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A role for glycosphingolipids in protein sorting
Sprong, H.
Publication date
2001
Link to publication
Citation for published version (APA):
Sprong, H. (2001). A role for glycosphingolipids in protein sorting.
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Chapterr 3
UDP-Galactose:ceramidee galactosyltransferase is a class I integral
membranee protein of the endoplasmic reticulum
Heinn Sprong, Boudewijn Kruithof, Richtje Leijendekker, Jan Willem Slot, Gerrit van Meer, andd Peter van der Sluijs
Summary y
UDP-galactosexeramidee galactosyltransferase (GalT-1) transfers UDP-galactose to ceramide too form the glycosphingolipid galactosylceramide. Galactosylceramide is the major constituent off myelin and is also highly enriched in many epithelial cells, where it is thought to play an importantt role in lipid and protein sorting. Although the biochemical pathways of glycosphingolipidd biosynthesis are relatively well understood, the localization of the enzymes involvedd in these processes has remained controversial. We here have raised antibodies against GalT-11 and shown by immunocytochemistry on ultrathin cryosections that the enzyme is localizedd to the endoplasmic reticulum and nuclear envelope but not to the Golgi apparatus or thee plasma membrane. In pulse-chase experiments, we have observed that newly synthesized GalT-11 remains sensitive to endoglycosidase H, confirming the results of the morphological localizationn experiments. In protease protection assays, we show that the largest part of the protein,, including the amino terminus, is oriented toward the lumen of the endoplasmic reticulum.. GalT-1 enzyme activity required import of UDP-galactose into the lumen of the endoplasmicc reticulum by a UDP-galactose translocator that is present in the Golgi apparatus off CHO cells but absent in CH01ec8 cells. Finally, we show that GalT-1 activity previously observedd in Golgi membrane fractions in vitro, in the absence of UDP-glucose, is caused by UDP-glucose:ceramidee glucosyltransferase. Therefore all galactosylceramide synthesis occurs byy GalT-1 in vivo in the lumen of the endoplasmic reticulum.
Introduction n
Glycosphingolipidss are enriched in the outer membrane leaflet of the plasma membrane of mostt eukaryotic cells, where they play a structural role in rigidifying and protecting the cell surface.. A remarkable property of glycosphingolipids is found in the myelin sheath of Schwannn cells where galactosylceramide (GalCer) and sulfatide are involved in axonal insulation,, myelin function, and stability (111, 112) (192). The enormous diversity in glycosidicc structure of glycosphingolipids suggests specific roles of individual glycosphingolipidss in cell physiology. Glycosphingolipids are involved in a variety of cellular processess including differentiation, cell-cell interaction, transmembrane signaling (193, 194), andd internalization of bacterial toxins (195) and viruses (196). Furthermore, glycosphingolipids aree thought to play a key role in the sorting of lipids and proteins to the apical plasma membranee domain of polarized epithelial cells (18, 116).
Althoughh the biochemical pathways of glycosphingolipid synthesis are relatively well established,, the intracellular localization and the topology of the enzymes involved in these pathwayss are incompletely understood. Until recently, these questions have largely been addressedd by measuring the activity of these enzymes in isolated subcellular fractions (122, 197-199).. The usefulness of such approaches, however, is limited, because removal of contaminatingg membranes has the caveat of selecting a subtraction of the membrane of interest.. Alternatively, enzymes associated with a given intracellular compartment may dissociatee from accessory factors, resulting in diminished activity or specificity. As a consequence,, the localization of some of the glycosphingolipid-synthesizing enzymes is not clear.. One of these enzymes is the UDP-galactose:ceramide galactosyltransferase. It catalyzes thee transfer of galactose from UDP-galactose (UDP-Gal) to ceramide, yielding GalCer (118). GalT-11 was recently cloned (125, 132, 133), and knockout studies have shown that there is onlyy one GalCer-synthesizing enzyme in the brain (111, 112). GalT-1 contains a carboxy-terminall KKVK sequence that may act as an endoplasmic reticulum (ER) retrieval signal, and thee lack of complex glycosylated oligosaccharide chains is consistent with an ER localization (132).. Still, a vast body of controversial results concerning the intracellular localization of GalT-11 has been reported. Biochemical enzyme assays on semipurified membranes and immunocytochemistryy suggest that GalCer synthesis occurs in the Golgi complex and ER (120, 122,, 123, 127, 200) and plasma membrane (128, 201-204).
Ass part of our ongoing efforts to define the molecular mechanism of glycosphingolipid-mediatedd intracellular protein and lipid sorting, we here investigated the cellular location of GalT-11 and its membrane topology. We raised antibodies against GalT-1 and show that it is a classs I integral membrane protein that is localized to the ER but not to the Golgi complex or thee plasma membrane. Importantly, we found that CGlcT (205) in addition to GlcCer also synthesizess GalCer from a short chain ceramide in vitro when assayed in the presence of UDP-Gal,, without UDP-glucose (UDP-Glc). This explains many of the ambiguities previously observedd forGalT-1 localization.
Results s
CharacterizationCharacterization ofGalT-1 antibodies
Too avoid difficulties associated with the use of enzyme assays on partially purified cell fractionss for localizing GalT-1, we first raised antibodies against the protein in rabbits. For this purpose,, we expressed four regions of GalT-1 as polyhistidine-tagged fusion proteins in
EscherichiaEscherichia coli. Three fusion proteins contained parts of the predicted luminal domain and
onee the putative cytoplasmic domain of the protein (Figure IA). Antisera were first tested for theirr ability to immunoprecipitate GalT-1 that was synthesized in an in vitro transcription
translationn system in the presence of microsomes. As shown in Figure IB, three of the four antiseraa recognized GalT-1 expression products, whereas the corresponding preimmune sera didd not. The antibodies detected at least three bands with different mobility in SDS-polyacrylamidee gels. The two lowest bands probably represent immature forms of the protein causedd by incomplete N-glycosylation or degradation products. The band with a molecular masss of approximately 54 kDa most likely is mature GalT-1, since it has the same molecular weightt as the major band we observed on Western blots of whole cell lysates and in pulse-chasee experiments (see below).
