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Vol. 20, Issue 13, 3088-3100, July 1, 2009
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*Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, Sendai, Miyagi 981-8558, Japan; and Core Research for Evolutional Science and Technology Program (CREST), Japan Science and Technology Agency (JST), Saitama, 332-0012, Japan
Submitted December 17, 2008;
Revised April 8, 2009;
Accepted April 29, 2009
Monitoring Editor: Benjamin S. Glick
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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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; Kono et al., 1998; Fukumoto et al., 1999; Kapitonov et al., 1999) provides an essential function in the biosynthesis of more complex ganglio-series gangliosides. In addition, GM3 itself forms lipid microdomains (rafts) that, with cholesterol and sphingomyelin, function as platforms for effective signal transduction in the plasma membrane (Simons and Toomre, 2000) and participate in the regulation of numerous cellular processes, such as proliferation, differentiation, and adhesion (Hakomori, 2004).
GM3 is also known to be involved in type 2 diabetes. GM3 levels are increased in the adipose tissue of Zucker fa/fa rats and ob/ob mice, which are typical rodent models of obesity (Tagami et al., 2002), and in SAT-I knockout mice insulin sensitivity is enhanced (Yamashita et al., 2003). In 3T3-L1 adipocytes, exposure to TNF-
increases the expression of GM3 (Tagami et al., 2002), and TNF--induced insulin resistance can be prevented by treatment with a glucosylceramide synthase inhibitor, D-PDMP, that decreases GM3 content (Tagami et al., 2002). The accumulation of GM3 in insulin resistance results in the dissociation of the insulin receptor from caveolae (Kabayama et al., 2005) after an electrostatic interaction between GM3 and a lysine residue (Lys-944) located just above the transmembrane of the insulin receptor (Kabayama et al., 2007). Overall, treatment with glucosylceramide synthase inhibitors significantly improves insulin sensitivity and glucose homeostasis in rodent models of obesity (Aerts et al., 2007; Zhao et al., 2007). Consequently, it is important to understand the regulation of GM3 biosynthesis as it relates to diabetes, as well as other pathological states.
The primary enzyme responsible for GM3 synthesis, SAT-I, transfers a sialic acid to lactosylceramide (LacCer) in the Golgi apparatus. The regulation of SAT-I expression at the transcriptional level has been partially clarified. All promoters of glycosyltransferase genes utilized in ganglioside synthesis are TATA-less and have no CCAAT box, but they do contain GC-rich sequences in their SP1- and AP2-binding sites. Indeed, it has been reported that mouse SAT-I genes are regulated by SP1 and AP2 (Xia et al., 2005), although the transcription factor regulation of human SAT-I, including the basal transcription machinery, is not understood (Kim et al., 2002; Zeng et al., 2003). However, CREB, the cAMP response element-binding protein, is known to be crucial for the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of human SAT-I mRNA in HL-60 and K562 cells (Choi et al., 2003, 2004).
Mouse SAT-I transcripts are classified into two types, mSAT-Ia and -Ib variants, according to the position of transcription initiation, exon 1 or 2 (Figure 1A; Kono et al., 1998; Kapitonov et al., 1999). In contrast, human SAT-I transcripts can be classified into three types, hSAT-Ia, -Ib, and -Ic variants, according to the position of transcription initiation, exon 1, 2, or 4 (see Figure 1B; Ishii et al., 1998; Kapitonov et al., 1999; Kim et al., 2001; Berselli et al., 2006). In addition, the structures of hSAT-Ia and -Ib mRNA are each further classified into two types: hSAT-Ia-1 and -Ia-2 variants and hSAT-Ib-1 and -Ib-2 variants, respectively, reflecting alternative splicing of exon 3 (Figure 1B; Kim et al., 2001). According to the first-AUG rule, translation is initiated at the AUG codon nearest the 5' end of an mRNA (Kozak, 2002). Thus, in the mSAT-Ib and hSAT-Ic variants, it has been assumed that the first AUG codons, M2 and M2', respectively, are the initiation codons (Figure 1; Kono et al., 1998; Berselli et al., 2006). However, in the mSAT-Ia, hSAT-Ia-1 and -Ia-2, and hSAT-Ib-1 and -Ib-2 variants, a second or third AUG, M3, has been considered as the initiation codon, based on speculation that the context presenting M1 and M2 is insufficient for either to be recognized by the ribosome as an initiation codon (Ishii et al., 1998; Kapitonov et al., 1999; Kim et al., 2001). Although the exact translational sites remain undetermined, the translational product from M3 (M3-SAT-I) has been used in many studies (Giraudo et al., 2001; Giraudo and Maccioni, 2003a,b; Uemura et al., 2006; Uliana et al., 2006; D'Angelo et al., 2007). Because the catalytic domain of the SAT-I protein exits on the lumen side of Golgi apparatus and M3-SAT-I can produce GM3 in vivo (Ishii et al., 1998), little attention has been given to the significance of the position of the initiation codon; however, the position of the initiation codon would determine the length of the cytoplasmic region of a SAT-I protein. For example, in the mouse SAT-I protein, the lengths of the cytoplasmic tails translated from M1, M2, and M3 are 69, 42, and 14 amino acids (aa), respectively (Figure 2). Notably, the length of M1-SAT-I is the greatest among the members of the sialyltransferase family (Figure 2).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The pSU180 [M1-SAT-I(N)-enhanced green fluorescent protein (EGFP)] plasmid encoding fusion proteins of the NH2-terminus of M1-SAT-I and EGFP was constructed using pSY14 and primers 5'-CCCCTATTGACGTCAATGACGG-3' and 5'-CCCGGGCAGGGTCCACATAGTGCATTC-3'. The resulting fragments were cloned into a pGEM-T Easy vector to generate pSU147. The 0.33-kb EcoRI-SmaI fragments of pSU147 were then cloned into the EcoRI-SmaI site of the pEGFP-N1 vector (BD Bioscience Clontech Laboratories, Palo Alto, CA) to generate pSU180. The pSU181 [M2-SAT-I(N)-EGFP] and pSU182 [M3-SAT-I(N)-EGFP] plasmids were created by similar procedures.
