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. Author manuscript; available in PMC: 2013 Nov 21.
Published in final edited form as: J Neurosci Res. 2011 Sep 9;89(12):10.1002/jnr.22738. doi: 10.1002/jnr.22738

GLUT-1 GLUCOSE TRANSPORTERS IN THE BLOOD-BRAIN BARRIER: DIFFERENTIAL PHOSPHORYLATION

Kavi Devraj 1,§, Marianne E Klinger 1, Roland L Myers 1, Ashwini Mokashi 2, Richard A Hawkins 2, Ian A Simpson 1,*
PMCID: PMC3835747  NIHMSID: NIHMS527292  PMID: 21910135

Abstract

Glucose is the primary metabolic fuel for the mammalian brain and a continuous supply is required to maintain normal CNS function. The transport of glucose across the blood-brain barrier (BBB) into the brain is mediated by the facilitative glucose transporter GLUT-1. Prior studies (Simpson et al. 2001) had revealed that the conformations of the GLUT-1 transporter were different in luminal (blood facing) and abluminal (brain facing) membranes of bovine cerebral endothelial cells, based on differential antibody recognition. In this study we have extended these observations and using a combination of 2D-PAGE/Western blotting and immunogold electron microscopy we determined that these different conformations are exhibited in vivo and arise from differential phosphorylation of GLUT-1 and not from alternative splicing or altered O- or N-linked glycosylation.

INTRODUCTION

Blood-Brain Barrier (BBB) is a specialized barrier made-up of microvascular endothelial cells that are held together by tight junction complexes that effectively restrict the paracellular diffusion of solutes. Consequently the passage of polar substituents is mediated by transendothelial transport. Glucose is the primary energy substrate for mammalian brain and a constant supply is necessary to maintain cerebral activity. The original studies of Dick and colleagues were the first to demonstrate the presence of the highly glycosylated (55 kDa) GLUT-1 glucose transporters in rat brain endothelial cells (Dick et al 1984). Earlier studies had demonstrated that this protein was also highly expressed in human erythrocytes where it mediated facilitated glucose transport and its presumed 2D structure is illustrated in Figure 1 (Blodgett and Carruthers 2008). Glucose transport across the BBB is not rate-limiting for cerebral metabolism under normal physiologic conditions; however under conditions of seizures, hypoxia, hypoxia-ischemia, chronic hypoglycemia it can become rate-limiting (see (Simpson et al 2007) for review. Analysis of the vectorial transport of glucose from blood to brain that encompasses the role of luminal to abluminal glucose transporters has also been long acknowledged as the appropriate mode to determine transport kinetics, however the molecular mechanism(s) underlying such transport have not been investigated (Cunningham et al 1986)(Choi et al 2001)(Simpson et al 2007).

Figure 1.

Figure 1

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GLUT-1 structure schematic (modified from Blodgett et al. 2008) to indicate primary antibody recognition sites. The structure of GLUT-1 glucose transporter showing the different regions of GLUT-1 from human erythrocytes presumed to be similar to the GLUT-1 at the blood-brain barrier. C-TER antibody recognizes the residues 472–492 at the C-terminus; the LOOP antibody recognizes the residues in the middle cytoplasmic loop from 231–246. H-SER antibody recognizes all the regions except the hydrophobic transmembrane domains. However, it may contain higher proportion of antibodies from the highly hydrophilic loop and C-terminus regions as shown for a similar whole sera antibody generated by Baldwin and colleagues (Davies et al. 1987).

In a previous study, we demonstrated that the GLUT-1 glucose transporter exists in two distinct forms in the luminal and abluminal membranes of the bovine blood-brain barrier (Simpson et al 2001). Western blot analysis using a rabbit polyclonal antibody raised against the C-terminal 20 amino acids (C-TER) of GLUT-1 indicated a relative luminal: abluminal distribution of GLUT-1 of 1:5 that was entirely comparable to estimates obtained by electron microscopy studies of Pardridge and colleagues in rat, rabbit, and human endothelial cells (Cornford et al 1993; Cornford et al 1995; Farrell and Pardridge 1991; Gerhart et al 1989). However, when the same studies were performed using an antibody raised against the purified human erythrocyte GLUT-1 (H-SER), the ratio of luminal: abluminal GLUT-1 distribution was 1:1 (Sogin and Hinkle 1980) (Wheeler et al 1982). From these observations it was proposed that accessibility to the C-terminal epitope was compromised in the luminal membranes suggesting an alternative conformation.

In this study, using the GLUT-1 antibodies described above and immuno-electron microscopy, we confirmed that the distinct luminal and abluminal conformations observed by Western blotting are retained in rat brain microvessels in situ. Moreover, using a combination of 2D gel electrophoresis, Western blotting and alkaline phosphatases, we can now attribute these differing conformations to differential phosphorylation and not to different protein sequences resulting from alternative splicing.

