Proc. Natl. Acad. Sci. USAVol. 93, pp. 11047-11052, October 1996Medical Sciences

Molecular identity and cellular distribution of advanced glycationendproduct receptors: Relationship of p60 to OST-48 and p90to 80K-H membrane proteins

(diabetes/vasculopathy/aging/endocytosis)

YONG MING LI*, ToMOKO MITSUHASHI*, DONALD WOJCIECHOWICZ*, NOBUYOSHI SHIMIZUt, JENNY LI*,ALAN STIrr*, CIJIANG HE*, DEBEN BANERJEEt, AND HELEN VLASSARA*§*The Picower Institute for Medical Research, 350 Community Drive, Manhasset, NY 11030; tSchool of Medicine, Keio University, 35 Shinanomachi, Shinjuku-Ku,Tokyo 160, Japan; and tLindsley F. Kimball Research Institute, New York Blood Center, 310 East 67 Street, New York, NY 10021

Communicated by Anthony Cerami, The Picower Institute for Medical Research, Manhassett, NY, June 11, 1996 (received for review April 12 1996)

ABSTRACT Advanced glycation endproducts (AGEs) arederivatives of nonenzymatic reactions between sugars and pro-tein or lipids, and together with AGE-specific receptors areinvolved in numerous pathogenic processes associated withaging and hyperglycemia. Two of the known AGE-binding pro-teins isolated from rat liver membranes, p60 and p90, have beenpartially sequenced.We now report that the N-terminal sequenceof p60 exhibits 95% identity to OST-48, a 48-kDa member of theoligosaccharyltransferase complex found in microsomal mem-branes, while sequence analysis of p90 revealed 73% and 85%identity to the N-terminal and internal sequences, respectively, ofhuman 80K-H, a 80- to 87-kDa protein substrate for proteinkinase C. AGE-ligand and Western analyses of purified oligo-saccharyltransferase complex, enriched rough endoplasmic re-ticulum, smooth endoplasmic reticulum, and plasma membranesfrom rat liver or RAW 264.7 macrophages yielded a singleprotein of 50 kDa recognized by both anti-p60 and anti-OST-48antibodies, and also exhibited AGE-specific binding. Immuno-precipitated OST-48 from rat rough endoplasmic reticulumfractions exhibited both AGE binding and immunoreactivity toan anti-p60 antibody. Immune IgG raised to recombinantOST-48 and 80K-H inhibited binding of AGE-bovine serumalbumin to cell membranes in a dose-dependent manner. Immu-nostaining and flow cytometry demonstrated the surface expres-sion of OST-48 and 80K-H on numerous cell types and tissues,including mononuclear, endothelial, renal, and brain neuronaland glial cells. We conclude that the AGE receptor componentsp60 and p90 are identical to OST-48, and 80K-H, respectively,and that they together contribute to the processing ofAGEs fromextra- and intracellular compartments and in the cellular re-sponses associated with these pathogenic substances.

Glucose and other reducing sugars react spontaneously with awide spectrum of proteins and lipids to initiate a posttransla-tional, nonenzymatic modification process called advancedglycation which yields a heterogeneous group of irreversibleadducts called advanced glycation endproducts (AGEs) (1).Numerous studies show that AGEs are important contributorsto the structural and functional alterations that occur insenescence and long-term diabetes (2-6).The removal of AGE-modified proteins is facilitated

through specific cell surface receptors identified first on cellsof the monocyte/macrophage lineage (7, 8) and subsequentlyon endothelial cells, mesangial cells, fibroblasts, and neuralcells (9, 10). These receptors were found to be distinct fromother receptors associated with the disposal of a variety ofenzymatically and chemically modified macromolecules, suchas the scavenger receptor and the mannose/fucose receptor (7,8, 11). In addition to the uptake and degradation of AGE-

modified proteins by macrophages, interactions betweenAGE-ligands, and receptors induce a range of biologicallyimportant responses, including chemotaxis, cell activation, andcytokine and growth factor secretion (12, 13). These propertieshave led to the hypothesis that the AGE receptor is likely toconsist of more than one component to participate in bothendocytic and cell-activation pathways, and thus playing animportant role in normal growth and tissue turnover.The first AGE-associated polypeptide, of -90 kDa, was iden-

