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J. Biol. Chem., Vol. 281, Issue 2, 896-904, January 13, 2006
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From the Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
Received for publication, October 18, 2005 , and in revised form, November 9, 2005.
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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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DPM is synthesized from dolichol phosphate and GDP-Man on the cytosolic surface of the ER membrane by DPM synthase (GDP-
-Man: dolichol-phosphate 
-mannosyltransferase; EC 2.4.1.83
[EC]
) and then is flipped onto the luminal side and used as a donor substrate. In lower eukaryotes, such as Saccharomyces cerevisiae and Trypanosoma brucei, DPM synthase consists of a single component (Dpm1p and TbDpm1, respectively) that possesses one predicted transmembrane region near the C terminus for anchoring to the ER membrane (11, 12). In contrast, the Dpm1 homologues of higher eukaryotes, namely fission yeast, fungi, and animals, have no transmembrane region (13), suggesting the existence of adapter molecules for membrane anchoring. In fact, we previously demonstrated that S. cerevisiae Dpm1 was able to complement two different DPM synthase-defective mutants, namely a Thy-1-negative class E mutant of mouse T-cell lymphoma and Chinese hamster ovary (CHO) Lec15 cells, whereas human DPM1 was only able to complement the class E mutant (14). We cloned a second component of DPM synthase, DPM2, which is the gene responsible for CHO Lec15 cells (15), and then further identified a third component, DPM3, in the biochemically purified active enzyme complex (16). Mammalian DPM1 was predicted to be a soluble protein and found to be associated with two small membrane proteins, DPM2 and DPM3. Our previous analyses using mammalian cells revealed that: (i) DPM2 stabilizes the expression of DPM3; (ii) DPM3 binds both DPM2 and DPM1; (iii) the N-terminal transmembrane regions of DPM3 are important for binding to DPM2; and (iv) the C-terminal hydrophilic region of DPM3 is important for binding to DPM1 (15, 16). Thus, DPM3 is most likely to be the anchoring molecule that tethers DPM1 to the ER membrane, although direct evidence was missing due to the lack of DPM3 mutant or knock-out cells. In the present study, we established a DPM3 mutant CHO cell line for the first time and addressed the biological functions of DPM3 using this mutant. Here we describe the essential role of the C-terminal coiled-coil domain of DPM3 and the fate of DPM1 in DPM3-defective mutant cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Flow Cytometric Analysis—Cells were stained with anti-CD59 (clone 5H8), anti-decay-accelerating factor (DAF; clone IA10) or anti-Chinese hamster urokinase plasminogen activator receptor (uPAR; clone 5D6) primary antibodies and then with a fluorescein isothiocyanate-conjugated anti-mouse IgG secondary antibody (ICN/Cappel). When cells were stained with a biotinylated antibody, phycoerythrin-streptavidin (Biomeda) was used as the secondary conjugate. Stained cells were analyzed using a FACScan (BD Biosciences).
Establishment of GPI-anchored Protein-defective CHO Mutant Cells—The parental CHO cell line F21 was established by stable transfection of the wild-type CHO K1 cells with cDNAs of GPI-anchored protein markers (human CD59 and DAF) and previously known GPI biosynthetic enzymes (DPM1, DPM2, SL15, PIG-A, PIG-L, PIG-V, PIG-B, PIG-N, PIG-F, GAA1, and PIG-U) as described previously (3). CHO F21 cells (3 x 107) were mutagenized with 1.2 µg/ml N-methyl-N'-nitro-N-nitrosoguanidine (Nacalai Tesque) for 2 days, cultured in fresh medium for 1 day, transferred to 12-well plates, and cultured for 6 days. Next, the cells were treated with 2.0 nM proaerolysin from Aeromonas hydrophila (Protox Biotech) for 3 days and 2.0 nM -toxin from Clostridium septicum (a gift from Dr. N. Sugimoto, Osaka University) for 3 days. Surviving cells were isolated by limiting dilution.