Point mutants of mSAT-Ia, mSAT-Ib, ks-M1-SAT-I, ks-M2-SAT-I, and M1-SAT-I(N)-EGFP were created by site-directed mutagenesis using the QuikChange kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions.
The primers used to amplify the deletion mutants of M1-SAT-I(N)-EGFP (
28-55) were 5'-TGCTCGGCAGGCAGGTGCCGCTGC-3' and 5'-ATGAGAAGACCCAGCTTGTTAATAAAAGACATC-3'. The antisense primers were treated with T4 polynucleotide kinase (Takara, Shiga, Japan). PCR was carried out with KOD plus (Toyobo, Osaka, Japan) in the presence of 0.2 mM dNTPs, 1 mM MgSO4, and 0.3 µM primers for 30 cycles. PCR products were ligated using a DNA Ligation Kit Ver.2.1 (Takara). Other deletion mutants were created by similar procedures.
To produce a retrovirus, the pSU213 (ks-M1-SAT-I/pBABE-puro) plasmid was constructed using pSY14. The 1.2-kDa BamHI-EcoRI fragment of pSY14 was cloned into the BamHI-EcoRI site of a pBABE-puro vector (Addgene, Cambridge, MA) to generate pSU213. The pSU214 (ks-M2-SAT-I/pBABE-puro), pSU215 (ks-M3-SAT-I/pBABE-puro), pSU216 (ks-M1-SAT-I M28/56A mutant/pBABE-puro), and pSU217 (ks-M2-SAT-I M29A mutant/pBABE-puro) plasmids were created by similar procedures.
Real-Time PCR
First-strand cDNA synthesis was performed using a First-Strand cDNA Synthesis kit for RT-PCR (AMV; Roche Diagnostics, Penzberg, Germany) and total RNA isolated from mouse tissues or a First Choice Human Total RNA Survey Panel (Applied Bioscience, Foster City, CA). Primers and probes (Taq-man probe) specific for mSAT-Ia (primers, 5'-TGCGAAGCCAAGCAGCG-3' and 5'-AGCAATCACTTCTCAGCTTTGCA-3'; probe, 5'-TGCCGAGCAATGCCAA-3'), mSAT-Ib (Mm00488232-m1), mSAT-I all (primers, 5'-GGCATCCTGCACGGACTAGA-3' and 5'-CGCACCCTCTGGGTAAGTCA-3'; probe CGATGTGGTAATAAGGTTGAACAGTGCGC-3'), hSAT-Ia-1 (primers, 5'-GGCCGAGGCACTGTGAAG-3' and 5'-TGCAGATTCCATGCAAGCA-3'; probe, 5'-AGTGGTGTGCAAAGC-3'), hSAT-Ia-2 (primers, 5'-CCGGCCGAGCAATGC-3' and 5'-GGCCTCGAGCAATCACTTCT-3'; probe, 5'-TGAGTACACCTATGTGAAAC-3'), hSAT-Ib-1 (primers, 5'-CATTTCTTCGTCTAGGCGAGAAA-3' and 5'-TGCAGATTCCATGCAAGCA-3'; probe, 5'-CAGGCACTGTGAAGAC-3'), hSAT-Ib-2 (primers, 5'-CATTTCTTCGTCTAGGCGAGAAA-3' and 5'-AGGCCTCGAGCAATCACTTC; probe, 5'-ACAGCAATGCCAAGTGAG-3'), rodent GAPDH (4308313), and 18S rRNA (Hs99999901-s1) were purchased from Applied Biosystems (Foster City, CA). A 2x PCR universal master mix (Applied Biosystems), containing a PCR buffer, MgCl2, dNTPs, and thermally stable AmpliTaq Gold DNA polymerase, was used in PCR reactions. In addition, the PCR reaction mixture contained forward and reverse primers at 0.9 µM, a 0.25 µM Taq-man probe, and 0.05 µg first-strand cDNA. RNase- and DNase-free water was added to a final volume of 10 µl. The PCR reaction mixture was incubated at 50°C for 2 min and at 95°C for 10 min and then run for 40 cycles at 95°C for 15 s and 60°C for 60 s on an Applied Biosystems 7500 Real-Time PCR System.