tified by AGE-specific affinity precipitation of cell surface pro-teins from the murine macrophage cell line RAW 264.7 (14, 15).Two AGE-binding proteins, designated p60 and p90, were iso-lated from rat liver membranes, with apparently novel N-terminalsequences (11). Antibodies raised against p60 and p90 recognizedcell surface determinants on rat monocytes, macrophages, as wellas on other cells (11). These antibodies inhibited AGE bindingand neutralized AGE-dependent responses of human, rat, andmurine cells (10), suggesting that the AGE receptor systeminvolves highly conserved proteins. Subsequently, additionalmembrane or soluble AGE-binding proteins have been identi-fied, including RAGE (receptor for advanced glycation end-product) (16), galectin-3 and galectin-4 (17), lactoferrin (18), andlysozyme (19), further expanding the number of proteins linkedto AGE processing.

Unlike glycated proteins, the synthesis of N-linked glycopro-teins depends on specific catalytic enzymes, among which theoligosaccharyltransferase (OST-48) complex plays a pivotal rolein the transfer of a high mannose oligosaccharide chain(GlcNAc2Man9Glc3) to asparagine residues of polypeptidestransported across the rough endoplasmic reticulum (RER)membrane (20). Of the three components of this complex,ribophorin I and II have been identified within the rough but notthe smooth ER or Golgi system, and OST-48 activity can beinhibited by an antibody to ribophorin I (20, 21). The thirdcomponent of the complex, a 48-kDa nonglycosylated proteintermed OST-48, is not recognized by antibodies to ribophorin Ior II, and although the canine and human cDNA and the avianOST-48 protein have been obtained, enzymatic activity has notbeen conclusively linked to this component nor has its localizationbeen noted other than in RER (21).The plasma membrane-associated protein 80K-H was first

purified and subsequently cloned from a human carcinoma cellline (22). This acidic and heterogeneous protein of 80 kDa inhuman and 87 kDa in rat cells is reported to be an efficientphosphorylation substrate for protein kinase C, thus it may belinked to important signal transduction-related cellular events(23).

Abbreviations: AGE, advanced glycation endproducts; ER, endoplas-mic reticulum; RER, rough ER; SER, smooth ER; BSA, bovine serumalbumin; FITC, fluroescein isothiocyanate; NC, nitrocellulose mem-brane.§To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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In this report we describe the identification of OST-48 and80K-H proteins as plasma membrane components correspond-ing to the p60 and p90 AGE-binding proteins, respectively.

MATERIALS AND METHODSChemicals, Reagents and Cell Lines. Bovine serum albumin

(BSA), chicken ovalbumin, D-glucose, sucrose, and protein A-conjugated Sepharose 4B were purchased from Sigma. Syntheticpeptides corresponding to the N-terminal portions of the p60 andp90 proteins were synthesized at the Protein Sequencing Facilityof The Rockefeller University, and antisera thereto generated inrabbits after conjugation of the peptides to keyhole limpethemocyanin. Antiserum to the C-terminal portion of canineOST-48 was developed as described (21). The native canineoligosaccharyltransferase complex was purified as previouslydescribed to approximate 90% purity on SDS/PAGE (20). Thehuman cell lines U937 (macrophage-like), Jurkat (T cell),U87MG (glioblastoma), and murine RAW 264.7 (macrophage)and A20 (B lymphoblast) were obtained from the American TypeCulture Collection. Human astroglial cells were prepared fromfetal human brain as described (24) and purity as measured bypositive staining of glial fibrillary acidic protein was >95%.Human umbilical vein endothelial cells were isolated from freshumbilical cords by collagenase digestion (25) and were charac-terized by von Willebrand factor immunostaining. Human pe-ripheral blood mononuclear cells were isolated from whole bloodas described (9). Mouse kidney glomerular mesangial cells wereprepared as described (26).AGE-BSA and chicken ovalbumin (AGE-ovalbumin) were

prepared as described (27). The level ofAGE modification wasdetermined by AGE ELISA (27). Iodination of AGE-BSAwith sodium 1251-labeled iodide (NEN) was performed by thelodo-Beads method (Pierce) (11). The specific radioactivity ofthe labeled 1251-AGE-BSA was 1000 cpm/ng protein.