Transfection of Cells—Cells were transfected by either electroporation or lipofection. For electroporation, CHO cells (4 x 106) suspended in 0.4 ml of culture medium were electroporated at 260 V and 1,000 microfarads with 10 µg of DNA using a Gene Pulser (Bio-Rad). For lipofection, CHO cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
Labeling of GPI Precursors and Dolichol-linked Oligosaccharides—For labeling with Man, CHO cells (1 x 106) were preincubated in medium containing 100 µg/ml glucose and 10 µg/ml tunicamycin (Sigma) for 1 h and then incubated in the same medium containing 40 µCi/ml D-[2-3H]Man (American Radiolabeled Chemicals) for 45 min. Lipids were extracted from the cells with chloroform:methanol (2:1, v/v) and then partitioned into water-saturated 1-butanol. The extracts were applied to a silica gel TLC plate (Merck), and developed with chloroform:methanol:water (10:10:3, v/v/v) to analyze the GPI anchor precursors. The radiolabeled lipids were detected using a Fuji BAS1500 image analyzer (Fuji Film).
For analysis of dolichol-linked N-glycan precursors, cells were metabolically labeled with [3H]Man in the absence of tunicamycin. Dolichol-linked oligosaccharides were extracted and hydrolyzed with 0.1 M HCl, 80% tetrahydrofuran at 65 °C for 30 min to release the oligosaccharides. After neutralization, the oligosaccharides were analyzed by TLC using 1-propanol:acetic acid:water (3:3:2, v/v/v) (3).
Cloning of Chinese Hamster DPM3 cDNA—The cDNA of Chinese hamster (Cricetulus griseus) DPM3 was cloned by degenerate reverse transcription-PCR using mRNA prepared from CHO K1 cells as a template and the following primers: forward, 5'-GACGAARTTARCRCAGTGGCTTTGGGGACT; reverse, 5'-TCGGGCCTCCWSKATCTGGCTCTGCAGCTC. The 5' and 3' sequences were obtained by 5' and 3' rapid amplification of cDNA ends (RACE) PCR. The GenBankTM accession number of Chinese hamster DPM3 cDNA is AB219149 [GenBank] .
Plasmids—Site-directed mutagenesis of pME/FLAG-DPM3 was carried out using a PCR-based method. To construct pME/3HSV-DPM3tail and pME/GST-DPM3tail, a DNA fragment encoding the C-terminal 35 amino acids (Gly58-Phe92) of DPM3 was amplified with a specific forward primer containing a SalI site (5'-AAGTCGACGGTGCAGGCTATCGTGTGGCCACTTTTCATG; the SalI site is underlined) and a reverse vector primer (5'-GTTAACAACAACAATTGCATTCAT) using pME/FLAG-DPM3 as a template and then was ligated into the pME/3HSV and pME/GST vectors, respectively. To prepare the chimeric construct pME/PIG-Ltm-DPM3tail, a DNA fragment encoding the N-terminal 30 amino acids (Met1-Ser30) of PIG-L was amplified with a forward SR primer (5'-TGACCCTGCTTGCTCAACTCTACG) and a specific reverse primer containing a SalI site (5'-AAGTCGACACTCTTCATTCGTTCTGAGGAG; the SalI site is underlined) using pME/py/PIG-L as a template, and the PCR product was used to replace the 3HSV sequence of pME/3HSV-DPM3tail. Human CHIP (C terminus of Hsc70-interacting protein) cDNA was amplified by PCR using a cDNA library prepared from Hep3B cells as a template.
