The critical interaction of the metallopeptidase PHEX with heparan sulfate proteoglycans

The critical interaction of the metallopeptidase PHEX with heparan sulfate proteoglycans

Author Barros, Nilana M. T. Autor UNIFESP Google Scholar
Nascimento, Fabio D. Autor UNIFESP Google Scholar
Oliveira, Vitor Autor UNIFESP Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Loisel, Thomas Google Scholar
Nader, Helena B. Autor UNIFESP Google Scholar
Boileau, Guy Google Scholar
Tersariol, Ivarne L. S. Google Scholar
Carmona, Adriana K. Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Enobia Pharma Inc
Univ Montreal
Univ Mogi das Cruzes
Abstract The PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X chromosome) identified as a mutated gene in patients with X-linked hypophosphatemia (XLH), encodes a protein (PHEX) that shows striking homologies to members of the M13 family of zinc metallopeptidases. in the present work the interaction of glycosaminoglycans with PHEX has been investigated by affinity chromatography, circular dichroism, protein intrinsic fluorescence analysis, hydrolysis of FRET substrates flow cytometry and confocal microscopy. PHEX was eluted from a heparin-Sepharose chromatography column at 0.8 M NaCl showing a strong interaction with heparin. Circular dichroism spectra and intrinsic fluorescence analysis showed that PHEX is protected by glycosaminoglycans against thermal denaturation. Heparin, heparan sulfate and chondroitin sulfate inhibited PHEX catalytic activity, however among them; heparin presented the highest inhibitory activity (K-i = 2.5 +/- 0.2 nM). Flow cytometry analysis showed that PHEX conjugated to Alexa Fluor 488 binds to the cell surface of CHO-K1, but did not bind to glycosaminoglycans defective cells CHO-745. Endogenous PHEX was detected at the cell surface of CHO-K1 colocalized with heparan sulfate proteoglycans, but was not found at the cell surface of glycosaminoglycans defective cells CHO-745. in permeabilized cells, PHEX was detected in endoplasmic reticulum of both cells. in addition, we observed that PHEX colocalizes with heparan sulfate at the cell surface of osteoblasts. This is the first report that the metallopeptidase PHEX is a heparin binding protein and that the interaction with GAGs modulates its enzymatic activity, protein stability and cellular trafficking. (c) 2008 Elsevier B.V. All rights reserved.
Keywords Phosphate-regulating gene with homologies to endopeptidase on the X chromosome (PHEX)
Metallopeptidase
Heparan sulfate
Heparin
Proteoglycans
Language English
Sponsor Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Date 2008-01-01
Published in International Journal of Biochemistry & Cell Biology. Oxford: Pergamon-Elsevier B.V., v. 40, n. 12, p. 2781-2792, 2008.
ISSN 1357-2725 (Sherpa/Romeo, impact factor)
Publisher Elsevier B.V.
Extent 2781-2792
Origin http://dx.doi.org/10.1016/j.biocel.2008.05.021
Access rights Closed access
Type Article
Web of Science ID WOS:000260125400014
URI http://repositorio.unifesp.br/handle/11600/30273

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