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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

2000; Elsevier BV; Volume: 275; Issue: 14 Linguagem: Inglês

10.1074/jbc.275.14.10623

1083-351X

Hanqing Mo, Harry C. Winter, Irwin Goldstein,

Carbohydrate Chemistry and Synthesis

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. 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. high-performance thin layer chromatography methyl 2-(N-morpholino)ethanesulfonic acid nitrophenyl polyacrylamide gel electrophoresis phosphate-buffered saline 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 (1.Goldstein I.J. Hughes R.C. Monsigny M. Osawa T. Sharon N. Nature. 1980; 285: 66Crossref Scopus (642) Google Scholar). They are ubiquitous in the biosphere, having been found in viruses, bacteria, fungi, plants, and animals (2.Kocourek J. Liener I.E. Sharon N. Goldtein I.J. The Lectins: Properties, Functions and Applications in Biology and Medicine. Academic Press, Orlando, FL1986: 1-32Crossref Google Scholar). 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 (3.Lin J.Y. Chou T.B. J. Biochem. 1984; 96: 35-40Crossref PubMed Scopus (64) Google Scholar), Grifola frondosa lectin is cytotoxic to Hela cells (4.Kawagishi H. Nomura A. Mizuno T. Kimura A. Chiba S. Biochim. Biophys. Acta. 1990; 1034: 247-252Crossref PubMed Scopus (123) Google Scholar), Agaricus bisporus lectin possesses antiproliferation activities against human colon cancer cell lines HT29, breast cancer cell lines MCF-7 (5.Yu L.G. Fernig D.J. Smith J.A. Milton J.D. Rhodes J.M. Cancer Res. 1993; 53: 4627-4632PubMed Google Scholar), and Tricholoma mongolicum lectin inhibits mouse mastocytoma P815 cells in vitro and sarcoma S-180 cells in vivo (6.Wang H.X. Ng T.B. Ooi V.E.C. Liu W.K. Chang S.T. Anticancer Res. 1997; 17: 419-424PubMed Google Scholar). Fungal lectins have recently been reviewed (7.Kawagishi H. Food Rev. Int. 1995; 11: 63-68Crossref Scopus (40) Google Scholar, 8.Guillot J. Konska G. Biochem. Syst. Ecol. 1997; 25: 203-230Crossref Scopus (117) Google Scholar, 9.Wang H.X. Ng T.B. Ooi V.E.C. Mycol. Res. 1998; 102: 897-906Crossref Scopus (164) Google Scholar). However, apart from the lectin from A. bisporus, which binds to Galβ1,3GalNAcα-Ser/Thr (T-disaccharide) (10.Irazoqui F.J. Vides M.A. Nores G.A. Glycobiology. 1999; 9: 59-64Crossref PubMed Scopus (32) Google Scholar), 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. 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 (11.Warren L. J. Biol. Chem. 1959; 234: 1971-1975Abstract Full Text PDF PubMed Google Scholar). 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. 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 concentration was determined by the method of Lowry et al. (12.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), using bovine serum albumin as a standard. Native gel electrophoresis using a 12.5% slab gel was carried out in alkaline buffer system (Tris/glycine, pH 8.3) (13.Davis B.J. Ann. N. Y. Acad. Sci. 1964; 121: 404-427Crossref PubMed Scopus (15957) Google Scholar). 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 (14.Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10481) Google Scholar). The hemagglutinating activity of the lectin was determined by a 2-fold serial dilution procedure using formaldehyde-treated (15.Nowak T.P. Barondes S.H. Biochim. Biophys. Acta. 1975; 393: 115-123Crossref PubMed Scopus (71) Google Scholar) human and rabbit erythrocytes as described previously (16.Crowley J.F. Goldstein I.J. Methods Enzymol. 1982; 83: 368-373Crossref PubMed Scopus (17) Google Scholar). The hemagglutination titer was defined as the reciprocal of the highest dilution still exhibiting hemagglutination. 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. 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 (17.Mo H.Q. Van Damme E.J.M. Peumans W.J. Goldstein I.J. J. Biol. Chem. 1994; 269: 7666-7673Abstract Full Text PDF PubMed Google Scholar,18.Mo H.Q. Goldstein I.J. Glycoconjugate J. 1994; 11: 424-431Crossref PubMed Scopus (11) Google Scholar). Tryptophan was estimated spectrophotometrically in 6 mguanidine hydrochloride (19.Edelhoch H. Biochemistry. 1967; 6: 1948-1954Crossref PubMed Scopus (3007) Google Scholar). 