Affinity Precipitation and Western Blotting—Cells were lysed in TEN buffer (1.0% Nonidet P-40, 50 mM Tris-HCl, pH 7.7, 5.0 mM EDTA, 150 mM NaCl, Complete protease inhibitor mixture (Roche Applied Science)), and cell debris was removed by ultracentrifugation (100,000 x g for 1 h). For immunoprecipitation of FLAG-tagged proteins, anti-FLAG M2-agarose (Sigma) was added to the supernatant and rotated at 4 °C for 2 h. For immunoprecipitation of HSV-tagged or GST-tagged proteins, anti-HSV (Novagen) or anti-GST (Amersham Biosciences) antibodies plus protein G-Sepharose (Amersham Biosciences) were added. The beads were collected and washed twice with TEN buffer. Next, the absorbed proteins were eluted with reducing SDS sample buffer. Aliquots of the samples were separated in a 10-20% gradient SDS-polyacrylamide gel and electroblotted onto a polyvinylidene difluoride membrane. The membrane was treated with a primary antibody and then with horseradish peroxidase-conjugated protein G (Bio-Rad). Detection was carried out using Western Lightning chemiluminescence reagents (PerkinElmer Life Sciences).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-toxin, both of which are GPI anchor-recognizing pore-forming cytolytic toxins (3). We found one mutant cell line, CHO2.38, in which surface expressions of the GPI-anchored proteins CD59, DAF, and uPAR were markedly decreased compared with the parental cells (Fig. 1A). The surface expressions of these GPI-anchored proteins were restored by transient transfection of DPM3 cDNA but not by transfection of DPM1 or DPM2 cDNA (Fig. 1B). To confirm the presence of a mutation in the DPM3 gene, we sequenced and compared the DPM3 mRNAs from CHO K1 and CHO2.38 cells. Chinese hamster DPM3 was found to encode 92 amino acid residues, as is the case for humans and mice, and showed 92% (85/92) and 97% (89/92) identities with these species, respectively. By comparison of the nucleotide sequences, we found an 8-base deletion at nucleotides 108-115 in the DPM3 cDNA from CHO2.38 cells (Fig. 1C). This mutation, which causes a frameshift, may completely abolish the function of DPM3. CHO2.38 cells stably transfected with pME/puro/DPM3 (CHO2.38/DPM3 cells) showed the same expression levels of surface GPI-anchored proteins as the parental CHO F21 cells (Fig. 1D). In vitro enzyme assays using microsomes revealed that CHO2.38 cells did not synthesize either DPM or Man-containing GPI anchor precursors (Fig. 1E, lane 3), whereas CHO2.38/DPM3 cells produced more DPM than CHO F21 cells (Fig. 1E, lane 2 versus lane 1). Furthermore, CHO2.38 cells did not synthesize the mature N-glycan precursor Dol-P-P-GlcNAc2Man9Glc3 and, instead, accumulated two immature precursors (Fig. 1F, lane 2). The upper large spot (Fig. 1F, lane 2) was GlcNAc2Man5, which was identical to the spot accumulated in CHO Lec35 cells (Fig. 1F, lane 3) (CHO Lec35 is a mutant cell line defective in the general usage of both DPM and dolichol-phosphate glucose (17)). The lower weak spot (Fig. 1F, lane 2) was probably GlcNAc2Man5Glc3. Taken together, these results indicate that the CHO2.38 cell line is a DPM3-defective mutant and that DPM3 is an essential component for DPM synthase activity.
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-helix. A comparison of the amino acid sequences of DPM3 homologues from various eukaryotes revealed four hydrophobic amino acid residues, Leu74, Ile78, Ala81, and Leu85, that were conserved (Fig. 2B) and present on the same side of the -helix (Fig. 2C). Thus, we introduced point mutations into three of these aliphatic residues, namely Leu74, Ile78, and Leu85, and analyzed the biological activities of the mutants. The constructs were FLAG-tagged at the N terminus, and their expressions were confirmed to be similar by Western blotting (data not shown). We transiently transfected CHO2.38 cells with the mutant constructs and analyzed their surface expressions of CD59 by FACS (Fig. 2D). The single-point mutants I78T and L85S and the double-point mutant I78T/L85S showed nearly the same activities as wild-type DPM3, whereas the single-point mutant L74S showed much lower restoration and the two double-point mutants L74S/I78T and L74S/L85S showed only slight restoration. The triple-point mutant L74S/I78T/L85S, designated DPM3(triple), completely lost its activity and was similar to DPM3
C, which lacked the C-terminal 27 amino acids (16). These results indicate that hydrophobic residues in the coiled-coil domain of DPM3 are very important for DPM synthase activity.