A A
300 0 aminoo acidBB
635 636 637 638
200--
976 9
--
46--PP I P 46--PP I P I
FigureFigure 1: Generation of antisera againstagainst GalT-1
A:A: Hydrophilicity profile of the
translatedtranslated cDNA sequence of GalT-1 generatedgenerated by the method of Kyte and DoolittleDoolittle with a seven-residue moving window.window. Horizontal bars below the hydrophilicityhydrophilicity plot denote parts of GalT-1GalT-1 that were expressed as
His-taggedtagged fusion proteins and used for antibodyantibody production in rabbits. B:B: Tran[35S]'labeled GalT-1 was producedproduced from GalT-lpcDNA3 in a coupledcoupled in vitro transcription-translationtranslation system in the presence of dogdog pancreas microsomes. The expression products were immunoprecipitated with the antisera 635-638635-638 (I), or the corresponding preimmune sera (P) and separated on a 10% SDS-polyacrylamidepolyacrylamide gel.
j ^ j j
ExpressionExpression ofGalT-1 in transfected and wild-type cells
Thee novel antibodies allowed us to characterize the molecular features of GalT-1 in more detail.. To obtain cells with high expression levels of GalT-1, we generated stable cell lines by transfectingg CHO (123) and CH01ec8 cells with the GalT-lpcDNA3. CHO cells were selected ass recipients because they do not express endogenous GalCer (206) and therefore represented ann ideal background for our studies. Both CHO and CH01ec8 cells transfected with GalT-1 (GalT-1-CHOO and GalT-l-CH01ec8) expressed a protein with a molecular mass of
otherr antisera (636 and 638) that reacted with GalT-1 by immunoprecipitation (Figure IB) also efficientlyy recognized the 54-kDa protein (not shown). In the nontransfected control CHO and CH01ec88 cell lines, this band was not detectable. Other studies describe an apparent molecular masss of 50-70 kDa (111, 131, 132), whereas the molecular mass of the conceptually translated GalT-11 cDNAis60kDa. // B
69-- ~ ~ ~ - 6 9
2°°-
97--mmmm
6 9.
oo o 00 0 CD Dc/ /
CO O co o CD D O O T — — LU U CD D LD D CO O CD DCf* *
O O T — — LU U CD Dt t
un n co o CD Dc/ /
o o T — — LU U CD D CO O CO O CD D CD D CO O CD D 4 6 -- - 4 646-FigureFigure 2: Expression ofGalT-1
A:A: Postnuclear supernatants of D6P2T and GalT-1-transfected CHO and CHOlec8 cells were
resolvedresolved on 10% SDS-polyacrylamide gels followed by Western blot detection with antiserum 635.635. A prominent band in the GalT-1-transfected cells comigrated with the 54-kDa band seen inin the rat oligodendrocyte D6P2T cell line B: HeLa cells were transfected with GalT-lpcDNA3 oror GalT-lmycpcDNA3 or mock-transfected with recombinant vaccinia virus. Five h after
transfection,transfection, the cells were metabolically labeled with Tran[35S]label, and GalT-1 was
immunoprecipitatedimmunoprecipitated with antibodies 635, 636, and 638 against GalT-1 or the 9E10 antibody againstagainst the Myc epitope tag (B). The position of molecular weight standards is indicated on the
left.left. Arrows point to mature GalT-1.
Too rule out the possibility that the 54-kDa band was due to some peculiarity of CHO cells or ourr antibodies, we analyzed the presence of endogenous GalT-1 in D6P2T cells, and we transfectedd Myc-tagged GalT-1 in a human cell line. The rat oligodendrocyte D6P2T cell line (207)) contains high levels of GalCer; accordingly, we expected appreciable expression levels off GalT-1. As shown in Figure 2A, the 54-kDa band that was seen in the CHO transfectants expressingg rat GalT-1 also represented the major form of GalT-1 in this nontransfected oligodendrocytee cell line. From quantitative Western blots, we estimated that initially the
expressionexpression in GalT-1-CHO and GalT-l-CH01ec8 cells was 4 and 13 times the endogenous GalT-11 in the D6P2T cells, respectively. As a second control experiment, we transiently
expressedd GalT-1 and the epitope-tagged version GalT-1 myc in HeLa cells using the recombinantt vaccinia T7 RNA polymerase system. Transfected cells were metabolically labeledd for 45 min, and GalT-1 was immunoprecipitated with the 9E10 antibody against the epitopee tag or antibodies against GalT-1. As shown in Figure 2B, the 9E10 antibody immunoprecipitated,, although less efficiently, the same bands as the rabbit antibodies that were raisedd against different regions of GalT-1. Upon comparing the bands immunoprecipitated fromm the GalT-1 and GalT-1 myc lysates with the rabbit antibodies, it is clear that insertion of thee Myc sequence altered the immunoprecipitation efficiency of some of the forms. The Myc
sequencee was inserted in the region to which antibody 635 was raised, possibly causing conformationall changes in the putative luminal domain that may affect the epitopes seen by the rabbitt antibodies. We therefore concluded that the apparent molecular mass of GalT-1 was -54 kDaa in our gel system. When the same samples were run on 12.5% (instead of 10%) SDS-polyacrylamidee gels, the immunoprecipitated GalT-1 bands shifted to 62 kDa (not shown).
BiosynthesisBiosynthesis and processing ofGalT-1
Too investigate the intracellular fate of GalT-1, GalT-1-CHO cells were pulse-labeled for 5 min withh Tran[ 5S]labeled amino acids and then chased for different periods of time. Besides some backgroundd bands (Figure 3), the same 54-kDa band was immunoprecipitated by antibody 635 ass detected on the Western blots. The antisera 636 and 638 also immunoprecipitated this band (nott shown), and we therefore interpreted it as GalT-1. A small but significant shift to a higher mobilityy form of GalT-1 occurred in the first hour after the pulse. Since this shift did not occur afterr Endo H treatment (see below), it represented processing of N-linked oligosaccharides of GalT-1.. Quantitative analysis of the 54-kDa band during the chase period showed that the half-lifee of GalT-1 was about 4 h. We next assessed the rate of GalT-1 degradation under steady statee conditions in GalT-1-CHO cells in the presence of 10 mM cycloheximide to inhibit proteinn synthesis. From the analysis of GalT-1 levels by quantitative Western blot (not shown), wee calculated a very similar half-life, confirming the results of the pulse-chase analysis.