MATERIALS AND METHODS

Materials

All chemicals and reagents were obtained from Sigma (St. Louis, MO) unless otherwise specified. TRIZOL reagent was obtained from Invitrogen Life Technology (Cincinnati, OH). The source of all the GLUT-1 antibodies has been described previously (Simpson et al 2001). The C-terminal antibody (C-TER) was raised in rabbit against the amino acids 472–492 of the human GLUT-1 C-terminus which are common to rat and bovine GLUT-1, the polyclonal loop antibody (LOOP) was raised in rabbit against residues 231–246 of the central cytoplasmic loop of human GLUT-1 (a gift from Dr. Anthony Carruthers). The antibody raised against the entire purified human erythrocyte GLUT-1 transporter (H-SER) is also a rabbit polyclonal antibody and recognizes a variety of epitopes across the GLUT-1 sequence, including the C-terminus (see Figure 1). PNGase F, Sialidase A, O-glycanase, and alkaline phosphatase were obtained from New England Biolabs, Inc. (Beverly, MA). Protein concentration for all the samples used in this study was performed with Bradford Coomassie Plus assay (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer's protocol. The detergents ASB-14 and DDM were obtained from Calbiochem (San Diego, CA), immobiline strips, ampholytes, Destreak™ (Hydroxyethyl disulphide), urea, thiourea and Deep Purple™ were obtained from GE Healthcare (Piscataway, NJ). Criterion™ precast gels and ReadyPrep ™ 2-D clean-up kit was obtained from Bio-Rad Laboratories Inc. (Hercules, CA). Chemiluminescence kit was obtained from Perkin Elmer Inc. (Billerica, MA). Water used for all the experiments was de-ionized proteomic grade Milli-Q water (Millipore, MA).

Bovine Brain Microvascular Endothelial Cell Isolation/Fractionation

Fresh bovine brains were obtained from Strauss Veal and Lamb International Inc. (Franklin, WI, Establishment # 2444) where the cows were killed for human consumption under US Department of Agriculture supervision. Microvascular endothelial cells were isolated and subsequently fractionated to obtain luminal and abluminal membrane vesicles as previously described (Sanchez del Pino et al 1992). Briefly, the microvessels isolated from bovine cerebral cortices as described by Pardridge et al. (Pardridge et al 1985) were digested with collagenase type IA (1800 units/g) to remove the basement membrane, pericytes, and glial fragments. The refined microvessels were homogenized and the luminal and abluminal membrane vessels were separated by discontinuous Ficoll gradient. The luminal and abluminal vesicles were ~ 80% pure as assessed using γ-glutamyl transpeptidase (GGT) as a luminal membrane marker and MeAIB transport (system A) activity as an abluminal membrane marker (Sanchez del Pino et al 1992; Sanchez del Pino et al 1995) and the vesicles were stored at −80°C.

SDS-PAGE and Western Blotting

Samples for Western blotting, SDS-PAGE were solubilized in SB buffer (2.3M Urea, 1.5% SDS, 15mM Tris, 100mM DTT, and 0.01% BPB) for 2 h at room temperature. Electrophoresis was performed at a constant 80 V for 3 h at room temperature on 10% Tris-HCL Bis-acrylamide gels. The separated proteins were transferred to nitrocellulose membrane (NC) at 4°C, 36 V constant for 16 h. Reversible Poncieu S staining was performed to confirm equal protein loading and was followed by Western blotting as described previously (Simpson et al 2001). The blots were visualized with the aid of an enhanced chemiluminiscence kit in a Fuji LAS-3000 system and the acquired 16-bit TIFF images were then analyzed using Multi Gauge software 3.0 (Fujifilm Life Science, Valhalla, NY). To account for gel-gel variation, the values from each sample lane were normalized to the luminal membrane sample as calibrator that was included on each gel. When stripping was performed, Restore™ Plus Western blot stripping buffer (Thermo Fisher Scientific, Rockford, IL) was used with the blots incubated for 1 h at room temperature followed by washes with PBS/Tween-20 (0.1%) before proceeding with the immunoblotting protocol.

Immuno-Electron Microscopy

Rats were transcardially perfused with 4% paraformaldehyde (Fisher Scientific, NJ)/0.01% glutaraldehyde (EM sciences, PA) and post-fixed O/N at 4°C in same solution. The tissue was cryoprotected by immersion successively in 10%, 20% and 30% sucrose/ PBS. Cryosections (16μm) were placed on Fisher Plus slides and stored at −80°C until ready to use. Slides were thawed, allowed to come to room temperature and all subsequent exposures were at room temperatures unless indicated to the contrary. The sections were circled with PAP pen, rinsed in PBS, and washed for 5 min × 4 on a rocker before incubation with 0.04% saponin (Sigma, MO) in PBS for 30 min followed by a 5 min exposure to 0.05M Glycine at pH 7.4. The sections were further rinsed in PBS, 4×5 min. The sections were blocked with 5% milk, 5% serum in PBS with for ~ 1.5 hours and rinsed in PBS, 4×5 min. Primary Antibody Incubation: All antibodies were diluted with 5% normal goat serum/PBS and incubated overnight. The GLUT-1 C-TER antibody was used at 1:1000 and H-SER at 1:500. After incubation slides rinsed with PBS, 4× 5min. Secondary Antibody Incubation: Sections were incubated with Alexa Fluronanogold Fab fragment of goat anti rabbit IgG (Nanoprobes, NY) at a 1:25 dilution in 5% milk/5%serum in PBS for 2 hours and then rinsed with PBS, 4×5min. Sections were then incubated with1% glutaraldhyde in PBS for 10 min, rinsed with dH20, 4×5min and then washed into 0.2 M citrate buffer, pH 7.4, 3X, 5 min before exposure to silver (Nanoprobes, NY) for 30 min in a darkroom. Upon completion, the slides were rinsed 3 × 5 min and then exposed to 0.5% OsO4 (Polysciences, PA) in 0.1M Phosphate Buffer pH 7.4 for 90 min and rinsed 3 × 5 min in dH20. Finally, the slides were stained with 2% uranyl acetate in 70% ethanol for 2 hours (Fisher Scientific, NJ), rinsed in 95% Ethanol for 10 min followed by 3× 10 min washes in 100% ethanol before being immersed in 1:1 100% ethanol: 100% Plastic (Polysciences, PA) for 16 h in the dark. The slides underwent two further changes of 100% plastic and then a third change in to 100% plastic to embed in the slide molds. 60–90 nm sections were cut using a Sorvall Porter-Blum MT2-B ultramicrotome and microscopy was performed on a Philips 400 electron microscope.

