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J. Biol. Chem., Vol. 279, Issue 14, 14256-14263, April 2, 2004
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From the Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
Received for publication, December 16, 2003 , and in revised form, January 13, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The enzyme involved in inositol deacylation of GPI-APs has not been characterized. Furthermore, the reason why the deacylation usually occurs is unclear. In order to identify a gene for the GPI inositol-deacylase, we isolated a mutant Chinese hamster ovary (CHO) cell line defective in inositol deacylation and cloned the gene responsible, PGAP1 (for Post GPI Attachment to Proteins 1). Rat PGAP1 is an ER membrane protein with a catalytic serine-containing motif that is conserved in a number of lipases.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasmids—The plasmids pME-Py (an expression vector with a polyoma origin of replication), pME-Py-FLAG-ALDH (ALDH, microsomal aldehyde dehydrogenase) (11), and pME-Py-GST-FLAG-hPIG-M were described previously (12, 13). A plasmid pME-Puro-PGAP1 was constructed by subcloning the XhoI/XbaI fragment of pME-Py-PGAP1 into the XhoI/XbaI site of pME-Puro. To fuse a FLAG tag at the N terminus of PGAP1, the rat DPM2 cDNA of pME-Py-FLAG-rDPM2 was replaced with a cDNA of PGAP1 whose start codon was replaced by a SalI recognition sequence. A glutathione S-transferase (GST)-tagged PGAP1 was constructed in a similar way using pME-Py-GST-rDPM2. We termed these plasmids pME-Py-FLAG-PGAP1 and pME-Py-GST-PGAP1, respectively.
To obtain the Bst1p expression vector pME-Py-BST1, we amplified BST1 with the primers 5'-GCGGCCGCCACCATGGGTATCAGGAGATTAGTTAGTGTT-3' (forward) and 5'-TCTAGACTAATGTATTGTTTCGAAAAATAGCAAC-3' (reverse) from a Saccharomyces cerevisiae genomic DNA library, and we cloned it into the pGEM-T Easy vector (Promega). The BST1 was sequenced and subcloned into the NotI/XbaI site of pME-Py.
We substituted serine 174 in PGAP1 with alanine by site-directed mutagenesis using a Quick Change Site-directed Mutagenesis Kit (Stratagene). After sequencing, the ClaI/BglI fragment of the mutated plasmid was used to replace the corresponding part of the wild-type pME-Py-PGAP1 to obtain pME-Py-PGAP1 (S174A).
To construct pME-Puro-FLAG-folate receptor 1 (FR1), we amplified the human FR1 sequence with primers 5'-CCAGAAGCTTGCTCAGACAAGGATTGCATGGG-3' (forward) and 5'-GGAGTGCGGCCGCTAGCTGAGCAGCCACAGCAGCA-3' (reverse) from human cDNA library. The HindIII/NotI-cut fragment was subcloned into HindIII/NotI-cut pME-Puro-FLAG-CD59 to replace CD59 with FR1.
Establishment of a GPI Inositol-deacylase-defective Cell Line, C10—3B2A cells (107) were treated with 0.4 mg/ml ethyl methanesulfonate for 1 day, followed by a 6-day culture without ethyl methanesulfonate. Cells were treated with 1 unit/ml PI-PLC (Molecular Probes) for 1 h at 37 °C, and the PI-PLC-resistant cells were enriched by auto-MACS (Miltenyi Biotec) using biotinylated anti-CD59 (5H8) and anti-DAF (IA10) antibodies and cloned by limiting dilution.