Pulsee (min) 5 5 5 5 5 5 30
Chasee (min) 0 5 30 60 120 240 960
Endoo H — + — +~ — +" — +~ ~ +" ~ +" "Z +"
97--
— —
-97
6 9 --
— —
- 6 9
mm^mm^ i^M» ^M> UPP* ^ ^ ^ ^ 4 6 -- mm mm - 4 6FigureFigure 3: Transport and processing ofGalT-1
GalT-1-CHOGalT-1-CHO cells were pulse-labeled for 5 min with Tran[35S]labeled amino acids and then chasedchased for different time intervals up to 4 h. After cell lysis, GalT-1 was immunoprecipitated withwith antiserum 635, and immunoprecipitates were treated with Endo H as described. Proteins werewere resolved under reducing conditions by SDS-PAGE on a 7.5% gel.
Thee intracellular transport of GalT-1 was further analyzed by assaying the acquisition of resistancee to Endo H cleavage of its N-linked oligosaccharide side chains. It has already been shownn by lectin blotting that the protein is a high mannose glycoprotein, although not all of the predictedd N-glycosylation sites appear to be used (132). N-Glycosylated proteins whose transportt is arrested in the ER or resident ER proteins containing high mannose sugars are susceptiblee to Endo H digestion, whereas glycoproteins that are transported beyond the medial Golgii generally possess N-glycans that are resistant to Endo H (208). It is shown in Figure 3 thatt GalT-1 remained Endo H-sensitive even with chase times up to 4 h. To exclude the possibilityy that very slow transport through the early biosynthetic pathway retarded the majorityy of newly synthesized GalT-1 in an Endo H-sensitive form, we also addressed its Endo HH sensitivity after an overnight chase. For this purpose, we labeled GalT-1-CHO cells for 30
minn and chased the protein for a period of 16 h. As shown in Figure 3, GalT-1 remained Endo H-sensitive,, suggesting that it is not transported through the Golgi complex.
ConfocalConfocal immunofluorescence microscopy
Thee finding that GalT-1 remained Endo H-sensitive suggested that the protein is not transportedd beyond the ER and possibly the cis-Golgi. The translated GalT-1 cDNA sequence containss a carboxyl-terminal KKVK sequence that may act as an ER retrieval signal, which alsoo suggests that GalT-1 is located in compartments of the early biosynthetic pathway. To extendd this observation to the morphological level, we next performed double label confocal immunofluorescencee microscopy on the GalT-1-transfected CHO cells using antibodies against GalT-1,, PDI (a resident protein of the ER lumen (209) and intermediate compartment), and a Golgii marker, CTR433 (210). As shown in Figure 4, the localization of GalT-1 (A, green) nearlyy completely overlapped with the diffuse reticular ER and nuclear envelope distribution of PDII (B, red). The Golgi complex was not labeled with the GalT-1 antibodies in these cells becausee the staining patterns of GalT-1 (C, red) and CTR 433 (D, green) were mutually exclusive.. To ascertain that the labeling of GalT-1 in the GalT-1-CHO cells was specific, we labeledd nontransfected control CHO cells lacking endogenous GalT-1 with GalT-1 and PDI antibodies.. As can be seen in Figure 4E (green channel), labeling with the GalT-1 antibody yieldedd a very faint signal in the CHO cells, while the staining pattern with the PDI antibody (F,, red) was identical in the GalT-1-CHO and CHO cells. In the oligodendrocytic D6P2T cell linee having high levels of endogenous GalT-1, we also found extensive colocalization of endogenouss GalT-1 (G, green) and PDI (H, red). Importantly, GalT-1 labeling was not present onn the plasma membrane and Golgi apparatus in the D6P2T cell line, suggesting that GalT-1 doess not localize to these compartments and is restricted to the ER.
UltrastructuralUltrastructural localization ofGalT-1
Althoughh the confocal immunofluorescence experiments suggested extensive overlapping distributionss of GalT-1 and PDI, this technique does not have the required resolution to unambiguouslyy demonstrate that GalT-1 colocalized with the ER marker. We therefore performedd immunogold electron microscopy with the GalT-1 antibody on ultrathin crysosectionss prepared from GalT-1-CHO cells. As shown in Figure 5A, the GalT-1 antibody heavilyy decorated the intracellular membranes in the perinuclear area. Most of the labeling occurredd on ER cisternae and the nuclear envelope. In accordance with the confocal immunofluorescencee experiments, we did not observe labeling of the Golgi apparatus (Figure 5B)) and the plasma membrane. Double label experiments also revealed extensive colocalizationn of 1 with PDI (Figure 5C). Occasionally we observed colabeling of GalT-11 and PDI in tubulovesicular structures at the cis-face of the Golgi complex. We next quantitativelyy addressed the distribution of GalT-1 on these crysosections, the results of which aree shown in Figure 5D. About 70% of the gold label was associated with the ER, and 23% wass associated with the nuclear envelope, whereas the labeling of plasma membrane and Golgi apparatuss was essentially negligible. These results confirm and extend the data from the light microscopyy experiments in which we showed that GalT-1 does not move beyond the ER. We alsoo performed immunolabeling of cryosections from the D6P2T cells; however, expression of GalT-11 was too low to discriminate specific labeling from background (not shown).
