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Purification and Characterization of a Neu5Acα2–6Galβ1–4Glc/GlcNAc-specific Lectin from the Fruiting Body of the Polypore Mushroom Polyporus squamosus *

  • Hanqing Mo
    Affiliations
    Department of Biological Chemistry, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0606
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  • Harry C. Winter
    Affiliations
    Department of Biological Chemistry, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0606
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  • Irwin J. Goldstein
    Correspondence
    To whom correspondence should be addressed: Dept. of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0606. Tel.: 734-763-3511; Fax: 734-763-4581
    Affiliations
    Department of Biological Chemistry, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0606
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  • Author Footnotes
    * This work was supported by Grant GM29470 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:April 07, 2000DOI:https://doi.org/10.1074/jbc.275.14.10623
      A lectin has been purified from the carpophores of the mushroom Polyporus squamosus by a combination of affinity chromatography on β-d-galactosyl-Synsorb and ion-exchange chromatography on DEAE-Sephacel. Gel filtration chromatography, SDS-polyacrylamide gel electrophoresis, and N-terminal amino acid sequencing indicated that the native lectin, designated P. squamosus agglutinin, is composed of two identical 28-kDa subunits associated by noncovalent bonds. P. squamosus agglutinin agglutinated human A, B, and O and rabbit red blood cells but precipitated only with human α2-macroglobulin, of many glycoproteins and polysaccharides tested. The detailed carbohydrate binding properties of the purified lectin were elucidated using three different approaches,i.e. precipitation inhibition assay (in solution binding assay), fluorescence quenching studies, and glycolipid binding by lectin staining on high-performance thin layer chromatography (solid-phase binding assay). Based on the results obtained by these assays, we conclude that although the P. squamosus lectin binds β-d-galactosides, it has an extended carbohydrate-combining site that exhibits highest specificity and affinity toward nonreducing terminal Neu5Acα2,6Galβ1,4Glc/GlcNAc (6′-sialylated type II chain) of N-glycans (2000-fold stronger than toward galactose). The strict specificity of the lectin for α2,6-linked sialic acid renders this lectin a valuable tool for glycobiological studies in biomedical and cancer research.
      HPTLC
      high-performance thin layer chromatography
      Me
      methyl
      MES
      2-(N-morpholino)ethanesulfonic acid
      NP
      nitrophenyl
      PAGE
      polyacrylamide gel electrophoresis
      PBS
      phosphate-buffered saline
      PSA
      P. squamosus agglutinin
      Lectins are proteins (or glycoproteins), other than antibodies and enzymes, that bind specifically and reversibly to carbohydrates, resulting in cell agglutination or precipitation of glycoconjugates (
      • Goldstein I.J.
      • Hughes R.C.
      • Monsigny M.
      • Osawa T.
      • Sharon N.
      ). They are ubiquitous in the biosphere, having been found in viruses, bacteria, fungi, plants, and animals (
      • Kocourek J.
      ).
      Lectins of known specificity recognizing sialic acid serve as valuable reagents in glycobiological research. They can be employed for the detection and preliminary characterization of sialic acid-containing glycoconjugates on the surface of cells and for assaying the incorporation of sialic acid into complex carbohydrates in biosynthetic studies. In their immobilized form, these lectins can be used for the resolution and isolation of sialic acid-containing glycoconjugates. Lectins are found in greatest quantity and are most readily purified from plant sources, especially higher plants, although relatively few sialic acid-binding lectins have been identified in the plant world (including fungi), which lacks sialic acid.
      During the last decade, there has been a growing interest in fungal lectins, largely due to the discovery that some of these lectins exhibit antitumor activities, e.g. Volvariella volvacea lectin shows antitumor activity against sarcoma S-180 cells (
      • Lin J.Y.
      • Chou T.B.
      ), Grifola frondosa lectin is cytotoxic to Hela cells (
      • Kawagishi H.
      • Nomura A.
      • Mizuno T.
      • Kimura A.
      • Chiba S.
      ), Agaricus bisporus lectin possesses antiproliferation activities against human colon cancer cell lines HT29, breast cancer cell lines MCF-7 (
      • Yu L.G.
      • Fernig D.J.
      • Smith J.A.
      • Milton J.D.
      • Rhodes J.M.
      ), and Tricholoma mongolicum lectin inhibits mouse mastocytoma P815 cells in vitro and sarcoma S-180 cells in vivo (
      • Wang H.X.
      • Ng T.B.
      • Ooi V.E.C.
      • Liu W.K.
      • Chang S.T.
      ). Fungal lectins have recently been reviewed (
      • Kawagishi H.
      ,
      • Guillot J.
      • Konska G.
      ,
      • Wang H.X.
      • Ng T.B.
      • Ooi V.E.C.
      ). However, apart from the lectin from A. bisporus, which binds to Galβ1,3GalNAcα-Ser/Thr (T-disaccharide) (
      • Irazoqui F.J.
      • Vides M.A.
      • Nores G.A.
      ), the detailed carbohydrate specificities of these fungal lectins have not been investigated in depth.
      We report herein the purification and characterization ofPolyporus squamosus lectin (designated PSA), a Neu5Acα2–6Galβ1–4Glc/GlcNAc-specific lectin present in the carpophores (fruiting bodies) of this member of the Polyporaceae family.

