment (Fig. 4, lanes 7–10). When the responsible DHAP-AT cDNA was transfected into RCDP type 2 fibroblasts, spots of the alkali-resistant lyso-forms appeared (Fig. 4, lanes 13 and 14). It is, therefore, evident that generation of 1-alkyl-2-acyl GPI-APs is defective in RCDP types 2 and 3.

Fig. 4.

Fig. 4.

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TLC analysis of alkali sensitivity of GPI mannolipids derived from fibroblasts from patients with RCDP types 2 and 3 and their complemented counterparts. Lanes 1 and 6, fibroblasts from a healthy control; lanes 2 and 7, NL-#2 (RCDP type 2); lanes 3 and 8, NL-#3 (RCDP type 2); lanes 4 and 9, NL-#5 (RCDP type 3); lanes 5 and 10, NL-#6 (RCDP type 3); lanes 11 and 13, NL-#2 complemented with DHAP-AT cDNA; lanes 12 and 14, NL-#3 complemented with DHAP-AT cDNA; lanes 1–5, 11, and 12, MeOH-treated cells; lanes 6–10, 13, and 14, KOH-treated cells.

GPI lipid remodeling to generate 1-alkyl-2-acyl GPI is defective in PEX5- and PEX19-mutant CHO cells and fibroblasts from patients with ZS

PEX5 recognizes the PTS1 signal and imports many proteins bearing a PTS1 into the peroxisome. A defect in PEX5 causes ZS. PEX16 and PEX19 are required for incorporation of peroxisomal membrane proteins into the membrane (3, 30–32). Defects in PEX16 and PEX19 also cause ZS (3, 33). First, we analyzed PEX5-defective CHO cells (Fig. 5A, lanes 2 and 5) and PEX19-defective CHO cells (Fig. 5A, lanes 3 and 6) (21, 34). Man-labeled GPIs generated in these mutant CHO cells disappeared after KOH treatment (Fig. 5A, lanes 5 and 6). Expression of PEX5 and PEX19 restored the production of alkali-resistant GPIs (Fig. 5A, lanes 9 and 10). Next, we tested fibroblast cell lines from patients with ZS (Fig. 5B). Cells from PEX5-defective (Fig. 5B, lanes 2 and 5) and PEX16-defective (Fig. 5B, lanes 3 and 6) patients did not produce alkali-resistant GPIs (Fig. 5B, lanes 3 and 6). These results indicate that PEX5, PEX16, and PEX19 are required for the generation of 1-alkyl-2-acyl GPI anchors and that patients with ZS are defective in generation of 1-alkyl-2-acyl GPI-APs.

Fig. 5.

Fig. 5.

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Only diacyl GPI intermediates were generated in CHO mutant cells defective in PEX5 or PEX19 and in fibroblasts from patients with ZS. A: Alkali sensitivity of GPI mannolipids derived from PEX5- and PEX19-defective mutant cells. Lanes 1 and 4, wild-type CHO-K1; lanes 2 and 5, ZP139 defective in PEX5; lanes 3 and 6, ZP119 defective in PEX19; lanes 7 and 9, ZP139 complemented with PEX5 cDNA; lanes 8 and 10, ZP119 complemented with PEX19 cDNA. Lanes 1, 2, 3, 7, and 8, MeOH-treated lipids; lanes 4, 5, 6, 9, and 10, KOH-treated lipids. B: Alkali-sensitivity of GPI mannolipids derived from ZS fibroblasts. Lanes 1 and 4, healthy control; lanes 2 and 5, ZS cells defective in PEX5; lanes 3 and 6, ZS cells defective in PEX16; lanes 1, 2, and 3, MeOH-treated cells; lanes 4, 5, and 6, KOH-treated cells.