FigureFigure 4: Confocal
immuno-fluorescencefluorescence microscopy of GalT-1 GalT-1
GalT-1-CHOGalT-1-CHO cells (A-D), nontransfectednontransfected CHO control cells (E,F),(E,F), and D6P2T cells (G,H) werewere labeled with rabbit anti GalT-1GalT-1 antibody (A, D, E, and G) andand mouse anti PDI (B, F, and H)H) or mouse anti-Golgi (C) antibodyantibody and counterstained with
5-5- ([4,6-dichlorotriazin-2-yl] amino)amino) fluorescein-labeled goat anti-rabbitanti-rabbit (A, D, E, G) and
indocarbocyanine-labeledindocarbocyanine-labeled goat anti-mouseanti-mouse antibodies (B, C, F,
andand H). Bar, 10 ptm. Colored versionversion of this figure is published inin Journal of Biological ChemistryChemistry (1998) 273, 25880-88. MembraneMembrane topology ofGalT-1
Havingg localized GalT-1 to the ER,, we next determined the membranee topology of GalT-1 in thiss compartment. GalT-1-CHO cellss were metabolically labeled withh Tran[35S]label for 10 min andd subsequently incubated for 5 minn in chase medium to allow completionn of nascent chains. Postnuclearr supernatants were incubatedd for 1 h with proteases inn the presence or absence of detergent.. GalT-1 was then immunoprecipitatedd with the 635 antibody.. In the absence of saponin,, treatment with proteinasee K or trypsin resulted inn a truncated protein. The results off this experiment are shown in Figuree 6A, from which we estimatedd that protease treatment resultedd in removal of a 4-kDa fragmentt from GalT-1. Because the GalT-1 antibody was raised against the amino-terminal portionn of the protein (Figure 1 A), we concluded that this part of GalT-1 must be present in the lumenn of sealed membranes in this experiment. When protease digestion was done in the presencee of saponin to permeabilize the membranes, GalT-1 was completely degraded (Figure 6A),, showing that it is intrinsically susceptible to trypsin and proteinase K.
CGlcTCGlcT is a dual specificity enzyme in vitro
InIn vitro substrate specificity assays have shown that GalT-1 has a marked preference for
2-hydroxyy fatty acid ceramide when compared with non-hydroxy fatty acid ceramide (118, 120, 201).. In vivo GalT-1, however, galactosylates both nonhydroxy fatty acid and 2-hydroxy fatty acidd ceramides, depending on their local availability (123). Interestingly, although CHO cells doo not produce GalCer, membranes from these cells in an in vitro assay converted the well characterizedd short chain fluorescent model substrate NBD-Cer to NBD-GalCer. In contrast to thee ER GalT-1, the GalT-1 activity fractionated at the density of Golgi membranes of CHO and Madin-Darbyy canine kidney cells was inhibited by UDP-Glc, l-phenyl-2-decanoylamino-3-morpholino-1-propanoll and was protease-sensitive (122, 123). Because the Golgi-associated GalT-11 activity shared several characteristics with CGlcT, we suspected that CGlcT might in factt be responsible for NBD-GalCer synthesis in vitro. To address this question, postnuclear supernatantss prepared from the CGlcT-negative GM95 cell line (205) were incubated with NBD-Cerr and UDP-Gal, and glycolipids produced in this assay were analyzed. As shown in Tablee I, GM95 cells did not synthesize NBD-GlcCer as expected. Interestingly, they also did nott produce the NBD-GalCer product. In contrast, in the MEB4 cell line from which the GM95 celll line was derived, we observed appreciable NBD-GalCer synthesis in the presence of UDP-Gal.. To show that the GlcCer-deficient phenotype of GM95 cells is due to the absence of only CGlcT,, we transfected GM95 cells with CGlcT and assayed synthesis of NBD-GlcCer and GalCerr in postnuclear supernatants prepared from these cells. In addition to NBD-GlcCer,, we also found considerable synthesis of NBD-GalCer in the presence of UDP-Gal (Tablee I). These results suggested that CGlcT could act as a dual specificity enzyme in vitro. Thiss idea was tested by directly comparing the amount of synthesized products when equimolarr UDP-Gal and UDP-Glc were added to postnuclear supernatants of MEB4 cells. As shownn in Table I, GlcCer as well as GalCer were produced in this experiment, but GalCer synthesiss was dramatically reduced in the presence of UDP-Glc, whereas GlcCer synthesis was increased. .
FigureFigure 5: Ultrastructural localization ofGalT-1
UltrathinUltrathin cryosections of GalT-1-CHOlec8 cells were incubated with antibody against GalT-1 andand PDI and labeled with 10-nm protein A gold. Top: Most of the label on these sections was associatedassociated with the ER (arrowhead) and nuclear envelope. Middle: Please note the absence of GalT-1GalT-1 labeling of the Golgi apparatus (G), nucleus and mitochondria (M). Bottom: GalT-1 colocalizedcolocalized with PDI to the ER. In this experiment, PDI and GalT-J detection was done with 5
andand 15 nm protein A gold,
respectively.respectively. Occasionally, colocalizationcolocalization of GalT-1 and PDIPDI is seen in tubulovesicular
structuresstructures atthe cis-side of the GolgiGolgi complex (asterisk). D: TheThe distribution of GalT-1
immunoreactivityimmunoreactivity in GalT-1-CHOCHO cells was quantitated fromfrom sections of 10 individual
cellscells (1500 gold particles counted)counted) and corrected for nonspecificnonspecific labeling (300 gold particlesparticles counted) on sections preparedprepared from nontransfected
%%
-N: :
»-fe^ai&afeafe; ; ; . &&-M -M
V V
IR RG G
N N
T r v nn Prnt K Figure 6: Membrane topology ofGalT-1
_ .. . , 112ÏL r l U l' [J GalT-l-CHO cells were labeled with
T i m ee ( m m ) 60 60 60 60 60 O Tran[35S]label for 15 min and chased for 5
S a p o n i nn - — + — + + min. Postnuclear supernatants were P r o t e a s ee — + + + + + incubated for 0 and 60 min at 10°C with
g g __ — trypsin (Tryp.) or proteinase K (Prot. K) in
thethe presence or absence of 0.5% saponin. GalT-1GalT-1 was immunoprecipitated with antiserumantiserum 635 and analyzed by 7.5% SDS-1&1& PAGE.