      MATERIALS AND METHODS

      Carpophores of P. squamosus (Huds.) Fr. were collected in late summer 1998 from a decaying Ulmus stump in Ann Arbor, Michigan. A voucher specimen (Goldstein (MICH) 27953) was deposited in the University of Michigan herbarium.
      Unless stated otherwise, saccharides, their derivatives, and glycoproteins (including fetuin, asialofetuin, transferrin, thyroglobulin, α2-macroglobulin, α1-acid glycoprotein, bovine mucin, etc.) were purchased from Sigma. Ovine submaxillary mucin was a gift of Dr. R. N. Knibbs (University of Michigan). Except for asialofetuin, asialoglycoproteins were prepared by heating the corresponding native glycoproteins in 0.1 mhydrochloric acid at 80 °C for 1 h, followed by dialysis and lyophilization; the removal of sialic acid was confirmed by the thiobarbituric acid assay (
      • Warren L.
      ).
      Neutral glycolipids and gangliosides were purchased from Matreya, Inc. (Pleasant Gap, PA), globopentaosyl ceramide (Forssman glycolipid) was a generous gift of Dr. S.-I. Hakomori (Biomembrane Institute, Seattle, WA), aluminum-backed HPTLC1sheets (HPTLC-Alufolien Kieselgel 60) were from E. Merck (Darmstadt, Germany), and EZ-Link NHS-LC-biotin (succinimidyl 6-(biotinamido) hexanoate) was a product of Pierce. Alkaline phosphatase-streptavidin was from Zymed Laboratories Inc. (San Francisco, CA).
      Galβ-O-(CH2)8CONH-Synsorb (β-d-galactosyl-Synsorb) was the product of Chembiomed Ltd. (Edmonton, Alberta, Canada), Bio-Gel P-150 (50–100 mesh) was from Bio-Rad, and DEAE-Sephacel was obtained from Amersham Pharmacia Biotech.
      Methyl 3-O-β-d-galactopyranosyl-2-acetamido-2-deoxy-β-d-glucopyranoside (Galβ1, 3GlcNAcβ1-OMe) and methyl 4-O-β-d-galactopyranosyl-2-acetamido-2-deoxy-β-d-glucopyranoside (Galβ1, 4GlcNAcβ1-OMe) were synthesized in this laboratory.
      Molecular mass standards used in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (i.e. BenchMark protein ladders) and alkaline phosphatase substrate package (i.e.5-bromo-4-chloro-3-indolylphosphate p-toluidine salt and nitroblue tetrazolium chloride) were from Life Technologies, Inc.

      Purification of the Lectin

      All procedures were conducted at 4 °C. The fruiting bodies from P. squamosus (fresh weight, 80 g) were homogenized and extracted overnight with 400 ml of extraction buffer (10 mm sodium phosphate, 0.15m NaCl, 0.135 mm CaCl2, 0.04% sodium azide, pH 7.2 (PBS), containing 10 mm thiourea, 0.25 mm phenylmethylsulfonyl fluoride, and 1 g/liter ascorbic acid). The homogenate was squeezed through two layers of cheesecloth and centrifuged at 10,000 × g for 30 min. To the supernatant solution was added solid ammonium sulfate to 25% saturation. After standing overnight, the precipitate was removed by centrifugation, and the supernatant solution was applied directly onto a β-d-galactosyl-Synsorb 100 column (18 × 1.2 cm; bed volume, 80 ml), which had been equilibrated with 10 mmPBS (pH 7.2) containing 1 m ammonium sulfate. The column was washed with the same solution until the absorbance at 280 nm of the effluent had fallen below 0.01. The affinity-adsorbed lectin was desorbed with 0.2 m lactose in 10 mm PBS, collected, dialyzed extensively against distilled water, and lyophilized (designated crude PSA). Approximately 18 mg of crude lectin was obtained from 80 g of fresh fruiting body.
      Crude lectin was reconstituted in 50 mm phosphate buffer, pH 7.8, and percolated slowly through a DEAE-Sephacel column (17 × 1.2 cm, bed volume 75 ml) preequilibrated and eluted with the same buffer (50 mm phosphate buffer, pH 7.8). The elution was monitored by absorbance at 280 nm until it become negligible, whereupon the adsorbed protein was eluted with 1 m sodium chloride. Both the DEAE-unbound (pass through) and DEAE-bound peak fractions were collected, dialyzed against distilled water, lyophilized, reconstituted in 10 mm PBS, pH 7.2, and tested for electrophoretic homogeneity and agglutination activity.

      Protein Estimation

      Protein concentration was determined by the method of Lowry et al. (
      • Lowry O.H.
      • Rosebrough N.J.
      • Farr A.L.
      • Randall R.J.
      ), using bovine serum albumin as a standard.

      PAGE and SDS-PAGE

      Native gel electrophoresis using a 12.5% slab gel was carried out in alkaline buffer system (Tris/glycine, pH 8.3) (
      • Davis B.J.
      ). SDS-PAGE in the presence and absence of β-mercaptoethanol using a 12.5% slab gel was conducted in Tris/tricine buffer system as described by Schagger and von Jagow (
      • Schagger H.
      • von Jagow G.
      ).

      Hemagglutination Assay

      The hemagglutinating activity of the lectin was determined by a 2-fold serial dilution procedure using formaldehyde-treated (
      • Nowak T.P.
      • Barondes S.H.
      ) human and rabbit erythrocytes as described previously (
      • Crowley J.F.
      • Goldstein I.J.
      ). The hemagglutination titer was defined as the reciprocal of the highest dilution still exhibiting hemagglutination.

      Molecular Mass and Molecular Structure

      The molecular mass and molecular structure of the purified PSA was determined by gel filtration and SDS-PAGE performed in the presence and absence of β-mercaptoethanol.
      Gel filtration chromatography of the lectin was carried out on a Bio-Gel P-150 column (1.45 × 120 cm; bed volume, 198 ml) operating at room temperature in PBS, pH 7.2, with or without 0.2m lactose, at a flow rate of 10 ml/h. Fractions of 10 min/tube (approximately 1.7 ml/tube) were collected and monitored atA 280. The column was calibrated with the following standard proteins: bovine γ-globulin (158 kDa), bovine serum albumin (67 kDa), chicken ovalbumin (45 kDa), equine myoglobin (17 kDa), and vitamin B-12 (1.35 kDa). Blue dextran 2000 was used for determination of the void volume of the column.

      Amino Acid Composition Analysis and N-terminal Sequence Analysis

      The amino acid composition and the N-terminal sequence of the purified lectin were analyzed by the Protein and Carbohydrate Structure Core facility on this campus as described previously (
      • Mo H.Q.
      • Van Damme E.J.M.
      • Peumans W.J.
      • Goldstein I.J.
      ,
      • Mo H.Q.
      • Goldstein I.J.
      ). Tryptophan was estimated spectrophotometrically in 6 mguanidine hydrochloride (
      • Edelhoch H.
      ). Sulfhydryl groups were estimated by release of 2-nitro-5-mercaptobenzoic acid (ε0 = 13, 600m−1 cm−1 at 410 nm) from 5,5′-dithiobis(2-nitrobenzoic acid).