Cell Culture
Chinese hamster ovary (CHO) cells and the amphotopic retroviral packaging cell line Phoenixeco (American Type Culture Collection, Manassas, VA) were cultured in the nutrient mixture F-12 HAM (N6558, Sigma, St. Louis. MO) and high-glucose DMEM (Invitrogen), supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin, respectively. CHO cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Stable transfectants were selected in the same medium used for culture in the presence of zeocin (Invitrogen).
Primary Cell Isolation and Immortalization
Two breeding pairs of SAT-I knockout mice were generous gifts from M. Saito (Pharmacodynamics, Meiji Pharmaceutical University, Tokyo, Japan; Tsukamoto et al., 2005). Mouse embryonic fibroblasts (MEFs) were derived and cultured from GM3 synthase knockout (–/–) embryos obtained by mating GM3 synthase (+/–) x GM3 synthase (+/–). Embryos were separated from the maternal tissues and yolk sack and then minced and incubated in 0.1% trypsin/002% EDTA (Sigma) at room temperature for 35 min. The supernatant was centrifuged at 300 x g for 5 min at 4°C, and the precipitations were resuspended in DMEM (D6046, Sigma) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin and seeded onto plastic plates for culture. To establish an immortalized MEF cell line, MEFs were transfected using Lipofectamine 2000 with SV40 Large T antigen/pcDNA3.1Zeo(+) (a gift from Dr. J. Gu, Division of Regulatory Glycobiology, Tohoku Pharmaceutical University, Sendai, Japan). SV40 Large T antigen–transfected cells were selected in the same medium used for culture in the presence of zeocin.
Preparation of Cell Lysates
Cells were washed twice with ice-cold phosphate-buffered saline (PBS), suspended in buffer A (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 4 M urea, 1 mM phenylmethylsulfonyl fluoride, and 1x Complete protease inhibitor mixture [EDTA-free]; Roche Diagnostics), and lysed by sonication. After removal of cell debris by centrifugation at 1000 x g for 3 min at 4°C, the supernatant (total cell lysates) was centrifuged at 100,000 x g for 1 h at 4°C. The precipitates (integral membrane proteins) were suspended in a 2x SDS sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and a trace amount of bromophenol blue) containing 5% 2-mercaptoethanol and incubated for 5 min at 37°C. Endoglycosidase H (Endo H) or Peptide: N-glycosidase F (PNGase F; New England Biolabs, Beverly, MA) digestion was performed on integral membrane proteins at 37°C for 1 h according to the manufacturer's recommended procedure.
Immunoblotting
Immunoblots were performed on integral membrane proteins prepared as described above. Proteins were separated by SDS-PAGE and transferred to an Immobilon polyvinylidene difluoride membrane (Millipore, Billerica, MA). The membrane was then incubated for 1 h with a 1:1000 dilution of anti-SAT-I antibodies that had been raised against GST or polyhistidine fusion proteins encompassing the COOH-terminal 55 aa (C-term; Uemura et al., 2006) or 267 aa (9129). After washing, the membrane was incubated for 1 h with a 1:5000 dilution of horseradish peroxidase–conjugated donkey anti-rabbit IgG F(ab')2 fragment (GE Healthcare Bio-Sciences, Piscataway, NJ). Labeling was detected using an ECL plus kit (GE Healthcare Bio-Sciences).
Detection of Endogenous Mouse SAT-I Isoforms
Mice (SAT-I+/+ and SAT-I–/–) were kept under deep anesthesia and briefly perfused with physiological saline. Brain tissues were isolated, minced, and homogenized in 0.25 M sucrose containing 1x Complete using Teflon homogenizer (10 strokes). After removal of nuclear and cell debris by centrifugation at 1000 x g for 10 min at 4°C, the supernatant was centrifuged at 8000 x g for 10 min at 4°C to remove mitochondria. Finally, the supernatant was centrifuged at 100,000 x g for 1 h at 4°C. The precipitates (microsome) were suspended with immunoprecipitation (IP) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM NaF, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, and 1x Complete) and incubated with protein A/G plus-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 4°C. After centrifugation, the supernatant was incubated with anti-SAT-I antibodies (9129) and protein A/G plus-agarose for 1 h at 4°C. The agarose beads were then washed four times with washing buffer (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, and 0.05% Tween20), and bound proteins were eluted with 2x SDS sample buffer containing 5% 2-mercaptoethanol and incubated for 5 min at 37°C. Immunoprecipitates were treated with PNGase F and analyzed by immunoblotting using anti-SAT-I antibodies (C-term) and Rabbit IgG TrueBlot (eBioscience, San Diego, CA).