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 assays were performed by a microprecipitation technique as described previously (17.Mo H.Q. Van Damme E.J.M. Peumans W.J. Goldstein I.J. J. Biol. Chem. 1994; 269: 7666-7673Abstract Full Text PDF PubMed Google Scholar). 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. 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. 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. 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. Ultraviolet absorbance spectra were recorded on a Shimadzu model UV160U spectrophotometer; fluorescence spectra were recorded on an ISA JodinYvon-Spex Fluoromax-2 spectrofluorometer. In a survey of mushrooms for hemagglutination activity by Pemberton (20.Pemberton R.L. Mycol. Res. 1994; 98: 277-290Crossref Scopus (44) Google Scholar), 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. 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. 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. 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 agglutininAmino acidMol %Residues/subunitAsx14.538Glx7.5320Ser6.5117Gly10.728His1.333Arg2.717Thr7.7220Ala9.6025Pro3.509Tyr2.948Val5.7115Met0.892Cys0.65aEstimated by 5,5′-dithiobis(2-nitrobenzoic acid) after denaturation (6 m guanidine HCl) and reduction.2Ile4.2511Leu7.6520Phe5.5214Lys4.9413Trp3.18bEstimated spectrophotometrically by the method of Edelhoch (19).8Total residues260Calculated molecular mass, 28,150–28,200 kDaa Estimated by 5,5′-dithiobis(2-nitrobenzoic acid) after denaturation (6 m guanidine HCl) and reduction.b Estimated spectrophotometrically by the method of Edelhoch (19.Edelhoch H. Biochemistry. 1967; 6: 1948-1954Crossref PubMed Scopus (3007) Google Scholar). Open table in a new tab 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 oligosaccharidesSugaraN-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).IC50bMinimum concentration required for 50% inhibition of the PSA/α2-macroglobulin precipitation reaction, unless otherwise noted.Relative potencymmGalactose30[1]GalαMe450.67GalβMe152.0p-nitrophenyl α-d-galactoside142.1p-nitrophenyl β-d-galactoside5.35.7d-Fucose400.752-Deoxy galactose640.47GalNAc100 (3.4%)cMaximum concentration tested (percentage of inhibition observed).<0.3l-Arabinose100 (15%)<0.3Me-β-l-arabinopyranoside100 (16%)<0.36-OMe-d-Gal330.9d-Talose (C-2 epimer)100 (0%)0d-Glucose (C-3 epimer)100 (0%)0d-Glucose (C-4 epimer)100 (0%)0Lactose4.07.5LacNAc4.37.0p-nitrophenyl β-lactoside3.58.6Galβ1,4GlcNAcβMe3.87.9Galβ1,3GlcNAcβMe6.05.0T disaccharide (Galβ1,3GalNAc)6.24.8Galβ1,6Gal4.86.3Galβ1,4Gal10 (15%)<3.0Galβ1,4Man5.06.0Galβ1,3Ara9.03.3Galβ1,4Fru (Lactulose)7.04.3Galβ1,4GlcOH (Lactitol)191.6Lactobionic acid (Galβ1,4GlcCOOH)850.35Galα1,4Gal40 (13%)<0.75Melibiose (Galα1,6Glc)800.38Raffinose (Galα1,6Glcα1,2Fru)740.41Galacturonic Acid100 (10%)<0.3Gal-6-SO317.51.7Gal-6-PO4100 (13.5%)<0.3NeuAcα2,6Lactose0.0161875NeuAcα2,6LacNAc0.0142143NeuAcα2,3Lactose0.1 (0%)≪300NeuAcα2,3LacNAc1.75 (0%)≪17N-acetylneuraminic acid200 (0%)0p-nitrophenyl-α-sialoside10 (0%)≪3Colominic 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). Open table in a new tab 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 glycosphingolipidsName (trivial name)Carbohydrate structureNeutral 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)GalNAcα1,3GalNAcβ1,3Galα1,4Galβ1,4Glcβ1,1CerGangliosides (acidic glycosphingolipids) MonosialogangliosidesGM3Neu5Acα2,3Galβ1,4Glcβ1,1CerGM2GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1CerGM1Galβ1,3GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1Cer DisialogangliosidesGD3Neu5Acα2,8Neu5Acα2,3Galβ1,4Glcβ1,1CerGD1aNeu5Acα2,3Galβ1,3GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1CerGD1bGalβ1,3GalNAcβ1,4(Neu5Acα2,8Neu5Acα2,3)Galβ1,4Glcβ1,1Cer Open table in a new tab 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 (21.Hassing G.S. Goldstein I.J. Eur. J. Biochem. 1970; 16: 549-556Crossref PubMed Scopus (76) Google Scholar). 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 (22.Dean B.R. Homer R.B. Biochim. Biophys. Acta. 1973; 322: 141-144Crossref PubMed Scopus (41) Google Scholar). 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 chan