      Quantitative Precipitation and Hapten Inhibition Assays

      Quantitative precipitation assays were performed by a microprecipitation technique as described previously (
      • Mo H.Q.
      • Van Damme E.J.M.
      • Peumans W.J.
      • Goldstein I.J.
      ). Briefly, varying amounts of glycoproteins or polysaccharides, ranging from 0 to 100 μg, were added to 10 μg of purified PSA in a total volume of 160 μl of PBS, pH 7.2. After incubation at 37 °C for 1 h, the reaction mixtures were stored at 4 °C for 48 h. The precipitates formed were centrifuged, washed three times with 150 μl of ice-cold PBS, dissolved in 0.05 m NaOH, and determined for protein content by Lowry's method using bovine serum albumin as standard.
      For hapten inhibition assays, increasing amounts of various haptenic saccharides were added to the reaction mixture consisting of 10 μg of the purified lectin and 5 μg of α2-macroglobulin in a final volume of 160 μl of PBS, pH 7.2. After incubation at 37 °C for 1 h and storage at 4 °C for 48 h, the precipitated proteins were centrifuged, washed, and determined. The percentage of inhibition was calculated, and inhibition curves were constructed. The minimum concentration of each haptenic sugar required for 50% inhibition was obtained from corresponding complete inhibition curves.

      Preparation of Biotin-Lectin Conjugate

      The biotinylation of the purified lectin was achieved using EZ-Link NHS-LC-Biotin according to the manufacturer's instructions, except that 0.2 mlactose was added to the reaction mixture to protect the carbohydrate-binding sites. After coupling, the lectin activity was ascertained by hemagglutination assay.

      HPTLC of Glycolipids and Lectin Staining

      Two identical sets of glycolipids were separated chromatographically in parallel on the same aluminum-backed silica gel 60 HPTLC plate (Merck, Darmstadt, Germany) using chloroform/methanol/water (65:25:4, by volume) for neutral glycolipids or chloroform/methanol/aqueous 0.25% KCl (50:40:10, by volume) for gangliosides as developing solvent. The reference chromatogram was chemically visualized by spraying the plate with orcinol reagent.
      The lectin staining was performed as follows: after drying, the plates were blocked by overlaying with 1% gelatin in PBS containing 0.1% NaN3 and incubating overnight at room temperature. The plates were then overlaid with biotin-labeled lectin diluted in PBS containing 1% bovine serum albumin and 0.05% Tween 20, and incubated at 37 °C for 2 h. After rinsing the plates five times with 10 mm Tris/HCl buffer, pH 9.5, containing 0.05% Tween 20, they were overlaid with alkaline phosphatase-streptavidin diluted with 0.1 m Tris/HCl, pH 9.5, containing 100 mm NaCl and 5 mm MgCl2, incubated at 37 °C for 1 h, washed five times with the same buffer, and finally visualized with 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium chloride.

      α2,6-Sialylation of Asialofetuin

      Asialofetuin, 2.5 mg (40 nmol) in 1.0 ml of 50 mm MES buffer, pH 6.0, containing 10 mm MnCl2, 0.15M NaCl, 10% glycerol, 2 μmol of CMP-sialic acid, and 10 milliunits of rat liver α2,6-sialyltransferase (Roche Molecular Biochemicals, 8 units/mg of protein) was incubated 24 h at 37o, followed by addition of another 1 μmol of CMP-sialic acid and 5 milliunits of sialyltransferase. After an additional 24 h of incubation, the reaction mixture was passed through a column (1 × 15 cm) of BioGel P-10. Fractions containing protein were pooled, dialyzed against distilled water, and lyophilized. The resialylated fetuin contained 3.1 mol of sialic acid/mol of protein, compared with a negligible amount (<0.1 mol of sialic acid/mol of protein) in the asialofetuin and approximately 10 mol of sialic acid/mol of protein in native fetuin.

      Absorbance and Fluorescence Spectra

      Ultraviolet absorbance spectra were recorded on a Shimadzu model UV160U spectrophotometer; fluorescence spectra were recorded on an ISA JodinYvon-Spex Fluoromax-2 spectrofluorometer.

      RESULTS

      Hemagglutinating Activity

      In a survey of mushrooms for hemagglutination activity by Pemberton (
      • Pemberton R.L.
      ), it was found that the extract of P. squamosus exhibited strong hemolytic activity. In the present study, by using hemolysis-resistant, formaldehyde-treated erythrocytes, we observed that the crude extract of P. squamosus contained a lectin(s) that agglutinated rabbit and human erythrocytes, irrespective of blood group type (type B erythrocytes were agglutinated slightly better than those of types A and O). The hemagglutinating activity was inhibited byd-galactose and d-galactose-related carbohydrates, such as d-fucose, l-arabinose, melibiose, and lactose.

      Purification of the Lectin

      Because the hemagglutination activity of the crude extract of P. squamosus was inhibited by d-galactose and lactose, β-d-galactosyl-Synsorb was used as an affinity absorbent for isolation of the lectin. As shown in Fig.1, upon nondenaturing PAGE at pH 8.3, the lectin preparation obtained from affinity chromatography on β-d-galactosyl-Synsorb showed two protein bands, which we later found could be separated from each other by ion exchange chromatography on DEAE-Sephacel in 0.05 m phosphate buffer, pH 7.8 (Fig. 1). Under these conditions, the major portion of the lectin activity was not retained on the DEAE column. This DEAE-unbound fraction was electrophoretically pure (designated purified PSA). The minimal concentration of the purified lectin required for the agglutination of formaldehyde-treated human type B erythrocytes was 0.6 μg/ml. Removal of divalent metal ions by extensive dialysis of this fraction against buffer containing 1.25 mm EDTA had little effect on its agglutination activity. The DEAE-bound fraction exhibited about 30 times lower agglutinating activity, 19 μg/ml being required for agglutination; therefore, in the present study, characterization of only the DEAE-unbound fraction was pursued.
      Figure thumbnail gr1
      Figure 1Native PAGE of PSA on a 12.5% gel in Tris/glycine running buffer at pH 8.3. Lane 1, crude lectin preparation from affinity chromatography on β-d-galactosyl-Synsorb containing two protein bands;lane 2, purified PSA from pass-through fraction from DEAE-Sephacel in 0.05 m phosphate buffer, pH 7.8;lane 3, The DEAE-bound fraction having low hemagglutinating activity.