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C. The cells were then lysed in 1.0% digitonin, and the FLAG-tagged proteins were immunoprecipitated with anti-FLAG agarose beads. FLAG-DPM3 proteins and co-immunoprecipitated 3HSV-DPM1 were detected by Western blotting. Although the three constructs of FLAG-DPM3 were expressed at similar levels (Fig. 3, top panel), 3HSV-DPM1 was only co-immunoprecipitated with FLAG-DPM3(wt) (Fig. 3, middle panel, lane 1) and not with FLAG-DPM3(triple) (Fig. 3, middle panel, lane 2) or FLAG-DPM3C (Fig. 3, middle panel, lane 3). The total expression level of 3HSV-DPM1 was about 3-fold higher after co-expression with FLAG-DPM3(wt) than after co-expression with FLAG-DPM3(triple) or FLAG-DPM3C (Fig. 3, bottom panel), suggesting that DPM1 expression was enhanced by DPM3. These results indicate that the cytosolic coiled-coil domain of DPM3 is essential for tethering DPM1 to the ER membrane. When 1.0% Nonidet P-40 was used for solubilization, co-immunoprecipitation of 3HSV-DPM1 with FLAG-DPM3(wt) was lost (data not shown), indicating that the association of these two molecules is maintained by a relatively weak hydrophobic interaction. Transmembrane Regions of DPM3 Are Not Essential for DPM Synthase—To address the function of the two transmembrane regions of DPM3, we replaced them with a transmembrane region from another membrane protein. For this, we chose PIG-L, which encodes GlcNAc-phosphatidylinositol N-deacetylase, the second-step enzyme in GPI biosynthesis, which is a type I transmembrane protein with a large C-terminal portion in the cytosol and a transmembrane region (Val3-Ala25) near the N terminus (18, 19). PIG-L is widely distributed in the ER membrane, and its ER localization is partly dependent upon the transmembrane region (19). We prepared a chimeric construct of the N-terminal portion (Met1-Ser30) of PIG-L, including the transmembrane region and the C-terminal tail (Gly58-Phe92) of DPM3, and designated it PIG-Ltm-DPM3tail. CHO2.38 cells were transiently transfected with pME/PIG-Ltm-DPM3tail(wt) or pME/PIG-Ltm-DPM3tail(triple), and their surface expressions of CD59 were analyzed by FACS. Interestingly, PIG-Ltm-DPM3tail(wt) restored the surface expression of CD59 similar to that of the wild-type DPM3 (Fig. 4A, panels 1 and 2). PIG-Ltm-DPM3tail(triple) and PIG-L showed no restoration, as expected (Fig. 4A, panels 3 and 4). These results suggest that the two transmembrane regions of DPM3 have no specific function in terms of the DPM synthesis required for the GPI anchor.
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DPM1 Is Degraded by the Proteasome in DPM3-defective Cells—The expression of DPM1 was enhanced by co-transfection of the wild-type DPM3 into CHO K1 cells (Fig. 3), suggesting that DPM3 stabilizes DPM1 on the ER membrane. To examine whether DPM1 is expressed in the absence of DPM3, we transiently transfected CHO2.38 cells with pME/3HSV-DPM1 in the presence or absence of pME/3HSV-DPM3. Rat microsomal aldehyde dehydrogenase (ALDH) was used as a control for transfection efficiency. After 2 days of culture, the cells were lysed, and the protein expression was analyzed by Western blotting. Unexpectedly, 3HSV-DPM1 was hardly detected in the absence of DPM3 (Fig. 5A, lane 2), whereas it was strongly expressed in the presence of DPM3 (Fig. 5A, lane 1). Similar experiments were carried out for DPM2. Unlike DPM1, DPM2 was stably expressed in the presence or absence of DPM3 (Fig. 5A, lanes 3 and 4).
