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J. Biol. Chem., Vol. 280, Issue 36, 31949-31956, September 9, 2005

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Heparin-binding Sites in Granulocyte-Macrophage Colony-stimulating Factor

LOCALIZATION AND REGULATION BY HISTIDINE IONIZATION^*

Adriano Sebollela, Thiago C. Cagliari¶, Gabriel S. C. S. Limaverde||, Alex Chapeaurouge**, Marcos H. F. Sorgine, Tatiana Coelho-Sampaio, Carlos H. I. Ramos, and Sérgio T. Ferreira

From the Programa de Bioquímica e Biofísica Celular, Instituto de Bioquímica Médica, ^||Laboratório de Modelagem e Dinmica Molecular, Instituto de Biofísica Carlos Chagas Filho, and Departamento de Histologia e Embriologia, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21940-590, Laboratório Nacional de Luz Síncrotron, Campinas, SP 13084-971l, ^¶Departamento de Bioquímica, Instituto de Biologia, UNICAMP, Campinas, SP, and ^**Departamento de Fisiologia e Farmacodinmica, Fiocruz, Rio de Janeiro, RJ 21045-900, Brazil

The biological activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) is modulated by the sulfated glycosaminoglycans (GAGs) heparan sulfate and heparin. However, the molecular mechanisms involved in such interactions are still not completely understood. We have proposed previously that helix C, one of the four -helices of human GM-CSF (hGM-CSF), contains a GAG-binding site in which positively charged residues are spatially positioned for interaction with the sulfate moieties of the GAGs (Wettreich, A., Sebollela, A., Carvalho, M. A., Azevedo, S. P., Borojevic, R., Ferreira, S. T., and Coelho-Sampaio, T. (1999) J. Biol. Chem. 274, 31468-31475). Protonation of two histidine residues (His⁸³ and His⁸⁷) in helix C of hGM-CSF appears to act as a pH-dependent molecular switch to control the interaction with GAGs. Based on these findings, we have now generated a triple mutant form of murine GM-CSF (mGM-CSF) in which three noncharged residues in helix C of the murine factor (Tyr⁸³, Gln⁸⁵, and Tyr⁸⁷) were replaced by the corresponding basic residues present in hGM-CSF (His⁸³, Lys⁸⁵, and His⁸⁷). Binding assays on heparin-Sepharose showed that, at acidic pH, the triple mutant mGM-CSF binds to immobilized heparin with significantly higher affinity than wild type (WT) mGM-CSF and that neither protein binds to the column at neutral pH. The fact that even WT mGM-CSF binds to heparin at acidic pH indicates the existence of a distinct, lower affinity heparin-binding site in the protein. Chemical modification of the single histidine residue (His¹⁵) located in helix A of WT mGM-CSF with diethyl pyrocarbonate totally abolished binding to immobilized heparin. Moreover, replacement of His¹⁵ for an alanine residue significantly reduced the affinity of mGM-CSF for heparin at pH 5.0 and completely blocked heparin binding to a synthetic peptide corresponding to helix A of GM-CSF. These results indicate a major role of histidine residues in the regulation of the binding of GM-CSF to GAGs, supporting the notion that an acidic microenvironment is required for GM-CSF-dependent regulation of target cells. In addition, our results provide insight into the molecular basis of the strict species specificity of the biological activity of GM-CSF.

Received for publication, May 16, 2005 , and in revised form, July 5, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 55-21-2562-6790; Fax: 55-21-2562-6789; E-mail: ferreira{at}bioqmed.ufrj.br.

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