Communication Vol. 260, No. 20. Issue of September 15, pp. 10897-10900,1985 THE JOURNAL OF BIOLOGICAL CHEMISTRY

0 1985 by The American Society of Biological Chemists, Inc. Printed in U.S.A.

Photoaffinity Labeling of the Human Erythrocyte Monosaccharide Transporter with an Aryl Azide Derivative of D-Glucose*

(Received for publication, December 31, 1984, and in revised form, May 15, 1985)

Michael F. Shanahanzg, B r i a n E. Wadzinski l , Joseph M. Lowndesll, and Arnold E. Ruoho l From the Departments of $Physiology, YPharmacology, and IIPhysiological Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706

A photoreactive, radioiodinated derivative of glu- cose, N-(4-iodoazidosalicyl)-6-amido-6-deoxygluco- pyranose (IASA-glc), has been synthesized and used as a photoaffinity label for the human erythrocyte monosaccharide transporter. Photoinactivation and photoinsertion are both light-dependent and result in a marked decrease in the absorption spectra of the compound. When [ '261]IASA-glc was photolyzed with erythrocyte ghost membranes, photoinsertion of ra- diolabel was observed in three major regions, spectrin, band 3, and a protein of 58,000 daltons located in the zone 4.5 region. Of the three regions which were pho- tolabeled, only labeling of polypeptides in the zone 4.5 region was partially blocked by D-glucose. In the non- iodinated form, N-(4-azidosalicyl)-6-amido-6-deoxy- glucopyranose inhibited the labeling of the transporter by [ '261]IASA-glc more effectively than D-glucose. The ability to synthesize this [ '261]containing photoprobe for the monosaccharide transporter at carrier-free lev- els offers several new advantages for investigating the structure of this transport protein in the erythrocyte.

A number of compounds have been utilized in recent years to label and identify the membrane components associated with monosaccharide transport in human erythrocytes. Dif- ferential and affinity labeling techniques as well as reconsti- tution for the most part have identified a broad molecular weight region of polypeptides on polyacrylamide gels (Mr = 45,000-65,000) as the transporter (see Ref. 1 for review; also Refs. 2-5). More recently, cytochalasin B, a potent inhibitor of glucose transport in these cells, has been demonstrated to be a useful photoaffinity label for this transport system in the red cell (3-5), as well as other tissues (6-10). Two laboratories have used this technique to probe the in situ structure of the glucose transporter in conjunction wtih enzymatic probes (11, 12). While all of the above methods have provided useful information concerning the transporter, they also possess a

* This work was supported by National Institutes of Health Grant AM 31335. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I To whom all correspondence should he addressed. Present ad- dress: Department of Physiology and Pharmacology, Southern Illinois University School of Medicine, Carbondale, IL 62901.

number of limitations. These limitations, which vary with the method employed, include such things as low specificity, high nonspecific background labeling, low efficiency of incorpora- tion, and low specific activity of radiotracer in the compounds being used.

Many of the problems encountered with photolabeling can often be overcome by the use of an orthoiodophenylazide functional group on the photoaffinity probe (13). In the pres- ent study we describe a simple method for the synthesis of a phenyl azide derivative of D-glucose, N-(4-azidosalicyl)-6- amido-6-deoxyglucopyranose (ASA-glc') and its carrier-free radioiodinated derivative ['251]IASA-glc. The results pre- sented in this report show that ['251]IASA-gl~ can be used for specific covalent labeling of the human erythrocyte monosac- charide transporter.

EXPERIMENTAL PROCEDURES

Materiak-Na'251 was from New England Nuclear. Electrophoresis reagents and molecular weight markers were purchased from Bio- Rad, except for Sequanal grade sodium lauryl sulfate which was from Pierce Chemical Co. 6-Amino-6-deoxy-~-glucose hydrochloride was purchased from United States Biochemical Co. N-Hydroxysuccini- midyl-4-azidosalicylic acid (NHS-ASA) was obtained from Pierce Chemical Co. All other reagents were purchased from Sigma. Thin layer chromatography was performed using Whatman type K5F silica gel plates for both analytical (type LKSDF) and preparative (type PLK5F) procedures. Outdated blood was provided by the American Red Cross Regional Blood Center, Madison, WI.