Northern Blotting

Total RNA was extracted using TRIZOL and RNA integrity was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., CA) and only samples with 28S/18S ratios > 1.8 were analyzed.

The samples were denatured using 1:2 ratio of RNA to loading buffer (50% formamide, 6% formaldehyde, 1X MOPS, 10% glycerol, 0.025 % BPB) at 65°C for 10 min and separated by electrophoresis on 1.2% agarose/2% formaldehyde gels at a constant 70 V for 2 h at RT. The gels were then transferred overnight at RT by capillary blotting in 20X SSC buffer (3.0M NaCl, 0.3M sodium citrate) onto a charged nylon membrane (Whatman, NJ), and fixed with ultraviolet light. Prehybridization was performed at 65°C for 2 h in a buffer containing 50% formamide, 5X SSC, 2% blocking reagent (Boehringer, Germany), 0.1% N-lauroylsarcosine, 50mM sodium phosphate (pH 7.0), and 7% SDS (Rayner et al 1994). Hybridization was performed overnight at 65°C in the prehybridization solution containing 100ng/ml of digoxigenin (DIG) labeled antisense GLUT-1 RNA probe complementary to bases 272–708 of the 2571-bp rat brain glucose transporter cDNA (Werner et al 1989), and was labeled with DIG-UTP by in vitro transcription with T7 polymerase according to manufacturer's protocol (Roche Applied Science, IN). Two, 5 min post-hybridization washes in 2X SSC/0.1% SDS at RT were followed by 2 × 15 min washes in 0.1X SSC/0.1% SDS at 65°C. All subsequent procedures were performed at room temperature. The nylon membranes were rinsed in wash buffer (pH 7.5; 0.1M maleic acid, 0.15M NaCl, 0.3% Tween 20) for 5 min followed by blocking in 1% blocking reagent (Boehringer, Germany) in the above wash buffer for 1 h and incubated with a mouse anti -digoxigenin Fab fragment conjugated to alkaline phosphatase (Roche Applied Science, IN) in blocking buffer for 45 min, followed by three 10 min washes in the wash buffer. The membranes were then equilibrated for 5 min with detection buffer (0.1 M Tris-HCl, 0.1 M NaCl, and pH 9.5) followed by immersion in the chemiluminescence substrate CDP-Star™ (Sigma, MO) for 5 min. Analysis of the chemilunescent signal was performed using a Fuji LAS-3000 system (Fujifilm Life Science, NY).

Slot Blots

Luminal and abluminal vesicles were first mildly sonicated to enable access to antibodies. Triplicate 2 μg samples that had been solubilized in SDS sample buffer (2.3M urea, 1.5% SDS, 15mM Tris, and 100mM DTT) or not for 2 h at room temperature were used. The samples were diluted to 100μl in PBS and captured under vacuum on a 0.45 μm nitrocellulose membrane (Pall Life Science, MI) on a 72-well slot-blot apparatus (Schleicher & Schuell, NH) and washed extensively with PBS. Immuno-blotting and quantitation of sample bands was performed as described above.

Deglycosylation

Enzymatic de-N-glycosylation was performed using Peptide N-Glycosidase F (PNGase F), an amidase that cleaves N-linked glycoproteins between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides. The reaction was performed by incubating 20 μg of total protein (in x PBS) with 8 μl of PNGase F (~60 IUB milliunits) in a total 30 μl volume for 10 h at 37°C on a rocker. After de-N-glycosylation, the O-linked carbohydrates were removed by adding a combination of Sialidase A and O-glycanase (2 μl of Sialidase A (10 mU), 2 μl of O-glycanase (2.5 mU).) to the above reaction volume of 30 μl and incubated for 1 hour at 37°C.

Dephosphorylation

Dephosphorylation was performed under denaturing conditions using calf intestinal alkaline phosphatase (CIP), which is able to release phosphate groups from tyrosine, serine and threonine residues. 20 μg of total luminal or abluminal membrane protein were incubated in 72 μl buffer (50mM NaCl, 10mM Tris.HCl, 10mM MgCl2, 1mM DTT; pH 7.9) containing 0.2% Saponin for 60 min at 37°C prior to addition of 80 units (8 μl) of CIP and the incubation continued for a further 60 min at 37°C. One unit is defined as the amount of enzyme that hydrolyzes 1 μmole of p-nitrophenylphosphate in 1 min at 37°C in a volume of 1 ml.

Two-Dimensional (2-D) Gel Electrophoresis

The protocol for 2-D electrophoresis was essentially as described in our recent methods paper (Devraj et al 2009). Briefly, 200 μg of each sample was solubilized in the SB buffer containing 2% DDM, 1% ASB-14, 5M urea, 2M thiourea, 10 mM TCEP, and 10 mM Tris (pH 8.0) for 2 h at room temperature followed by ultracentrifugation at 100,000×g for 30 min. The resultant supernatant (100 μl) was incubated with 10 μl of 200 mM 4-Vinyl Pyridine (4-VP) in the dark for 1 h at RT and the alkylation reaction was stopped by the addition of equimolar DTT. The alkylated membrane samples were further processed prior to isoelectric focusing (IEF) separation on a 11 cm immobiline 3–11 pH gradient strip as described in detail in Devraj et al, 2009. Following separation the IEF strips were equilibrated in 6M urea, 0.375 M Tris at pH 8.8, 10% SDS, 20% glycerol and SDS–PAGE was carried out in a Bio-Rad Criterion electrophoresis unit on 10% Criterion Tris-HCl acrylamide precast gels for 2 h at room temperature, 100 V constant.