Cloning of PGAP1 cDNA—C10 cells (1.2 x 108) were mixed with 360 µg each of a rat C6 glioma cDNA library (10) and pcDNA-PyT(ori-) plasmids (10) in 4.8 ml of Opti-MEM I and electroporated in 12 cuvettes at 960 microfarads and 300 V using Gene Pulser (Bio-Rad). After 1 day, cells (107/ml) were treated with 25 mg/ml Pronase (Sigma) in Opti-MEM I for 2 h at 37 °C. Transfected cells were resuspended in Opti-MEM I at 2 x 107/ml 1.5 days after the Pronase treatment, treated with 1 unit/ml of PI-PLC for 1 h at 37 °C, and then stained with a biotinylated anti-CD59 antibody (5H8) and phycoerythrin-conjugated streptavidin (Biomeda). Approximately 6,000 cells with low negative CD59 staining were collected using a cell sorter (FACS-Vantage, BD Biosciences). Plasmids (2 x 105 clones) were recovered from these cells by Hirt's method (14). Pooled plasmids (15 µg) were transfected with 45 µg of pcDNA-PyT(ori-) plasmids into 1.8 x 108 C10 cells, as described above, in 9 cuvettes. After another cycle of cell sorting and plasmid recovery, the plasmids were electroporated into Escherichia coli and fractionated to 576 pools (20-30 clones/pool). One of these pools restored PI-PLC sensitivity in C10 cells, and 3 positive colonies were obtained from this pool.
Human PGAP1 cDNA—Human PGAP1 cDNA was amplified by PCR using forward (5'-CTCGAGCCACCATGTTTCTTCACTCAGTTAATCTCTG-3') and reverse (5'-GCGGCCGCTTACATAAAGTTGCATAATGCATGG-3') primers. The primers were designed based on the sequences of FLJ12377(DDBJ/GenBankTM/EMBL accession number AK022439 [GenBank] ) and IMAGE:1676930 (3') (DDBJ/GenBankTM/EMBL accession number AI076615 [GenBank] ), respectively.
PI-PLC Sensitivity Assay—Cells (106) were treated with or without 50 µl of 1 unit/ml PI-PLC in Opti-MEM I (Invitrogen) for 1-1.5 h at 37 °C. The cells were stained for CD59 and DAF and analyzed with a FACSCalibur (BD Biosciences).
Triton X-114 Partitioning of FLAG-tagged CD59—C10 cells (2 x 106) were transiently transfected with 3.2 µg of pME-Py-FLAG-CD59 (15) using LipofectAMINE 2000 (Invitrogen). Transfected cells (106) were treated with or without 100 µl of 1 unit/ml PI-PLC for 1 h at 37 °C, lysed in 1 ml of 2% Triton X-114 (Nacalai Tesque) in Opti-MEM I for 10 min on ice, and then incubated for 15 min at 37 °C with vigorous mixing. The cell lysates were separated into aqueous and detergent phases by centrifugation at 1,000 x g for 10 min at 25 °C. FLAG-tagged CD59 was immunoprecipitated from each fraction using anti-FLAG beads (Sigma) and analyzed by SDS-PAGE/Western blotting.
Alkaline Hydroxylamine Treatment of FLAG-FR1—3B2A and C10 cells were transfected with FLAG-FR1 expression plasmid. After 2 days, cells (106) were treated with or without 1 M hydroxylamine (Sigma) in 0.1 M triethylamine, pH 11.5, overnight at 4 °C. After neutralization, the membranes were collected by centrifugation at 100,000 x g for 1 h at 4 °C, washed, and resuspended in 100 µl of PBS by sonication, treated with or without 3 units/ml of PI-PLC for 1.5 h at 37 °C. FLAG-FR1 proteins were subjected to Triton X-114 partitioning and analyzed by SDS-PAGE/Western blotting as described above.
Subcellular Localization of PGAP1—CHO cells (4 x 107) transfected with pME-Py-FLAG-PGAP1 were fractionated, and the activities of plasma membrane, Golgi, and ER marker enzymes were assayed as described previously (16). FLAG-PGAP1 was extracted and immunoprecipitated from each fraction using anti-FLAG beads and then analyzed by SDS-PAGE/Western blotting. The intensities of the FLAG-PGAP1 bands were quantified using a LAS1000 image analyzer (Fujifilm, Tokyo, Japan).