      Molecular Mass and Molecular Structure

      Upon gel filtration chromatography on Bio-Gel P-150, the purified lectin eluted as a single, symmetric peak, irrespective of the presence of 0.2m lactose, at an elution volume corresponding to an apparent molecular mass of 52 kDa (not shown). On the other hand, upon SDS-PAGE, with or without β-mercaptoethanol, purified PSA gave a single band with an apparent mass of 28 kDa (Fig.2). Taken together, these data suggest that at neutral pH, the lectin exists as a homodimer of 28-kDa subunits associated by noncovalent bonds. No neutral carbohydrate was detected using the phenol-sulfuric acid assay.
      Figure thumbnail gr2
      Figure 2SDS-PAGE of purified PSA on a 12.5% slab gel in Tris/Tricine running buffer, pH 8.3, showing a single band with an apparent subunit mass of 28 kDa. The same result was obtained in the presence of β-mercaptoethanol. Left lane, purified PSA; right lane, molecular mass standards (BenchMark Protein Ladders) from Life Technologies, Inc.

      Amino Acid Composition and N-terminal Amino Acid Sequence

      As shown in Table I, purified PSA contains an extremely high proportion of hydrophobic amino acids (Ala, Ile, Leu, Val, and Phe) that account for one-third of the total amino acids, high contents of acidic and hydroxyl amino acids (Asx and Glx account for 22%; Ser and Thr, 14%), and relatively high amounts (11.5%) of aromatic amino acids, accounting for the high absorbency at 280 nm of the lectin (A 1% = 29.0). The lectin also contains two residues each of methionine and cysteine. No free sulfhydryl groups were detected by reaction with 5,5′-dithiobis(2-nitrobenzoic acid); together with the observation that β-mercaptoethanol has no effect on SDS-PAGE migration, the presence of an intrachain disulfide linkage is indicated. A single N-terminal amino acid sequence, H2N+-PFEGHGIYHIPSVNTANVRI, was determined. A search of the protein data base revealed no significant homology of this N-terminal sequence to any sequence in the data base.
      Table IAmino acid composition of purified P. squamosus agglutinin
      Amino acidMol %Residues/subunit
      Asx14.538
      Glx7.5320
      Ser6.5117
      Gly10.728
      His1.333
      Arg2.717
      Thr7.7220
      Ala9.6025
      Pro3.509
      Tyr2.948
      Val5.7115
      Met0.892
      Cys0.65
      Estimated by 5,5′-dithiobis(2-nitrobenzoic acid) after denaturation (6 m guanidine HCl) and reduction.
      2
      Ile4.2511
      Leu7.6520
      Phe5.5214
      Lys4.9413
      Trp3.18
      Estimated spectrophotometrically by the method of Edelhoch (19).
      8
      Total residues260
      Calculated molecular mass, 28,150–28,200 kDa
      a Estimated by 5,5′-dithiobis(2-nitrobenzoic acid) after denaturation (6 m guanidine HCl) and reduction.
      b Estimated spectrophotometrically by the method of Edelhoch (
      • Edelhoch H.
      ).

      Quantitative Precipitation and Precipitation Inhibition

      Inasmuch as the hemagglutinating activity of the crude extract of P. squamosus was specifically inhibited by galactose and the lectin was initially isolated by affinity chromatography on β-d-galactosyl-Synsorb, various glycoproteins were chemically desialylated to expose their penultimated-galactosyl residues and tested for their ability to precipitate the lectin. However, none of these asialoglycoproteins formed a detectable precipitate with the lectin. The galactomannan fromCassia alata, which contains multiple terminal α-d-galactosyl residues, also failed to precipitate with the lectin. To our great surprise, of the many native glycoproteins tested, including fetuin, transferrin, thyroglobulin, α1-acid glycoprotein, bovine mucin, and ovine submaxillary mucin, only human α2-macroglobulin, but not its desialylated form, gave a pronounced precipitation reaction with the lectin. Therefore, human α2-macroglobulin was employed as a precipitant in the inhibition assays.
      The results of sugar hapten inhibition are shown in TableII. Among the monosaccharides tested, only d-galactose, its derivatives, andd-galactose-related carbohydrates (i.e. d-fucose and l-arabinose) were inhibitory, whereas epimers of d-galactose (i.e. d-talose (C-2 epimer), d-gulose (C-3 epimer), and d-glucose (C-4 epimer)) were all noninhibitory up to 100 mm. However, the most striking observation was that both Neu5Acα2, 6Galβ1,4Glc (6′-sialyllactose) and Neu5Acα2,6Galβ1,4GlcNAc (6′-sialylLacNAc), but not their α2,3 isomers, were very strong inhibitors, being 2000-fold more inhibitory than d-galactose and 250–300 times stronger than lactose and LacNAc. Neither free N-acetylneuraminic acid, its p-nitrophenyl glycoside, nor its α2,8-linked polymer (colominic acid) was inhibitory.
      Table IIInhibition of precipitation of P. squamosus agglutinin with α2-macroglobulin by oligosaccharides
      Sugar
      N-Acetyl-glucosamine,d-arabinose, 2-deoxy-ribose, l-fucose,d-mannose, l- and d-rhamnose,l- and d-ribose, l- andd-xylose, cellobiose, chitobiose, gentiobiose, maltose, isomaltose, sucrose and trehalose were all noninhibitory up to 200 mm (cellobiose to saturation).
      IC50
      Minimum concentration required for 50% inhibition of the PSA/α2-macroglobulin precipitation reaction, unless otherwise noted.
      Relative potency
      mm
      Galactose30[1]
      GalαMe450.67
      GalβMe152.0
      p-nitrophenyl α-d-galactoside142.1
      p-nitrophenyl β-d-galactoside5.35.7
      d-Fucose400.75
      2-Deoxy galactose640.47
      GalNAc100 (3.4%)
      Maximum concentration tested (percentage of inhibition observed).
      <0.3
      l-Arabinose100 (15%)<0.3
      Me-β-l-arabinopyranoside100 (16%)<0.3
      6-OMe-d-Gal330.9
      d-Talose (C-2 epimer)100 (0%)0
      d-Glucose (C-3 epimer)100 (0%)0
      d-Glucose (C-4 epimer)100 (0%)0
      Lactose4.07.5
      LacNAc4.37.0
      p-nitrophenyl β-lactoside3.58.6
      Galβ1,4GlcNAcβMe3.87.9
      Galβ1,3GlcNAcβMe6.05.0
      T disaccharide (Galβ1,3GalNAc)6.24.8
      Galβ1,6Gal4.86.3
      Galβ1,4Gal10 (15%)<3.0
      Galβ1,4Man5.06.0
      Galβ1,3Ara9.03.3
      Galβ1,4Fru (Lactulose)7.04.3
      Galβ1,4GlcOH (Lactitol)191.6
      Lactobionic acid (Galβ1,4GlcCOOH)850.35
      Galα1,4Gal40 (13%)<0.75
      Melibiose (Galα1,6Glc)800.38
      Raffinose (Galα1,6Glcα1,2Fru)740.41
      Galacturonic Acid100 (10%)<0.3
      Gal-6-SO317.51.7
      Gal-6-PO4100 (13.5%)<0.3
      NeuAcα2,6Lactose0.0161875
      NeuAcα2,6LacNAc0.0142143
      NeuAcα2,3Lactose0.1 (0%)≪300
      NeuAcα2,3LacNAc1.75 (0%)≪17
      N-acetylneuraminic acid200 (0%)0
      p-nitrophenyl-α-sialoside10 (0%)≪3
      Colominic acid1 mg/ml(0%)
      a N-Acetyl-glucosamine,d-arabinose, 2-deoxy-ribose, l-fucose,d-mannose, l- and d-rhamnose,l- and d-ribose, l- andd-xylose, cellobiose, chitobiose, gentiobiose, maltose, isomaltose, sucrose and trehalose were all noninhibitory up to 200 mm (cellobiose to saturation).
      b Minimum concentration required for 50% inhibition of the PSA/α2-macroglobulin precipitation reaction, unless otherwise noted.
      c Maximum concentration tested (percentage of inhibition observed).