GalT-1GalT-1 requires translocation of UDP-Gal into the ER
Thee predicted membrane topology of GalT-1 and the results of the protease protection assays suggestedd that the active site of GalT-1 is oriented toward the lumen of the ER. Therefore, GalT-11 enzyme activity should require UDP-Gal import. To test this requirement, postnuclear supernatantss were prepared from GalT-l-CHO cells and the GalT-l-CH01ec8 cell line, which iss deficient in UDP-Gal import into the Golgi apparatus (134). An enzyme assay with NBD-Cerr and UDP-Gal in the absence or presence of saponin to render membranes permeable to UDP-Gall showed that the specific activity of both postnuclear supernatants was comparable (Figuree 7B). In contrast, when the enzyme assay was carried out on intact membranes, the GalT-11 activity in GalT-l-CH01ec8 cells was much lopwer than in the GalT-l-CHO cells (Figuree 7A), which suggests that UDP-Gal import was limiting.
Cells s NBD-lipid d
conditions s Noo UDP-sugars s
UDP-Gal l UDP-Glc c Bothh UDP-sugars s pmol/mgpmol/mg protein MEB4 4 GM95 5 GM95--CGlcT T SM M GlcCer r GalCer r SM M GlcCer r GalCer r SM M GlcCer r GalCer r a ND,, not detectable: a 52 2 35 5 NDa a 62 2 ND D ND D 53 3 17 7 ND D fluorescentt signal 52 2 35 5 35 5 63 3 ND D ND D 53 3 22 2 5 5 27 7 413 3 ND D 64 4 ND D ND D 54 4 85 5 ND D 35 5 406 6 2 2 69 9 ND D ND D 59 9 76 6 1 1 correspondingg to less than 0.5 pmol.
TableTable I: GalCer synthesis by CGlcT in vitro
PostnuclearPostnuclear supernatants prepared from MEB4 cells, CGlcT-deficient GM95 cells, and CGlcT-transfectedCGlcT-transfected GM95 cells were incubated with 50 \iM NBD-Cer and 2 mM UDP-Gal, 2 mMmM UDP-Glc, or 2 mM of both for I h at 37°C. NBD-lipids were analyzed as described under
MaterialsMaterials and methods and expressed as pmol/mg protein.h.. Data are means of two independentindependent experiments (n = 4). Standarddeviation was less than 2%.
Finally,, we investigated the dependence of NBD-GalCer synthesis on UDP-Gal concentration. Iff UDP-Gal import is limiting for GalCer synthesis, saturation kinetics would be predicted for NBD-GalCerr formation in the GalT-l-CHO cells. As shown in Figure 7C, NBD-GalCer
synthesiss indeed increased over a UDP-Gal concentration range that occurs in cytosol. In contrast,, a much lower, nearly linear increase was observed in the GalT-l-CH01ec8 cells, whichh was probably caused by leakage of the membranes or by GalT-1 activity of CGlcT as discussedd above. Subtraction of this background signal from the GalT-1-CHO data points showss that saturation of the UDP-Gal translocator already occurred at 0.5 mM UDP-Gal. Thus NBD-GalCerr synthesis is critically dependent on an active UDP-Gal transporter. Interestingly, ourr results also document that the UDP-Gal translocator, which originally was identified in the Golgii apparatus, is required and sufficient for UDP-Gal import into the lumen of the ER to servee with NBD-Cer as substrates for GalT-1.
—— 60 E E 11 50 a. a. fc40 fc40 O O CO O 99 30 Q Q CO O 22
20-A 20-A
oo GalT-1-CHO .. GalT-1-CHOIec8 600 80 0 10 20 30 40 50 60 timee (min) time (min)FigureFigure 7: GalT-1 activity is dependent onon UDP-Gal translocation in the ER
PostnuclearPostnuclear supernatants prepared from GalT-1-CHOGalT-1-CHO cells (open symbols) and GalT-1-CHOlec8GalT-1-CHOlec8 cells (closed symbols) werewere incubated with NBD-Cer and
UDP-GalGal for different periods of time in the absenceabsence (A) or presence of saponin (B), andand NBD-GalCer synthesis was analyzed.analyzed. Synthesis of NBD-GalCer is dependentdependent on UDP-Gal concentration (C).(C). When the experiment was carried outout in the presence of NBD-GlcCer,
1.00 1.5 2.0 synthesis of lactosylceramide was UDP-Galactosee (mM) virtually lost in CHOlec8 (not shown), as
reportedreported for untransfected CHOlec8 cells (122). Although we showed that GalT-l-CHOlec8 cellscells expressed about 3 times more GalT-1 than the GalT-1-CHO cells, the transfected cells graduallygradually lose expression as we previously observed for the GalT-1-CHO cells. This explains whywhy the GalT-1 enzyme activity of the GalT-l-CHOlec8 postnuclear supernatant in the presencepresence of saponin was not directly related to the expression of the protein. UDP-Glc was
Discussion n
Thee availability of high affinity antibodies against GalT-1 has greatly facilitated the analysis of thee protein by allowing us to rigorously establish its biosynthesis and maturation, intracellular localization,, and membrane topology. GalT-1 was not detectable by Western blot analysis in CHOO and CH01ec8 cells using the GalT-1 antibodies, extending previous observations that CHOO cells do not contain GalCer. Using antibodies raised against three different regions of GalT-1,, we consistently detected mature GalT-1 in four different expression systems as a band withh an apparent molecular mass of 54 kDa. In order to separate mature and newly synthesized GalT-1,, we used 10% SDS-polyacrylamide gels. This caused a downward shift of 10 kDa in apparentt molecular mass as compared with analyzing the protein on 12.5% gels and with the sizee of the protein recently reported by others (131). The resolution of the latter separating systemm is insufficient to visualize the relatively small differences in molecular weight during biosynthesiss of the protein. Importantly, the immunoreactive 54-kDa band was also detected withh the 9E10 antibody in cells transfected with GalT-lmyc, and we identified it in the nontransfectedd rat Schwann cell line D6P2T expressing high levels of GalCer. Possibly, the highh content of hydrophobic amino acids in the luminal portion of GalT-1 is responsible for the anomalouss behavior of the protein on SDS-polyacrylamide gels.
