Interchain and intrachain disulphide bonds in human platelet glycoprotein IIb. Localization of the epitopes for several monoclonal antibodies

Biochemical Journal - Tập 261 Số 2 - Trang 551-560 - 1989
Juan J. Calvete1, M V Alvarez1, Germán Rivas1, C. L. Hew2, Agnes Henschen3, José González-Rodrı́guez1
1Instituto de Química Física, Consejo Superior de Investigaciones Científicas, Serrano, 119, 28006 Madrid, Spain.
2Department of Clinical Biochemistry, Banting Institute, University of Toronto, Toronto, Ontario, Canada M5G IL5
3Max-Planck-Institut für Biochemie, D-8033 Martinsried/Miinchen, Federal Republic of Germany

Tóm tắt

The single interchain disulphide bond in platelet glycoprotein IIb (GPIIb) is accessible to extracellular reductants, and selective cleavage does not liberate GPIIb alpha from platelet plasma membrane, confirming that non-covalent interactions contribute to maintaining attachment of this subunit to the membrane. Eosin-maleimide labelling of isolated GPIIb after selective cleavage of this interchain disulphide bond, followed by full reduction and alkylation, CNBr cleavage, and analysis of the cleavage products allowed us to establish that this interchain disulphide bridge is formed between GPIIb beta (GPIIb beta-subunit) Cys-9 and GPIIb alpha Cys-826, and this conclusion was confirmed by independent routes. The other two cysteines of GPIIb beta (Cys-14 and Cys-19) form the single intrachain disulphide bond in this subunit. Last, the intrachain disulphides in GPIIb alpha (GPIIb alpha-subunit) are distributed in four main peptide domains which are not disulphide-bonded among themselves. The linear epitope for monoclonal antibody M1 is localized between Pro-4 and Met-24 (or Met-31) of GPIIb beta. The linear epitope for M3 is situated between Cys-826 and the C-terminus of GPIIb alpha. The M4 epitope is also linear and localized somewhere between residues 115 and 285 of GPIIb alpha. Finally, the epitopes for M5 and M6 are somewhere between Cys-608 and Met-704, within a 35 kDa membrane-bound chymotryptic product of digestion of GPIIb in whole platelets. The N-terminal amino acid sequences determined for eight different cleavage products of GPIIb alpha and GPIIb beta agree with the corresponding amino acid sequences predicted by cDNA sequence for human-erythroleukaemic-cell GPIIb [Poncz, Eisman, Heindenreich, Silver, Vilaire, Surrey, Schwartz & Bennett (1987) J. Biol. Chem. 262, 8476-8482].

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Biochemical Journal

Tập 261 Số 2

551-560

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