B. simplicifolia I ISOLECTINS
bohydrate of the original glycoprotein. Further details of this work, including the properties of the lectin and the nature of the glycopeptide linkages, will be published elsewhere. 15 Acknowledgment We arc gratefulto the MedicalResearch Councilfor financialsupport. 15 A. K. Allen, N. N. Desai, A. Neuberger, and J. M. Creeth,Biochem. J. (1978), in press.
 Bandeiraea simplicifolia I I s o l e c t i n s
By LEE A. MURPHY and IRWIN J. GOLDSTEIN The seeds of Bandeiraea simplicifolia contain a family of five aD-galactosyl-binding isolectins (BS I) J as well as an N-acetyl-Dglucosamine-binding lectin (BS II),=' which is reviewed elsewhere in this volume. 3 The five a-D-galactosyl-binding isolectins--BSI (A4, A3B, A2B2, AB3, B4)--are tetrameric structures composed of two different glycoprotein subunits designated A and B. The carbohydrate binding specificities of the two subunits differs significantly: the A subunit exhibits a primary specificity for a-o-GalNAcp but also reacts with aD-Galp units whereas the B subunit shows a sharp specificity toward a-D-Galp residues. ~The natural mixture of isolectins will agglutinate type B and A1 human red blood cells4; the natural mixture in the presence of D-GalNAc will agglutinate only type B. 1 BS I isolectin (B4) is specific for type B human red blood cells and BS I isolectin (A4) is specific for cells of type A1. ~ The natural isolectin mixture in the presence of o-GalNAc or purified BS I (B4) can serve as a valuable tool for detecting terminal nonreducing a-linked D-galactosyl residues in polysaccharides, glycolipids, and glycoproteins. 1 L. A. Murphy and I. J. Goldstein, J. Biol. Chem. 252, 4739 (1977). '-'P. N. Shankar Iyer, K. D. Wilkinson, and I. J. Goldstein, Arch. Biochem. Biophys. 177, 330 (1976). 3 S. Ebisu and I. J. Goldstein, this volume . 4 C. E. Hayes and I. J. Goldstein, J. Biol. Chem. 249, 1904 (1974).
Assay Methods The B. simplicifolia I isolectins can be assayed by hemagglutination or precipitin methods. The quantitative precipitin assay is the more sensitive and precise method; however, the hemagglutination assay is rapid and convenient. Hemagglutination Procedure. Hemagglutination assays are conducted by serial dilution of lectin solution with 0.9% saline l0 mM phosphate pH 7.2, using a hemagglutination plate and a 0.025-/A microdiluter [Cooke Engineering Co.]. A drop of a 2% suspension of type A, or B erythrocytes is added to each dilution, and the hemagglutination plate is covered and kept at room temperature for 1 hr. The titer is the reciprocal of the highest dilution showing detectable agglutination. Quantitative Precipitin Analysis. A microprecipitin technique ~ using 40-60/zg of purified BS I gives very precise and reproducible results. The precipitin curve is set up in conical 3-ml centrifuge tubes by combining a constant amount of lectin with various concentrations of biopolymer (1-400 p.g) in PBS (0.1 M phosphate, 0.15 M NaCl, 0.04% NaNa, 0.1 mM Mn '-'+, 0.1 mM Mg, and 0.1 mM Ca "+) pH 7.0 to a final'volume of 0.5 ml. The tubes are incubated for 48 hr at 25 °. At the end of this time the tubes are centrifuged at 3000g for 15 min, and the supernatant solution is carefully removed using a drawn-out Pasteur pipette. The precipitates are washed twice with PBS (0.2 ml). The washed precipitates are dissolved in 0.05 N NaOH containing l0 mM methyl a-D-Galp (0.3 ml), and protein is determined by a semimicro Lowry procedure. 6 Sugar inhibition of the precipitin reaction is carried out by adding increasing amounts of sugar or sugar derivative to duplicate tubes containing BS | lectin (50 p.g) in PBS. After a 15-min preincubation period, the precipitin reaction is initiated by the addition of guaran (guar gum) (5/~g) to give a final volume of 0.5 ml. Purification of BS I Isolectins Principle A partially purified B. simplicifolia extract is passed over a column of Bio-Gel-melibionate. After elution of inert protein, the five BS I isolectins are displaced by 5 mM methyl a-D-GaSp .4 Alternately, the five individual BS I isolectins may be prepared. BS I isolectin (A4) and (AaB) are purified on a Bio-Gel-melibionate column by sequential elution with different conL. L. So and I. J. Goidstein,J. Biol. Chem. 242, 1617(1967). R. Mage and S. Dray, J. Immunol. 95,523 (1963).