Surface expression of urokinase-type plasminogen activator receptor, a GPI-AP, significantly increase when alkylphospholipid biosynthesis is defective

We next asked if a defect in peroxisome-dependent alkylphospholipid biosynthesis affects any properties of GPI-APs other than lipid remodeling to 1-alkyl-2-acyl form. We focused on urokinase-type plasminogen activator receptor (uPAR), an endogenous GPI-AP of CHO cells. We used NRel-4 and NRel-1 cells defective in DHAP-AT and alkyl-DHAP synthase, respectively (19, 20). The surface expression of uPAR on NRel-4 cells was 4-times of that on wild-type CHO-K1 cells and returned to 1.5-times of normal level by transfection of DHAP-AT cDNA (Fig. 6A). Similarly, uPAR on NZel-1 cells was 2-times of that on CHO-K1 cells and returned to 1.4-times of normal level by transfection of alkyl-DHAP synthase cDNA (Fig. 6A). To see if the increased surface expression of uPAR was due to an increase in total cellular uPAR level or to a skew in a ratio of surface to intracellular uPAR fractions, we used confocal fluorescence microscopy. The total cellular level of uPAR was higher in NRel-4 and NZel-1 cells than in CHO-K1 cells and was substantially decreased after transfection of DHAP-AT and alkyl-DHAP synthase cDNAs, respectively (Fig. 6B). We assessed the amounts of uPAR and some nonGPI-APs, such as calnexin, α-tubulin, glyceraldehydephosphate dehydrogenase, and caveolin-1, with Western blotting before and after PI-PLC treatment (Fig. 6C). Most of the uPAR was lost by PI-PLC treatment, indicating that most cellular uPAR was on the cell surface (Fig. 6C, top panel, lanes 1–5 vs. lanes 6–10). Levels of uPAR were clearly higher in NRel-4 and NZel-1 cells than in parental CHO cells (Fig. 6C, lanes 2 and 4 vs. lane 1) and were substantially decreased after transfection of cDNAs responsible for the mutants (Fig. 6C, lanes 3 and 5). In contrast, expression levels of calnexin, α-tubulin, and glyceraldehydephosphate dehydrogenase were not changed with or without alkyl phospholipids synthesis (Fig. 6C, three middle panels). Consistent with a previous report that levels of GPI-APs and caveolin-1 are inversely correlated in CHO cells (35), we found that levels of caveolin-1 were decreased in NRel-4 and NZel-1 cells and were increased after transfection of responsible cDNAs (Fig. 6C, bottom panel). These results suggest that the defective alkylphospholipid synthesis causes increased surface expression of uPAR probably due to slowed turnover. Whether this remarkable phenotype is due to the diacyl form GPI anchor itself, to a lack of alkyl/alkenyl phospholipids such as plasmalogens, or both is yet to be determined.

Fig. 6.

Fig. 6.

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Increased expression of uPAR in CHO cells defective in DHAP-AT or alkyl-DHAP synthase. A: FACS analysis of endogenous GPI-AP, uPAR in CHO mutant cells defective in DHAP-AT (NRel-4 cells, top), or alkyl-DHAP synthase (NZel-1 cells, bottom) and their complemented counterparts. Mutant cells were transfected with DHAP-AT or alkyl-DHAP synthase cDNA stained 72 h later for uPAR and analyzed for FACS. Dark gray areas, CHO-K1 transfected with an empty vector; dotted lines, mutant cells transfected with an empty vector; solid lines, complemented counterparts; light gray areas, isotype control staining of mutant cells. B: Confocal immunofluorescence microscopic analysis of uPAR in CHO mutant NRel-4 and NZel-1 cells. Cells were fixed with paraformaldehyde, permeabilized with 0.1% saponin, and stained for uPAR. The uPAR staining in two mutant cells was stronger (left middle and bottom) than in wild-type CHO-K1 cells (right top). The uPAR expression in mutant cells was reduced after transfection with DHAP-AT or alkyl-DHAP synthase cDNA (right middle and bottom). C: Western blotting of uPAR and nonGPI-APs in CHO mutant NRel-4 and NZel-1 cells before and after PI-PLC treatment. Cells (1 × 106) were harvested with EDTA, incubated with or without 1 U/ml of PI-PLC in 100 μl of Ham F-12 medium for 2 h at 20°C, and subjected to SDS/PAGE and Western blotting under nonreducing conditions for uPAR (top) and Caveolin-1 (bottom) and under reducing conditions for Calnexin, α-Tubulin, and GAPDH (middle).