Preparation of Plasma Membranes-Washed human erythrocytes and membrane ghosts were prepared by the method of Steck and Kant (14). Ghost membranes in 5 mM sodium phosphate buffer (5P8 buffer) were used the same day for photolabeling experiments. The pellets were isolated by centrifugation at 35,000 X g for 40 min and washed twice with 5P8 buffer. All procedures were carried out a t 4 "C unless noted otherwise.

Synthesis of ASA-glc-6-Amino-6-deoxy-o-glucose HCI (85.6 mg, 0.4 mmol) was dissolved in 2.5 ml of dimethyl formamide followed by the addition of 55.3 pl (0.4 mmol) of triethylamine to neutralize the solution. NHS-ASA (120 mg, 0.44 mmol) in 0.83 ml of dimethyl formamide was then added to the mixture. The vial containing the NHS-ASA was rinsed twice with 0.5 ml of dimethyl formamide and combined with the reaction mixture. The reaction was allowed to proceed for 48 h at room temperature with constant stirring. At the end of this time the dimethyl formamide was removed by vacuum drying. The oily residue was dissolved in 1.5 ml of absolute ethanol and further purified by preparative thick layer (1-mm) chromatog- raphy on silica gel plates developed with butano1:acetic acidwater (5:l:l). The major UV-positive hand (RF = 0.75) was removed and extracted 4 times with 2 ml of ethanol using a scintered-glass funnel. The solvent was removed from the combined extracts on a rotory evaporator under reduced pressure. The residue was washed in 2 ml of methanol/benzene ( l : l , v/v) and centrifuged. The supernatant was removed, its volume was reduced to 1 ml by drying, and it was centrifuged again. The white solids recovered from both centrifuga- tions were combined and dried under N,; yield, 90%; m.p. 183-185 "C; IR (KBr): 2143 cm" (azide) and 3400 cm" (broad -OH); NMR (deuterodimethyl sulfoxide): 6 6.05 ( t , lH, aromatic H para to hy- droxyl), 6 6.25 (d, l H , aromatic H ortho to hydroxyl), 6 7.73 (d, 1H, aromatic H meta to hydroxyl).

Synthesis of f251/IASA-glc--Iodination of ASA-glc was performed using the method of Hunter and Greenwood (15). 10 pl of 0.1 N HC1

' The abbreviations used are: ASA-glc, N-(4-azidosalicyl)-6-amido- 6-deoxyglucopyranose; IASA-glc, N-(4-iodoazidosalicyl)-6-amido-6- deoxyglucopyranose; NHS-ASA, N-hydroxysuccinimidyl-4-azidosal- icylic acid; 5P8, 5 mM sodium phosphate buffer, pH 8.0; NaDodS04- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

10897

Photoaffinity Labeling of the D-Glucose Transporter

cy 0

DMF 48 HOURS

ROOM TEMP.

~npw-~-(& ASA-D-GLUCOSE

OH I 1 0.5M No-PHOSPHATE 0UPFLR pH 7.4

CHLORAMINE-T

No1" I

OH

FIG. 1. Schematic representation of the synthesis of ASA- glc and ['2"I]IASA-glc. DMF, dimethyl formamide.

was added to a vial containing 10 pl of NalZ5I (1 mCi) in 10 p1 of 0.1 M NaOH, followed by the addition of 20 p1 (100 nmol) of ASA-glc solution (1.7 mg/ml in 0.5 M sodium phosphate buffer, pH 7.4). To this solution 20 pl of chloramine-T (1.14 mg/ml in phosphate buffer) was added and the reaction was allowed to proceed for 1 min, followed by quenching with 50 pl of 5% sodium metahisulfite. Initial purifica- tion of ['251]IASA-glc was performed by thin layer chromatography using ethyl acetate/isopropyl alcohol/water (65:22:11, v/v). The ra- dioactive product was identified on the plate by autoradiography and extracted from the silica gel 4 times with 1 ml of 95% ethanol. The volume of the extract was reduced to 100 pl by drying under N P . ASA- glc (125 pg) was added and the mixture was rechromatographed in hutanol/acetic acid/water (5:1:1, v/v). A subsequent autoradiogram indicated that the radioiodinated product migrated slightly ahead of the UV-positive ASA-glc. This established the separation of [lZ5I] IASA-glc from ASA-glc. The radioiodinated product was removed from the plate, extracted 4 times with 1 ml of ethanol and the volume reduced to a final concentration of 0.05 pCi/pl by drying under Nz. The product was obtained carrier-free at a theoretical specific activity of 2200 Ci/mmol.