      HPTLC of Glycolipids and Lectin Staining

      A panel of neutral glycosphingolipids and gangliosides with well defined carbohydrate structures (Table III) was also used to investigate the binding specificity of purified PSA. The chromatograms are shown in Fig. 3. Of the neutral glycosphingolipids tested, only lactosylceramide was bound by the lectin. This result is in good agreement with the results obtained by precipitation inhibition assays, in which lactose is a fairly good inhibitor of the PSA/α2-macroglobulin precipitation reaction, but oligosaccharides similar to those found in the other neutral glycolipids were not inhibitory. On the other hand, all gangliosides tested failed to react with the lectin. It is especially noteworthy that GM3, which contains the lactosylceramide moiety but is substituted by α2,3-linked Neu5Ac at the β-linked galactosyl residue, also failed to react with the lectin, further confirming that this lectin is exclusively specific for α2,6-linked Neu5Ac.
      Table IIIThe carbohydrate structures of neutral and acidic glycosphingolipids
      Name (trivial name)Carbohydrate structure
      Neutral glycosphingolipids
       Lactosyl ceramide
       (Lac-cer, CDH)Galβ1,4Glcβ1,1Cer
       Globotriosyl ceramide
       (Gb3, Ceramide trihexoside, CTH)Galα1,4Galβ1,4Glcβ1,1Cer
       Globotetraosyl ceramide
       (Globoside, Gb4)GalNAcβ1,3Galα1,4Galβ1,4Glcβ1,1Cer
       Globopentaosyl ceramide
       (Forssman glycolipid, Gb5)GalNAcα1,3GalNAcβ1,3Galα1,4Galβ1,4Glcβ1,1Cer
      Gangliosides (acidic glycosphingolipids)
       Monosialogangliosides
      GM3Neu5Acα2,3Galβ1,4Glcβ1,1Cer
      GM2GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1Cer
      GM1Galβ1,3GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1Cer
       Disialogangliosides
      GD3Neu5Acα2,8Neu5Acα2,3Galβ1,4Glcβ1,1Cer
      GD1aNeu5Acα2,3Galβ1,3GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1Cer
      GD1bGalβ1,3GalNAcβ1,4(Neu5Acα2,8Neu5Acα2,3)Galβ1,4Glcβ1,1Cer
      Figure thumbnail gr3
      Figure 3HPTLC of glycolipids and lectin staining. Two identical sets of glycolipids were separated in parallel on the same aluminum-backed silica gel 60 HPTLC plate. Solvents were chloroform/methanol/water (65:25:4, by volume) (A) and chloroform/methanol/0.25% KCl (50:40:10, by volume) (B). The reference chromatograms were visualized chemically by orcinol reagent; lectin staining was conducted as described under “Materials and Methods.” A, neutral glycosphingolipids (for structures, see Table ). Lane 1, lactosyl ceramide;lane 2, globotriosyl ceramide (note: the two bands are due to heterogeneity of the lipid moiety; the upper band containes nonhydroxy fatty acid side chains, and the lower band contains mostly hydroxylated fatty acid side chains); lane 3, globotetraosyl ceramide; lane 4, globopentaosyl ceramide; lanes 5 and 6, mixture of the four glycolipids. Lanes 1–5 were detected by orcinol reagent, and lane 6 was stained with biotinylated PSA. Among neutral glycolipids tested, only lactosyl ceramide reacted with the lectin. B, acidic glycosphingolipids (gangliosides; see Table for structures).Lanes 1 and 3, monosialoganglioside mixture including GM3, GM2, and GM1; lanes 2 and 4,disialoganglioside mixture containing GD3, GD1a, and GD1b. Lanes 1 and 2 were visualized by orcinol spray, andlanes 3 and 4 were stained with biotinylated PSA. None of the gangliosides tested (all have α2,3-linked Neu5Ac, but lack α2,6-linked Neu5Ac) were bound by the lectin, further confirming the results obtained by precipitation inhibition assays that PSA is exclusively specific for α2,6-linked Neu5Ac.