Pulse-chase Analysis of DAF—C10 and RD3 cells were incubated for 30 min at 37 °C in methionine- and cysteine-free Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% dialyzed fetal bovine serum, pulse-labeled with 100 µCi/ml [35S]methionine and -cysteine (Amersham Biosciences) for 10 min at 37 °C, and then chased with cold 1.5 mM methionine and 1.2 mM cysteine for 0-60 min at 37 °C. Cells were washed with ice-cold PBS and lysed in 0.6 ml of RIPA buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS) containing "complete protease inhibitor mixture" (Roche Applied Science). DAF was immunoprecipitated using a monoclonal anti-DAF antibody (IA10) and protein-A-Sepharose (Amersham Biosciences). 35S-Labeled DAF was separated by SDS-PAGE under reducing conditions and analyzed using a BAS 1000 analyzer (Fujifilm).
Membrane Orientation of PGAP1 Protein—CHO cells were transiently transfected with pME-Py-GST-PGAP1 or pME-Py-GST-FLAG-hPIG-M and replated on gelatin-coated coverslips after 1 day of culture. After 2 days, cells were fixed with 4% paraformaldehyde in PBS for 15 min and then incubated in 40 mM NH4Cl in PBS for 20 min. Nonspecific binding sites were blocked with 1% bovine serum albumin in PBS for 30 min. For complete permeabilization, 0.1% Triton X-100 was added to the blocking solution and incubated for 30 min at room temperature. For selective permeabilization of the plasma membrane, cells were incubated in 5 µg/ml digitonin (Wako) for 30 min. The cells were then incubated with a rabbit anti-BiP antibody (ABR) or goat anti-GST antibody (Amersham Biosciences) followed by rhodamine-conjugated donkey anti-rabbit IgG (Chemicon) or Alexa 488-conjugated donkey anti-goat IgG (Molecular Probes) antibodies. The cells were observed under a fluorescent microscope (BX-50, Olympus) equipped with a CCD camera (VB-6010, Keyence).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We confirmed the PI-PLC resistance of C10 cells by FACS. The intensities of CD59 and DAF on C10 mutant cells were similar to those of wild-type 3B2A cells and were not affected by PI-PLC treatment (Fig. 1A, a and b), whereas the intensities on 3B2A cells were decreased by 90-95% (c and d).
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We found two bands of smaller molecular size (arrows a and b). Band a was detected in both cell lines and was hydrophilic (lanes 3 and 7). It may be FLAG-CD59 that failed to become GPI-anchored due to the overexpression. Band b was hydrophobic, PI-PLC resistant, and only seen in C10 mutant cells (lanes 6 and 8). This would be an immature GPI-anchored FLAG-CD59 bearing acylated inositol.
To obtain evidence that the PI-PLC resistance of GPI-APs on C10 mutant cells is indeed due to the inositol acylation, we eliminated the acyl chain by treating C10 cells with alkaline hydroxylamine (17) and then tested whether GPI-APs became PI-PLC-sensitive. We transfected C10 and 3B2A cells with FLAG-tagged FR1, GPI-AP. Some of the FLAG-FR1 was lost after alkaline hydroxylamine treatment, most likely because FR1 with GPI-anchor having diacylglycerol was released and because FR1 with GPI-anchor having alkylacylglycerol remained membrane-bound. We analyzed PI-PLC sensitivity of the remaining FLAG-FR1 by Triton X-114 partitioning. Although FLAG-FR1 on alkali buffer-treated C10 cells was resistant to PI-PLC (Fig. 2, lanes 5-8), the majority of FLAG-FR1 became PI-PLC-sensitive after alkaline hydroxylamine treatment (lanes 1-4). FLAG-FR1 on 3B2A was sensitive to PI-PLC without alkaline hydroxylamine treatment as expected (lanes 9-12). These results support that the inositol acylation of GPI-APs on C10 mutant cells was the basis for PI-PLC resistance.