Preparation ofMembrane Fractions. Preparation ofER andGolgi fractions were performed as described (28). Briefly,livers were removed from male Sprague-Dawley rats afterperfusion, minced, and homogenized in 0.25 M sucrose con-taining 5 mM Tris-HCl (pH 7.4) and 1 mM MgCl2. Thehomogenate was centrifuged at 10,000 x g for 10 min and thesupernatant was centrifuged again at 50,000 x g for 60 min toisolate the total microsomal fraction. The pellet was suspendedin a mixture of 0.25 M and 2.5 M sucrose adjusted to arefractive index of 1.392. A 10 ml aliquot of the microsomalsuspension was overlaid on 10 ml of 2 M sucrose, and adiscontinuous sucrose gradient composed of 1.1, 0.6, and 0.25M sucrose was overlaid. After centrifugation for 16 h at82,000 x g, the materials at the 0.6/1.1, 1.1/1.37, and 1.37/2.0M sucrose interfaces were collected and termed enrichedGolgi, smooth ER (SER), and RER fractions, respectively,and fractions were resuspended in 0.25 M sucrose. To isolatethe plasma membrane fraction (29), liver homogenate wascentrifuged at 280 x g for 5 min and the supernatant wascentrifuged again at 1550 x g for 10 min. The pellet wasresuspended in 35 ml of 1.42 M sucrose, and 4 ml of 0.25 Msucrose was overlaid. After centrifugation for 1 h at 82,000 xg, the interface was collected as an enriched plasma membranefraction. Membrane proteins were solubilized by adding 0.5%deoxycholic acid. All samples were stored at -20°C. Using anantibody raised against the p90 N-terminal peptide sequence(11), p90 was repurified from rat liver membrane fractions byDEAE chromatography followed by two-dimensional PAGE.The N-terminal and internal sequences were determined bymicrosequencing at The Rockefeller University.

Ligand Blotting and Dot Blotting. Ligand blotting was carriedout as described (30). Briefly, proteins were transferred to anitrocellulose membrane, blocked with 1% BSA in PBS for 1 h,and blotted with 4 x 106 cpm 1251-AGE-BSA for 1 h at 4°C.Finally, the blots were rinsed three times with washing buffer,air-dried, and exposed to X-ray film for 2-24 h. Dot blotting wasperformed similarly as above after immobilizing protein samplesin 1 x sample buffer directly onto nitrocellulose membranes.

Immunoblotting and Immunoprecipitation. Samples wereelectrophoresed and transferred onto nitrocellulose membranesas described above. The membranes were blocked with 2% drymilk in washing buffer for 1 h, washed twice, and incubated withprimary antibodies or control antibodies (1:500) in washing bufferfor 1 h. The membranes were then incubated with 1:5000 alkalinephosphatase-conjugated anti-rabbit IgG (Sigma). After threefinal washes, bands were visualized by incubating with substratebuffer for 5-15 min. Solubilized rat RER membrane proteins(100 jig) were incubated with 5 ,ul anti-OST-48 C terminalpeptide antiserum (21) or normal rabbit serum in 100 ,lI PBSbuffer for 2 h at 4°C on a shaker. After three washes with PBSbuffer, 20 ,lI of a protein A-conjugated Sepharose 4B slurry wasadded to each sample and the mixture was incubated overnight.The Sepharose beads were washed three times, and the immu-noprecipited proteins were dissociated by adding 50 ,l reducingSDS sample buffer and boiling for 3 min. The supernatants wereanalyzed by SDS/PAGE.

Expression of Recombinant Proteins and Production of An-tisera. The coding sequence for the canine OST-48 matureprotein was amplified from a plasmid containing OST-48 cDNA(21) by PCR, cloned into a pCR II vector (Invitrogen), andsubcloned into a pET 23a expression vector (Novagen). ThecDNA for human 80K-H mature protein (22) was directly sub-cloned into a pET 23a vector. Both clones were transformed intoEscherichia coli, and protein expression was induced by isopropylf3-D-thiogaloctoside following the supplier's protocol. Recombi-nant proteins found in inclusion bodies were subsequently puri-fied by urea extraction. The identities of the expressed proteinswere confirmed by sequencing the first 20 N-terminal amino acidsat North Shore University Hospital. Purified recombinant pro-teins were injected into rabbits with adjuvant, and antisera againstrecombinant OST-48 and 80K-H were isolated after 3 months.IgG fractions were purified by Protein-G columns (Pharmacia).The specificity of these antibodies was confirmed by Western blotand titers were above 1:10,000 as estimated using a conventionalELISA.

