Biological and biochemical properties of Phaseolus vulgaris isolectins.

Affinity-purified phytohemagglutinin from red kidney bean resolves into five isolectins by SP-Sephadex ion exchange chromatography. Recoveries ranging from 30 to 130 mg of protein for each isolectin are easily achieved. The isolectins have similar amino acid compositions which differ only in threonine, lysine, and arginine. A distinguishing feature of the amino acid composition is the total lack of sulfur-containing amino acids. Each isolectin contains about 4% mannose and 2.2% N-acetyl-D-glucosamine. All isolectins on electrophoresis form single protein bands under denaturing and nondenaturing conditions in polyacrylamide gels, and all have apparent subunit molecular weights of 33,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isolectins are also homogeneous by ultracentrifugation and have apparent native molecular weights of 115,000 +/- 4,130, suggesting tetrameric quaternary structures. Whereas 80% of the starting erythroagglutinin activity is recovered, one of the five isolectins possesses 50% of that original activity. As sequentially eluted from the ion exchange column, each isolectin displays progressively higher erythroagglutinin and lower lymphocyte mitogenic activities. Based on their relative biological activities, the isolectins are assigned the structures L4, L3E1, L2E2, L1E3, and E4, where L and E represent lymphocyte- and erythrocyte-reactive subunits, respectively, and the subscripts represent the proposed subunit composition.

Affinity-purified phytohemagglutinin from red kidney bean resolves into five isolectins by SP-Sephadex ion exchange chromatography.
Recoveries ranging from 30 to 130 mg of protein for each isolectin are easily achieved. The isolectins have similar amino acid compositions which differ only in threonine, lysine, and arginine. A distinguishing feature of the amino acid composition is the total lack of sulfur-containing amino acids. Each isolectin contains about 4% mannose and 2.2% N-acetyl-o-glucosamine.
All isolectins on electrophoresis form single protein bands under denaturing and nondenaturing conditions in polyacrylamide gels, and all have apparent subunit molecular weights of 33,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
The isolectins are also homogeneous by ultracentrifupation and have apparent native molecular weights of 115,000 rf: 4,130, suggesting tetrameric quaternary structures. Whereas 80% of the starting erythroagglutinin activity is recovered, one of the five isolectins possesses 50% of that original activity. As sequentially eluted from the ion exchange column, each isolectin displays progressively higher erythroagglutinin and lower lymphocyte mitogenic activities. Based on their relative biological activities, the isolectins are assigned the structures L,, L,,E,, L,E,, L,Es, and E,, where L and E represent lymphocyte-and erythrocyte-reactive subunits, respectively, and the subscripts represent the proposed subunit composition.
Phytohemagglutinin, the lectin extract from red kidney bean (Phaseohs uulgaris), contains potent, cell-agglutinating, and mitogenic activities. The clarification of the biochemical basis of PHA' action is complicated by the fact that PHA is a mixture of several proteins with different biological activities. Allen et al. (1) and Weber and associates (2,3)  Studies of protein structure by Weber et al. (2,3) suggest that LPHA is a tetramer of four identical subunits. To explain the mixed erythrocyte-lymphocyte reactivity of HPHA, Yachnin and co-workers (4,5) postulated that HPHA is a mixture of three to four proteins, isomitogens, each a tetramer consisting of varying proportions of the lymphocyte-specific subunit of LPHA and an erythrocyte-specific subunit unique to HPHA. Using affinity chromatography, we previously isolated a mixture of five PHA proteins which contained all the red cellagglutinating and lymphocyte-stimulating activity of red kidney beans (6). These five carbohydrate-binding proteins have now been separated and recovered in high yield by ion exchange chromatography and their individual properties examined. Our experiments show that these lectins have similar physical and chemical properties, yet differ in their relative biological activities (7). We suggest that the family of Phaseolus vulgaris lectins be called isolectins.

Affinity
Chromatography-A mixture of five proteins, which contain all the red cell-agglutinating and lymphocyte-stimulating activitv of red kidnev beans was obtained from 100 e of red kidnev beans by affinity chromatography on thyroglobulin conjugated to'sepharose, as previous1.v described (6). The affinity-purified proteins were concentrated by ultrafiltration (PM-10 membrane, Amicon) to a protein concentration of 30 to 75 mglml, stored at -19, and called PHA-mix.
Ion Exchange Chromatography-Forty-five grams of preswollen and desassed SP-Senhadex (Pharmacia) was uacked in a column (2.5 x 45 cm) under a pressure of30 cm of water and washed with several liters of 0.05 M potassium nhosnhate (pH 6.0) buffer. PHA-mix isolated from a saline extract bf 160 g of red kidney beans was dialyzed for 60 h against several changes of 0.05 M potassium phosphate (pH 6.0) buffer, added to the column, and eluted with the same buffer at a flow rate of 24 ml/h. After 640 ml, the elution buffer was changed to a linear gradient of 2 liters of 0.05 M potassium phosphate (pH 6.01, and 2 liters of the same buffer containing 0.188 M NaCl. Fractions (25 ml) were monitored for protein absorbance (280 nm) and for salt by conductance.
Fractions from the center absorbance peaks were nooled and concentrated bv nressure ultrafiltration, stored at a concentration of 10 to 75 m"g/ml, and called sequentially L,, L,E,, LrEz, LIE:,, and Eq bv their order of elution (8).

Polyacrylamide
Gel Electrophoresis -Electrophoresis was performed on a 1.5-mm flat gel of 7% acrylamide, 0.4% bisacrylamide (9) at 5" with methyl green as the tracking dye. Electronhoresis was at a current of 40 mk until the tracking dye had migrated 11 cm. The gels were then stained with Coomassie brilliant blue R250 (10) and destained as described (6).