DISCUSSION

The primary finding of this study is that cells from patients with RCDP and ZS are defective in biosynthesis of 1-alkyl-2-acyl GPIs and express only diacyl GPI-APs. Many proteins are anchored by GPI to the cell surface, and the majority of proteins in mammalian cells contain 1-alkyl-2-acyl GPI (36). It is known that changes in the fine structure of the lipid portion of GPI-APs lead to altered properties of the GPI-APs and various abnormalities. When removal of the inositol-linked acyl chain from nascent GPI-APs does not occur due to a defect in the inositol-deacylase PGAP1, transport of GPI-APs from the ER to the Golgi apparatus is delayed, and sensitivity to PI-specific phospholipase C is lost (37). Pgap1-knockout mice have high rates of perinatal death due to craniofacial deformity (in particular otocephaly), growth retardation, and male infertility (38). The mutant mouse strain oto characterized by otocephaly is due to mutation in Pgap1 (39). Another example is GPI fatty acid remodeling occurring in the Golgi, in which an unsaturated fatty acid at the sn2 position is exchanged with saturated, stearic acid. When the fatty acid remodeling does not occur due to a defect in PGAP3, which is involved in removal of the sn2-linked unsaturated fatty acid, GPI-APs are present in the detergent-sensitive fraction rather than in the detergent-resistant membrane fraction after extraction with a cold nonionic detergent such as Triton X-100 (40). Pgap3-knockout mice have a number of abnormal phenotypes, including growth retardation and immune dysfunction (41). Overall, it is possible that a lack of 1-alkyl-2-acyl GPI-APs accounts for some of the clinical symptoms of ZS and RCDP.

Mammalian cells elaborate 1-alkyl-2-acyl GPI-APs from diacyl PI via at least two events. The first event, which occurs in the third biosynthetic intermediate GlcN-acyl-PI, is replacement of a diacyl glycerol moiety or a diacyl glycerol phosphate moiety (17, 18). In CHO cells and mouse T-lymphoma cell lines, approximately 60% of GPIs are converted to the 1-alkyl-2-acyl form at this stage (17). Because fatty acid composition of the diacyl form of GlcN-acyl-PI was different from those of earlier GPI intermediates and PI, even the diacyl form must be subjected to replacement reaction (18). Therefore, a putative donor lipid for the remodeling presumably comprises 60% of the 1-alkyl-2-acyl form and 40% of the diacyl form (18). The exact reaction mechanism of this GPI lipid remodeling is yet to be elucidated. The second event occurs during the following biosynthetic step(s), in which enrichment of the 1-alkyl-2-acyl form proceeds further, leading to occupation of 80–90% of cell surface GPI-APs in CHO cells (18). A possible mechanism might be selection by biosynthetic enzymes, selective compartmentalization of the 1-alkyl-2-acyl form, or, although less likely, degradation of diacyl GPI intermediates.

To understand the functional significance of 1-alkyl-2-acyl GPI-APs, the enzyme required for the GPI lipid remodeling needs to be identified and disrupted. If the gene for the putative enzyme that catalyzes diacyl to 1-alkyl-2-acyl exchange in GlcN-acyl-PI is disrupted in mice, such mice would not express 1-alkyl-2-acyl GPI-APs but would have normal plasmalogens; this should allow us to determine the phenotypes that result from defective generation of 1-alkyl-2-acyl GPI-APs. Until this is achieved, information derived from the cells of patients with RCDP types 2 and 3 and CHO mutant cell lines defective in DHAP-AT and alkyl-DHAP synthase is useful. Cells from patients with RCDP types 2 and 3 are defective only in alkyl-phospholipid biosynthesis, resulting in defects in several end-products, namely, plasmalogens, platelet activating factor, and 1-alkyl-2-acyl GPI-APs, whereas cells from patients with RCDP type 1 and ZS are also defective in other peroxisomal pathways. We found that the surface expression of uPAR is 2- to 4-times elevated on mutant cells defective in DHAP-AT or alkyl-DHAP synthase (Fig. 6). It was reported that these mutant CHO cells are inefficient in the transport of cholesterol for the cell surface or endocytic compartment to the ER (42). Fibroblasts from patients with RCDP types 2 and 3 display a number of morphological and cell biological abnormalities, such as the common presence of enlarged and multipolar cells, dilated ER and Golgi cisternae, a reduced number of caveolae associated with a 50–60% reduction in caveolin 1, accumulation of cholesterol in perinuclear structures, an increased level of clathrin, and inefficient endocytosis of transferrin (43). Whether the elevated uPAR expression is related to these cellular abnormalities, including affected cholesterol trafficking, is unclear.