GalT-11 is synthesized as a 60-kDa precursor protein that reached its mature form within a periodd of 60 min. The small decrease in molecular weight that we observed by SDS-PAGE duringg the first 30 min after the pulse showed that the protein was subject to oligosaccharide trimmingg in the ER. This was confirmed in Endo H-treated samples in which we found that GalT-11 remained Endo H-sensitive and where no such decrease occurred. The fact that GalT-1 didd not become Endo H-resistant even after a 16-h chase showed that the protein does not pass throughh the Golgi apparatus. A quantitative morphological analysis at the ultrastructural level ascertainedd that GalT-1 is retained within the ER and the nuclear envelope. Using this sensitive technique,, we did not detect GalT-1 in other intracellular compartments including the Golgi apparatus. .
GalT-11 behaved as a type I transmembrane protein. Protease treatment of intact membranes producedd a truncated protein with a molecular mass of about 50 kDa, which could still be immunoprecipitatedd with an antibody directed against the amino-terminal portion of the protein,, suggesting that the amino terminus was intact and oriented toward the lumen of the organelle.. Thus, a carboxyl-terminal region of 4 kDa appeared to be exposed to the cytosol, whichh is in excellent agreement with the length of the predicted cytoplasmic tail of 49 amino acidss and the presence of an arginine and various lysines close to the membrane. The carboxyl terminuss also contains the -KKVK sequence that most likely retains GaIT-1 in the ER by actingg as a cytosolic ER retrieval signal (211). Although most of the GalT-1 labeling on the cryosectionss was associated with the ER, it is possible that some GalT-1 is present in the intermediatee compartment between the ER and the cis-Golgi. From here, it could be recycled backk to the ER by retrograde transport.
Becausee we showed here that GalT-1 is localized to the ER, the previously reported GalT-1 activityy in Golgi fractions (122, 123, 127, 189) must be accounted for by a different enzyme. Wee here found that the CGlcT-deficient cell mutant GM95 not only lacked the ability to synthesizee GlcCer but also failed to synthesize GalCer under low UDP-Glc conditions in vitro. Retransfectionn of CGlcT in this cell line restored the ability to synthesize GlcCer and, in vitro, GalCer.. Thus, in vitro in the absence of UDP-Glc, CGlcT is capable of transferring Gal from UDP-Gall to ceramide. The ceramide galactosyltransferase mechanism of CGlcT seems to be unrelatedd to that of GalT-1, since CGlcT and GalT-1 do not share significant sequence homologyy and they are localized to cellular compartments with entirely different redox and
ionicc compositions. GalT-1 is exposed to the interior of the ER, whereas CGlcT most likely residess on the cytoplasmic surface of the Golgi apparatus. It is unlikely that CGlcT synthesizes GalCerr in vivo, since all mammalian cells express CGlcT, but no GalCer is observed unless the cellss express GalT-1 as well. The Km of CGlcT for UDP-Glc is at least 200 times lower than forr UDP-Gal when assayed in vitro.
Synthesiss of GalCer in membranes from GalT-l-CH01ec8 cells was greatly stimulated as comparedd with control cells when the membranes were permeabilized with saponin upon the additionn of UDP-Gal. As GalT-l-CH01ec8 cells are deficient in the UDP-Gal translocator in thee Golgi apparatus (134), we conclude that the active site of GalT-1 must reside on the luminall side of the ER and that the Golgi UDP-Gal translocator is responsible for the translocationn of UDP-Gal into the ER as well. Because antibodies against this translocator are nott available, its intracellular distribution can only be inferred from functional in vitro assays. Thiss question may soon be solved, since the cDNA of a UDP-Gal translocator that complementss the genetic defect of cells with a phenotype similar to that of CH01ec8 has been clonedd (212).
Inn contrast to all other glycosyltransferases identified in glycosphingolipid synthesis, GalT-1 is locatedd in the ER and not in the Golgi. It is most closely related to the glucuronyltransferase familyy of ER enzymes (133). The presence of newly synthesized GalCer in the luminal leaflet off the ER membrane is potentially interesting in terms of domain formation and sorting of (glycosylphosphatidylinositol-anchored)) proteins. However, because lipids rapidly translocate acrosss the ER membrane, GalCer has access to the cytosolic surface.
Interestingly,, CGlcT, the other enzyme generating a monoglycosylceramide, synthesizes its productt GlcCer on the cytosolic surface and not, like all others, to the lumen of the Golgi. Whilee GalCer and GlcCer may translocate across the Golgi membrane to serve as substrates for higherr glycolipid synthesis (122), the finding of a GalCer and GlcCer transfer protein (213, 214)) suggests that these glycolipids can reach the cytosolic surface of other membranes where theyy may fulfil yet unknown functions. In addition, subsequent specific translocation across the apicall membrane of epithelial cells (27) could contribute to epithelial lipid polarity, the enrichmentt of glycolipids on the apical cell surface.
Inn conclusion, we have shown that rat GalT-1 is a type I integral membrane protein exclusively localizedd to the endoplasmic reticulum. In mice, the enzyme is essential for the formation of functionall myelin (111, 112). In humans, the enzyme is responsible for high levels of galactolipidss in numerous epithelia as well. It will be interesting to see how the specific featuress of GalT-1 relate to its functions and those of its product GalCer in the organism.
Acknowledgements s
Drs.. Stephen Fuller (EMBL, Heidelberg, Germany), Yoshio Hirabayashi (RIKEN, Saitama, Japan),, Brian Popko (University of North Carolina, Chapel Hill, NC) and Michel Bornens (Institutee Curie, Paris, France) generously provided reagents. We thank Ineke Braakman (AMC,, University of Amsterdam, Amsterdam, The Netherlands) for valuable advice and our colleaguess in the Department of Cell Biology for as always helpful comments.