      Spectral and Fluorescence Studies

      Changes in absorbance or fluorescence spectra were examined as probes for binding studies. It had been observed that p-nitrophenyl α-d-mannoside, upon binding to the mannose/glucose-specific lectin concanavalin A, undergoes a small but definite spectral change with a maximum decrease in absorbance at 317 nm, apparently due to interaction of an aromatic residue with the chromophore (
      • Hassing G.S.
      • Goldstein I.J.
      ). Accordingly, we looked for such a spectral change with both pNPβGal and pNPβLac upon reaction with PSA, but none was observed (data not shown). Another indication of the interaction of an aromatic aglycon with residues in the lectin is the complete quenching of fluorescence of methylumbelliferyl α-d-mannoside (excitation at 320 nm, emission peak at 375 nm) upon binding to concanavalin A (
      • Dean B.R.
      • Homer R.B.
      ). Again, however, fluorescence of methylumbelliferyl β-d-galactoside was unaffected by titration with PSA, suggesting that an aromatic residue of the protein is not in a position near the binding site to interact with the chromophore or fluorophore of these β-galactosides.
      We also examined the effects of ligands on the intrinsic fluorescence of tryptophanyl residues of the protein, as measured by excitation at 280 nm and emission at 300–400 nm. Free monosaccharides, free oligosaccharides, or methyl glycosides had no effect on the intrinsic fluorescence peak at 340 nm. As expected because of its strong UV absorbance, all p-nitrophenyl glycosides quenched intrinsic fluorescence and caused a slight shift in the emission peak toward longer wavelengths. However, we observed that quenching bypNPβGal or pNPβLac was significantly greater than that caused by a nonreactive p-nitrophenyl glycoside, such as p-nitrophenyl α-d-mannoside, at the same concentration. Furthermore, addition of competing oligosaccharides, such as lactose or LacNAc, reversed this additional quenching in a saturable manner, indicating that it is caused by thep-nitrophenyl β-galactosides binding in the vicinity of a fluorophoric group on the lectin. We thus refer to this phenomenon as “specific quenching.”
      Thus, intrinsic fluorescence titration of oligosaccharide ligands in the presence of a fixed amount of pNPβGal yielded apparent inhibition constants (IC50 values) for the titrant from a plot of 1/ΔF versus 1/[L], where ΔF is the increase in peak fluorescence caused by the titrant at a concentration of [L], in a manner analogous to Lineweaver-Burk plots. The ordinate intercept gives the reciprocal of the maximum fluorescence change ΔF max (in arbitrary fluorescence units), which is a function of concentration and binding affinity of the quenching probe. TableIV summarizes data for several oligosaccharides and chromophoric probes. At a lectin concentration of 0.09 mg/ml (3.2 μm in monomers) and pNPβGal of 100 μm, the maximum fluorescence change was approximately 120,000 units, as compared with a total quenching of about 800,000, or about 15% of the total quenching. The use ofpNPαGal at the same concentration also caused a specific quenching signal, but the ΔF max was only about 40,000 units, consistent with the weaker interaction of the lectin with α-galactosides.
      Table IVInhibition of pNP-glycoside quenching of intrinsic fluorescence of P. squamosus agglutinin by ligands
      QuencherLigandK iΔF max(specific quenching)
      μmkilounits
      pNPβGalSialα2,6Lac1.88135
      Sial-Lac
      Mixed α2,3 and α2,6-linked from human milk (∼80% α2,6).
      2.08123
      Sialα2,3LacNR
      NR, no reaction.
      Sialα2,6LacNAc1.38138
      Sialα2,3LacNAc
      10% of ΔF given by Sialα2,6LacNAc at 5 μm; double reciprocal plot extrapolated below origin.
      10% of ΔF given by Sialα2,6LacNAc at 5 μm; double reciprocal plot extrapolated below origin.
      Lactose105119
      LacNAc128116
      LacNAc-βOMe96114
      Lac-N-biose-βOMe227126
      pNPαGalLactose5842
      pNPβLacLactose11187
      pNPβLacNAcLactose25996
      pNPα/βManLactoseNR
      NR, no reaction.
      0
      Intrinsic fluorescence of 0.1 mg/ml of purified high-activity PSA in 1 ml of PBS + 0.1 mm p-nitrophenyl glycoside was measured with excitation at 280 nm, peak emission at approximately 342 nm. Values of K i and ΔF maxwere obtained from plots of 1/ΔF vs. 1/[L].
      a Mixed α2,3 and α2,6-linked from human milk (∼80% α2,6).
      b 10% of ΔF given by Sialα2,6LacNAc at 5 μm; double reciprocal plot extrapolated below origin.
      c NR, no reaction.