Dhap-at-knockout mice display a variety of phenotypes, including male infertility, defects in eye development, cataract, optic nerve hypoplasia, and cerebellum anomalies including impaired Purkinje cell innervation, defective myelination, and disturbances in paranode organization (44, 45). Patients with RCDP types 2 and 3 have craniofacial dysmorphism, cataract, severe psychomotor retardation, as well as rhizomelia and chondrodysplasia punctata (3, 10–12). It is tempting to speculate that the lack of a particular 1-alkyl-2-acyl GPI-AP is responsible for some of these abnormalities. For example, abnormal alkaline phosphatase, a GPI-AP that is expressed on the membrane of osteoblasts and that is secreted to the blood during bone formation (14, 46–50), might be responsible for the abnormal bone formation or chondrodysplasia. Delayed cerebellar development is a hallmark in RCDP and ZS patients, and these patients suffer from severe neonatal neurodegenerative disorders. The severity is related to the importance of peroxisomes in the maturation of the central nervous system. Peroxisomes are abundant at the termini of developing neurons and have been implicated in the early determination of neural polarity (51, 52). Although the mechanisms of neuropathology are unknown, ZS leads to loss of essential peroxisomal function and causes problems in cerebellum formation. Neural cell adhesion molecule, a GPI-AP, is expressed on the surface of neural cells and contributes to cell polarization (53). The abnormal diacyl form GPI might be causally related to the neurodegenerative phenotypes. Although the functional importance of the alkyl-acyl form of GPI-APs is yet to be determined, our results suggest the possibility that the absence of alkyl-acyl GPI contributes to some of the symptoms of RCDP and ZS.

Acknowledgments

The authors thank Drs. Y. Miyauchi and K. Okumoto for cells; K. Kinoshita, K. Miyanagi, and Y. Onoe for excellent technical assistance; K. Nakamura for help with cell sorting; and Drs. N. Inoue and Y. Morita for helpful discussions.

Footnotes

Abbreviations:

alkyl-DHAP synthase
alkyl-dihydroxyacetone phosphate synthase
DHAP-AT
dihydroxyacetone phosphate acyltransferase
GlcN
glucosamine
Man
mannose
GlcNAc
N-acetyl glucosamine
GPI
glycosylphosphatidylinositol
GPI-AP
GPI-anchored protein
GST
glutathione S-transferase
PI
phosphatidylinositol
PTS
peroxisome targeting signal
RCDP
rhizomelic chondrodysplasia punctata
uPAR
urokinase-type plasminogen activator receptor
ZS
Zellweger syndrome

This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the Osaka University Global COE program.