Antibody Blocking Study. U87MG cells were grown inDMEMwith 10% fetal calf serum, and a membrane fraction was preparedas described (31). Membrane protein (2.7 gg) was dot-blottedonto Immobilon-P filters (Millipore). The blots were preblockedwith binding buffer (Hank's balanced salt solution with Ca2+,Mg2+, 25 mM Hepes, and 0.5 mg/ml BSA) and then incubatedwith various dilutions of rabbit anti-rOST-48 anti-r8OK-H, orcontrol rabbit IgG (Sigma). Subsequently, 5 jig 125I-AGE-BSAwas added to each dot and incubated at room temperature for 90min.Flow Cytometric Analysis. Cells were first incubated with

antiserum or preimmune serum (100 pll, diluted 1:50 with PBS)and then with 1:100 fluroescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG F(ab')2 (Cappel Laborato-ries). Stained cells were examined in a flow cytometer (CoulterInstruments) (32).

Immunocytochemistry. All cultures were grown to confluencyon chamber slides, washed in cold PBS, and fixed for 20 min in4% paraformaldehyde in PBS. Following incubation with the IgGfraction of anti-rOST-48 or anti-r8OK-H for 18 h at 4°C, the cellswere exposed to biotinylated secondary antibody (Amersham)for 1 h then to streptavidin FITC for a further 1 h. Normal rabbitIgG was used as a negative control.

RESULTSThe original, partial sequences of AGE-binding proteins p60and p90 purified from rat liver membranes failed to showsignificant homology to any known proteins (11). A recentsearch against nonredundant protein data bases at the Na-tional Center for Biotechnological Information (34) showedthat the N-terminal sequence of rat p60 shares a high homol-ogy with the deduced protein sequence of OST-48, a 48-kDaprotein subunit of the canine pancreas oligosaccharyltrans-ferase complex (21). Partial N-terminal sequence comparisons

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A. N-TerminalRat AGEBP p6O X G P R T L V L L D N L N V R D T H X L F FCanine OST-48 S - - - - - - - - - - - - L -E - --Avian OST-50 S - - - S - - - - G - - - L - - - - S - - -Hum OST-48 s - - - - - - - - - - - - - - S - - -

B. N-TerminalRatAGEBP p9O X I V K L P D M V S L X DHuman 80 K-H V--- R - R G - - - T -

C.JntemalRatAGEBP p9O X P P Y D B E T E A I I D A A Q E A R N K

Human80K-H N- - - Q - Q - F - - - - - - - - - -

FIG. 1. Partial amino acid sequence comparisons of AGE-bindingproteins (AGEBP) p60 and p90 with OST-48 and 80K-H, respectively.(A) The N-terminal sequence of rat p60 is aligned with the N-terminalsequences of canine (21), human,1 and avian (35) OST-48, indicatinga 90%, 90%, and 95% sequence identity, respectively. (B) TheN-terminal sequence of rat p90 is aligned with the N-terminal ofhuman 80K-H, indicating a 73% identity. (C) The internal sequenceof rat p90 aligned with the corresponding sequences of human 80K-H(85% identity).

between rat p60 and canine, avian (35), and human OST-481are shown in Fig. LA, indicating a sequence identity of 90%,90%, and 95%, respectively.When the canine oligosaccharyltransferase complex puri-

fied from pancreas RER (20) and an irrelevant protein such asinsulin were immobilized on a nitrocellulose membrane (NC)and incubated with 125I-AGE-BSA, the RER oligosaccharyl-transferase complex, but not insulin, bound 125I-AGE-ligand ina manner proportional to the amount of adsorbed protein (Fig.24). Binding could be inhibited by excess unlabeled AGE-BSA, which suggested the presence of epitopes interactingspecifically with AGEs in this fraction. When enriched rat liverRER, SER, and Golgi membrane fractions were subjected toSDS/PAGE and stained with antiserum to the N-terminalpeptide of AGE-binding protein p60 (Fig. 2B, lanes 1-3) orantiserum to the C terminal peptide of OST-48 (Fig. 2B, lanes4-6), both antibodies recognized a single --50-kDa proteinwhich is abundant in RER, less so in SER, and absent fromGolgi. Binding of 125I-AGE-BSA to immobilized RER wasreadily inhibited in the presence of 50-fold excess unlabeledAGE-BSA or AGE-ovalbumin, but not by unmodified BSA or

¶ Data from N. Miyajima (1994), GenBank accession no. D29957.