Materialss and methods
Materials Materials
Reagentss used in this study were from commercial sources and described in previous papers originatingg from this laboratory (27, 122, 123).
CellCell culture and transfection
Chinesee hamster ovary (CHO) cells, CH01ec8 cells (ATCC, Rockville, MD), and HeLa cells weree cultured as described before (215). D6P2T cells, MEB4 cells, and CGlcT-deficient GM95 cellss were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Transientt expression in HeLa cells was done with recombinant vaccinia T7 RNA polymerase, andd protein expression was analyzed 5 h after infection (215). CH01ec8 cells were transfected withh GalT-lpcDNA3 (123) using the calcium phosphate procedure (216). Stable cell lines were obtainedd by subcloning individual colonies. Positive clones were selected by measuring GalT-1 enzymee activity as described (123). Transfected GalT-1-CHO (123) and GalT-l-CH01ec8 cells weree cultured in minimal essential medium containing 10% fetal calf serum and 0.5 mg/ml geneticin.. Protein expression was induced by 5 mM sodium butyrate (Fluka, Buchs, Germany) 14-166 h prior to all experiments (217).
PlasmidPlasmid construction
Specificc regions of GalT-1 (Figure 1) were amplified in PCR reactions using GalT-lpcDNA3 (123)) as template and the following primer sets: for 635, 5'-CGG GAT CCA AAA TCA TCA TTGG TGC CGC CAA TATG-3' (forward) and 5-GGG AATTCA TCA TTG GGG TCA ACC AGTT AGC AG-3' (reverse); for 636, 5-CGG GAT CCC CTG CTG AAG TCG GAG CGC CTG-3** (forward) and 5'-GGG AAT TCG TTA GCA ATG TCT TCT GAC AGA TAC-3' (reverse);; for 637, 5'-CGG ATC CAG AAA AGT CAA AAG TCT GTT CTA G-3' (forward) andd 5'-GGG AAT TCA TTT TAC CTT TTT TTC ATG TTT AAT ATG-3' (reverse); for 638, 5'-CGGG GAT CCGT CAA GTA TCT GTC AGA AGA CAT TGC-3' (forward) and 5-GGG AATT TCG AAC GGA GGT GAT GGG CTC C-3' (reverse). PCR products were ligated betweenn the BamHI and EcoRI sites of pRSET-A (Invitrogen, Leek, The Netherlands). A c-Mycc epitope was spliced between Ser69 and Leu70 of GalT-1 by separately amplifying the 5' andd 3' region of its cDNA in PCR reactions using GalT-lpcDNA3 as template and the followingg primer sets: for the 5' region, 5'-CGT CAA TGG GAG TTT GTT TTG GCA C-3' (forward)) and 5*-CTC TTC CGA TAT CAG CTT CTG TTC CTC GCT GTA GTG ATT AGA TGGG GTC AAT GTC TC-3' (reverse); for the 3' region, 5-GAG GAA CAG AAG CTG ATA TCGG GAA GAG GAT CTA CTC CAG CGA TAC CCA GGG-3' (forward) and 5'-GGT CAA GGAA AGG CAC GGG GGA G-3' (reverse). PCR products were ligated into pGEMT-easy (Promega,, Madison, WI). The 3' region was released with EcoRV and Spel and ligated betweenn the EcoRV and Spel sites of 5' region pGEMT-easy. GalT-lmyc was released with Hindllll and Xbal and inserted in pcDNA3 (Invitrogen). A CGlcT cDNA was generously providedd by Yoshio Hirabayashi and ligated in the NotI site of pcDNA3 to produce CGlcTpcDNA3.. All constructs made by PCR were confirmed by sequencing both strands.
Antibodies Antibodies
pRSET-AA constructs were transformed into Escherichia coli BL21(DE3)pLysS (Novagen, Madison,, WI) and used for fusion protein production. Fusion proteins were insoluble and purifiedd under denaturing conditions on nickel-nitrilotriacetic acid columns (Qiagen, Leusden, Thee Netherlands) according to the vendor's instructions. His6-GalT-l fusion proteins were dialyzedd against PBS and injected into New Zealand White rabbits. Microsomal proteins preparedd from GalT-1-CHO cells were separated on preparative 10% SDS-polyacrylamide gels,, transferred to polyvinylidene difluoride membranes and used for affinity purification of antibodies.. The mouse monoclonal antibodies 1D3 against PDI and CTR433 against a Golgi proteinn (210) were generous gifts of Stephen Fuller and Michel Bomens, respectively. The mousee monoclonal antibody 9E10 against the Myc epitope was described previously (218).
InIn vitro transcription and translation
GalT-11 was synthesized from GalT-lpcDNA3 in a coupled T7 RNA polymerase transcription-translationn system (Promega) in the presence of rabbit reticulocyte lysate, dog pancreas microsomess (Promega), and Tran[35S]label (ICN, Costa Mesa, CA) as described previously (219).. The translation mixture was diluted with PBS containing 1% Triton X-100 and spun for 100 min at 13,000 rpm in a microcentrifuge at 4°C, after which GalT-1 was immunoprecipitated fromm the supernatant.
MetabolicMetabolic labeling
GalT-1-CHOO Cells were washed with PBS and methionine- and cysteine-free minimal essentiall medium containing 20 mM Hepes, pH 7.4 (pulse medium). The cells were subsequentlyy incubated for 30 min in pulse medium and labeled with 250 /iCi/ml Tran[[ SJlabel for 5 or 15 min at 37°C. Cells were washed and chased at 37°C in growth mediumm containing 5 mM methionine, 5 mM cysteine, and 20 mM Hepes, pH 7.4. After differentt periods of chase time, the cells were lysed in PBS, 1% TX-100, and GalT-1 was immunoprecipitatedd from detergent lysates as described below. HeLa cells were depleted as describedd for GalT-1-CHO cells and labeled for 45 min with 150 /iCi/ml Tran[35S]label. Cells weree next detergent-lysed and processed for immunoprecipitation.