REFERENCES

  • 1.Wanders R. J., Ferdinandusse S., Brites P., Kemp S. 2010. Peroxisomes, lipid metabolism and lipotoxicity. Biochim. Biophys. Acta. 1801: 272–280 [DOI] [PubMed] [Google Scholar]
  • 2.Wanders R. J., Waterham H. R. 2006. Peroxisomal disorders: the single peroxisomal enzyme deficiencies. Biochim. Biophys. Acta. 1763: 1707–1720 [DOI] [PubMed] [Google Scholar]
  • 3.Steinberg S. J., Dodt G., Raymond G. V., Braverman N. E., Moser A. B., Moser H. W. 2006. Peroxisome biogenesis disorders. Biochim. Biophys. Acta. 1763: 1733–1748 [DOI] [PubMed] [Google Scholar]
  • 4.Fujiki Y. 2000. Peroxisome biogenesis and peroxisome biogenesis disorders. FEBS Lett. 476: 42–46 [DOI] [PubMed] [Google Scholar]
  • 5.Fujiki Y., Okumoto K., Kinoshita N., Ghaedi K. 2006. Lessons from peroxisome-deficient Chinese hamster ovary (CHO) cell mutants. Biochim. Biophys. Acta. 1763: 1374–1381 [DOI] [PubMed] [Google Scholar]
  • 6.Fujiki Y. 2011. Peroxisome biogenesis disorders. In Encyclopedia of Life Sciences. John Wiley and Sons, Hoboken, NJ. 1–9. [Google Scholar]
  • 7.Shimozawa N., Tsukamoto T., Suzuki Y., Orii T., Shirayoshi Y., Mori T., Fujiki Y. 1992. A human gene responsible for Zellweger syndrome that affects peroxisome assembly. Science. 255: 1132–1134 [DOI] [PubMed] [Google Scholar]
  • 8.Goldfischer S., Moore C. L., Johnson A. B., Spiro A. J., Valsamis M. P., Wisniewski H. K., Ritch R. H., Norton W. T., Rapin I., Gartner L. M. 1973. Peroxisomal and mitochondrial defects in the cerebro-hepato-renal syndrome. Science. 182: 62–64 [DOI] [PubMed] [Google Scholar]
  • 9.Shimozawa N. 2011. Molecular and clinical findings and diagnostic flowchart of peroxisomal diseases. Brain Dev. 33: 770–776 [DOI] [PubMed] [Google Scholar]
  • 10.Wanders R. J., Schumacher H., Heikoop J., Schutgens R. B., Tager J. M. 1992. Human dihydroxyacetonephosphate acyltransferase deficiency: a new peroxisomal disorder. J. Inherit. Metab. Dis. 15: 389–391 [DOI] [PubMed] [Google Scholar]
  • 11.Wanders R. J., Dekker C., Hovarth V. A., Schutgens R. B., Tager J. M., Van Laer P., Lecoutere D. 1994. Human alkyldihydroxyacetonephosphate synthase deficiency: a new peroxisomal disorder. J. Inherit. Metab. Dis. 17: 315–318 [DOI] [PubMed] [Google Scholar]
  • 12.Barr D. G., Kirk J. M., al Howasi M., Wanders R. J., Schutgens R. B. 1993. Rhizomelic chondrodysplasia punctata with isolated DHAP-AT deficiency. Arch. Dis. Child. 68: 415–417 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.McConville M. J., Menon A. K. 2000. Recent developments in the cell biology and biochemistry of glycosylphosphatidylinositol lipids [review] Mol. Membr. Biol. 17: 1–16 [DOI] [PubMed] [Google Scholar]
  • 14.Ikezawa H. 2002. Glycosylphosphatidylinositol (GPI)-anchored proteins. Biol. Pharm. Bull. 25: 409–417 [DOI] [PubMed] [Google Scholar]
  • 15.Kinoshita T., Fujita M., Maeda Y. 2008. Biosynthesis, remodelling and functions of mammalian GPI-anchored proteins: recent progress. J. Biochem. 144: 287–294 [DOI] [PubMed] [Google Scholar]
  • 16.Fujita M., Kinoshita T. 2010. Structural remodeling of GPI anchors during biosynthesis and after attachment to proteins. FEBS Lett. 584: 1670–1677 [DOI] [PubMed] [Google Scholar]
  • 17.Houjou T., Hayakawa J., Watanabe R., Tashima Y., Maeda Y., Kinoshita T., Taguchi R. 2007. Changes in molecular species profiles of glycosylphosphatidylinositol anchor precursors in early stages of biosynthesis. J. Lipid Res. 48: 1599–1606 [DOI] [PubMed] [Google Scholar]