Immunofluorescence Microscopy
Transfected cells were cultured on coverglass and then fixed for 15 min with 3.7% formaldehyde in PBS at room temperature. After rinsing with PBS, the cells were permeabilized, if necessary, by 0.5% SDS or 0.5% Triton X-100 in PBS, treated with Image-iT FX Signal Enhancer (Invitrogen) for 30 min, and then incubated for 1 h with anti-SAT-I (C-term), anti-KDEL (StressGen Bioreagents, Victoria, BC, Canada), anti-GM130 (BD Bioscience, Franklin Lakes, NJ), or anti-hemagglutinin (HA) antibodies (InvivoGen, San Diego, CA; diluted 1:100 with blocking solution). After washing three times in PBS, the cells were incubated for 30 min with Alexa 488–conjugated anti-rabbit IgG antibodies (Invitrogen) or Alexa 594–conjugated anti-mouse IgG antibodies (Invitrogen) diluted in blocking solution to 5 µg/ml. Coverslips were washed in PBS three times, mounted on glass slides using ProLong Gold antifade reagent (Invitrogen), and analyzed by fluorescence microscopy FV1000 (Olympus, Tokyo, Japan).
Pulse Chase and IP
CHO cells stably expressing ks-M1-SAT-I, ks-M2-SAT-I, ks-M3-SAT-I, and ks-M1-SAT-I R11/12S were cultured in DMEM Nutrient Mixture F-12 HAM (D8437, Sigma) with or without 200 µM chloroquine. The culture medium was then changed to DMEM without methionine (Met)/cysteine (Cys) (21013–024, Invitrogen) with or without 200 µM chloroquine and incubated at 37°C for 1h. Cells were pulse-labeled with [35S]Met/[35S]Cys (27.5 mCi/ml EXPRESS protein labeling mix; Perkin Elmer Life Science, Waltham, MA) for 30 min and chased with unlabeled Met (final concentration, 0.5 mg/ml) and Cys (final concentration, 0.1 mg/ml) in DMEM containing 10% fetal bovine serum with or without 200 µM chloroquine. At predetermined times, cells were suspended with RIPA buffer (50 mM Tris-HCl, pH 7.2, 500 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5%. sodium deoxycholate, 10 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, and 1x Complete) and kept on ice. Cells were disrupted by bath sonication, and debris was removed by centrifugation at 20,000 x g for 3 min at 4°C. Cell lysates with equal radioactivity were incubated with anti-SAT-I antibodies (C-term) and protein A/G plus-agarose overnight at 4°C. After two washes with RIPA buffer and one with 10 mM Tris-HCl, pH 7.5, agarose beads were suspended in a 2x SDS sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and a trace amount of bromophenol blue) containing 5% 2-mercaptoethanol and incubated for 5 min at 37°C. Precipitates were treated with PNGase F and separated by SDS-PAGE. Radioactivities associated with SAT-I were quantified using a Bio-Imaging Analyzer BAS5000 (Fuji Photo Film, Tokyo, Japan).
Phoenix Retroviral Expression System
At 24 h before transfection, Phoenixeco cells were seeded at 0.5 x 106 cells per 60-mm dish. Phoenixeco cells were transfected with ks-M1-SAT-I, ks-M2-SAT-I, ks-M3-SAT-I, ks-M1-SAT-I M28/56A, and ksM2-SAT-I M29A/pBABE-puro plasmids using Lipofectamine 2000. Approximately 32 h after transfection, the culture temperature was shifted from 37 to 32°C to maintain virus stability. The retrovirus-containing supernatant was harvested 48 and 72 h after transfection and centrifuged at 200 x g for 3 min to remove living cells. The retroviral supernatant was either used immediately for experiments or snap-frozen in liquid nitrogen and stored at –80°C for later applications. For the retroviral transduction of immortalized SAT-I knockout cells, 4 ml of the retroviral supernatant containing 10 µg/ml polybrene (Sigma) was added to 0.25 x 106 cells in a 60-mm dish, and the cells were cultured at 32°C. After 24 h, the culture temperature was shifted from 32 to 37°C, and the cells were incubated for 48 h.
Assay of Enzymatic Activity In Vitro
SAT-I activity was performed as described previously (Uemura et al., 2006).