Photoaffinity Labeling-Conditions for photoaffinity labeling of membranes with ['z51]IASA-glc were as follows. Membranes were first preincuhated in 15-ml Corex centrifuge tubes in the presence of either of wglucose (0.4 M ) or ASA-glucose (lo-' M ) for 1 h. Following preincubation, 0.2-0.3 pCi of ['251]IASA-glc was added to each tube and incubated for an additional 1-h period in the dark. Immediately before photolysis, 1-5 ml of buffer containing 1% mercaptoethanol was added as a scavenger to each tube. The diluted mixtures were photolyzed in the Corex tubes for 5 s in ice water at 10 cm from a 1- kilowatt high pressure mercury lamp AH-6 (Advanced Radiation, Santa Clara, CA). The membranes were then centrifuged a t 30,000 X g for 45 min and washed once with 1-5 ml of 5P8 buffer. The final

WAVELENGTH, nm FIG. 2. Photodecomposition of ASA-glc. An 80 p~ aqueous

solution of ASA-glc was exposed to various times of irradiation: a, control; b, 3 s; c, 5 s; d, 10 s; and e, 30 s. Samples were then placed in 3-ml quartz cuvettes (1-cm) and the scanning absorbance spectra was determined.

pellets were then solubilized in gel electrophoresis sample buffer (5) and samples (80-100 pg of protein) were layered onto gels for electro- phoresis. Under our present photolysis conditions, no detectable photocross-linking of membrane proteins to each other is evident for up to 30 s.

Electrophoresis-Electrophoresis was performed on 12% gels using the modified Laemmli buffer system of Giulian et al. (16). Following electrophoresis, gels were fixed, stained, sliced, and assayed by scin- tillation counting as previously described (4). For analysis of ['*'I] IASA-glc photolabeling experiments, stained gels were first dried on a slab-gel dryer, then exposed to Kodak X-Omat film with a Cronex Hi-Plus intensifier screen (DuPont) at -100 "C for the time indicated. Autoradiographs were scanned using a Bio-Med (Fullerton, CA) 504XL Soft Laser Scanning densitometer (633 nm) with Apple 2e computer integration. Peak heights for gel lanes from the same experiment were normalized using spectrin band heights to account for slight differences in load and nonspecific labeling. The Coomassie Blue protein staining patterns for these red cell polypeptides were typical for this gel system. These staining patterns were essentially unchanged for both control and treated membranes following photol- ysis.

Other Procedures-Protein determinations were performed accord- ing to the modified Lowry procedure reported by Peterson (17).

RESULTS

Photodecomposition of ASA-glc-In order to produce a pho- toactive analog of D-glucose suitable for photoaffinity labeling of the erythrocyte glucose transporter, we used a heterobi- functional photoactivable compound, NHS-ASA, originally reported by Ji and Ji (18), coupled to 6-amino-6-deoxygluco- samine to form ASA-glc. This compound was then radioiodi- nated using the Hunter-Greenwood method (15). The synthe- sis scheme and structures are presented in Fig. 1. The optical absorption spectra following light-sensitive activation of the aryl azide is shown in Fig. 2. A substantial photosensitivity is