Deep Purple™ Staining/2-D Western Blotting

Initially, parallel gels were run to obtain the IEF/SDS-PAGE patterns by staining with Deep Purple™ total protein. Briefly, solution containing 7.5% (v/v) acetic acid/ 10% (v/v) methanol was used to fix the gels for 1 h at room temperature followed by gentle wash in a solution containing 200 mM sodium carbonate for 30 min. Finally, the gels were incubated in Deep Purple™ stain at 1:200 dilution in proteomic grade Milli-Q water for 1 h at room temperature with gentle agitation. To reduce background, the gels were washed with 7.5% (v/v) acetic acid twice for 15 min before imaging them on the Typhoon TM 9400 scanner at 500V PMT (Photo Multiplier Tube) and 50 micron resolution by excitation with green laser (532 nm)/emission with 610 bandpass filter. The protocol for Western blotting/image acquisition was as described under 1-D SDS-PAGE/Western blotting.

RESULTS

GLUT-1 conformations in the BBB

Our previous study had demonstrated the presence of distinct forms of the GLUT-1 glucose transporter in the luminal and abluminal membranes isolated from bovine endothelial cell (Simpson et al 2001). These observations are confirmed in Figure 2 which illustrates Western blots for GLUT-1 in bovine luminal and abluminal membranes using the two antibodies: C-TER, a rabbit antibody raised against the 20 C-terminal amino acids of GLUT-1 and H-SER, a rabbit antisera that was raised against the purified human erythrocyte GLUT-1 protein (see Methods). These blots clearly demonstrate that the two antibodies interact differently with the transporter present in the two membranes such that the ratio of luminal: abluminal staining with the C-TER antibody is 1:2 compared to 1:1 with the H-SER antibody. The disparity in the C-terminal interaction with the luminal membranes was greater the more dilute the C-terminal antisera i.e.1:5,000 vs 1:30,000 (data not shown). We were able to confirm that this differential interaction resulting from C-TER antibody dilution could also be observed in vivo, by immunoelectron microscopy, where the ratios of GLUT-1 in luminal to abluminal of 1:4 were observed when using the C-TER antibody. Such values are entirely comparable to those observed by Farrel et al. These experiments confirmed our previous findings and therefore the quantitation of results is not shown (Simpson et al 2001). However, when we used the C-terminal antibody at 1:1000 and H sera antibody at 1:500 dilution as is illustrated in Figure 2B and quantitated in Figure 2C, the differential interactions of the antibodies were essentially eliminated. The number of particles in each membrane was determined for the entire cross-section of the respective vessels. Given the variation in numbers of transporters detected in the individual vessels, we performed the statistical analysis on the logarithmic transformation of the numbers of gold particles/cell in the respective membranes. The vessels analyzed were taken from the inferior colliculus; n >10 from each of three separate animals. The inferior colliculus was chosen because in situ hybridization indicated that GLUT-1 vessel density was high in that region (Vannucci et al 1997). No statistical differences could be determined between the values obtained for the respective membranes with either antibody, nor were any statistical differences observed between luminal and intracellular GLUT-1 concentrations. However both fractions were statistically different from the abluminal fraction (p<0.001), with the ratio of luminal: intracellular: abluminal at 1:1:2.45.

Figure 2.

Figure 2

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A) The Western blot for GLUT-1 using two different antibodies C-TER and H-SER on the luminal (L) and abluminal (A) membranes of the bovine BBB showing the differential staining in the two membrane fractions with the two antibodies when equal amount of the total protein was loaded on to the SDS-PAGE gels confirming our previous report (Simpson et al 2001). B) The electron micrograph showing the equivalent interaction of the two antibodies C-TER and H-SER at the luminal and abluminal membranes of the rat brain microvessels. C) The quantification of the number of transporters in the luminal (L), intracellular (I), and abluminal membranes (A). The statistical analysis was performed on natural logarithmic transformation of the numbers of gold particles/cell (Ln particle #) in the respective membranes. The vessels analyzed were taken from the inferior colliculus; n 10–16 from each of three separate animals.

However, certain caveats must be considered with this and any other EM data regarding GLUT-1 localization because not only are the data dependent on antibody concentration but all observations are also subject to the fixation conditions employed. This is clearly illustrated in Figure 3 where the effects of differing glutaraldehyde concentrations (0.01 vs 0.05%) are compared on the ability of H-SER to interact with GLUT-1. At the higher glutaraldehyde concentration, labeling of abluminal transporters is lost whereas the luminal transporters appear to be enhanced. The effect of the perfused glutaraldehyde on the abluminal transporter clearly confirms that the conformations of the luminal and abluminal GLUT-1 transporters are different, particularly as the glutaraldehyde must pass through the luminal membrane before gaining access to the abluminal membrane. These findings suggest that the GLUT-1 conformations result from either alternative splice variants, or post-translational modifications.

Figure 3.

Figure 3

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Effect of glutaraldehyde concentration on epitope access of GLUT-1 H-SER antibody. Electron micrograph showing that at higher glutaraldehyde concentration, labeling of abluminal GLUT-1 transporters is lost whereas the luminal transporters appear to be enhanced.