Lipid Analysis
Cells were washed twice with PBS, and total lipids were extracted from the cells with chloroform/methanol (1/1 and 1/2 vol/vol, successively). Samples were equalized for protein concentration, which was determined using a BCA kit (Pierce Chemical, Rockford, IL). The total lipid extract was dissolved in 6 ml of diisopropyl ether/butanol (3/2) and added to 3 ml of 50 mM NaCl. The mixture was vortexed for 1 min and centrifuged at 700 x g for 5 min. The upper layer was removed, and the ganglioside-containing lower layer was washed with 6 ml diisopropyl ether/butanol (3/2) again. The lower layer was diluted with 5 ml of 50 mM NaCl solution and then applied to a Sep-Pak C18 reverse-phase cartridge (Waters Associates, Milford, MA). The cartridge was washed with 40 ml water, and lipids were eluted with 10 ml methanol and chloroform/methanol (1/1, vol/vol), successively. Gangliosides were separated by thin-layer chromatography (TLC) using chloroform/methanol/0.2% CaCl2 (55/45/10, vol/vol/vol) and detected with an orcinol-sulfuric acid reagent. Standard compounds were purchased from Matreya (Pleasant Gap, PA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), so transcriptional variants other than mSAT-Ia and -Ib may be present. In human tissues, expression levels of hSAT-Ia-1 and -Ia-2 were high in brain, thymus, lung, spleen, testis, skeletal muscle, thyroid, ovary, and cervix (Figure 3B). In contrast, the expression levels of the hSAT-Ib-1 and -Ib-2 variants were very low in all tissues. We could not examine the tissue distribution of the hSAT-Ic variant because of difficulties in the design of specific primers and probes for real-time PCR measurements. The observed tissue distribution of the human SAT-I transcripts was consistent with a previous report of a Northern blot analysis of human SAT-I (Ishii et al., 1998). Moreover, our analyses revealed that in all tissues the hSAT-Ia-2 variant exhibited the highest relative levels. These results suggest that the transcription of human SAT-I is initiated mainly from exon 1 and not from exon 2, and that exon 3 is efficiently spliced. Thus, coexpression of the SAT-I variants that utilize M1 and M2 as their first AUGs was found in both mouse and human tissues (Figures 1 and 3).
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), samples of integral membrane fractions were also treated with PNGase F before immunoblotting. Interestingly, the broad bands in mSAT-Ia–transfected cells shifted to 46, 40, and 36 kDa, whereas the broad band from mSAT-Ib–transfected cells resolved into bands at 40 and 36 kDa (Figure 4B). These results suggest that the band of 46 kDa in mSAT-Ia–transfected cells is a SAT-I protein translated from M1 (M1-SAT-I), and the 40- and 36-kDa bands in both mSAT-Ia– and mSAT-Ib–transfected cells are SAT-I proteins translated from M2 and M3 (M2-SAT-I and M 3-SAT-I), respectively. Accordingly, site-directed mutagenesis of Met (M2 and M3 in mSAT-Ia and -Ib, respectively) to Ala mutants (mSAT-Ia M28/56A and mSAT-Ib M29A) was performed. Integral membrane fractions were prepared from these mutant-transfected cells and subjected to immunoblotting with anti-SAT-I antibodies. As shown in Figure 4C, the bands of 40 and 36 kDa in the mSAT-Ia M28/58A-transfected cells, and the band of 36 kDa in the mSAT-Ib M29A-transfected cells disappeared, indicating that mSAT-Ia and mSAT-Ib produce three and two independently initiated proteins, respectively. This supports the finding from the tissue distribution analysis above (Figure 3A) that M1-SAT-I, M2-SAT-I, and M3-SAT-I isoforms may coexist in mouse cells and tissues.
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Next, to confirm the expression pattern of endogenous SAT-I isoforms, we purified SAT-I proteins from brain tissue of SAT-I+/+ and SAT-I–/– mice by IP using anti-SAT-I antibodies. Because the expression levels of mSAT-Ia and mSAT-Ib were high in brain tissue (Figure 3A), we expected that three SAT-I isoforms would be detected. Three bands equivalent to the three isoforms were detected only in the SAT-I+/+ mice (Figure 4F). The expression level of M3-SAT-I was slightly higher than the other isoforms. These results firmly support the contention that the three SAT-I isoforms endogenously coexist in tissues after leaky scanning from mRNA variants of mSAT-Ia and -Ib.