  • 18.Kanzawa N., Maeda Y., Ogiso H., Murakami Y., Taguchi R., Kinoshita T. 2009. Peroxisome dependency of alkyl-containing GPI-anchor biosynthesis in the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA. 106: 17711–17716 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Nagan N., Hajra A. K., Larkins L. K., Lazarow P., Purdue P. E., Rizzo W. B., Zoeller R. A. 1998. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol. Biochem. J. 332: 273–279 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Nagan N., Hajra A. K., Das A. K., Moser H. W., Moser A., Lazarow P., Purdue P. E., Zoeller R. A. 1997. A fibroblast cell line defective in alkyl-dihydroxyacetone phosphate synthase: a novel defect in plasmalogen biosynthesis. Proc. Natl. Acad. Sci. USA. 94: 4475–4480 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Otera H., Okumoto K., Tateishi K., Ikoma Y., Matsuda E., Nishimura M., Tsukamoto T., Osumi T., Ohashi K., Higuchi O., et al. 1998. Peroxisome targeting signal type 1 (PTS1) receptor is involved in import of both PTS1 and PTS2: studies with PEX5-defective CHO cell mutants. Mol. Cell. Biol. 18: 388–399 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Mukai S., Ghaedi K., Fujiki Y. 2002. Intracellular localization, function, and dysfunction of the peroxisome-targeting signal type 2 receptor, Pex7p, in mammalian cells. J. Biol. Chem. 277: 9548–9561 [DOI] [PubMed] [Google Scholar]
  • 23.Kinoshita N., Ghaedi K., Shimozawa N., Wanders R. J., Matsuzono Y., Imanaka T., Okumoto K., Suzuki Y., Kondo N., Fujiki Y. 1998. Newly identified Chinese hamster ovary cell mutants are defective in biogenesis of peroxisomal membrane vesicles (Peroxisomal ghosts), representing a novel complementation group in mammals. J. Biol. Chem. 273: 24122–24130 [DOI] [PubMed] [Google Scholar]
  • 24.Shimozawa N., Suzuki Y., Zhang Z., Miura K., Matsumoto A., Nagaya M., Castillo-Taucher S., Kondo N. 1999. A novel nonsense mutation of the PEX7 gene in a patient with rhizomelic chondrodysplasia punctata. J. Hum. Genet. 44: 123–125 [DOI] [PubMed] [Google Scholar]
  • 25.Braverman N., Steel G., Obie C., Moser A., Moser H., Gould S. J., Valle D. 1997. Human PEX7 encodes the peroxisomal PTS2 receptor and is responsible for rhizomelic chondrodysplasia punctata. Nat. Genet. 15: 369–376 [DOI] [PubMed] [Google Scholar]
  • 26.Motley A. M., Hettema E. H., Hogenhout E. M., Brites P., ten Asbroek A. L., Wijburg F. A., Baas F., Heijmans H. S., Tabak H. F., Wanders R. J., et al. 1997. Rhizomelic chondrodysplasia punctata is a peroxisomal protein targeting disease caused by a non-functional PTS2 receptor. Nat. Genet. 15: 377–380 [DOI] [PubMed] [Google Scholar]
  • 27.Purdue P. E., Zhang J. W., Skoneczny M., Lazarow P. B. 1997. Rhizomelic chondrodysplasia punctata is caused by deficiency of human PEX7, a homologue of the yeast PTS2 receptor. Nat. Genet. 15: 381–384 [DOI] [PubMed] [Google Scholar]
  • 28.Serrano A. A., Schenkman S., Yoshida N., Mehlert A., Richardson J. M., Ferguson M. A. 1995. The lipid structure of the glycosylphosphatidylinositol-anchored mucin-like sialic acid acceptors of Trypanosoma cruzi changes during parasite differentiation from epimastigotes to infective metacyclic trypomastigote forms. J. Biol. Chem. 270: 27244–27253 [DOI] [PubMed] [Google Scholar]
  • 29.Fontaine T., Magnin T., Melhert A., Lamont D., Latge J. P., Ferguson M. A. 2003. Structures of the glycosylphosphatidylinositol membrane anchors from Aspergillus fumigatus membrane proteins. Glycobiology. 13: 169–177 [DOI] [PubMed] [Google Scholar]
  • 30.Titorenko V. I., Mullen R. T. 2006. Peroxisome biogenesis: the peroxisomal endomembrane system and the role of the ER. J. Cell Biol. 174: 11–17 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Fujiki Y., Matsuzono Y., Matsuzaki T., Fransen M. 2006. Import of peroxisomal membrane proteins: the interplay of Pex3p- and Pex19p-mediated interactions. Biochim. Biophys. Acta. 1763: 1639–1646 [DOI] [PubMed] [Google Scholar]