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by the synthetic early glycation product, 1-deoxy-1-propylamino fructose (Amadori product), ruling out nonspe-cific binding or possible interactions with early glycationstructures present in the AGE preparations (Fig. 2C). Uponimmunoprecipitation of OST-48 from rat liver RER mem-brane fractions using either anti-OST-48 or normal rabbitserum, followed by SDS/PAGE and transfer onto NC mem-branes for parallel Western and ligand blot analysis, a similarsingle -50-kDa protein species was identified by both methodsin the anti-OST-48-precipitated material, but not in normalrabbit serum-precipitated samples (Fig. 2D), further indicatingthat a single molecule recognizable by anti-OST-48 possessedAGE-specific binding property.Thus far, OST-48 has been described only as a RER

membrane protein (20). To investigate its expression on thecell surface, plasma membranes were isolated and enrichedfrom rat liver using sucrose gradient centrifugation. Proteinswere separated by SDS/PAGE then electrotransferred ontoNC membranes and subjected to 125I-AGE-ligand and Westernanalysis using antibodies to either the p60 N-terminal peptideor the OST-48 C-terminal peptide. Both anti-p60 and anti-OST-48, but not normal rabbit serum, recognized with equalavidity a single protein of identical size, '50 kDa (Fig. 3, lanes3, 4, 5) which corresponded to an area with strong AGE-binding activity (Fig. 3, lane 2), likely to represent the previ-ously reported AGE-binding protein p60.To expand on whether p60/OST-48 and p90/80K-H pro-

teins in fact participate in the binding and processing ofAGEs,the corresponding recombinant proteins were obtained afterPCR amplification of the coding sequence, subcloned into apET 23a expression vector, and expressed in E. coli. Followingconfirmation of the correct sequence identities of the ex-pressed proteins by protein sequencing, they were used to raisepolyclonal antisera. In each case, antisera were tested againstrOST-48 and r8OK-H immobilized on microtiter plates andwere found to recognize the corresponding antigen with a titerof above 1:10,000 for both antisera.To test whether anti-rOST-48 and anti-r8OK-H recognized any

plasma membrane components corresponding to the AGE-binding proteins p60 and p90 found on macrophage/monocytes(11), Western analysis was performed on murine RAW 264.7 cellmembrane fractions. A single polypeptide with molecular weightof -50 kDa was visualized by anti-rOST-48 serum (Fig. 4, lane 2)and another of '80 kDa by anti-r8OK-H serum (lane 4), while no

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FIG. 2. AGE-modified protein binding to canine and rat liver microsomal OST-48. (A) Ligand dot-blot analysis of purified canine RERoligosaccharyltransferase complex or a control peptide (insulin). Dot blots were incubated with 125I-AGE-BSA (1 jig/ml) in the presence (+) orabsence (-) of 150-fold excess of unlabeled AGE-BSA. (B) Western analysis of rat liver microsomal RER (lanes 1 and 4), SER (lanes 2 and 5),and Golgi (lanes 3 and 6) membrane fractions using anti-N-terminal p60 antibody (lanes 1-3) or anti-C-terminal OST-48 antibody (lanes 4-6). (C)Competitive inhibition of 1251-AGE-BSA (1 ,ug/ml) binding to rat liver RER immobilized on NC membranes at 10 and 100 ,ug/dot by 100-foldexcess unlabeled AGE-BSA, AGE-ovalbumin, D-glucose (100 mM), Amadori product (1-deoxy-1-propylamino fructose, 1 mM), or 100-fold excessnative BSA. (D) Immunoprecipitation of rat liver RER OST-48 by anti-C-terminal OST-48 antibody and subjected to Western analysis usinganti-OST-48 antibody or nonimmune rabbit serum (NRS), and to ligand blot analysis using 125I-AGE-BSA.