In general, the first AUG codon is recognized as an initiation codon by the ribosome, a phenomenon known as the first-AUG rule (Kozak, 2002). However, the first AUG codon is sometimes skipped, and a downstream AUG codon is then recognized as an initiation codon; this is known as a leaky scanning system (Kozak, 2002). In mammals, the optimal context for recognition of the AUG start codon is GCCRCCAUGG. Within this motif, the purine nucleotide (R = A or G) in position –3 is the most highly conserved and the most functionally important. The G in position + 4 is also highly conserved and, especially in the absence of A in position –3, makes a strong contribution to recognition. When the first AUG residue is presented in a weak context, especially lacking both R in position –3 and G in position + 4, some ribosomes will initiate at that point, but most continue scanning and then initiate transcription further downstream. Such leaky scanning enables the production of two distinct proteins from one mRNA. Positions –3 and + 4 in mSAT-Ia are occupied by A and C, respectively (Figure 5A, mSAT-Ia WT). To identify the optimal context for recognition of the M1 site in mSAT-Ia, CHO cells were transfected with mutants that replaced the A in position –3 with G, C, or U (Figure 5A, mSAT-Ia m1, m2, and m3, respectively), and immunoblots were performed. In cells carrying mSAT-Ia m2 or m3, the expression of M1-SAT-I decreased and the expression of M2-SAT-I increased, whereas the band pattern for the mSAT-Ia m1 cells was similar to that for mSAT-Ia wild-type (WT) cells (Figure 5B). These results suggest that for initiation, A or G is the optimum residue for position –3 in mSAT-Ia (Kozak, 2002). Next, CHO cells were transfected with mutants that replaced the C in position + 4 with G, A, or U (Figure 5A, mSAT-Ia m4, m5, and m6, respectively) and examined the cells by immunoblotting. Only in cells transfected with mSAT-Ia m4 did the expression levels of M2-SAT-I and M3-SAT-I decrease slightly, indicating that G at position + 4 is optimum for efficient initiation (Figure 5B, top panel). Nevertheless, leaky scanning of the mSAT-Ia m4 mutant, which would be expected to represent the optimal condition, was not completely eliminated. We next created a mutant (Figure 5A, mSAT-Ia m7) in which the nucleotide sequence upstream of M1 was replaced with GCCACC (ks), and expressed it in CHO cells. In immunoblots of cells carrying mSAT-Ia m7, the M2-SAT-I band disappeared, and the amount of M3-SAT-I decreased slightly (Figure 5B, bottom panel). In addition, the band pattern of the mSAT-Ia m8 mutant, in which the C in position +4 of mSAT-Ia m7 was replaced with G, was similar to that of the mSAT-Ia m7 mutant. These results suggest that the nucleotide at position +4 is not restricted if the nucleotide sequence before M1 is GCCACC. On the other hand, the expression level of M3-SAT-I also decreased remarkably in cells carrying the mSAT-Ib m1 in which the nucleotide sequence from position –6 to –1 at the M2 site was switched for GCCACC (Figure 5C). These results suggest that the entire sequence from position –6 to –1 is important for recognition of the initiation codon in mSAT-I variants. Subsequently, to investigate posttranslational regulation of each SAT-I isoform, we used constructs in which GCCACC was inserted before M1, M2, or M3 to minimize leaky scanning.
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), but they partially colocalized with GM130, a cis-Golgi marker protein (Figure 6, D and E). Thus, M1-SAT-I is localized in the ER, whereas M2- and M3-SAT-I exhibit a distinct localization, mainly in the trans-Golgi.
Selective Retrograde Transport of M1-SAT-I from the Golgi Apparatus to the ER
To clarify whether the cytoplasmic tails of the SAT-I proteins influenced their subcellular localization, we stably expressed in CHO cells a fusion protein for the NH2-terminus of M1-, M2-, or M3-SAT-I containing the transmembrane region and EGFP (M1-SAT-I(N)-EGFP, M2-SAT-I(N)-EGFP, and M3-SAT-I(N)-EGFP; Figure 7, A and B). M1-SAT-I(N)-EGFP was localized in the ER, whereas M2-/M3-SAT-I(N)-EGFP was localized in the Golgi apparatus (Figure 7B), indicating that the cytoplasmic tail of each of the SAT-I isoforms defines its subcellular localization.
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12–26 and 12–21 deletion mutants localized in the Golgi apparatus, although other mutants remained in the ER, suggesting the presence of ER-targeting signals in the amino acid residues between 12 and 21 (Figure 7D).
As shown in Figure 8A, the NH2-terminal amino acid sequence of SAT-I is highly conserved between mice and humans. Multiple arginine residues, especially Arg-11, Arg-12, and Arg-17, are conserved in both species, corresponding to the region essential for ER targeting. R-based motifs located on the cytoplasmic side of a transmembrane protein reportedly function as retrograde transport signals (Michelsen et al., 2005). We generated R11S, R12S, and R11/12S M1-SAT-I(N)-EGFP mutants by site-directed mutagenesis and examined their subcellular localizations. R11/12S double-substitution mutant resulted in a shift of localization from the ER to the Golgi apparatus, although R11S and R12S single mutants were localized in the ER (Figure 8B). The RR motif is well known as an R-based motif (Schutze et al., 1994), yet if only the RR motif functioned as an ER-targeting signal in M1-SAT-I, then the R11S and R12S single mutants would shift from the ER to the Golgi apparatus (Schutze et al., 1994). However, as demonstrated in Figure 8B, these single mutants remained in the ER. We also tried to determine the role of Arg-17 in the ER targeting of M1-SAT-I, by generating and expressing R17S, R11/17S, or R12/17S M1-SAT-I(N)-EGFP mutants. As expected, R11/17S and R12/17S mutants shifted their localization to the Golgi apparatus, whereas the R17S mutant stayed in the ER (Figure 8B). These results indicate that not only the RR motif but also RXXXXR and RXXXXXR motifs function as ER-targeting signals of M1-SAT-I.
