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Vol. 17, Issue 3, 1410-1420, March 2006
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* Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan;
Department of Metabolome, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan
Submitted November 2, 2005;
Revised December 27, 2005;
Accepted January 4, 2006
Monitoring Editor: Howard Riezman
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Kinoshita and Inoue, 2000; Ikezawa, 2002). The GPI anchor is assembled on PI and transferred to proteins in the ER. Newly synthesized GPI-APs are transported to the plasma membrane by vesicular trafficking. GPI-APs are concentrated in microdomains in the plasma membrane, so-called rafts, which are enriched with cholesterol and sphingolipids (Brown and Rose, 1992; Simons and Ikonen, 1997; Simons and Toomre, 2000). GPI-APs are incorporated into rafts in the Golgi during their transport to the cell surface (Brown and Rose, 1992). The rafts are thought to act as signaling platforms in which molecules are assembled according to extracellular stimuli and transduce raft-dependent intracellular signals (Brown and Rose, 1992; Simons and Ikonen, 1997; Simons and Toomre, 2000), to mediate apical sorting of GPI-APs in epithelial cells (Brown and Rose, 1992; Mayor and Riezman, 2004; Paladino et al., 2004), and to regulate the endocytic trafficking (Chatterjee et al., 2001; Mayor and Riezman, 2004). Therefore, it is important to clarify the mechanisms by which GPI-APs are transported to the cell surface and integrated into rafts.
The GPI biosynthetic pathway has been extensively studied and many of the genes involved have been identified in mammalian, yeast, and other systems (Kinoshita and Inoue, 2000). On the other hand, little is known, especially in mammalian cells, about which molecules and transport vesicles mediate the transport of GPI-APs and their integration into rafts. In yeast, GPI-APs are transported in ER-derived vesicles that differ from those for other secretory proteins, and the Rab GTPase Ypt1p and tethering factors Uso1p, Sec34p, and Sec35p are required for sorting of GPI-APs upon their exit from the ER (Morsomme and Riezman, 2002; Morsomme et al., 2003). The cargo receptor molecules Emp24p, Erv25p, and their family members are also required for efficient sorting and transport of GPI-APs (Schimmoller et al., 1995; Muniz et al., 2000). Moreover, trafficking of GPI-APs from the ER to the Golgi requires ongoing ceramide synthesis (Skrzypek et al., 1997; Sutterlin et al., 1997). In mammalian cells, several cholesterol and sphingolipid depletion experiments have indicated the importance of their association with rafts for the endocytic pathway of GPI-APs (Mayor et al., 1998; Chatterjee et al., 2001; Mayor and Riezman, 2004). VIP17/MAL is the only molecule known to interact biochemically with GPI-APs and is required for apical sorting of GPI-APs in polarized cells (Cheong et al., 1999; Martin-Belmonte et al., 2000). Studies of cells from caveolin-1 knockout mice have revealed that caveolin-1 affects the distribution of GPI-APs (Sotgia et al., 2002). Thus, the sorting mechanism for GPI-APs is unique and specific, presumably due to the characteristics of GPI.