      DISCUSSION

      By definition, a lectin is a sugar-binding protein or glycoprotein of nonimmune origin that agglutinates cells and/or precipitates glycoconjugates (
      • Goldstein I.J.
      • Hughes R.C.
      • Monsigny M.
      • Osawa T.
      • Sharon N.
      ). In order to form detectable precipitate, however, both the lectin and the glycoconjugate must be multivalent as well as in an appropriate stoichiometric ratio.
      Of all the sialoglycoproteins we assayed for their ability to form a precipitate with the P. squamosus agglutinin, only human α2-macroglobulin precipitated the lectin. Human α2-macroglobulin is composed of four identical subunits, each of which contains 1451 amino acid residues and eightN-linked sugar chains (
      • Sottrup-Jensen L.
      • Stepanik T.M.
      • Kristensen T.
      • Wierzbicki D.M.
      • Jones C.M.
      • Lonblad P.B.
      • Magnusson S.
      • Petersen T.E.
      ) that are 70% sialylated, exclusively in Neu5Acα2,6 linkage (
      • Hanaoka K.
      • Pritchett T.J.
      • Takasaki S.
      • Kochibe N.
      • Sabesan S.
      • Paulson J.C.
      • Kobata A.
      ). On the other hand, most native glycoproteins, including α1-acid glycoprotein, fetuin, transferrin, and thyroglobulin, contain both α2,3- and α2,6-linked N-acetylneuraminic acids. In addition to the prevalence of α2,3-linked sialic acids at the nonreducing termini of these glycoproteins, many α2,6-linked Neu5Ac residues occur not at the nonreducing termini, but rather attached to the penultimate GlcNAc residues of the tri- and tetraantennary structures, thus precluding lectin binding. It is noteworthy that these α2,6-linked sialic acid groups are also resistant to cleavage by neuraminidase fromClostridium perfringens (
      • Tamura T.
      • Wadhwa M.S.
      • Rice K.G.
      ). To further confirm the specificity of PSA for terminal α2,6-linked Neu5Ac, we resialylated asialofetuin using rat liver α2,6-sialyltransferase, which sialylates specifically at the 6-position of nonreducing terminal β1,4-linked galactose (
      • Wlasichuk K.B.
      • Kashem M.A.
      • Nikrad P.V.
      • Bird P.
      • Jiang C.
      • Venot A.P.
      ). Despite incorporating only 3 Neu5Ac groups per molecule, this resialylated fetuin precipitated PSA much more strongly than either asialofetuin or fully sialylated native fetuin.
      Taking these facts into consideration, it is understandable why the native glycoproteins tested, except for α2-macroglobulin, failed to precipitate with PSA. In fact, we observed that these native glycoproteins inhibited the PSA/α2-macroglobulin precipitation reaction (data not shown), indicating that they do contain a few nonreducing terminal Neu5Acα2,6Galβ1,4Glc/GlcNAc structures, but not a sufficient number to act as a multivalent precipitating ligand for the lectin.
      To elucidate the detailed carbohydrate binding specificity of purified PSA, precipitation inhibition assays were carried out using α2-macroglobulin as the precipitant. The data in Table IIpermit some conclusions to be drawn regarding the specificity of the lectin. The 2-, 3-, and 4-hydroxyl groups in the galactopyranose ring are evidently important in binding, because the epimers in these positions (d-talose, d-gulose, andd-glucose, respectively) are unreactive. The reactivity of β-d-galactopyranosides, compared with freed-galactose, indicates a preference for the β-d-pyranose form, which is predominantly in the4C1 chair conformation (
      • Lehmann J.
      ). Becaused-gulose and d-glucose in solution equilibrium are 60–70% in the same conformation and anomeric configuration, their lack of reactivity indicates that the 3- and 4-hydroxyl groups in the equatorial and axial epimer, respectively, are essential for binding. The conclusion regarding the 2-epimer, d-talose is less clear; it exists in the β-d-pyranose form only to the extent of 29% (
      • Lehmann J.
      ), and the two chair conformations are approximately energetically equivalent. However, its complete lack of reactivity at 100 mm, together with the poor reactivity of 2-deoxy-d-galactose and GalNAc, indicate that the 2-hydroxyl group is also important in binding. The fact thatd-fucose (i.e. 6-deoxy galactose) and 6-O-methyl-d-galactose are nearly equal tod-galactose in inhibitory potentcy suggests that the 6-hydroxyl group is not involved, either as a hydrogen bond donor or acceptor. However, the poor reactivities of l-arabinose and methyl β-l-arabinopyranoside are enigmatic. The free pentose in solution is approximately 61% in the α-l-pyranose form (
      • Lehmann J.
      ), which is homomorphous with β-d-galactose except for the lack of the hydroxymethyl group on C-5. This suggests that the methylene or methyl carbon (C-6) of d-galactose or d-fucose, respectively, contributes significantly to the binding. However, the effect of the hydroxylmethyl group on C-5 may be indirect, e.g. by its stabilizing effect of the 4C1 chair conformation of the pyranose ring.
      Both methyl β-d-galactopyranoside andp-nitrophenyl β-d-galactopyranoside are three times more potent inhibitors than the corresponding α-glycosides, as indicated both by precipitin inhibition and extent of specific fluorescence quenching, suggesting that PSA has an anomeric preference for the β-configuration. This β-anomeric preference is further confirmed by the oligosaccharides tested, of which most with nonreducing terminal β-galactosyl residues (e.g. lactose and N-acetyllactosamine) are 3–8-fold more potent inhibitors than d-galactose and 10–20-fold better than α-galactosyl-terminated disaccharides (e.g. melibiose and raffinose). A major exception is Galβ1,4Gal, which is a relatively poor inhibitor. This could be explained by the fact that in Galβ1,4Gal, the nonreducing terminal galactosyl residue is β-linked to an axial C-4 oxygen atom, causing the two hexose residues to lie at a considerably sharper planar angle to each other than inequatorial oxygen-linked disaccharides, such as lactose, LacNAc, and Galβ1,4Man, increasing the possibility of steric hindrance of the hydroxyl groups involved in binding. The corresponding α-digalactoside is also a very poor inhibitor, probably because of the bulk of the substituent in the α-position. In neither case could the reducing galactose moiety occupy the binding site, because of the necessity of a free C-4 hydroxyl group.
      The substitution of the C-6 hydroxyl group of the nonreducing terminal β-galactosyl residue with α 2,6-linkedN-acetylneuraminic acid increased the inhibitory potency by 3 orders of magnitude (2000-fold) as compared with galactose and 250–300 times compared with the parent sugars (i.e. lactose and LacNAc). However, free N-acetylneuraminic acid,p-nitrophenyl α-sialoside, and the Neu5Acα 2,8 polymer colominic acid did not react, whereas galactose derivatives having an acidic function near the C-6 position (d-galacturonic acid,d-galactose 6-sulfate, and d-galactose 6-phosphate) are moderate to very weak inhibitors. Furthermore, the addition of sialic acid to the C-3 position of the nonreducing terminal galactose abolished the lectin binding almost totally. This is in contrast to S. nigra agglutinin, a plant lectin isolated from elderberry (Sambucus nigra) bark, which recognizes not only the terminal Neu5Acα2,6Gal/GalNAc sequence but also the α2,3-linked isomer, to a 100-fold smaller extent (