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). To confirm this observation, we created various deletion mutants of the M1-SAT-I(N)-EGFP WT sequence and examined their subcellular localization (Table 1). Only the 28–55 mutant (distance: 25 aa) changed its localization from the ER to the Golgi apparatus. This result indicates that the RRXXXXR motif requires a distance of at least 30 aa from the membrane. Here, we use the term "functional distance" to describe the space essential for R-based motifs to function as a retrograde transport signal. We next examined the localization of the single deletion mutants M1-SAT-I(N)-EGFP R11S, R12S, and R17S to determine whether the functional distances among RR, RXXXXR, and RXXXXXR were different. As summarized in Table 1, the functional distances of M1-SAT-I(N)-EGFP R11S (SRXXXXR), R12S (RSXXXXR), and R17S (RRXXXXS) were 40, 45, and 25 aa, respectively. These results indicate that the functional distances of the RXXXXR and RXXXXXR motifs are longer than that of the RR motif. In addition, the functional distance of the R17S (RR motif) single mutant is similar to that of WT M1-SAT-I. Thus, the sequences of the R-based motifs greatly affect the functional distance.
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). Accordingly, we examined whether M2-SAT-I and the M1-SAT-I R11/12S mutants were also sorted to lysosomes for degradation. SAT-I–expressing cells were treated with or without 0.2 mM chloroquine, a lysosomal protein degradation inhibitor, for 24 h, and then the cells were labeled with [35S]Met/Cys for 30 min and incubated for 3 h with excessive amounts of unlabeled Met and Cys. SAT-I proteins were immunoprecipitated from cell lysates using anti-SAT-I antibodies. Before analysis by SDS-PAGE, immunoprecipitated proteins were treated with PNGase F to remove N-glycans. As expected, the disappearance of M2-SAT-I and its M1-SAT-I R11/12S mutant was inhibited by treatment with chloroquine (Figure 10B). These results suggest that M2-SAT-I is sorted to lysosomes for degradation, whereas M1-SAT-I and M3-SAT-I escape this degradation pathway.
Reduction of the In Vivo GM3 Synthesis of M1-SAT-I by ER Targeting
To clarify whether differences in intracellular dynamics among SAT-I isoforms affect GM3 synthesis activity, we examined their activities in vitro and in vivo. The introduction of ks-M1-SAT-I or ks-M2-SAT-I into CHO cells led to the production of M3-SAT-I by leaky scanning (Figure 6B). To completely suppress leaky scanning, we constructed ks-M1-SAT-I M28/56A and ks-M2-SAT-I M29A mutants. The subcellular localization and stability of these mutants were similar to those of the WT sequences (data not shown). To compare the activities of SAT-I isoforms and their mutants, we determined the activities in vitro using lysates from CHO-K1 cells transiently expressing the protein or a mutant as enzyme sources. The activity of ks-M3-SAT-I was about three fold higher than that of either ks-M1-SAT-I or ks-M2-SAT-I (Figure 11A). The activity of the ks-M1-SAT-I M28/56A mutant was similar to that of ks-M1-SAT-I, whereas the activity of the ks-M2-SAT-I M29 mutant was decreased, to 
50%, relative to the activity of ks-M2-SAT-I (Figure 11A).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), the RXXXXR and RXXXXXR sequences identified in this study are clearly new consensus sequences. ER targeting restricts M1-SAT-I transport to the Golgi apparatus, leading to the suppression of its GM3 synthesis activities in vivo. On the other hand, although both M2-SAT-I and M3-SAT-I were found to be localized in the Golgi apparatus, we observed distinct differences in their protein stabilities. Further analysis revealed that M2-SAT-I is sorted to lysosomes via the Golgi for degradation, whereas M3-SAT-I remains relatively stable in the Golgi apparatus. Observations regarding the tissue distributions of mouse and human mRNA variants also determined that SAT-I isoforms with different properties coexist in most tissues. Thus, we speculate that changes in the relative composition and/or intracellular trafficking of these isoforms are important for the regulation of GM3 synthesis.
There are many reports indicating that a single mRNA can produce several independently initiated proteins via leaky scanning (Kozak, 2002). This phenomenon often results from a suboptimal context of the first AUGstart codon. There are, however, rare instances of leaky scanning occurring despite the first AUG being presented in a strong context (R–3 and G+4; Kozak, 2002). Such a result can occur when the first AUG codon is too close to the 5'-end to be efficiently recognized. Most leaky scanning of mouse SAT-I can be explained by a suboptimal context. However, the production of M3-SAT-I from mSAT-Ia and mSAT-Ib was not completely abolished when the first AUG was replaced with an optimal sequence by site-directed mutagenesis. Because mSAT-Ia and mSAT-Ib were inserted into an expression vector for mammalian cells [pcDNA3.1 Zeo(+)], the possibility that the first AUG is too close to the 5'-end to be recognized as the initiation codon could not be confirmed. Thus, M3-SAT-I may be produced by context-independent leaky scanning, although the mechanism remains unclear.