B. simplicifolia I ISOLECTINS
centrations of D-GalNAc. BS I isolectins (A2B2), (AB3), and (B4) are separated according to their affinity for a column of type A + H hog gastric mucin, insolubilized by treatment with N-carboxyanhydroleucine. J
Procedure All procedures are carred out at 4 °. Extraction and Ammonium Sulfate Fractionation. One hundred grams of finely ground B. simplicifolia seeds (Calbiochem, La Jolla, California) are extracted three times (1 hr) with 400 ml of methanol containing 20 mg of butylated hydroxytoluene and filtered. The filtrate is discarded. Polyvinylpolypyrrolidone (6 g) is added to the dry, methanol-extracted meal, and the mixture is extracted by stirring for 2 hr with 300 ml of PBS made 0.1 M in o-galactose. The extract is centrifuged at 10,000 g for 20 min, and the sediment is reextracted as before. Solid ammonium sulfate is added to the pooled supernatants to give 40% saturation. After stirring gently for 1 hr, precipitated proteins are removed by centrifugation at 9000 g for 20 min and discarded. The isolectins are precipitated by adding solid ammonium sulfate to 75% saturation. The precipitate is collected by centrifugation, dissolved in PBS (80 ml), and dialyzed against four changes of PBS to remove o-galactose. A broad ammonium sulfate fractionation (40--75%) is used in order to facilitate the simultaneous preparation of B. simplicifolia II lectin. 2 A 55-75% ammonium sulfate fractionation is preferable if the preparation of BS II is not desired. 4 Preparation of the Affinity Columns. The Bio-Gel-melibionate affinity column is prepared by coupling melibionic acid to aminoethyl Bio-Gel P-300 at pH 4.7 using the water-soluble carbodiimide EDC [1-ethyl-3-(3dimethylaminopropyl)carbodiimide HCI]. 4 The type A + H blood group substance from hog gastric mucin is insolubilized by treatment with N-carboxyanhydroleucine and mixed with 5 parts of Celite to make a column with good flow properties. 7 Affinity Chromatography of the BS I Isolectins. The 40-75% ammonium sulfate fraction in PBS is applied to the Bio-Gel-melibionate column (2 x 30 cm) at 30 ml/hour. When the absorbance at 280 nm is <0.04, a solution of 5 mM methyl o~-D-Galp in PBS is added to elute the five BS I isolectins. Fractions having an absorbance at 280 nm > 0.1 are combined, concentrated by membrane ultrafiltration (Amicon Corporation PM-10 membrane), and dialyzed against several changes of PBS to remove methyl a-o-Galp. 4 An o~-o-Galp-specific reagent may be prepared 7M. E. Kaplan and E. A. Kabat, J. Exp. Med. 123, 1061 (1966).