In order to further confirm the EM data that the relative accessibility of the respective antibody epitopes was retained in the membranes in their native state, we performed a simple slot blot assay where luminal and abluminal membranes were either sonicated to expose the epitopes or exposed to the urea/SDS solubilizing mixture, blotted onto nitrocellulose and the exposed to the respective antibodies. As illustrated in Figure 4, the luminal: abluminal ratio for the binding of the C-terminal binding is essentially unchanged with that observed in the denatured membranes. Although the ability of the C-terminal antibody to fully recognize the luminal transporter at higher concentrations would suggests that the C-terminus is modified such that the affinity is reduced rather than result of alternative splicing, it was clearly important to rule out the possibility of a splice variant. An RNA probe corresponding to N-terminal GLUT-1 sequence was developed for Northern blot analysis and total RNA prepared from whole rat brain homogenate and primary cultured rat astrocytes were used as controls to ascertain the splice variants in brain microvessels. The data presented in Figure 5 indicates that no variants could be detected and the only transcript was ~2.8kb that was present in bovine and rat brain microvessels which is consistent with the observations of (Birnbaum et al 1986).

Figure 4.

Figure 4

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A) Slot blots for GLUT-1 performed on the luminal (L) and abluminal (A) samples using the two antibodies C-TER and H-SER under native and denatured conditions as described in the experimental section. In both conditions, with C-TER antibody there is an increased staining in the abluminal fraction whereas with H-SER the two fractions have equal staining. B) The quantitation of the blots show that the state of denaturation does not bring the ratio between the luminal and abluminal samples with the C-TER closer to that with H-SER suggesting an absence of conformational changes in GLUT-1. * Represents significance (p-value < 0.05) with a two-tailed unpaired t-test for the ratio with C-TER compared to the H-SER.

Figure 5.

Figure 5

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Northern blot for GLUT-1 showing the presence of a single 2.8kb mRNA transcript and no splice variants in the various samples: RB (rat brain homogenate), AS (primary cultured astrocytes at P0 passage), UB (undigested bovine brain microvessels), BB (primary cultured bovine brain microvascular endothelial cells at 6th passage), UR (undigested rat brain microvessels), R2 (primary cultured rat brain microvessels at 2nd passage).

Deglycosylation/1-D Western Blotting

After eliminating the possibility of splice variants the question of differential glycosylation of GLUT-1 in the respective membranes was considered. The initial study of Birnbaum et al had indicated that marked differences in the extent of glycosylation at the first extracellular loop (N=45, asparigine residue) accounted for the differing molecular weights for GLUT-1 observed in endothelial cells and human erythrocytes (Mr ~55kDa) and GLUT-1 present in brain astrocytes (Mr ~ 45kDa) but no information exists regarding any distinction between luminal and abluminal membranes (Birnbaum et al 1986).

The luminal and abluminal membranes were N-deglycosylated with PNGase F, an endoglycosidase that cleaves asparagine-linked (N-linked) oligosaccharides from glycoproteins and in Figure 6A illustrates that GLUT-1 is deglycosylated from 55 kDa to ~ 42 kDa as previously observed (Birnbaum et al 1986)(Kumagai et al 1994). However, the deglycosylation had no effect on the relative affinities of the respective antibodies for the transporters in the luminal and abluminal membranes and thus N-linked glycosylation was not responsible for the apparent differences. The presence of O-glycosylation was examined using the enzyme combination of sialidase A and O-glycanase on the residual N-deglycosylated membranes. Again no significant changes were observed in GLUT-1 staining (with either antibody) between the luminal and abluminal membranes after O-deglycosylation (Figure 6B), implying that O-glycosylation is not a potential modification in GLUT-1 at the BBB.

Figure 6.

Figure 6

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A1) Western blot for GLUT-1 on the luminal (L) and abluminal (A) membranes using the two antibodies C-TER and H-SER after N-deglycosylation comparing it to the native glycosylated state. The blots show a reduction in the molecular weight from 55kD to around 42kD as expected (with both antibodies) after deglycosylation. A2) The quantitation of these blots indicates that N-glycosylation does not bring the ratio between the luminal and abluminal samples with the C-TER closer to that with H-SER suggesting that additional post-translational modifications are responsible for the differences in GLUT-1 with the two antibodies. B1) Western blot for GLUT-1 on the luminal (L) and abluminal (A) membrane fractions after O-deglycosylation using the C-TER and H-SER antibodies. O-deglycosylation was performed with enzymes listed in the experimental section after first performing N-deglycosylation, which was used as a control for comparison. The blots do not indicate any staining differences between the luminal and abluminal samples after O-deglycosylation when compared to the starting N-deglycosylated samples with either antibody. B2) The quantitation of the above blots indicates that O-deglycosylation does not bring the ratio between the luminal and abluminal samples with the C-TER closer to that with H-SER similar indicatting that O-glycosylation is not responsible for the differences in GLUT-1 with the two antibodies. * Represents significance (p-value < 0.05) with a two-tailed unpaired t-test for the ratio with C-TER compared to the H-SER.

Dephosphorylation/1-D Western Blotting

Phosphorylation was an obvious candidate for potential post-translational modification of GLUT-1, as the C-terminus appears to be a prime target for modification with two consensus casein kinase phosphorylation sites, one PKC site, and an additional serine at the third amino acid from the C-terminus phosphorylation. However, of these, only the PKC site has been confirmed as a minor modification of GLUT-1 in human erythrocytes (Witters et al 1985).

We therefore performed dephosphorylation of luminal and abluminal membranes with alkaline phosphatase as described in the experimental section. In these studies we have included an additional LOOP antibody raised against amino acids 231–246 in the large intracellular loop of GLUT-1 (Figure 1), which has been shown to reveal interactions between the loop and the C-terminus in erythrocyte GLUT-1 (Blodgett and Carruthers 2008). Western blotting the membranes after dephosphorylation has illustrated (Figure 7) that abluminal/luminal GLUT-1 staining is increased with the C-TER antibody compared to the native controls. The LOOP antibody however showed an increased staining in the luminal membrane in the native state antibody staining but after dephosphorylation the staining was equivalent between these two membrane fractions. In these particular membranes staining with H-sera was equivalent in native state and upon dephosphorylation the abluminal: luminal signal ratio was only slightly increased. However overall these data clearly suggest that phosphorylation is indeed modulating these antibody interactions.