We have been studying genes that play roles in GPI-AP transport and behavior on the cell surface. Previously, we reported one such gene, designated PGAP1 (Post-GPI-Attachment to Proteins 1; Tanaka et al., 2004). PGAP1 is a deacylase that removes a palmitate from the inositol of GPI-APs in the ER immediately after attachment of GPI to proteins. In PGAP1-deficient cells, transport of GPI-APs from the ER to the Golgi was delayed (Tanaka et al., 2004). Here, we report the establishment of new mutant cell lines whose surface expressions of GPI-APs were greatly decreased despite normal biosynthesis of GPI-APs in the ER, and the identification of the PGAP2 gene responsible for this defect. Analyses of the mutant phenotype provide further insights into the mechanisms by which GPI-APs are correctly processed during transport and expressed on the cell surface.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Cloning of PGAP2 cDNA
C84* cells (3 x 108) were suspended in 4 ml of Opti-MEM I (Invitrogen) containing 300 µg each of a rat C6 glioma cDNA library and pcDNA-PyT (ori-) plasmids (Nakamura et al., 1997), divided into 10 cuvettes and electroporated at 280 V and 960 µF using a Gene Pulser (Bio-Rad, Richmond, CA). At 2 d after the transfection, the cells were stained with a biotinylated anti-CD59 antibody and phycoerythrin-conjugated streptavidin. Cells with restored surface expression of CD59 were collected by a cell sorter. Plasmids were recovered from these cells by Hirt's method (Hirt, 1967). After another cycle of cell sorting and plasmid recovery, we analyzed 960 clones and obtained 1 positive clone. The rat UGT gene was also identified by a similar expression cloning experiment based on the restoration of GD3 expression on C84* cells.
To clone hamster PGAP2, a partial cDNA fragment was amplified by PCR from a CHO cDNA library (a gift from Dr. O. Kuge, Kyushu University) using the degenerate primers deF1 (5'-GCCTTCGCCTAYTGGAAYCAYTA) and deR1 (5'-TCCCACCAGGCYGTCATRTGRAA). Based on this amplified sequence, hamster PGAP2-specific primers were designed. The remaining 5' and 3' regions of the cDNA were amplified by nested-PCR using vector- and PGAP2-specific primers.
FACS Analyses for GPI-APs and Transmembrane Proteins
AM-B cells were cotransfected with either a mock or PGAP2 expression vector and the indicated plasmids. Cells (5 x 106) were suspended in 0.4 ml of Opti-MEM I and electroporated with 10 µg of each of the plasmids at 260 V and 960 µF using a Gene Pulser. FLAG-tagged-CD59-TM was produced by replacing the GPI attachment signal with the transmembrane (TM) domain of CD46 (Hong et al., 2002). At 2 d after the transfection, the cells were stained with mouse anti-FLAG M2 (Sigma), mouse anti-placental alkaline phosphatase (PLAP; Sigma), mouse anti-CD25 (BD Biosciences, San Jose, CA) or mouse anti-P75 antibodies (Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University) followed by an FITC-conjugated anti-mouse IgG antibody, and analyzed by FACS.
Immunofluorescence Microscopy
NRK cells in 0.4 ml of Opti-MEM I were electroporated with 10 µg each of plasmids containing rat PGAP2 cDNA and myc-tagged DPM2 at 250 V and 960 µF using a Gene Pulser. Cells were incubated with or without 5 µg/ml BFA (MP Biomedicals, Irvine, CA) for 10 min, and then fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min. After quenching with 40 mM ammonium chloride in PBS, the cells were permeabilized with PBS containing 0.1% Triton-X (TX)-100 and 2.5% goat serum for 1 h at room temperature and stained with rabbit anti-PGAP2, mouse anti-GM130 (BD Biosciences) or mouse anti-myc (9E10) antibodies followed by an Alexa 488-conjugated donkey anti-mouse IgG antibody (Molecular Probes, Eugene, OR) or Alexa 594-conjugated goat anti-rabbit IgG antibody (Molecular Probes). The hybridoma producing the 9E10 antibody was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the Department of Biological Sciences, University of Iowa. The rabbit anti-PGAP2 serum was raised against the C-terminal peptide (DFGNKELLITSQPEEKRF) that is conserved among hamsters, rats, mice, and humans. The pictures were taken by BX50 microscope (Olympus, Tokyo, Japan) and VB-6010 CCD camera (Keyence, Osaka, Japan).