Because DPM1 was hardly expressed in DPM3-defective CHO2.38 cells, we speculated that it was rapidly degraded after translation. To elucidate this, we stably transfected CHO K1 and CHO2.38 cells with pME/puro/3HSV-DPM1 to generate CHO K1/3HSV-DPM1 and CHO2.38/3HSV-DPM1 cells, respectively, and analyzed their 3HSV-DPM1expression levels. DPM1 was strongly expressed in CHO K1/3HSV-DPM1 cells (Fig. 5B, lane 1) but was hardly expressed in CHO2.38/3HSV-DPM1 cells (Fig. 5B, lane 2), as expected. When a DPM3 cDNA was transiently transfected into CHO2.38/3HSV-DPM1 cells, the expression of DPM1 was recovered to some extent (Fig. 5B, lane 3), indicating that the cDNA of DPM1 was maintained in the cells but the protein was degraded.
Next, we speculated that DPM1 expressed in CHO2.38 cells may be dissociated from the ER membrane and degraded by the proteasome, because DPM1 is predicted to be located on the cytosolic surface of the ER membrane. To test this hypothesis, we cultured CHO K1/3HSV-DPM1 and CHO2.38/3HSV-DPM1 cells in the presence of protease inhibitors. In CHO K1/3HSV-DPM1 cells, the expression levels of 3HSV-DPM1 remained unchanged following the addition of lactacystin, an inhibitor of the proteasome, or leupeptin, an inhibitor of lysosomal proteases (Fig. 5C, lanes 1-3). In contrast, in CHO2.38/3HSV-DPM1 cells, the expression of 3HSV-DPM1 was restored after the addition of lactacystin (Fig. 5C, lane 5) but not leupeptin (Fig. 5C, lane 6). These results strongly suggest that DPM1 is highly unstable and degraded by the proteasome in the cytosol when DPM3 is absent. It is worth noting that CD59 expression was not restored at all in CHO2.38/3HSV-DPM1 cells following the treatment with lactacystin (Fig. 5D, panels 2 and 4), although the level of DPM1 protein was similar to that in CHO2.38/3HSV-DPM1 cells transiently transfected with DPM3 cDNA (Fig. 5C, lane 5, versus Fig. 5B, lane 3). These results suggest that the coiled-coil domain of DPM3 is necessary not only for the stabilization of DPM1 but also for its enzyme activity.
Free DPM1 Strongly Interacts with CHIP, a Chaperone-dependent E3 Ubiquitin Ligase—There are several hundred known candidates for E3 ubiquitin ligases in mammalian genomes, and these define the specificity of the target protein degradation by the proteasome. To explore which ubiquitin ligase is responsible for the degradation of DPM1, we focused on CHIP, which was originally discovered as a co-chaperone containing a tetratricopeptide repeat domain (20) and later identified to be a chaperone-dependent U-box-type E3 ubiquitin ligase (21, 22). We transiently transfected CHO2.38 cells with GST-CHIP and either 3HSV-ALDH as a control or 3HSV-DPM1. GST-CHIP was strongly expressed in all transfectants (Fig. 6, bottom panel). 3HSV-ALDH was also strongly expressed in the presence or absence of lactacystin (Fig. 6, top panel, lanes 1-4; lanes 3 and 4 contain 10% of the loading of lanes 1 and 2, respectively). However, very little GST-CHIP co-immunoprecipitated with 3HSV-ALDH (Fig. 6, middle panel, lanes 1 and 2), and it was hardly detected in the lanes with the 10% loadings (Fig. 6, middle panel, lanes 3 and 4). In contrast to ALDH, the expression of 3HSV-DPM1 was very weak in the absence of lactacystin and increased after the addition of lactacystin (Fig. 6, top panel, lanes 5 and 6, respectively). Despite its weak expression, GST-CHIP was much more strongly co-immunoprecipitated with 3HSV-DPM1 than with ALDH (Fig. 6, middle panel, lanes 5 and 6 versus lanes 3 and 4). These results strongly suggest that free DPM1 is ubiquitinated by CHIP.