  • 32.Matsuzaki T., Fujiki Y. 2008. The peroxisomal membrane protein import receptor Pex3p is directly transported to peroxisomes by a novel Pex19p- and Pex16p-dependent pathway. J. Cell Biol. 183: 1275–1286 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Ebberink M. S., Kofster J., Wanders R. J., Waterham H. R. 2010. Spectrum of PEX6 mutations in Zellweger syndrome spectrum patients. Hum. Mutat. 31: E1058–E1070 [DOI] [PubMed] [Google Scholar]
  • 34.Matsuzono Y., Kinoshita N., Tamura S., Shimozawa N., Hamasaki M., Ghaedi K., Wanders R. J., Suzuki Y., Kondo N., Fujiki Y. 1999. Human PEX19: cDNA cloning by functional complementation, mutation analysis in a patient with Zellweger syndrome, and potential role in peroxisomal membrane assembly. Proc. Natl. Acad. Sci. USA. 96: 2116–2121 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Abrami L., Fivaz M., Kobayashi T., Kinoshita T., Parton R. G., van der Goot F. G. 2001. Cross-talk between caveolae and glycosylphosphatidylinositol-rich domains. J. Biol. Chem. 276: 30729–30736 [DOI] [PubMed] [Google Scholar]
  • 36.Chatterjee S., Mayor S. 2001. The GPI-anchor and protein sorting. Cell. Mol. Life Sci. 58: 1969–1987 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Tanaka S., Maeda Y., Tashima Y., Kinoshita T. 2004. Inositol deacylation of glycosylphosphatidylinositol-anchored proteins is mediated by mammalian PGAP1 and yeast Bst1p. J. Biol. Chem. 279: 14256–14263 [DOI] [PubMed] [Google Scholar]
  • 38.Ueda Y., Yamaguchi R., Ikawa M., Okabe M., Morii E., Maeda Y., Kinoshita T. 2007. PGAP1 knock-out mice show otocephaly and male infertility. J. Biol. Chem. 282: 30373–30380 [DOI] [PubMed] [Google Scholar]
  • 39.Zoltewicz J. S., Ashique A. M., Choe Y., Lee G., Taylor S., Phamluong K., Solloway M., Peterson A. S. 2009. Wnt signaling is regulated by endoplasmic reticulum retention. PLoS ONE. 4: e6191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Maeda Y., Tashima Y., Houjou T., Fujita M., Yoko-o T., Jigami Y., Taguchi R., Kinoshita T. 2007. Fatty acid remodeling of GPI-anchored proteins is required for their raft association. Mol. Biol. Cell. 18: 1497–1506 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Murakami H., Wang Y., Hasuwa H., Maeda Y., Kinoshita T., Murakami Y. 2011. Enhanced response of T lymphocytes from Pgap3 knockout mouse: insight into roles of fatty acid remodeling of GPI anchored proteins. Biochem. Biophys. Res. Commun. 417: 1235–1241 [DOI] [PubMed] [Google Scholar]
  • 42.Munn N. J., Arnio E., Liu D., Zoeller R. A., Liscum L. 2003. Deficiency in ethanolamine plasmalogen leads to altered cholesterol transport. J. Lipid Res. 44: 182–192 [DOI] [PubMed] [Google Scholar]
  • 43.Thai T. P., Rodemer C., Jauch A., Hunziker A., Moser A., Gorgas K., Just W. W. 2001. Impaired membrane traffic in defective ether lipid biosynthesis. Hum. Mol. Genet. 10: 127–136 [DOI] [PubMed] [Google Scholar]
  • 44.Rodemer C., Thai T. P., Brugger B., Kaercher T., Werner H., Nave K. A., Wieland F., Gorgas K., Just W. W. 2003. Inactivation of ether lipid biosynthesis causes male infertility, defects in eye development and optic nerve hypoplasia in mice. Hum. Mol. Genet. 12: 1881–1895 [DOI] [PubMed] [Google Scholar]
  • 45.Teigler A., Komljenovic D., Draguhn A., Gorgas K., Just W. W. 2009. Defects in myelination, paranode organization and Purkinje cell innervation in the ether lipid-deficient mouse cerebellum. Hum. Mol. Genet. 18: 1897–1908 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Fedde K. N., Lane C. C., Whyte M. P. 1988. Alkaline phosphatase is an ectoenzyme that acts on micromolar concentrations of natural substrates at physiologic pH in human osteosarcoma (SAOS-2) cells. Arch. Biochem. Biophys. 264: 400–409 [DOI] [PubMed] [Google Scholar]