      • Shibuya N.
      • Goldstein I.J.
      • Broekaert W.F.
      • Nsimba-Lubaki M.
      • Peeters B.
      • Peumans W.J.
      ). The exclusive specificity of PSA is consistent with the C-3 equatorial hydroxyl group of the terminal galactosyl group being a critical locus, so that its substitution abrogates the lectin binding, probably due to steric effects. However, the C-6 hydroxyl group of the d-pyranose ring is not involved in lectin binding, so that substitution at that position of the galactose is tolerated. Furthermore, sialylation at the C-6 position evidently creates additional interactions via hydrogen bonds and/or charge interactions between the carboxylate group and positively charged amino acids in the vicinity of the carbohydrate-binding site of the lectin. However, because free sialic acid, or sialosides, its polymer, or its 2,3-substituted form does not react, the lectin does not appear to have an independent binding site for sialic acid and cannot be considered as anN-acetylneuraminic acid-binding lectin.
      A lectin with similar affinity was isolated from tubers ofTrichosanthes japonica (Cucurbitaceae) (
      • Yamashita K.
      • Umetsu K.
      • Suzuki T.
      • Ohkura T.
      ). That lectin also recognized β-galactosyl residues and was greatly enhanced by α2,6-sialylation but blocked by α2,3 sialylation. However, that lectin did not react with the Galβ1,3GlcNAc group, showed tolerance for C-2 epimerization of the galactose (i.e. d-talose), and had little or no preference for β-galactosides versus α-galactosides. Those workers also observed that 6-sulfated lactose and 6′-sialyllacto-N-neotetraose strongly interacted with the lectin. We have tested neither of these latter compounds, but would expect them to react at least as well as lactose and 6′-sialyllactose, respectively. We did, however, observe that galactose 6-sulfate is equivalent in its inhibitory potency to galactose. On the other hand, a lectin isolated from a related polypore mushroom, Laetiporus sulfureus, appears to be completely different in its molecular structure, amino acid composition, and carbohydrate binding specificity (
      • Konska G.
      • Guillot J.
      • Dusser M.
      • Damez M.
      • Botton B.
      ).
      The inhibitory potentcy of LacNAcβOMe and the isomeric Galβ1,3GlcNAcβOMe are similar, suggesting that PSA would recognize Neu5Acα2,6Galβ1,3GlcNAc as well as it does Neu5Acα2,6Galβ1,4GlcNAc. However, until an authentic sample of this sialylated β1,3 oligosaccharide is assayed, we can only speculate on its recognition by the mushroom lectin.
      Of equal importance is the finding that ovine submaxillary mucin, which contains a prodigious number of Neu5Acα2, 6GalNAcα1-Ser/Thr moieties per molecule, is neither a precipitant nor an inhibitor of PSA, in contrast to S. nigraagglutinin, which requires only a sialylα2,6Gal/GalNAc moiety (
      • Shibuya N.
      • Goldstein I.J.
      • Broekaert W.F.
      • Nsimba-Lubaki M.
      • Peeters B.
      • Peumans W.J.
      ). This observation is consistent with the preference of PSA forthree structural features lacking in the mucinO-linked glycans: a β-galactosidic linkage (cf.GalβOMe versus GalαOMe), the necessity of a free equatorial 2-OH group on the galactosyl residue (cf.galactose versus GalNAc, 2-deoxy galactose, ord-talose), and the preference for an additional sugar at the β-galactoside linkage (cf. lactose or LacNAcversus GalβOMe).
      The relative inhibitory constants of oligosaccharides tested in the specific fluorescence quenching assay (Table IV) are consistent with those observed by precipitin inhibition (Table II), although absolute values are lower in the former case. This difference is understandable, because the reporter ligand in the fluorescence assay is monovalent, whereas in the precipitin assay, it is a polyvalent ligand leading to precipitation of an extensive network of lectin and glycoprotein molecules, requiring higher concentrations of a given competitive monovalent ligand to cause inhibition. Preliminary measurements of ligand binding using isothermal titration calorimetry showed PSA to bind methyl-β-galactoside, lactose, and Neu5Acα2,6Lac withK d values of 393, 92, and 0.59 μm, respectively (data not shown), values that are also 30–40-fold lower than the IC50 values for precipitation inhibition (TableII) but comparable to K i values for specific fluorescence quenching (Table IV). Calorimetric studies with PSA, as well as further studies of this specific fluorescence quenching phenomenon, which we have observed with several lectins, are in progress and will be published subsequently.
      In conclusion, the P. squamosus agglutinin possesses an extended carbohydrate-combining site with strict specificity and high affinity for nonreducing terminal Neu5Acα2,6Galβ1,4Glc(NAc) residues. Thus, it appears that PSA has a binding site that accommodates three carbohydrate moieties. This specificity could make this lectin an invaluable tool for glycobiological studies, especially for cancer research and diagnosis. For example, it has been well documented in NIH3T3 (or FR3T3) cells transformed withras oncogene that there is an increased β-galactoside α-2,6-sialyltransferase activity (
      • Le Marer N.
      • Laudet V.
      • Svensson E.C.
      • Cazlaris H.
      • Van Hille B.
      • Lagrou C.
      • Stehelin D.
      • Montreuil J.
      • Verbert A.
      • Delannoy P.
      ,
      • Vandamme V.
      • Cazlaris H.
      • Le Marer N.
      • Laudet V.
      • Lagrou C.
      • Verbert A.
      • Delannoy P.
      ) and a concomitant decreased CMP-Neu5Ac:Galβ1,3GalNAc α-2,3-sialyltransferase activity (
      • Delannoy P.
      • Pelczar H.
      • Vandamme V.
      • Verbert A.
      ). Furthermore, some tumors, e.g. hepatocellular carcinoma (
      • Pousset D.
      • Piller V.
      • Bureaud N.
      • Monsigny M.
      • Piller F.
      ) and human colorectal tumors (
      • Dall'Olio F.
      • Malagolini N.
      • Di Stefano G.
      • Minni F.
      • Marrano D.
      • Serafini-Cessi F.
      ,
      • Dall'Olio F.
      • Malagolini N.
      • Serafini-Cessi F.
      ), express a high level of α2,6-sialylation of N-acetyllactosaminic sequences on their cell surface, which is correlated with high metastatic potential (
      • Dall'Olio F.
      • Malagolini N.
      • Di Stefano G.
      • Ciambella M.
      • Serafini-Cessi F.
      ,
      • Le Marer N.
      • Stehelin D.
      ).

      Acknowledgments

      We thank Dr. Robert Fogel of the University of Michigan Herbarium for identifying the P. squamosusmushroom and depositing a voucher specimen, David Carruthers for allowing us to harvest the P. squamosus mushroom from his property, and Dr. Michael Marletta and his group for permission and assistance in use of the spectrofluorometer.

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