Photoaffinity Labeling of the n-Glucosc Transportcr 10899

8

1

1

0 R f

1

FIG. 3. Partial protection of ['2sIIIASA-glc photoincorpo- ration hy 1)-glucose. A. A 5-dav autoradiogram o f a I'A(;F: gel o f eryt hrocvte mc~ml)r;lne proteins photolaheled with ["~'I]IASA-glc (:%.X nM) in the ahsenre (-), or presence (+) o f 480 mM Ibglucose. 8. densitometer recordings of the autoradiograms in A . T h e uppvr t r o w represents the rrppcJr gel lane (-glc): the h r v r fmcr represents the lower gel lane (+glc). Arrows indicate molecular weight markers (Bin- Rad): phosphorylase h (92,500); bovine serum alhnmin (66,200): oval- I)rrmin (45,000): carbonic anhydrase ( 3 1 , 0 0 0 ) : sovhean trvpsin inhib- itor (21.500): and Ivsozvme (14.400).

exhibited by this compound as illustrated by the decrease in the ahsorpt ion spectra with increasing time of phot,olysis. The 270 nm hand rapidly disappears during the first 5 s of irradia-

R t 1

10900 Photoaffinity Labeling of the D-Glucose Transporter

under "Experimental Procedures") indicates that ASA-glc inhibits incorporation of label by 51% in the transporter region with little or no effect on incorporation into the other regions of labeling.

DISCUSSION

The results of experiments presented in this report suggest that ['251]IASA-g1~ is a useful photoaffinity probe of the human erythrocyte monosaccharide transporter. Photoacti- vation of this compound resulted in its covalent incorporation into erythrocyte membrane polypeptides, as evidenced by the fact that the compound is not displaced under the harsh denaturing conditions of NaDodS04-gel electrophoresis. Fur- thermore, at low concentrations, IASA-glc specifically labels polypeptides in the region of zone 4.5, previously identified as containing the glucose transporter (1-5), and this incorpora- tion can be partially blocked in the presence of D-glUCOSe. While spectrin is also labeled at this concentration, it appears to be nonspecific in nature since no protection was observed in the presence of either D-glucose or ASA-glc.

This new photoprobe offers a number of novel features for exploring the structure of the glucose transporter. First, the ligand can be synthesized with lZ5I at carrier-free levels. No previously reported affinity probe for this transport system contains this high energy isotope in carrier-free form, al- though both a low specific activity, diiodinated form of ASA- glc (19) and a tritiated aryl azide derivative of D-glUCOSe have previously been reported (20). The efficacy of the compound described in the present report is evident. For example, it allows detection of very low levels of incorporated radioligand. Thus, low concentrations of the photoprobe may be used which should result in an increase in the specific binding to the transporter, with a concomitant lowering of nonspecific binding. Secondly, it offers the advantage of a rapid and convenient means for detection by autoradiography. Probes such as ["H]cytochalasin B, while highly specific, require either long time periods for fluorography or else the more expensive, laborious task of slicing and scintillation counting of gels.

Our results suggest that this photoactive analog of D-glucose has a greater affinity for the transporter than D-glucose. In the experiments involving protection against ['251]IASA-glc photoincorporation (Figs. 3 and 4), ASA-glc M ) was more effective than D-glucose (480 mM) in protecting against [12s1]IASA-glc photolabeling. The derivatization of the glucose moiety results in a ligand of greater hydrophobicity than D- glucose itself which is reflected by a 30-fold decrease in aqueous solubility of ASA-glc relative to D-glUCOSe.2 While we have no direct evidence, we suspect that the observed increase

M. F. Shanahan, and B. E. Wadzinski, unpublished observation.

in affinity of the transporter for ASA-glc compared to D- glucose may be related to the increased hydrophobicity of the glucose analog.

This photolabel should prove to be a useful tool for further probing both structural and functional aspects of the mono- saccharide transport system in the human erythrocyte and possibly other cell systems.

Acknowledgments-We thank Dr. Richard L. Moss and Gary G. Giulian for the use of their scanning densitometer and helpful sug- gestions.

Note Added in Proof-Since the submission of the present report for publication, a study using a similar approach has been published (Weber, T. M., and Eichholz, A. (1985) Biochim. Biophys. Acta 8 1 2 , 503-511). These authors report that a diiodinated aryl azide derivative of D-glUCOSe (19) competitively inhibits glucose transport in intact erythrocytes. In addition, following photolysis this compound specif- ically labels bands 4.51 and 6 in concentration dependent, D-glucose protectable manner.

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