SDS-Polyacrylamide
Gel Electrophoresis-Protein subunit molecstrength. The separated protein fractions contained from 30 to ular weights were determined by SDS-polyacrylamide gel electrophoresis (10) on a 0.75mm slab of SDS-polyacrylamide gel, with 130 mg of protein each (Table I) stain, showed that more than 95% of the white blood cells were lymphocytes, with less than one red blood cell and ten platelets per five lymphocytes.
The cultures and viability tests were then performed as previously described (6). All samples were more than 95% viable.
Assay of Incorporation of ZsHIThymidine-The incorporation of PHlthvmidine was assaved after 56 h as ureviouslv described (6)  In order to correct for decomposition, quantities determined for threonine and serine were increased 5 and lo%, respectively.
The absence of cysteic acid and methionine sulfone was confirmed after performic acid oxidation (17). The tryptophan content was determined spectrophotometrically (18

Zon Exchange
Chromatography- The PHA-mix proteins obtained by affinity chromatography are resolved into five fractions by SP-Sephadex ion exchange chromatography (Fig. 1). L,, in the first absorbance peak, is eluted with the equilibration buffer. Four other absorbance peaks, L,E,, L2E2, L,E3, and E4, are eluted by a shallow gradient of increasing ionic  the isolectins differ primarily in content of threonine, lysine, and arginine (Table II). Despite the differences in individual basic amino acids, the total basic amino acid content is identical for each of the isolectins. The compositions also are notable for the lack of sulfur-containing amino acids.

Carbohydrate Composition-
The carbohydrate compositions of the isolectins are also similar (Table II). Their only neutral sugar is mannose, which constitutes from 3.4 to 4.3 g/ 100 g of protein. The only other sugar was glucosamine (which is assumed to be N-acetyl-n-glucosamine) and constitutes 2.0 to 2.4 g/100 g of protein. The ratio of mannose to N-acetyl-nglucosamine of all five isolectins was 2.25 + 0.08. toins were identified by gas-liquid and high pressure liquid chromatography.
Positive identification of amino acid derivatives was made for residues 1, 3, 6, 8, 9, 11, 14, 16, 17, and 18. The residues identified are identical with the NH,-terminal sequence previously found for a polypeptide isolated from denatured HPHA by isoelectric focusing and believed to be the erythrocyte-specific subunit (23). In addition, the absence of identifiable residues at positions 2, 4, 5, and 7 is consistent with the presence of the labile residues, serine and threonine, previously proposed for these positions. DISCUSSION

NH,-terminal
Amino Acid Sequence-E4 was subjected to Whereas PHA is a standard reagent for cell biology and automated Edman degradations, and the phenylthiohydan-membrane biochemistry studies, its biological and biochemi- activities been studied. In addition, the contribution of each active protein to the collective activity of the PHA family has not been assessed.
We have described a simple two-step procedure for the isolation of five isolectins from red kidney beans. The individual isolectins are isolated in high yield and contain all the red cellagglutinating and lymphocyte-stimulating activity of the red kidney bean extracts. Each isolectin is homogeneous by discontinuous and SDS-polyacrylamide gel electrophoresis and by sedimentation equilibrium.
E, satisfies the additional criteria of homogeneity, since it chromatographs reproducibly on ion exchange chromatography and contains a single NH,terminal amino acid sequence. The conditions of isolation are mild as shown by quantitative and reproducible recoveries of the erythroagglutinin-and lymphocyte-stimulating activities. The L, isolated by our procedure is probably identical with the potent lymphocyte mitogen-leukoagglutinin with low erythroagglutinating activity isolated from commercial PHA (1, 3) or isolated from kidney beans (22). In agreement with previous investigators, we have found similar biological activity, amino acid composition, sedimentation coefficient, chromatographic and electrophoretic characteristics, and native and subunit molecular weights for L,.
Our isolation of pure E, proves the existence of that hypothetical isolectin of four apparently identical E subunits having the highest erythroagglutinating activity of the PHA family (4, 5). The low, but measurable mitogenic activity of E, as well as the low erythroagglutinating activity of L, suggest that, while the theorized PHA subunits are highly reactive with their respective cell type, the interaction is not absolutely specific. In addition to E,, we have proved the existence and established the biological activities of the theorized isolectins L,,E,, L,E,, and L,E,,. The spectrum of biological activities of the isolectins is consistent with the hypothesis that the isolectins are tetramers of varying proportions of L and E subunits (subunit proportions indicated by subscripts). The recovery of the PHA activity as five separate and distinct native isolectins is demonstrated for the first time and provides an opportunity to examine the PHA subunit relationships and structures directly (8).
In contrast to their different biological activities, the isolectins have similar physicochemical properties and structural homology. They all bind avidly to the carbohydrate moiety of thyroglobulin, elute from SP-Sephadex at similar ionic strengths, migrate similarly in electrophoretic fields and have similar isoelectric points2 In addition, they have identical sedimentation coefficients and native and subunit molecular weights. Partial NH,-and COOH-terminal amino acid analyses and peptide maps lead to the conclusion that limited regions of structural difference are responsible for the different cellular interactions (23, 24).
The different biological actions of the isolectins may reflect different affinities or specificities for oligosaccharide receptors at the cell surface. For example, isolectins have different affinities for the glycoproteins fetuin (25) and thyroglobulin (6). Enzymatically modified glycopeptide from thyroglobulin possesses different inhibitory activity of PHA-induced lymphocyte stimulation and of PHA-induced erythroagglutination