Subcellular Fractionation
We established AM-B cells that were responsive to doxycycline by stable cotransfection with pTRE2-puro-rat PGAP2, constructed by subcloning rat PGAP2 into the pTRE2pur vector (BD Biosciences), and pUHrT 62–1 (a gift from Dr. W. Hillen, Erlangen University; Urlinger et al., 2000). PGAP2 was induced with 0.05 µg/ml doxycycline (the lowest concentration able to fully recover the cell surface expression of GPI-APs) for 1 d and the cells were then cultured without doxycycline for another 1 d. Cells (1 x 108) were suspended in 1.5 ml of a buffer (9.6% sucrose, 20 mM HEPES-NaOH, pH 7.4, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride [PMSF]) and destroyed by nitrogen cavitation (300 psi, 4°C, 30 min). The cell lysate was centrifuged at 10,000 rpm at 4°C for 10 min and the supernatant was placed on top of a continuous sucrose gradient (20–50%). After centrifugation at 35,000 rpm (SW41 rotor) at 4°C for 19 h, 1-ml fractions were collected from the top and analyzed by SDS-PAGE and Western blotting. The primary antibodies used were anti-human transferrin receptor (TfR; Zymed Laboratories, South San Francisco, CA), anti-caveolin-1 (BD Biosciences), anti-ribophorin II (Santa Cruz Biotechnology, Santa Cruz, CA), anti-KDEL receptor (Stressgen, Victoria, British Columbia, Canada), anti-syntaxin 6 (Stressgen), anti-GS27 (Stressgen), anti-GS28 (Stressgen), anti-DAF (IA10) and anti-PGAP2, whereas the secondary antibodies were horseradish peroxidase (HRP)-conjugated anti-mouse IgG, HRP-conjugated anti-rabbit IgG, and HRP-conjugated anti-goat IgG (all from Amersham Bioscience, Piscataway, NJ).
Pulse-Chase Metabolic Labeling
Cells (2 x 106) were pre-incubated for 30 min at 37°C in methionine- and cysteine-free DMEM (Invitrogen) containing 25 mM HEPES-NaOH, pH 7.5, and 10% dialyzed FCS, pulsed with 100 µCi/ml [35S]methionine and [35S]cysteine (Amersham Bioscience) for 10 min at 37°C and then chased after adding cold 0.7 mM methionine and 0.3 mM cysteine. After each chase period, the culture medium and cell lysate were prepared (see Supplementary Information). Both the lysates and culture media were incubated with a monoclonal anti-DAF antibody (IA10) and protein A-Sepharose (Amersham Bioscience) that had been precoated with a nonlabeled CHO-K1 cell lysate. Immunoprecipitates were separated by SDS-PAGE and detected using a BAS 1000 analyzer (Fujifilm, Tokyo, Japan).
LC/ESIMS/MS Analysis of the GPI Anchor of CD59 Secreted from AM-B Cells
We generated AM-B cells expressing CD59 with tandem His, FLAG, GST, and FLAG-tags at the N-terminus (HFGF-CD59). The expressed HFGF-CD59 was captured from the culture medium by glutathione Sepharose 4B (Amersham Bioscience) and eluted with a solution consisting of 20 mM reduced glutathione, 30 mM Tris, and 150 mM NaCl. The purified HFGF-CD59 was subjected to SDS-PAGE and extracted after in-gel digestion with trypsin. The extracted peptides were directly analyzed by LC/ESIMS/MS using a Shimadzu LC-10ADvp LC system and an ion trap mass spectrometer LCQ (Thermo Finnigan, Waltham, MA) equipped with an electrospray ion source. A Unison C18 (Imtakt, Kyoto, Japan) reverse phase nano LC column (0.1 x 150 mm, 3 µm) was used for chromatographic separation of the peptides. A gradient solvent system was used at a flow rate of 500 nl/min with splitting. The LC was equipped with a 2-µl sample loop, and 2 µl of the sample was loaded using a PAL auto sampler (CTC Analytics, Zwingen, Switzerland). The mobile phase consisted of acetonitrile/water (5:95) containing 0.05% formic acid (solvent A) and acetonitrile/H2O (90:10) containing 0.05% formic acid (solvent B). The gradient conditions were 100% A for 0–10 min, followed by a linear gradient from 95%A/5% B to 40% A/60% B for 10–110 min. The mass spectrometer was operated in the positive ion mode with data-dependent MS/MS. The mass range of the instrument was set at m/z 400-1500, and a scan speed of 1000 Da/s was used. The ion spray voltage was set at 5500 V in the positive ion mode. Nitrogen was used as the curtain gas and helium was used as the collision gas.