R-based motifs function as retrograde transport signals when positioned distally from a lipid bilayer. Several reports suggest that the minimum distance would be between 16 and 46 Å (Shikano and Li, 2003). Our results also indicate that a distance of between 24 and 47 aa from the lipid bilayer is necessary for the functioning of R-based motifs, a distance we refer to as the "functional distance." For example, the ER-exporting signal R/K(X)R/K, located just above the transmembrane domain in many glycosyltransferases, cannot function as a retrograde transport signal (Giraudo and Maccioni, 2003a). R-based motifs need to interact with COP I subunits, β-COP and 
-COP, to function as a signaling sequence, and binding affinities to β-COP and -COP depend on the sequences of R-based motifs (Michelsen et al., 2007). We also confirmed that M1-SAT-I WT, but not R11/12S mutant, interacts with β-COP and/or -COP (data not shown). Thus, we speculate that a functional distance is essential for R-based motifs to interact with β-COP and -COP. On the basis of this idea, we also assume that for a variety of R-based motifs, the distinct binding affinities to β-COP and -COP influence the functional distance. In other words, the functional distance is longer if the R-based motif has weak binding affinity. As shown in Table 1, RXXXXR and RXXXXXR motifs require a long range to function, relative to the RR motif. These results suggest that the binding affinities of RXXXXR and RXXXXXR motifs to β-COP and -COP are weaker than that of the RR motif. In conclusion, we propose that the functional distance should be the criterion that defines the affinity of R-based motifs to β-COP and -COP.
M2-SAT-I and the M1-SAT-I R11/12S mutant are both sorted to lysosomes via the Golgi and rapidly degraded (Figure 10A). M3-SAT-I is more stable than M2-SAT-I or the M1-SAT-I R11/12S mutant, indicating that the N-terminus of M2-SAT-I (1-28 aa) may be involved in trafficking to the lysosomes. Many membrane proteins are known to be sorted to the lysosomes when carrying multiple monoubiquitins or short polyubiquitin chains (Pelham, 2004; Piper and Luzio, 2007). It might be possible that the lysine residues present in the M2-SAT-I confer the lower stability to this protein when compared with M3-SAT-I. In contrast, M3-SAT-I remains stable in the trans-Golgi by escaping the degradation pathway (Figure 10A). Based on the Golgi maturation model (Losev et al., 2006; Matsuura-Tokita et al., 2006), this result suggests that M3-SAT-I undergoes retrograde transport between the Golgi cisternae. Most N-glycans on M3-SAT-I are of the Endo H–resistant, mature type, whereas most N-glycans on M2-SAT-I are of the Endo H–sensitive, high-mannose type. This suggests further that the retrograde transport cycle between the Golgi cisternae is involved in the maturation of N-glycans on SAT-I proteins.
Most M1-SAT-I is targeted to the ER by R-based motifs, but a small population of M1-SAT-I is sorted to the Golgi apparatus, which participates in GM3 synthesis (Figure 11D). To escape ER targeting, it would be necessary for the R-based motif containing M1-SAT-I to be masked. Such masking has been proposed as a mechanism utilized by ATP-binding cassette proteins. ATP-sensitive K+ channels are octameric complexes that consist of four pore-forming, inwardly rectifying subunits (Kir6.1/2) and four sulfonylurea-binding β subunits (SUR1/2A/2B) belonging to the ATP-binding cassette family. Each subunit contains an R-based motif, and unassembled subunits or incompletely assembled complexes stay in the ER. During the process of heteromultimeric assembly, R-based motifs are masked, which permits transport of the complex to the cell surface (Zerangue et al., 1999). The R-based motif in Kir6.2 is sterically masked by the SUR1 subunit. In contrast, the R-based motif in SUR1 can be inactivated by the recruitment of 14-3-3 (Heusser et al., 2006). For SAT-I, proteins involved in masking R-based motifs have not been identified. However, the affinity of the NH2-terminus of M1-SAT-I for masking proteins is expected to be lower than that for β- and/or -COP. Consequently, M1-SAT-I is predominantly remains in the ER and then sorted slowly to the Golgi. The significance of the slow sorting of M1-SAT-I must be a subject for future research. Currently, we assume that there are two possibilities, either M1-SAT-I functions as a means to stably supply GM3, or it has a distinct function in the ER. In regards to the latter function, Protein O-fucosyltransferase 1 (OFUT1), an enzyme that glycosylates the epidermal growth factor-like domains of Notch, is localized in the ER by the C-terminal KDEL-like sequence and displays a Notch chaperone activity (Okajima et al., 2005).
In the presented study, we were able to demonstrate distinct intracellular dynamics for several SAT-I isoforms and possible regulatory mechanisms for in vivo GM3 synthesis (Figure 12). Further work will be required to confirm whether various pathological conditions alter the composition and/or intracellular trafficking of endogenous SAT-I isoforms, and thereby affect GM3 synthesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Jin-ichi Inokuchi (jin{at}tohoku-pharm.ac.jp).
Abbreviations used: aa, amino acid residues; CHO, Chinese hamster ovary; Cys, cysteine; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; Endo H, endoglycosidase H; ks, kozak sequence; LacCer, lactosylceramide; Met, methionine; PNGase F, peptide: N-glycosidase F; 5'-UTR, 5'-untranslated region.
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