CellCell fractionation
Cellss were washed, gently scraped in 250 mM sucrose, 10 mM Hepes, 1 mM EDTA-NaOH, pHH 7.2 (homogenization buffer), and broken by 12-14 passages through a 25-gauge needle. A postnuclearr supernatant was prepared by centrifugation for 15 min at 375 x g. Protein concentrationss were adjusted to 1.0 mg of protein/ml using the BCA assay (Pierce). For some experiments,, postnuclear supernatants were layered over 1 ml of 0.4 M sucrose, 1 ml of 1.25 M sucrosee in homogenization buffer and centrifuged for 30 min at 50,000 rpm in a SW60 rotor. Membraness were retrieved from the 0.4/1.25 M sucrose interface and used for blot purification off antibodies against GalT-1.
ProteaseProtease protection assay
Fiftyy fil (50 fig) of postnuclear supernatant prepared from metabolically labeled GalT-1-CHO cellss was incubated with 0.1 mg/ml of proteinase K or trypsin for 60 min at 10°C in the presencee or absence of 0.5% saponin. Samples were transferred to ice, and the reaction was stoppedd by adding phenylmethylsulfonyl fluoride (2.5 mg/ml), leupeptin (0.25 mg/ml), aprotininn (0.25 mg/ml), and pepstatin A (0.25 mg/ml) to the indicated final concentrations. Membraness were solubilized in 0.5% saponin, and GalT-1 was immunoprecipitated from the detergentt lysates in the presence of protease inhibitors.
ImmunoprecipitationImmunoprecipitation and endoglycosidase H digestion
Antibodiess were prebound to protein A-Sephacryl CL4B beads, and immunoprecipitations weree done exactly as described (219). Immunoprecipitates were resuspended in 50 fi\ of endoglycosidasee H (Endo H) buffer (50 mM sodium citrate, pH 5.5, 20 mM EDTA, 0.1 M 2-mercaptoethanol,, 0.1% SDS containing 1 /ig/ml of chymostatin, leupeptin, aprotinin, pepstatin andd 0.5 mM phenylmethylsulfonyl fluoride). Samples were split into two equal aliquots, one of whichh received 3 milliunits of Endo H, and both tubes were incubated for 6 h at 30°C and processedd for SDS-PAGE.
SDS-PAGESDS-PAGE and Western blot
Afterr the addition of 4x sample buffer (chapter 2), samples were heated for 5 min at 95°C and resolvedd by SDS-PAGE on 10% minigels. Gels were analyzed by fluorography or a STORM 8600 Phosphorlmager using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).
Forr Western blotting, polyvinylidene difluoride transfers were blocked for 90 min in PBS, 5% Protifarr (Nutricia, Zoetermeer, The Netherlands), 0.2% Tween 20 (blotto). Primary antibody incubationss were done for 60 min in blotto. Detection was with horseradish peroxidase-conjugatedd goat anti-rabbit IgG using enhanced chemi luminescence (Amersham Pharmacia Biotech,, Little Chalfont, United Kingdom). For quantitative Western blots, detection was done withh 125I-protein A and Phosphorlmager analysis with ImageQuant software.
GlycosphingolipidGlycosphingolipid synthesis
Cellss were homogenized, and postnuclear supernatant was prepared as described above. Unless statedd otherwise, postnuclear supernatants were incubated for various periods of time at 37°C withh 1% bovine serum albumin, 2 mM UDP-Glc, 2.0 mM UDP-Gal, 2 mM MgCl2, 2 mM
MnCl2,, and 50 (iM NBD-ceramide (NBD-Cer). At the end of the incubation period, lipids were
extractedd as described (122). Samples were dried under nitrogen and applied to TLC plates usingg chloroform/methanol (2:1, v/v). Fluorescent lipids were separated by two-dimensional thinn layer chromatography, identified by comparison with standards, and quantitated as describedd (122, 123).
ImmunofluorescenceImmunofluorescence microscopy
Cellss were grown on coverslips to 40-60% confluency. The cells were fixed with 3% paraformaldehyde;; quenched in PBS, 50 mM NH4CI; and incubated for 1 h in PBS, 0.5% bovinee serum albumin, 0.1% saponin (blocking buffer). The cells were labeled with affinity-purifiedd antibody 635 against GalT-1, the mouse monoclonal antibody 1D3 against PDI, or the mousee monoclonal antibody CTR433 against a Golgi protein. After 30 min, the coverslips weree washed for four periods of 10 min with blocking buffer and counterstained for 15 min withh 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein-labeled goat anti-rabbit IgG and indocarbocyanine-labeledd goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove,, PA). The coverslips were mounted in Mowiol and examined with a Leica confocal microscopee (Leica, Heidelberg, Germany) attached to a Leica microscope using separate filters forr each fluorochrome viewed: for 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein, ex = 488 nm andd em = 515 LP; for indocarbocyanine, ex = 568 nm and em = 585 LP. Singly labeled cells weree examined to exclude the possibility that bleed-through occurred for the given confocal conditions.. Images were imported into Adobe Photoshop and printed on a Tektronix dye sublimationn printer.
ImmunoelectronImmunoelectron microscopy
Forr immunogold electron microscopy, cells were fixed with a mixture of 2% paraformaldehyde andd 0.2% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4. After 2 h at room temperature,, the cells were scraped, embedded in 10% gelatin, and stored for ultracryotomy as describedd (220). Cryosections were prepared as described (221) and labeled with rabbit GalT-1 antibody,, and with the monoclonal 1D3 antibody against PDI, followed by protein A gold. For singlee labeling of GalT-1, we used 10- or 15-nm protein A gold. For double label experiments, GalT-11 was detected with 15-nm protein A gold, and PDI was detected with 5-nm protein A gold.. A swine anti-mouse antibody was used to enhance binding of protein A gold to sections labeledd with monoclonal antibodies. The intracellular distribution of gold label was quantitated byy counting the gold particles associated with-identifiable organelles on sections prepared from GalT-1-CHOO cells (n = 10). Data were corrected for nonspecific labeling of sections prepared fromm nontransfected control CHO cells (n = 10).