Figure 7.

Figure 7

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A) Western blot for luminal (L) and abluminal (A) fractions after dephosphorylation with alkaline phosphatase along with the samples in their native state using the C-TER, LOOP, and H-SER antibodies. A LOOP antibody was included to obtain the phosphorylation changes in the loop and C-terminal domains as an interaction between these domains has been reported for erythrocyte GLUT-1 (Blodgett et al 2007). B) The quantitation of the above Western blots indicates an increase in the abluminal to luminal ratio with the C-TER antibody after dephosphorylation, and reversal to equivalent staining with the LOOP antibody similar to that with H-SER antibody suggesting phosphorylation as a potential modification in GLUT-1 at the BBB. * Represents significance (p-value < 0.05) when the abluminal to luminal ratio is compared to H-SER antibody in each state and # represents significance (p-value < 0.05) of the ratio in the dephosphorylated compared to the native state for each antibody.

2-D Experiments

To further resolve these phosphorylations, we employed the novel methodology of two-dimensional gel electrophoresis on the luminal and abluminal membranes proteins (Devraj et al 2009). Deep purple total protein stains obtained on luminal and abluminal membranes reveal the effective IEF and SDS-PAGE patterns of separation of the membrane proteins with minimal horizontal and vertical streaking and the markedly different composition of the two membranes. Having established that GLUT-1 remains soluble and could be detected on 2-D maps without undergoing precipitation, we proceeded to identify the various GLUT-1 species present in the respective membranes before and after dephosphorylation. These studies are illustrated in Figure 8 and reveal the presences of at least 3 different GLUT-1 forms with differing isoelectric points. The trends in GLUT-1 signal intensity for the three antibodies (stripped from the same blot) are similar to those 1-D Western blot (Figure 7) and indicate the C-terminal antibody more readily detecting the most acidic isoform. In the subsequent experiments (Figure 9), we assessed the effects of dephosphorylation on the relative mobility of different GLUT-1 forms. Based upon the amino acid sequence for bovine GLUT-1 and using Scansite 2.0 tool (http://scansite.mit.edu/calc_mw_pi.html), the predicted pI for the native unmodified form of GLUT-1 was 8.84, and this form appears to be detected in the 2-D blots of Figure 9, by all the three antibodies in the abluminal membranes. Using the same program a single site phosphorylation would predict a pI of 8.55 which would appear to correspond to a minor component in the abluminal membrane whereas for two sites of phosphorylation a pI of 8.10 is predicted which appears to correspond to a major GLUT-1 component detected in the luminal membrane and runs at higher Mr.

Figure 8.

Figure 8

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A) 2-D Deep Purple total protein stain for luminal and abluminal fractions that indicates an IEF pattern with minimal vertical and horizontal streaking. B) 2-D Western blots of luminal and abluminal samples for parallel gels using the three GLUT-1 antibodies (C-TER, LOOP, and H-SER), stripping the same gel twice as described in the experimental section to obtain the three blots. The blots indicate multiple GLUT-1 positive spots in the pI range of 8–10, indicating the presence of different GLUT-1 species in these two membrane fractions.

Figure 9.

Figure 9

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2-D Western blots of luminal and abluminal samples in the native and dephosphorylated state after stripping the stripping the same gel twice as described in the experimental section to obtain the three blots.

Assuming that H-sera is comparable to the antibody of Baldwin and colleagues (Davies et al 1990), it is likely to be comprised of IgGs that would recognize the C-terminus and the loop regions, thus should replicate all the GLUT-1 spots observed with C-TER and the LOOP antibody and in addition would also recognize other epitopes. The specific sites recognized by each of the antibodies are presented in Figure 1. The apparent affinity of the three GLUT-1 antibodies: C-TER, LOOP, and H-SER in the luminal and abluminal membranes and their affinities after dephosphorylation as deduced from the Western blot data (Figure 7) are presented in the Tables 1A and 1B respectively. A schematic composite of the 2-D blots of Figure 9 is presented in Figure 10. From this schematic and the above tables, different scenarios for GLUT-1 structure and function at the luminal and abluminal membranes of the blood-brain barrier are possible. While, we cannot account for all of those scenarios, one model that fits with most of the features of GLUT-1 at the BBB observed in the current study along with its inconsistencies is described below.

Table 1.

A) Primary antibody affinity of the abluminal membrane compared to the luminal membrane. B) Primary antibody affinity of the dephosphorylated membranes compared to the native state.

A)
1°Ab Affinity (Abluminal compared to Luminal in the native state)
C-TER ↑
LOOP ↓
H-SER ↔
B)
1°Ab Affinity (Dephosphorylation compared to native state)
LUMINAL ABLUMINAL
C-TER ↔ ↑
LOOP ↓ ↔
H-SER ↔ ↔
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Figure 10.

Figure 10

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A schematic for 2-D blots that shows the various GLUT-1 positive spots of Figure 9. The spots in red are detected by the C-TER and H-SER antibodies. The blue spot may be a real spot with a PTM or may be an artifactual spot spread out from the native pI spot at 9.0. All the three antibodies detect the spots in black.