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immunoreactive species were observed with corresponding pre-immune serum (Fig. 4, lanes land 3).The surface expression of OST-48 was determined by flow

cytometric analysis of various cell lines of murine and humanhematopoietic lineage, using anti-rOST-48 or anti-p60 N-terminal peptide antibodies. RAW 264.7, U937, and Jurkat,but not A20, cells stained positively with anti-p60 serumcompared with background staining with preimmune serum(Fig.5 B, C, D, andA). Moreover, to confirm specificity, whensynthetic p60 N-terminal peptide was added to the incubationmixture containing anti-p60 N-terminal peptide antibodies tostain Jurkat cells (Fc receptor negative), fluorescence intensitywas markedly diminished (Fig. SD). Consistent with the pos-itive staining of hematopoietic cells with anti-N-terminal p60peptide antibody, anti-rOST-48 antiserum positively stainedRAW 264.7 cells (Fig. SE).

In addition to and consistent with the binding specificity ofOST-48 for AGE moieties, anti-rOST-48 IgG in a dose-dependent manner blocked binding of 125I-AGE-BSA to plasmamembrane fractions from U87MG glial cells immobilized on NCmembranes, whereas the control IgG did not (Fig. 6).To determine whether cells from other tissues and different

species contained cell surface epitopes similar to OST-48,indirect immunofluorescence was performed with non-permeabilized human peripheral mononuclear cells, humanumbilical vein endothelial cells, mouse mesangial cells, andprimary human astroglial cells. Using anti-rOST-48, we ob-served a pattern of diffusely punctate staining on most cells(Fig. 7A, C, E, and G), further confirming the presence of thisprotein on the cell surface.Based on an identical isolation procedure employing AGE-

affinity chromatography as previously described, or aftertwo-dimensional gel electrophoresis of freshly prepared ratliver membrane extracts and Western analysis using a previ-ously characterized anti-p90 antiserum (11), a protein of-80-90 kDa was obtained with the identical N-terminal se-quence to that reported previously (11). After the purificationprocedure which involved detergent solubilization under re-ducing conditions, this protein no longer carried AGE-bindingactivity, as observed previously. Protein sequence comparisonsof proteolytic digests from this polypeptide with recent data atthe National Center for Biotechnological Information re-vealed a 73% homology of the N terminus, and a 85% internalsequence homology, to 80K-H, a 80- to 87-kDa substrate forprotein kinase C (23) cloned from the human epithelial cellline Ca9-22 (22) (Fig. 1 B and C). To further characterize theproperties of this protein, recombinant human 80K-H(r8OK-H) was expressed in E. coli as described above forOST-48. The identity of expressed r8OK-H was confirmed byprotein sequencing and recognition by anti-p90 antibody byWestern blot analysis; however, r8OK-H remained insolubleand showed no AGE-binding activity in this form. ImmuneIgG raised against r8OK-H recognized a single '80-kDaprotein on immobilized plasma membrane extracts (Fig. 4), aswell as on the cell surface of the murine macrophage line 264.7by flow cytometry analysis (Fig. 5F). The anti-r8OK-H IgG

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FIG. 3. Expression of immunoreactive OST-48 on liver plasma mem-brane by Western analysis. Enriched plasma membrane fractions weresubjected to reducing SDS/PAGE. Protein staining with amido black(lane 1), ligand blotting with 1251-AGE-BSA (lane 2), or Western blottingwith anti-C-terminal OST-48 serum (lane 3), with anti-N-terminal p60serum (lane 4), or with nonimmune rabbit serum (lane 5).

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FIG. 4. Expression of immunoreactive OST-48 and 80K-H onmacrophage-like RAW 264.7 cell membrane by Western analysis.Membrane proteins from RAW cells were subjected to reducingSDS/PAGE and electrotransfer onto NC filters. Western blot analysiswere performed using nonimmune rabbit serum, anti-rOST-48, oranti-r8OK-H serum.

prevented in a dose-dependent manner 1251-AGE-ligand bind-ing to U87MG cell membrane extracts (Fig. 6).

Significant anti-r8OK-H immunoreactivity was also detectedon human peripheral blood mononuclear cells, human umbil-ical vein endothelial cells, murine mesangial cells, and humanastroglial cells (Fig. 7 B, D, F, H, and J). In contrast, nonim-mune IgG was without effect either in AGE-binding inhibitionstudies (Figs. 5 and 6) or immunostaining (data not shown).

