Mechanism of human lymphocyte stimulation by concanavalin A: Role of valence and surface binding sites

J. R. Wands, D. K. Podolsky, K. J. Isselbacher

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Abstract

A monovalent form of concanavalin A(m Con A) has been prepared to determine the importance of valence for human lymphocyte surface binding and subsequent lymphocyte stimulation as measured by blast transformation and cytotoxicity. Concanavalin A (Con A) was fragmented by a proteolytic process and the m Con A derivative was isolated by elution with an ascending D glucose gradient on a Sephadex G 200 column. The molecular weight of m Con A was 18,000 by sodium dodecyl sulfate polyacrylamide electrophoresis. Equilibrium dialysis with α methyl D glucoside and subsequent Scatchard plot analysis revealed an association constant (Ka) of 1.2 x 103 liters/mol and a valence of 1.1. Incubation of lymphocytes with 125I labeled m Con A demonstrated surface binding at 1.21 x 106 molecules per cell, which was comparable to the binding of [3H]Con A (1.02 x 106 molecules per cell). However, in contrast to the intact lectin, m Con A had a markedly reduced capacity to agglutinate rabbit erythrocytes and human lymphocytes and did not stimulate lymphocyte blast transformation or cytotoxicity at 1 and 10 μg/ml. Finally, pretreatment of lymphocytes with m Con A blocked blast transformation induced by Con A. These observations demonstrate that m Con A binds to lymphocyte surface receptors but does not stimulate blast transformation or cytotoxicity, suggesting that Con A must bridge binding sites on the lymphocyte surface to induce lymphocyte activation.

Original languageEnglish (US)
Pages (from-to)2118-2122
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume73
Issue number6
StatePublished - 1976

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Lymphocyte Activation
Concanavalin A
Binding Sites
Lymphocytes
Lectins
Sodium Dodecyl Sulfate
Electrophoresis
Dialysis
Erythrocytes
Molecular Weight
Rabbits
Glucose

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

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title = "Mechanism of human lymphocyte stimulation by concanavalin A: Role of valence and surface binding sites",
abstract = "A monovalent form of concanavalin A(m Con A) has been prepared to determine the importance of valence for human lymphocyte surface binding and subsequent lymphocyte stimulation as measured by blast transformation and cytotoxicity. Concanavalin A (Con A) was fragmented by a proteolytic process and the m Con A derivative was isolated by elution with an ascending D glucose gradient on a Sephadex G 200 column. The molecular weight of m Con A was 18,000 by sodium dodecyl sulfate polyacrylamide electrophoresis. Equilibrium dialysis with α methyl D glucoside and subsequent Scatchard plot analysis revealed an association constant (Ka) of 1.2 x 103 liters/mol and a valence of 1.1. Incubation of lymphocytes with 125I labeled m Con A demonstrated surface binding at 1.21 x 106 molecules per cell, which was comparable to the binding of [3H]Con A (1.02 x 106 molecules per cell). However, in contrast to the intact lectin, m Con A had a markedly reduced capacity to agglutinate rabbit erythrocytes and human lymphocytes and did not stimulate lymphocyte blast transformation or cytotoxicity at 1 and 10 μg/ml. Finally, pretreatment of lymphocytes with m Con A blocked blast transformation induced by Con A. These observations demonstrate that m Con A binds to lymphocyte surface receptors but does not stimulate blast transformation or cytotoxicity, suggesting that Con A must bridge binding sites on the lymphocyte surface to induce lymphocyte activation.",
author = "Wands, {J. R.} and Podolsky, {D. K.} and Isselbacher, {K. J.}",
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T1 - Mechanism of human lymphocyte stimulation by concanavalin A

T2 - Role of valence and surface binding sites

AU - Wands, J. R.

AU - Podolsky, D. K.

AU - Isselbacher, K. J.

PY - 1976

Y1 - 1976

N2 - A monovalent form of concanavalin A(m Con A) has been prepared to determine the importance of valence for human lymphocyte surface binding and subsequent lymphocyte stimulation as measured by blast transformation and cytotoxicity. Concanavalin A (Con A) was fragmented by a proteolytic process and the m Con A derivative was isolated by elution with an ascending D glucose gradient on a Sephadex G 200 column. The molecular weight of m Con A was 18,000 by sodium dodecyl sulfate polyacrylamide electrophoresis. Equilibrium dialysis with α methyl D glucoside and subsequent Scatchard plot analysis revealed an association constant (Ka) of 1.2 x 103 liters/mol and a valence of 1.1. Incubation of lymphocytes with 125I labeled m Con A demonstrated surface binding at 1.21 x 106 molecules per cell, which was comparable to the binding of [3H]Con A (1.02 x 106 molecules per cell). However, in contrast to the intact lectin, m Con A had a markedly reduced capacity to agglutinate rabbit erythrocytes and human lymphocytes and did not stimulate lymphocyte blast transformation or cytotoxicity at 1 and 10 μg/ml. Finally, pretreatment of lymphocytes with m Con A blocked blast transformation induced by Con A. These observations demonstrate that m Con A binds to lymphocyte surface receptors but does not stimulate blast transformation or cytotoxicity, suggesting that Con A must bridge binding sites on the lymphocyte surface to induce lymphocyte activation.

AB - A monovalent form of concanavalin A(m Con A) has been prepared to determine the importance of valence for human lymphocyte surface binding and subsequent lymphocyte stimulation as measured by blast transformation and cytotoxicity. Concanavalin A (Con A) was fragmented by a proteolytic process and the m Con A derivative was isolated by elution with an ascending D glucose gradient on a Sephadex G 200 column. The molecular weight of m Con A was 18,000 by sodium dodecyl sulfate polyacrylamide electrophoresis. Equilibrium dialysis with α methyl D glucoside and subsequent Scatchard plot analysis revealed an association constant (Ka) of 1.2 x 103 liters/mol and a valence of 1.1. Incubation of lymphocytes with 125I labeled m Con A demonstrated surface binding at 1.21 x 106 molecules per cell, which was comparable to the binding of [3H]Con A (1.02 x 106 molecules per cell). However, in contrast to the intact lectin, m Con A had a markedly reduced capacity to agglutinate rabbit erythrocytes and human lymphocytes and did not stimulate lymphocyte blast transformation or cytotoxicity at 1 and 10 μg/ml. Finally, pretreatment of lymphocytes with m Con A blocked blast transformation induced by Con A. These observations demonstrate that m Con A binds to lymphocyte surface receptors but does not stimulate blast transformation or cytotoxicity, suggesting that Con A must bridge binding sites on the lymphocyte surface to induce lymphocyte activation.

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