The native pI GLUT-1 predicted from the amino acid sequence spot is pI 8.9 and is detected by each antibody in both the luminal and abluminal membranes. However, staining with the C-terminal antibody also clearly reveals an apparently more prominent spot with a pI ~ 8.0 in the luminal membranes that runs at a slightly higher Mr but is barely detectable in the abluminal membranes which is confirmed by the interactions of H-sera. Upon dephoshorylation, the pI 8.0 spot appears to collapse into a spot that is at approximately pH 8.7 and the native protein at pH8.9. The loop antibody barely detects the pI 8.0 isoform but is able to recognize both pI 8.7 and 9.0 proteins in the dephosphorylated luminal membranes. In the abluminal membranes three isoforms at ~pIs 8.5, 8.7, 8.9 are detected which upon dephosphorylation all essentially collapse to pI 8.9 isoform.

DISCUSSION

Glucose is the primary energy source for the mammalian brain and the transport of glucose across the BBB is the first step in its utilization by the brain. In the many studies, including our own, that have investigated the kinetics of transendothelial glucose transport across the blood-brain barrier, it is generally assumed that the distribution of glucose transporters between the luminal and abluminal membranes are equal. However, early electron microscopy studies by Pardridge and colleagues had indicated an asymmetry in the membrane localization of the GLUT-1 glucose transporters in rat endothelial cells that suggested the distribution of GLUT-1 transporters between luminal: intracellular: abluminal was 11%: 45%: 44% respectively, and a comparable distribution was subsequently confirmed in human and rabbit brain endothelial cells (Cornford et al 1993; Cornford et al 1994; Farrell and Pardridge 1991). Whereas others had suggested the distribution of GLUT-1 between the luminal and abluminal was much closer (Gerhart et al 1989). In our earlier study on bovine brain endothelial cells, it was suggested that this apparent asymmetry might result in part from differences in the apparent affinities of the GLUT-1 C-terminal antibodies for this epitope that were dependent on the specific membrane environment (Simpson et al 2001). In the current study we have attempted to systematically investigate the distribution of GLUT-1 between the luminal and abluminal membranes of bovine brain endothelial cells and to determine what if any structural differences in GLUT-1 might exist in the transporters in the respective membranes.

In our slot blot studies, as well as the EM studies, we were able to demonstrate that these distinctions in the relative interactions of the respective antibodies were retained even when the transporters were in their respective native membranes. Moreover, we were able to essentially reproduce the observations of Farell et al regarding the apparent distribution of GLUT-1 between the luminal, abluminal and intracellular membranes in the rat if we employed a dilute form of the C-terminal antibody (Farrell and Pardridge 1991). However, when we increased the concentration of the C-terminal antibody to 1:1000, the distinction in the GLUT-1 distribution observed between the luminal and abluminal membranes relative to that observed with H-sera effectively disappeared and based on EM data the ratio of luminal to abluminal GLUT-1 was 1:2.45. We drew two conclusions from these observations: first there was a difference in the relative affinity of the C-terminal antibody for GLUT-1 derived from the luminal and abluminal GLUT-1 in both rat and bovine brain endothelial cells and second that the epitope although with reduced affinity for the C-TER antibody in the luminal membranes remained and thus was not likely to result from alternative splicing which we subsequently confirmed (See Figure 5).

Differences in the molecular weight for GLUT-1 in endothelial cells and human erythrocytes and GLUT-1 found in most other cells was first noted by Birnbaum upon cloning the so called “brain form” of GLUT-1. The GLUT-1 in endothelial cells has an apparent Mr of ~55 kDa whereas the other cells within the brain express a form with an Mr of ~45 kDa. GLUT-1 is N-glycosylated at asparagine 45 which is located in the first extracellular loop of this twelve membrane spanning protein (Mueckler et al 1985) and differences in the apparent molecular weight of the transporter in BBB and human erythrocyte and other cells results from difference in the extent of N-glycosylation (Birnbaum, 1986)(Kumagai et al 1994). To this point nobody had determined whether the glycosylation might be different between luminal and abluminal membranes or whether it could account for the different antibody interaction. Western blots on native and N-deglycosylated luminal and abluminal fractions confirmed the presence of N-glycosylation, however, the quantitative analysis did not indicate altered N-glycosylation as being responsible for the differences in antibody cross-reactivity between the two forms. Subsequently, we investigated the same N-deglycosylated membranes for O-glycosylation by using a combination of Sialidase A and O-glycanase to subsequently remove O-linked glycosyl residues. We did not observe either a change in the molecular weight or antibody cross-reactivity of luminal or abluminal membranes following this treatment. This is consistent with the observations of Kumagai et al and Kasanicki and colleagues who failed to detect O-glycosylation (Kumagai et al 1994)(Kasanicki et al 1987). This data is also consistent with predictions provided by the software- NetOGlyc (http://www.cbs.dtu.dk/services/NetOGlyc) that indicates no potential O-glycosylation sites reside in the GLUT-1 loop sequence (207–271) and only two potential sites (478 T [53%], 490 S [51%]) reside in the C-terminal sequence (451–492) that are just above the threshold (50%). However, it should be noted that O-glycosylation is heterogeneous and therefore complete removal of all potential structures would require a combination of additional enzymes not employed in the current study. Therefore we cannot completely eliminate a role for O-glycosylation in GLUT-1 modulation, particularly given that such modification. would not necessarily alter the pI for the protein.