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Regarding the human homologue, we found that the sequence of the first 591 amino acids of the hypothetical protein FLJ12377was 90% identical with that of PGAP1. However, the predicted sequence of the C-terminal portion was not similar to PGAP1, and the entire sequence was shorter than that of PGAP1. The C-terminal sequence of FLJ12377was found in genomic sequence of human PGAP1 gene (NCBI data base), suggesting that it was a product of alternative splicing. We also found ESTs whose sequences are homologous to the C-terminal portion of PGAP1. To obtain the full-length sequence of the human homologue, we amplified it from a human cDNA library by PCR and determined the sequence (DDBJ/GenBankTM/EMBL accession number AB128038 [GenBank] ). The identity of the predicted amino acid sequences of human and rat PGAP1 was 90%, and their lipase consensus sequences were identical (Fig. 3B).
S. cerevisiae Bst1p Is an Orthologue of PGAP1—We searched the data bases for homologues of PGAP1 and found an S. cerevisiae homologue of PGAP1 that was known as BST1 (Fig. 3B, DDBJ/GenBankTM/EMBL accession number NC_001138 [GenBank] ) (18, 19). The amino acid identity of rat PGAP1 and yeast Bst1p was 18%, and the lipase consensus sequence was conserved. To examine whether BST1 was a functional homologue, we transiently transfected C10 mutant cells with BST1 cDNA and tested the PI-PLC sensitivity. We found a partial restoration of the PI-PLC sensitivity of the transfectants, indicating that Bst1p is an orthologue of PGAP1 (Fig. 4b).
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45 kDa) is transported to the Golgi and accepts multiple O-glycans and modification of the N-glycan to become mature DAF (75 kDa). In C10 mutant cells, the amount of mature DAF gradually increased with a slow decrease in the amount of the ER form of DAF during the chase period (Fig. 8A, lanes 1-5). In contrast, in RD3 rescued cells, the increase in the mature DAF and decrease in the ER form of DAF were faster than those in C10 mutant cells (lanes 6-10). Parental 3B2A cells showed basically the same kinetics as RD3 cells (data not shown). Quantification of the data showed that the C10 mutant cells had a clear delay in the maturation of DAF; half the DAF matured within 20 min in RD3 rescued cells, while 60 min was required in C10 cells (Fig. 8B), indicating that the defect in the inositol deacylation affects the maturation of DAF. The molecular sizes of the mature DAF in C10 and RD3 cells were similar. Therefore, the extent of the glycosylation was not affected. We then analyzed total DAF in the whole cell lysates by Western blotting and found a significant accumulation of the ER form of DAF in C10 cells (Fig. 8C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The major findings in the present study are as follows: 1) that PGAP1 encodes an ER-associated GPI inositol-deacylase, thereby providing a molecular basis for the report that inositol deacylation occurs in the ER soon after the generation of GPI-APs (6); and 2) that inositol deacylation is important for efficient ER-to-Golgi transport of GPI-APs.
A Role for Inositol Deacylation in the ER-to-Golgi Transport of GPI-APs—In PGAP1-deficient mutant cells, the ER-to-Golgi transport of DAF as measured by the generation of O-glycosylated Golgi form of DAF was clearly delayed. The time necessary for 50% of DAF to mature was 20 min in wild-type cells, while it was 60 min in the mutant cells. In S. cerevisiae, the BST1 gene is homologous to PGAP1 (18). PI-PLC sensitivity in PGAP1-deficient CHO cells was partially restored by transfection of BST1, showing that Bst1p is a functional homologue of PGAP1. Consistent with this result, maturation of Gas1p, a GPI-anchored protein whose maturation is dependent upon the transport to the Golgi (21), was delayed in bst1-deleted yeast (19), like DAF in C10 mutant cells.
BST1 (Bypass of Sec Thirteen 1) gene was identified by screening mutations that suppress lethality caused by down-regulation of SEC13 gene, a gene for a component of COPII vesicle coatmers (18). In sec13 mutant cells, normally deacylated GPI-APs may not be incorporated into COPII vesicles and may accumulate in the ER, possibly around the zone where COPII vesicles are generated. The fact that the simultaneous defect in BST1 rescued the lethality caused by down-regulation of SEC13 suggested that inositol-acylated GPI-APs may have been transported from the ER via some alternative way.