from this isolectin mixture by the addition of D-GaINAc to the lectin protein in a 5:1 (w/w) ratio.SThe yield ofB. simplicifolia I isolectins varies from 0.1 to 0.3% of the dry meal. 1.4 In order to separate the five individual isolectins, a modification of the above protocol is used. After the Bio-Gel-melibionate column is washed free of unbound protein, BS I (A4) is eluted using PBS containing 1.4 mM D-GalNAc; (A3B) is eluted by increasing the D-GaINAc concentration to 6.8 mM; and (A2B2, AB3, B4) are eluted with 5 mM methyl a-D-Galp. The B. simplicifolia I isolectins (A2B2, AB3, B,) fractions are pooled, concentrated by membrane ultrafiltration, and dialyzed against PBS until free of sugar. The dialyzed BS I isolectins (A2B2, AB3, B4) are applied to a column of insolubilized type A + H hog mucin mixed with five parts of Celite (1.5 × 30 cm). BS I (B4) passes through the column, whereas BS I (AB3) is retarded slightly as determined by gel electrophoresis. BS I (A2B2) is eluted from the column using 50 mM methyl a-D-Ga[o. Isolectin fractions are pooled, concentrated by membrane ultrafiltration, dialyzed against PBS until free of sugar, and stored at - 2 0 ° until used.' Several alternatives to the Bio-Gel-melibionate column for preparation of the BS I isolectin mixture are now available. Reductive amination of the reaction product formed between melibiose and aminoethyl Bio-Gel P- 150 using sodium cyanoborohydride gives a matrix with properties similar to those of the Bio-Gel-melibionate column. 9 Guaran, which forms a precipitate with the BS I lectin, has been polymefized and converted to insoluble beads by cross-linking a 2% solution of purified guaran with epichlorohydrin.'° Guaran has also been immobilized in a polyacrylamide gel." Copolymerization of acrylamide, N , N ' - m e t h y l e n e bisacrylamide, and allyl a-o-galactopyranoside produces a column that appears to give very low yields of BS I lectin.'" Purity of BS I Isolectins Polyacrylamide gel electrophoresis of the five BS I isolectins gives one band at p H 4.3 and five main bands at p H 8.9.1'4 Polyacrylamide gel electrophore sis at p H 8.6 in the presence of sodium dodecyl sulfate (S DS) gives two bands corresponding to the A and B subunits. 1Polyacrylamide disc gelelectrophoresis of the individual BS I isolectins gives one band at 4.3, one s W. J. Judd, L. A. Murphy, I. J. Goldstein,L. Campbell, and M. E. Nichols, Transfusion, in press. ~'R. J. Bauves, and G. R. Gray, J. Biol. Chem. 252, 57 (1977). ,0 j. L6nrlgrenand I. J. Goldstein,FEBS Lett. 68, 31 (1976). ~' M. Horisberger,Carbohydr. Res. 53, 231 (1977). '~ V. H6rej~iand J. Kocourek,Biochim. Biophys. Acta 297, 346 (1973).
B. simplicifolia I ISOLECTINS
main band at 8.9 corresponding to one of the five seen with the natural mixture ofBS I isolectins, and one BS I (A4, B4) or two bands BS I (A3B, A2B2, AB3) at pH 8.6 in the presence of SDS. 1Analysis of the five BS I isolectins by immunoelectrophoresis with antibody to the crude seed proteins, gel filtration on Sephadex G-200 superfine, and sedimentation analysis gives evidence for only one homogeneous protein component. 4
Properties of the BS I Isolectins The five BS I isolectins are tetrameric structures with molecular weights of approximately 114,0004 composed of two different glycoprotein subunits having molecular weights of 32,000 (A) and 33,000 (B). 1 Glycoproteins, the BS I isolectin mixture, contain approximately l0 mol of mannose, 2 mol of fucose, 2 tool of xylose, and 4 mol of glucosamine per subunit. 4 The A and B subunits of BS I each contain one cysteine residue and exhibit few differences in their amino acid composition with the exception of methionine. The A subunit does not contain methionine whereas the B subunit contains one residue. 1 The BS I isolectins will form precipitates with guaran, type B substance, and type A substance but not with larch arabinoglactan. 4 Addition of o-GalNAc to the natural BS I isolectin mixture restricts the formation of precipitates to guaran and type B substance. BS I (A4) will form precipitates with guaran, type B, and type A substance but not larch arabinogalactan, whereas BS I (B4) will form precipitates only with guaran and type B substance. Inhibition of the precipitation of BS I (A4) or BS I (B4, AB3) and guaran by sugars of low molecular weight shows which sugars are most complementary to the A and B combining sites. Methyl a-o-GalNAcp was the best inhibitor tested in the BS I (A4)-guaran system, being 3.5 times more potent than o-GalNAc, 18 times better than methyl a-D-Galp, 50 times better than o-galactose, and 90 times better than methyl fl-D-Galp. In the BS I (B4, AB3)-guaran system, methyl a-v-Galp proved to be the best inhibitor, being 3.5 times more effective than o-galactose, 9 times more effective than methyl fl-D-Galp, and more than 90 times more effective than o-GalNAc or methyl a-I>GalNAcp. From the precipitation and inhibition studies, we conclude that BS I (B4) and the natural BS I isolectin mixture in the presence of o-GalNAc is specific for a-o-galactopyranosyl determinants and BS I (A4) reacts best with a-o-GalNAcp residues but will cross-react with a-D-Galp. 1 Acknowledgment This work was supported by USPHS grants AM-10171 and 6M-00187.