However, given these observations, the major focus of the current study was then to determine what role phosphorylation might play in the modulation of GLUT-1 antibody affinity. We elected to employ enzymatic dephosphorylation followed by 1-D Western blots to initially determine whether the antibody cross reactivity was altered in the respective membranes after dephosphorylation (Spillantini et al 1998)(Blodgett and Carruthers 2008). In these studies we included an additional antibody that was raised against the intracellular loop (LOOP antibody) in our analysis as our collaborators have demonstrated an interaction between the loop and C-terminal regions of the GLUT-1 in erythrocytes (Blodgett and Carruthers 2008). Indeed, our results show that dephosphorylation restores equivalent signal intensity with the LOOP antibody between the luminal and abluminal membranes similar to that observed with H-SER antibody, by apparently unmasking the double phosphorylated form. However, with the C-TER antibody we noticed a further increase in the abluminal: luminal signal ratio, resulting from an apparent increase in signal in the abluminal membrane. To confirm these observations, we performed two dimensional electrophoresis followed by Western blots using our recently established protocol for membrane proteins (Devraj et al 2009).

As indicated in results, the predicted pI for the native unmodified form of GLUT-1 was 8.84, and a single site phosphorylation would predict a pI of 8.55 for GLUT-1 whereas for two sites of phosphorylation a pI of 8.10 would be predicted. Each of these intermediates has been detected in either luminal or abluminal membranes. In the luminal membranes GLUT-1 would appear to be predominantly in the double phosphorylated form and this is a conformation that is readily recognized by the C-terminal antibody but not by the antibody directed against the loop sequence. We are assuming that H-sera is comprised of IgGs that would recognize the C-terminus, the loop region and various other epitopes and thus should detect all the GLUT-1 isoforms (Davies et al 1990). It clearly detects both forms in the luminal membranes, however the extent of the respective interactions is not as restricted as the C-TER, which appears to recognize the pI 9.0 isoform more readily than pI 8.0 and requires higher concentrations to enable complete detection relative to the H-SER. The majority of the GLUT-1 is present in the non-phosphorylated form in the abluminal membranes and thus most readily detected by all the antibodies, thus explaining the apparent differential interactions originally observed (Simpson et al 2001). The apparent changes in affinity of the antibodies after dephosphorylation can be explained by conformational changes that can result in a reduced access to the antibodies. Such a scenario has been shown to exist in erythrocytes by Carruthers and collegues (Blodgett et al 2007), where an antibody to the C-terminus of GLUT-1 lost its ability to bind to the transporter in the presence of ATP and it could be envisaged that phosphorylation could elicit the same changes. Further, this effect was dependent on the loop region of the transporter suggesting an interaction between the loop and the C-terminus of GLUT-1. Such an interaction might also exist for GLUT-1 in the BBB where the altered affinity of the C-TER as well as LOOP antibody changes are mediated by phosphorylation rather than ATP.

The concept of modulated GLUT1 activity is not novel as studies by Baldwin and colleagues and Ismail-Beigi and colleagues have reported enhanced glucose transport activity and the increased exposure of exofacial GLUT 1 transporters at the plasma membrane in clone 9 liver cells that can be induced by exposure to osmotic or metabolic stress by a mechanism that was distinct from translocation from the intracellular pool (Barros et al. 2001; Hamrahian et al. 1999). Furthermore this activation of transport appears to be mediated by AMP-kinase that is activated either by reducing ATP levels by exposing cells to hypoxia or metabolic poisons such as azide or cyanide or direct activation with AMP analogues such as 5 - aminoimidazole-4-carboxamide-1-D-ribofuranoside (AICAR). There are two consensus sites for AMP kinase phosphorylation on GLUT1, Ser95 and Ser225, the latter being close to the binding site for the loop antibody (see Figure 1). However, in recent studies by Jing and Ismail-Beigi, the concomitant activation of AMP- kinase activity and glucose transport activity in C 9 cells was not apparently associated with by an increase in glucose transporter phosphorylation when GLUT1 was immunoprecipitated from activated cells and analyzed using anti-phosphoserine antibodies REFS. Teleologically, such a mechanism in endothelial cells would be attractive as it has the potential to reflect decreases in ATP resulting from local energy expenditure and respond by increasing glucose transport.

In summary, the predominant isoform of GLUT-1 in the luminal membrane has pI a 8.0 which suggests it has multiple phosphorylations and is clearly distinct from its counterpart in the abluminal membranes that is clearly less phosphorylated. These phosphorylations clearly alter the conformation of the proteins whether denatured or when in their respective membranes as demonstrated by the ability of the specific antibodies to interact. The important questions that remain are obviously: 1) what are the sites of phosphorylation? 2) what are the consequences of these modifications? and 3) how are these modulations regulated? The possibility of modulating transport activity by phosphorylation is attractive because it would enable for changes in transport activity to be mediated more rapidly than can be achieved by a translocation mechanism involving recruitment of intracellular transporters and could be more responsive to local cerebral demands.

In the earlier study by Simpson et al it was suggested that there were more cytochalasin B binding sites (a specific inhibitor of facilitative glucose transporters) in the luminal membrane than in the abluminal membrane suggesting more glucose transporters, which was further supported by an apparent greater glucose transport activity (Simpson et al 2001). In the light of the current data, it would suggest that the phosphorylation must therefore lead to an activation of transport activity, however, this would still not account for the increased cytochalasin B binding sites. Thus, the presence of another glucose transporter still remains a possibility to account for the missing transporters and is not unprecedented, as GLUT-3 has been detected in canine microvessels (Gerhart et al 1995). The facilitative glucose transporter family has grown to 14 members of which GLUTs 1–4 and X are able to bind cytochalasin B transporter and the focus of our ongoing studies is to determine which are present in the bovine endothelial cells.

ACKNOWLEDGEMENTS

This work was supported by the NIH blood-brain barrier RO-1 grant (NS41405) and the Pennsylvania Department of Health CURE award. We would also like to thank Dr. Anthony Carruthers for the many helpful discussions.

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