Emp24p (22) and Erp1p (23), members of p24 family, are thought to be components of cargo receptors for GPI-anchored protein Gas1p in S. cerevisiae. Both emp24 and erp1 mutations are also sec13 bypass (18, 23). Taken together with a fact that surface transport of GPI-APs is essential for growth of S. cerevisiae, it is suggested that association of GPI-APs with their cargo receptors that would cause accumulation of GPI-APs in the ER is involved in the lethal effect of sec13 mutation. It is therefore possible that the molecular basis of sec13 bypass by bst1 mutation is that the acyl chain remaining on inositol of GPI-APs in bst1 mutant yeast might interfere with the efficient association with the cargo receptors, resulting in a lack of accumulation of GPI-APs. Thus, Bst1p/PGAP1-mediated inositol deacylation may be required for efficient incorporation of GPI-APs into COPII transport vesicles through the association with the cargo receptors.
GPI Inositol Deacylation in Trypanosoma brucei Versus Mammals/S. cerevisiae—In mammalian cells and the yeast S. cerevisiae, an acyl chain is added to the 2-position of inositol early in the GPI biosynthetic pathway (24). The acylation is mediated by PIG-W in mammalian cells (5) and by Bwt1p, the PIG-W orthologue, in S. cerevisiae (25). The acceptor substrate for these acyltransferases is glucosaminyl-phosphatidylinositol (26, 27). Once added, the acyl chain remains until transfer of GPI to proteins. In African trypanosomes, T. brucei, inositol acylation and deacylation reactions are more complex, differ from those in mammalian and yeast systems, and differ between the bloodstream and procyclic forms. In T. brucei, the acyl chain is added one step later, because it is first added to mannosyl-glucosaminyl-phosphatidylinositol (28, 29). However, the palmitoyl chain can be removed and added again at any of the later steps (28). The direct precursor of the GPI-anchor in bloodstream parasites is not acylated, whereas in procyclic form parasites, the direct GPI-anchor precursor is acylated.
GPIdeAc gene that partially accounts for inositol deacylase activity in T. brucei has been cloned (30). GPIdeAc consisting of 558 amino acids has an N-terminal signal peptide but not a transmembrane domain. It is homologous to mammalian acyloxyacyl hydrolase that removes fatty acids from bacterial lipopolysaccharides. GPIdeAc is much smaller than PGAP1 and has no significant sequence homology with PGAP1. GPIdeAc is hydrophilic whereas PGAP1 has multiple transmembrane domains. Therefore, GPIdeAc and PGAP1 are not homologous.
We found a sequence that has significant homology with PGAP1 in the T. brucei genome data base. Cloning and knock out or knock down of this gene are required to determine whether it is an inositol deacylase that accounts for the inositol deacylation activity remaining in the GPIdeAc knock out T. brucei (30).
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FOOTNOTES |
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* This work was supported by grants from the Ministry of Education, Science, Sports, Culture and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
To whom correspondence should be addressed. Tel.: 81-6-6879-8328; Fax: 81-6-6875-5233; E-mail: tkinoshi{at}biken.osaka-u.ac.jp.
1 The abbreviations used are: GPI, glycosylphosphatidylinositol; GPI-APs, GPI-anchored proteins; ER, endoplasmic reticulum; PI-PLC, phosphatidylinositol-specific phospholipase C; GST, glutathione S-transferase; CHO, Chinese hamster ovary; ALDH, aldehyde dehydrogenase; PBS, phosphate-buffered saline; DAF, decay-accelerating factor; FACS, fluorescence-activated cell sorter.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-mannosidase II for the Golgi apparatus (B) and dolichol-phosphate-mannose synthase for the ER (C)). The distribution of FLAG-PGAP1 was analyzed by SDS-PAGE/Western blotting using monoclonal anti-FLAG antibodies.