TX-114 Partitioning
Cells were lysed in 1 ml of a buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF) containing 2% TX-114 (Nakalai Tesque, Kyoto, Japan) for 15 min on ice. After centrifugation at 15,000 rpm at 4°C for 15 min, the supernatant was incubated for 10 min at 37°C. Samples were separated into aqueous and detergent phases by centrifugation at 5600 x g at 37°C for 7 min. After separation, aliquots of buffer and 12% TX-114 were added to the detergent and aqueous phases, respectively, such that all the fractions were under the same conditions.
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-D-glucoside, 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF) and agitated for 1 h at 4°C. After centrifugation at 15,000 rpm at 4°C for 15 min, the supernatants were incubated with anti-FLAG (M2) beads overnight. The immunoprecipitates were washed twice with 0.5 ml of buffer B (30 mM 1-octyl--D-glucoside, 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA) and twice with 0.5 ml of buffer C (20 mM Tris-HCl, pH 7.4, 0.1% Nonidet P-40). FLAG-mEGFP-GPI was eluted with 100 µl of 0.1 M ammonium acetate containing 1 mg/ml FLAG-peptide (Sigma), 0.03% Nonidet P-40, and 5% 1-propanol four times (total volume, 400 µl). For phospholipase A2 (PLA2) treatment, immunoprecipitates bound to anti-FLAG beads obtained from 3B2A cell lysates were washed with buffers B and C, suspended in buffer D (100 mM Tris-HCl, pH 7.4, 6 mM CaCl2, 0.1% Nonidet P-40, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF) and treated with 75 U of PLA2 (Sigma) at 37°C overnight. The beads were washed with buffers B and C, and PLA2-treated FLAG-mEGFP-GPI was eluted as described above. A 200-µl aliquot of the eluate was loaded onto an Octyl-FF column (Amersham Bioscience) and chromatographed in ÄKTA (Amersham Bioscience) at a flow rate of 0.5 ml/min using a 5–40% gradient of 1-propanol. Fractions of 0.7 ml were collected, dried, subjected to SDS-PAGE, and analyzed by Western blotting using an anti-FLAG (M2) antibody and an HRP-conjugated anti-mouse IgG antibody.
Supplementary Data
Supplementary data are available at MBC online.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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, p75 (NGFR), and FLAG-tagged-VSVG as examples of TM proteins. The levels of all the GPI-APs were greatly diminished in the absence of PGAP2, whereas the TM proteins were expressed at comparable levels in the presence or absence of PGAP2 (Figure 2B). These data demonstrated that the defective surface expression was common and specific to GPI-APs.
Cloning and Characterization of PGAP2
We isolated a rat PGAP2 cDNA that restored the surface expression of CD59 on C84 cells by expression cloning and obtained hamster PGAP2 from a CHO cDNA library by PCR. Furthermore, we identified human PGAP2 with 95% amino acid identity to rat PGAP2 in the NR database (NCBI). The rat, hamster, and human PGAP2 cDNAs each encoded 254 amino acids (Figure 2C). Rat FGF receptor-activating gene 1 (FRAG1) (Lorenzi et al., 1996) appeared to be identical to PGAP2, except for several C-terminal amino acids. Alternatively spliced PGAP2 cDNAs that lacked nucleotides encoding four amino acids (VSQE) were present in Chinese hamsters and humans (Figure 2C, 
2). These shorter isoforms were functional, as assessed by their restoration of CD59 surface expression upon transfection into mutant cells (unpublished data).