DISCUSSIONSeveral studies have provided strong evidence for the existenceof AGE-specific receptors, the distribution and properties ofwhich are apparently widespread among cell types and well

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FIG. 5. Demonstration by flow cytometry of surface immunore-activity to p60, OST-48, and 80K-H by antibodies either to theN-terminal peptide of rat p60 (A-D), to rOST-48 (E), or to r80 K-H(F). Hematopoietic cell lines include B-lymphoblastoid A20 (A),human monocytic U937 (B), murine macrophage-like RAW 264.7 (C,E, and F), and human T-lymphocytic Jurkat cells (D). Preimmunerabbit serum (a, solid line) was used as a control to compare withspecific staining with antiserum (b, dotted line), while in the Jurkat cellexperiment, soluble N-terminal p60 peptide was added to the stainingmixture to adsorb anti-N-terminal p60 (c, dashed line). Cells werestained with FITC-conjugated anti-rabbit IgG [F(ab')2].

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FIG. 6. Inhibitory effect of anti-rOST and anti-r8OK-H IgG on1251-AGE-BSA binding by U87MG plasma cell membrane. Immobi-lized cell membrane extracts were blocked with binding buffer andpreincubated with immune or pre-immune rabbit IgG (columns: 1, 150,tg; 2, 250 ,ug; 3, 500 ,ug IgG/ml) prior to adding 12-I-AGE-BSA in thepresence or absence of 100-fold excess unlabeled AGE-BSA. Data are

expressed as percent of maximal control binding and represent themean ± SD of triplicate experiments.

conserved among species (6, 7, ¶). Data from our earlier worksuggested that at least two distinct AGE-binding proteinspurified from rat liver plasma membrane are components ofthis receptor, an '50- to 60-kDa protein termed p60 and an-80- to 90-kDa protein termed p90 (11). The initial sequenceshave now been confirmed, and although initially reported asunique proteins, subsequent comparisons to recent sequencedata bases revealed that p60 is identical to OST-48 (21), andp90 is identical to 80K-H, a substrate for protein kinase C (22).

In addition to sequence homology, further experimental evi-dence supports the identity of p60 as OST-48. First of all,

AGE-specific binding activity was found in both the purifiedOST-48 and in RER membrane fractions, and colocalized withina single '50-kDa protein species recognized by antibodies raisedagainst the synthetic peptides of p60 and OST-48, as well asagainst rOST-48. Also, a single AGE-binding protein of similarsize could be immunoprecipitated from microsomal membranesby anti-OST-48 antibody.

Second, OST-48 is now also found in plasma membraneextracts, comigrating with a species of identical molecular weightwhich bound AGE-ligand and crossreacted with antibody to asynthetic N-terminal peptide of p60. A similar '50-kDa immu-noreactive protein species was identified on macrophage-like cellline RAW 264.7, on intact macrophages, or on human monocytesand lymphocytes, also known to express p60 (10, 11, 15). Flowcytometry analysis further suggested that the N-terminal portionof OST-48 may represent the extracellular domain of this mem-brane protein, since these cells were also positively stained withanti-p60 N-terminal peptide antibodies. Anti-rOST-48 antibodyblocked the binding, of AGE-BSA to cell membrane extractsindependently of, or in combination with, anti-r8OK-H, establish-ing a closer structure/function identity for these molecules asactive participants in AGE-ligand binding.More importantly, the findings indicate that OST-48, though

initially identified as part of a complex catalyzing the transfer ofhigh mannose oligosaccharides onto asparagine-acceptor sites ofproteins within the RER (20), also exists on the cell surface andfunctions as an AGE-protein transporter, thus having a dualfunction. Although the enzymatic function or role of OST-48 hasnot been clearly demonstrated, duality of function is not incon-sistent with that of other examples, including the mannose-6-phosphate receptor, also known as the insulin-like growth factorII receptor, which apparently participates in both intracellular aswell as in surface-specific functions (37). The C-terminal segmentof OST-48 contains a di-lysine motif (two lysines positioned threeand four residues from the C terminus) which has been proposedas an ER retention sequence (38). However, a protein data basesearch indicated that not onlyER proteins but also certain plasmamembrane proteins contain this specific motif, including the