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-mannosidase and Golgi -mannosidases I and II. These residues are not essential for the viability of cultured mammalian cells but are important for quality control of glycoprotein production in the ER. It has been reported that the maturation, transport, and degradation of nascent glycoproteins in the ER are delayed in DPM synthase-defective cells (23). Recently, ER degradation-enhancing -mannosidase-like protein (EDEM) was found to play a key role in ER-associated degradation by recognition of the terminal 1,2-linked Man residue in the GlcNAc2Man8 structure, which is derived from DPM (24). In humans, genetic diseases caused by mutations in DPM synthase are known and classified into congenital disorder of glycosylation type Ie (CDG-Ie). These patients show accumulation of the N-glycan precursor with GlcNAc2Man5 (25, 26), similar to the case with CHO2.38 cells (Fig. 1F). To date, mutations that cause CDG-Ie have been found only in DPM1 and not in DPM2 or DPM3. Because DPM3 and DPM2 are essential for enzyme activity (Fig. 1E), CDG-Ie patients harboring mutations in these subcomponents may be identified in the future. Defects in DPM synthesis also affect GPI anchor biosynthesis, because the three core Man residues of GPI are derived from DPM (Fig. 1A). Knock-out mice for PIG-A, which encodes the catalytic subunit of GPI-GlcNAc-transferase, the first-step enzyme of GPI anchor biosynthesis, show embryonic lethality (27). This observation suggests that complete loss of DPM synthase activity may cause death in early embryogenesis. Specific defects in protein O-mannosyltransferase, which is also DPM-dependent, cause certain types of congenital muscular dystrophies with neuronal migration disorders, such as Walker-Warburg syndrome (28). Therefore, taken together, DPM synthesis is critical for multicellular functions.
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DPM3 appeared to be a limiting factor for the formation of the DPM synthase complex, because the enzyme activity was enhanced by overexpression of DPM3 in CHO2.38 cells (Fig. 1E). When the amount of DPM3 is limited or depleted, excess DPM1 may be degraded by the proteasome, as demonstrated in CHO2.38 cells (Fig. 5). Although DPM1 does not have a transmembrane region, it does possess a hydrophobic peptide sequence near the C terminus that functions in its binding to DPM3 (15, 16). Thus, DPM1 dissociated from DPM3 must bear the hydrophobic peptide on the surface of the molecule, which could be a binding target for cytosolic chaperones. We demonstrated that free DPM1 formed a complex with an E3 ubiquitin ligase, CHIP (Fig. 6), which is known to bind an E2 ligase through its U-box domain and is also able to bind the cytosolic chaperones Hsc70/Hsp70 or Hsp90 through its tetratricopeptide repeat domain. It has been reported that CHIP accelerates the degradation of several cytosolic and membrane proteins and also that CHIP prefers misfolded proteins over correctly folded proteins (30, 31). The interaction between DPM3 and DPM1 must be relatively weak, because it was stable in digitonin but labile in Nonidet P-40 (Fig. 3), suggesting that some intracellular stimuli may dissociate these two molecules under physiological conditions. It has been reported that cAMP-dependent protein kinase regulates mammalian DPM synthase activity by phosphorylating a serine residue in DPM1 (32, 33). The conformational changes of DPM1 caused by such post-translational modifications may trigger dissociation of the DPM synthase complex.
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FOOTNOTES |
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* This work was supported by grants from the Ministry of Education, Sports, Science, Culture, and Technology of Japan and the Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama, 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. 
1 To whom correspondence should be addressed: Dept. of Immunoregulation, Research Inst. for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-8328; Fax: 81-6-6875-5233; E-mail: tkinoshi{at}biken.osaka-u.ac.jp.
2 The abbreviations used are: DPM, dolichol phosphate mannose; ALDH, aldehyde dehydrogenase; CDG-Ie, congenital disorder of glycosylation type Ie; CHIP, C terminus of Hsc70-interacting protein; CHO, Chinese hamster ovary; DAF, decay-accelerating factor; ER, endoplasmic reticulum; FACS, fluorescence-activated cell sorter; GPI, glycosylphosphatidylinositol; GST, glutathione S-transferase; HSV, herpes simplex virus; PIG, phosphatidylinositol glycan; uPAR, urokinase plasminogen activator receptor.
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ACKNOWLEDGMENTS |
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