We showed that PGAP2 was the gene responsible for the AM-B cell defect by mRNA analysis (Supplementary Figure 1).
Next, we analyzed the subcellular localization of PGAP2. Rabbit polyclonal anti-rat PGAP2 antibodies were raised against the C-terminal peptide. Because we were unable to detect endogenous PGAP2 protein, we transfected NRK cells with the rat PGAP2 cDNA and myc-tagged DPM2, an ER marker protein (Maeda et al., 1998), followed by staining with anti-PGAP2 and anti-GM130 or anti-myc antibodies. PGAP2 was clearly stained in the Golgi identified by the Golgi matrix marker protein GM130 and to a lesser extent in the ER (Figure 3A). PGAP2 was not localized at the ER-Golgi intermediate compartment stained with anti-GS27 antibody (unpublished data). Brefeldin A (BFA) treatment, which is known to cause rapid redistribution of Golgi proteins into the ER, altered the localization of PGAP2 to the ER, where it colocalized with DPM2 (Figure 3A), confirming that PGAP2 was localized in the Golgi. Because PGAP2 was overexpressed in these experiments and overexpression can sometimes cause mislocalization of proteins, we repeated the analysis using the Tet-on system to regulate the expression of PGAP2. Even when the expression level was only slightly above the detection limit, the staining pattern did not change (unpublished data). Finally, we analyzed the localization of PGAP2 expressed at a low level under the Tet-on system by subcellular fractionation using sucrose-density gradient ultracentrifugation. PGAP2 fractionation was most similar to that of GS28, a cis-Golgi protein that recycles between the Golgi and ER or Golgi cisternae (Subramaniam et al., 1996). The distribution of PGAP2 also partially coincided with ribophorin II, an ER protein (Figure 3B). Although quantitative measurement of the ratio of PGAP2 in the ER to that in the Golgi has been difficult, these results indicated that PGAP2 was expressed in the Golgi at a high density and in the ER at a lower density.
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Low Surface Expression of GPI-APs Is Due to Rapid Secretion
To understand the basis of the low surface expressions of GPI-APs, we analyzed the stability and transport kinetics of GPI-APs by pulse-chase metabolic labeling experiments. DAF is highly O-glycosylated and sialylated during maturation in the Golgi (Lublin et al., 1986), such that the ER form can be distinguished from the mature form by its molecular size. In mutant AM-B cells, DAF matured with glycosylation at a rate similar to that in wild-type BTP2 cells (AM-B cells permanently transfected with PGAP2). Subsequently, however, DAF was rapidly secreted from AM-B cells into the culture medium, whereas almost no DAF was secreted from BTP2 cells (Figure 4). These results showed that DAF was not degraded in the cells but secreted into the culture medium, following normal transport kinetics from the ER to the Golgi and presumably to the plasma membrane. It was observed that the secreted DAF was smaller than the mature DAF. The molecular sizes of the secreted and cell-associated DAFs were almost identical after sialidase treatment (unpublished data), indicating that the decreased size was due to partial loss of sialic acids after secretion. CD59 was also secreted from AM-B cells into the medium (Supplementary Figure 2). These results indicated that the low surface expression of GPI-APs in PGAP2-deficient cells was due to secretion of GPI-APs into the medium.
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Secretion of GPI-APs Is Due to Cleavage between Inositol and Phosphate
To examine whether the GPI-APs secreted from the mutant AM-B cells retained their GPI-anchor, we used Clostridium septicum -toxin as a probe, because it is known to selectively bind to the GPI anchor (Gordon et al., 1999; Hong et al., 2002). DAF was precipitated by -toxin from the culture medium of AM-B cells as efficiently as by an anti-DAF antibody (Supplementary Figure 3A). Furthermore, metabolic pulse labeling with [2-3H]D-mannose confirmed that the CD59 secreted from AM-B cells retained its GPI anchor (Supplementary Figure 3B). These results indicated that GPI-APs secreted from PGAP2-defective mutant cells contained part of the GPI-anchor.