FIG. 7. Immunocytochemical analysis for cell surface expression of OST-48 (Upper) and 80K-H (Lower). Murine RAW (A and B), humanperipheral mononuclear cells (C and D), human umbilical endothelial cells (E and F), human astroglial cells U87MG (G and H), and murinemesangial cells (I andJ) were stained with the IgG fraction of anti-rOST, anti-r8OK-H, or pre-immune IgG, and then with the biotinylated secondaryantibody and streptavidin-FITC. (A-D, G, and H, X400; E, F, I, and J, X200.)

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Proc. Natl. Acad. Sci. USA 93 (1996)

constant region of the T-cell receptor. This argues that othermechanisms may be involved in determining ER retention ortrafficking from the ER compartment to the cell surface.

After establishing that AGE-binding protein p90 was ho-mologous to 80K-H (22), specific immune IgG preparedagainst the recombinant form of the human protein readilycrossreacted with the N-terminal peptide of rat liver p90.Similar epitopes were identified on macrophage membraneextracts and on the surface of various cell types and tissues ofdiverse species, including human arterial tissue, rat retina,mesangial and tubular cells of mouse kidney, and tyramadl-neurons and glial cells of human brain (data not shown), inagreement with earlier evidence of the well-conserved natureof the AGE receptor system (7). This acidic protein, which hasan apparent mass of 87 kDa in rat and '80 kDa in human cells,contains many short repeats and several phosphorylation andglycosylation sites. Although also present in the cytosol, 45%of the total cellular 80K-H is membrane-bound and becomesparticularly prominent during cellular mitogenic activation(23). Of note, anti-r8OK-H IgG also inhibited the binding ofAGE onto cell membrane extracts. Yet, our data thus far donot directly support inherent AGE-binding properties of80K-H per se (17). It is thus possible that p90/80K-H mayparticipate in receptor stabilization by association with otherreceptor components.More importantly, this protein may be involved in AGE-

mediated cellular activation (6). The 80K-H protein is alreadydescribed as a prominent kinase C-mediated phosphorylationsubstrate found in numerous cells (22, 23). This points towardan active role of 80K-H in AGE receptor signaling andautoregulation under conditions requiring enhanced receptorefficiency via transmission of intracellular signals.

It has been suggested that AGE receptor upregulationoccurs via an autocrine pathway involving enhanced tumornecrosis factor a (12) and tumor necrosis factor a-receptorexpression (39). Among these, a 35-kDa AGE-binding proteinisolated from bovine lung referred to as RAGE (16) oramphoterin-binding protein (40), also has been linked to geneactivation and enhanced oxidant stress mediated by AGEs(41). We found neither sequence nor immunological homologybetween RAGE and either OST-48 or 80K-H, using anantibody raised against the N-terminal peptide of RAGE (datanot shown). However, a number of AGE-binding proteins arelikely to exist which interact with the heterogeneous popula-tion of AGE structures. This may account for the incompleteblocking of ligand binding to plasma membranes by antibodiesto OST-48 and 80K-H. Furthermore, we have not ascertainedwhether OST-48 or 80K-H exhibit binding specificities forother ligands, such as acetyl- or malonaldehyde-modifiedproteins. However, we concur with findings suggesting thatcertain classes of AGE moieties may interact with other cellsurface receptor proteins known for their nonspecific bindingto negatively charged compounds, such as the scavengerreceptors (36). This property is known to be shared by theAGE receptor (8) and numerous other proteins.Taken together, the data presented here suggest that two

highly conserved and widely distributed protein molecules,already linked to other functions, may be key regulators ofcellular interactions with the naturally forming extra- andintracellular products of advanced glycation. These observa-tions provide an important step in further probing novelaspects of this universally damaging process, in both diabeticand normal adult human tissues.We thank Dr. Reid Gilmore (University of Massachusetts Medical

School) for providing antibody and cDNA for canine OST-48, Ms. LillianSellati, and Dr. Robert Pergolizzi (North Shore University Hospital) forprotein sequencing. These studies were supported in part by NationalInstitutes of Health Grants AG06943 and AG09453 to H.V. and aResearch Award from American Diabetes Association to Y.M.L.

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