  • 47.Hooper N. M. 1997. Glycosyl-phosphatidylinositol anchored membrane enzymes. Clin. Chim. Acta. 266: 3–12 [DOI] [PubMed] [Google Scholar]
  • 48.Bellows C. G., Aubin J. E., Heersche J. N. 1991. Initiation and progression of mineralization of bone nodules formed in vitro: the role of alkaline phosphatase and organic phosphate. Bone Miner. 14: 27–40 [DOI] [PubMed] [Google Scholar]
  • 49.Barling P. M., Gupta D. K., Lim C. E. 1999. Involvement of phosphodiesterase I in mineralization: histochemical studies using antler from red deer (Cervus elaphus) as a model. Calcif. Tissue Int. 65: 384–389 [DOI] [PubMed] [Google Scholar]
  • 50.Wennberg C., Hessle L., Lundberg P., Mauro S., Narisawa S., Lerner U. H., Millan J. L. 2000. Functional characterization of osteoblasts and osteoclasts from alkaline phosphatase knockout mice. J. Bone Miner. Res. 15: 1879–1888 [DOI] [PubMed] [Google Scholar]
  • 51.Ishikawa T., Kawai C., Sano M., Minatogawa Y. 2001. Peroxisomes exist in growth cones and move anterogradely and retrogradely in neurites of PC12D cells. Exp. Cell Res. 266: 260–269 [DOI] [PubMed] [Google Scholar]
  • 52.Bradke F., Dotti C. G. 1997. Neuronal polarity: vectorial cytoplasmic flow precedes axon formation. Neuron. 19: 1175–1186 [DOI] [PubMed] [Google Scholar]
  • 53.Powell S. K., Cunningham B. A., Edelman G. M., Rodriguez-Boulan E. 1991. Targeting of transmembrane and GPI-anchored forms of N-CAM to opposite domains of a polarized epithelial cell. Nature. 353: 76–77 [DOI] [PubMed] [Google Scholar]
  • 54.Honsho M., Tamura S., Shimozawa N., Suzuki Y., Kondo N., Fujiki Y. 1998. Mutation in PEX16 is causal in the peroxisome-deficient Zellweger syndrome of complementation group D. Am. J. Hum. Genet. 63: 1622–1630 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Blasco M. A., Lee H. W., Hande M. P., Samper E., Lansdorp P. M., DePinho R. A., Greider C. W. 1997. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell. 91: 25–34 [DOI] [PubMed] [Google Scholar]
  • 56.Bodnar A. G., Ouellette M., Frolkis M., Holt S. E., Chiu C. P., Morin G. B., Harley C. B., Shay J. W., Lichtsteiner S., Wright W. E. 1998. Extension of life-span by introduction of telomerase into normal human cells. Science. 279: 349–352 [DOI] [PubMed] [Google Scholar]
  • 57.Manfredi J. J., Prives C. 1994. The transforming activity of simian virus 40 large tumor antigen. Biochim. Biophys. Acta. 1198: 65–83 [DOI] [PubMed] [Google Scholar]
  • 58.Counter C. M., Hahn W. C., Wei W., Caddle S. D., Beijersbergen R. L., Lansdorp P. M., Sedivy J. M., Weinberg R. A. 1998. Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization. Proc. Natl. Acad. Sci. USA. 95: 14723–14728 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Taguchi R., Houjou T., Nakanishi H., Yamazaki T., Ishida M., Imagawa M., Shimizu T. 2005. Focused lipidomics by tandem mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 823: 26–36 [DOI] [PubMed] [Google Scholar]
  • 60.Hong Y., Maeda Y., Watanabe R., Ohishi K., Mishkind M., Riezman H., Kinoshita T. 1999. Pig-n, a mammalian homologue of yeast Mcd4p, is involved in transferring phosphoethanolamine to the first mannose of the glycosylphosphatidylinositol. J. Biol. Chem. 274: 35099–35106 [DOI] [PubMed] [Google Scholar]
  • 61.Sutterlin C., Horvath A., Gerold P., Schwarz R. T., Wang Y., Dreyfuss M., Riezman H. 1997. Identification of a species-specific inhibitor of glycosylphosphatidylinositol synthesis. EMBO J. 16: 6374–6383 [DOI] [PMC free article] [PubMed] [Google Scholar]

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