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). These results indicated that the backbone structure of GPI was the same as the previously reported common structure of surface GPI-APs and that all four peaks were generated by hydrolysis between inositol and phosphate by phosphodiesterase activity, such as a PLD.
DAF Is Released from the Membrane at the Cell Surface
Next, we examined where GPI-APs are released from the membrane in the mutant cells. AM-B cells were metabolically labeled in the presence of BFA at 37°C to block transport beyond the mixed ER/Golgi system. Chases were performed under different conditions as indicated in the top panel of Figure 6A. TX-114 phase separation assays were used to distinguish membrane-bound GPI-APs from cleaved soluble GPI-APs. In the case of chasing at 37°C without BFA, the majority of DAF was secreted into the medium (Figure 6A, condition 2) and partitioned into the aqueous phase (unpublished data), as expected. During chasing for 1.5 h at 37°C in the presence of BFA, DAF was retained in the cells and partitioned into the detergent phase (condition 1) and no secretion was observed (unpublished data). Under condition 1, intracellular DAF seemed to be partially sialylated. To examine whether the cleavage/release occurred at the trans-Golgi network (TGN), chasing was performed at 19.5°C, which blocks transport from the TGN to the plasma membrane. Under this condition, mature DAF was partitioned into the detergent phase (condition 3). To eliminate the possibility that 19.5°C was too cold for the cleavage enzyme, the chase at 19.5°C was followed by incubation for 2 h at 37°C in the presence of BFA (condition 4; Miller et al., 1992; Strous et al., 1993). This incubation did not cause secretion of DAF into the culture medium and mature DAF was again partitioned into the detergent phase (condition 4). These data indicated that DAF was not released during transport from the ER to the TGN.
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). After the pulse for metabolic labeling, AM-B cells were chased for 40 min in the absence or presence of tannic acid. In the presence of tannic acid, intracellular mature DAF was separated into the detergent phase, indicating that DAF was membrane-bound (Figure 6B). Taken together with the kinetics data showing that the secretion started soon after the arrival of DAF at the cell surface (Figure 4), these results strongly suggested that DAF was released from the membrane at the cell surface of PGAP2-deficient cells.
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), a furin cleavage site (Nakayama, 1997), a FLAG tag, modified EGFP, and a GPI attachment signal. Because of the temperature-sensitivity of the extracellular domain of VSVGtsO45, the reporter protein was retained in the ER at 40°C and only left the ER for transport to the TGN once the temperature was shifted to a permissive temperature below 32°C, although the temperature restriction and exit efficiency from the ER were not as good as those of full-length VSVGtsO45 (our unpublished observations). In the TGN, furin cleaved the 90-kDa reporter protein to yield a smaller protein of 30 kDa, FLAG-mEGFP-GPI, such that the post-Golgi reporter protein could be distinguished from the ER/Golgilocalized protein (Figure 7A, lane 3). To allow accumulation of the furin-cleaved reporter protein in the TGN in C84 cells, incubation at 19.5°C for 4 h that allowed transport to the TGN but inhibited exit from the TGN, was followed by incubation for 1.5 h at 37°C in the presence of BFA for complete proteolysis by furin (Figure 7A, lane 5). FLAG-mEGFP-GPIs accumulated in C84 cells and expressed on the surface of wild-type cells were collected using anti-FLAG antibody-conjugated beads. We analyzed the hydrophobicity of the proteins by fractionation in octyl-Sepharose using elution with a 1-propanol gradient. Wild-type FLAG-mEGFP-GPI harboring two lipid chains was eluted in fractions 15–18 (Figure 7B, top panel). Lyso-GPI harboring one lipid chain prepared from wild-type FLAG-mEGFP-GPI by PLA2 treatment was eluted in fractions 8–14 (middle panel). FLAG-mEGFP-GPI from C84 cells (bottom panel) was eluted in similar fractions to the PLA2-treated lyso-GPI but clearly different fractions from wild-type GPI, strongly suggesting that the FLAG-mEGFP-GPI in PGAP2-deficient cells had one lipid chain. The amount of FLAG-mEGFP-GPI from C84 cells was not sufficient to determine its structure by MS analysis. These results suggested that lyso-GPI-APs were produced by a putative Golgi/ER-resident PLA in the absence of PGAP2, transported to the cell surface, cleaved by a PLD and secreted. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Characteristics of PGAP2
PGAP2 encoded a protein of 254 or 250 amino acids. Rat PGAP2 appeared to be identical to FRAG1 (Lorenzi et al., 1996). In a rat osteosarcoma cell line, chromosomal rearrangement produced a fusion protein of fibroblast growth factor receptor 2 and FRAG1 with constitutively elevated tyrosine kinase activity. Because PGAP2 does not contain any known domains, it is difficult to predict its functions from the amino acid sequence. PGAP2s are conserved among eukaryotes. The PGAP2 homologue in Saccharomyces cerevisiae, Cwh43p, is much larger than mammalian PGAP2 and has an extradomain of 
700 amino acids (Martin-Yken et al., 2001). The yeast mutant cwh43-2, originally isolated for its Calcofluor White hypersensitivity, displays several cell wall defects, which notably include increased release of -1,6-glucan, Cwp1p, which is a GPI-AP, and Pir2p, which is covalently attached to the cell wall (Martin-Yken et al., 2001). Because many GPI-APs, such as Cwp1p, Cwp2p, Gas1p, and Dcw1p, are involved in cell wall biogenesis (Kitagaki et al., 2002; Frieman and Cormack, 2003), we assume that the secretion of GPI-APs from the cwh43-2 yeast mutant occurs in a manner similar to that in our PGAP2-deficient cells, thereby resulting in defective cell wall biogenesis.
Candidate PLD Acting on GPI-APs
We showed that the GPI-APs in our PGAP2-deficient cells were subjected to sequential modifications/cleavages in intracellular organelles and on the plasma membrane. The first event, namely intracellular modification/cleavage, is most likely to occur in the Golgi, because most of the PGAP2 protein is localized in the Golgi. The second event, namely digestion by a PLD on the plasma membrane, must be dependent on the first event, because such rapid and efficient cleavage was not observed in normal cells. The substrate of the PLD appeared to be lyso-GPI. The basis for why the second event only occurs in PGAP2-deficient cells may be that the PLD cleaves lyso-GPI-APs but not normal GPI-APs. Alternatively, normal GPI-APs localized in rafts may be prevented from encountering the PLD, whereas lyso-GPI-APs may lose their specific localization and be susceptible to the cleavage. GPI-PLD is the only enzyme known to cleave GPI-APs and exists abundantly in serum. It is known that GPI-PLD cannot act on surface GPI-APs under physiological conditions (Scallon et al., 1991; Deeg and Davitz, 1995). We examined whether GPI-PLD present in FCS was responsible for the second event. The secretion of DAF was slower under serum-free conditions and the addition of FCS or recombinant GPI-PLD to the serum-free medium accelerated the secretion (our unpublished results). However, PGAP2-deficient cells cultured in serum-free medium for more than 1 mo showed only a small increase in the surface expression of GPI-APs (our unpublished results). Moreover, bathophenanthroline, a membrane-impermeable inhibitor of GPI-PLD, did not inhibit the secretion of DAF (our unpublished results). These results clearly indicate that another PLD that cleaves lyso-GPI must be expressed in CHO cells. Recently, it was reported that enzymes in a family of nucleotide pyrophosphatases/phosphodiesterases have lyso-PLD activity that is resistant to metal-chelating reagents, such as bathophenanthroline (Scallon et al., 1991; Tokumura et al., 2002), although whether these enzymes cleave lyso-GPI-APs has not yet been determined.
Possible Lipid Remodeling of GPI and PGAP2
How does PGAP2 deficiency cause the first event? We consider that this point can be discussed in terms of the lipid remodeling of GPI. Both 1-alkyl, 2-acyl PI, and diacyl PI are used in mammalian GPI-APs and in both cases two saturated alkyl/acyl chains are usually involved. In particular, a stearoyl (C18:0) group is mainly used in the sn-2 position (McConville and Ferguson, 1993; Brewis et al., 1995; Treumann et al., 1995; Ikezawa, 2002). Saturated chains are compatible with the liquid ordered phase of rafts in which GPI-APs are enriched (Schroeder et al., 1994; Ahmed et al., 1997; Subczynski and Kusumi, 2003). In contrast, intracellular PI, from which GPI is synthesized, usually has an unsaturated chain in the sn-2 position and the major species is sn-1-stearoyl (C18:0)-sn-2-arachidonoyl (C20:4)-PI (Kerwin et al., 1994). On the basis of the structural differences between free PI and PI in GPI-APs, we propose that lipid remodeling occurs in GPI-APs in mammalian cells. Lipid remodeling of GPI is well known to occur in S. cerevisiae (Reggiori et al., 1997; Sipos et al., 1997) and Trypanosoma brucei (Masterson et al., 1990; Morita et al., 2000; Morita and Englund, 2001). In S. cerevisiae, diacylglycerol-containing GPI is remodeled into ceramide-containing GPI or, in some cases, into diacylglycerol with a long fatty acyl chain (C26:0) in the sn-2 position (Reggiori et al., 1997; Sipos et al., 1997). In T. brucei, GPIs of variant surface glycoproteins have been well characterized and their lipid moieties are converted to dimyristoyl-PI by putative remodelases with PLA-like activity before attachment to the proteins (Morita et al., 2000). Although little is known about the precise mechanism, exchange of the sn-2 acyl chain occurs in both organisms. We assume a similar reaction may occur in mammalian cells, such that the unsaturated sn-2 acyl chain is replaced by a saturated acyl chain. In this process, PGAP2 and a putative PLA may cooperate as the remodelase and a defect in PGAP2 may cause cleavage by PLA without replacement of the chain. Further studies are required to test these possibilities.
Secretion of GPI-anchored NCAMs during Differentiation of Myoblasts
A previous study reported that GPI-anchored NCAMs expressed on the myoblast cell line C2C12 were released from the cell surface during myoblast differentiation (Mukasa et al., 1995). This release was observed even when the cells were cultured in the absence of FCS and was resistant to EDTA and phenanthroline. Moreover, the secretion was catalyzed by PLD-like cleavage. This situation is quite similar to that seen in our PGAP2-deficient cells. Although the biological significance of the secretion of GPI-anchored NCAMs remains unresolved, it is very interesting to examine whether the secretion is caused by down-regulation of PGAP2. We could not detect any endogenous PGAP2 in C2C12 cells by either Western blotting or immunofluorescence microscopy with polyclonal antibodies (our unpublished results). These findings may reflect that PGAP2 is not abundant and therefore that its down-regulation may mimic the situation of PGAP2-deficiency. Future studies investigating whether the expression of PGAP2 is regulated and whether overexpression of PGAP2 disturbs the differentiation of C2C12 cells will provide clues for how PGAP2 works in vivo.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: BFA, brefeldin A; CHO, Chinese hamster ovary; GPI-AP, glycosylphosphatidylinositol-anchored protein; PGAP, post-GPI-attachment to proteins; PLA, phospholipase A; PLD, phospholipase D; TGN, trans-Golgi network.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Yusuke Maeda (ymaeda{at}biken.osaka-u.ac.jp) or Taroh Kinoshita (tkinoshi{at}biken.osaka-u.ac.jp).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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