Back to Search
Start Over
The critical interaction of the metallopeptidase PHEX with heparan sulfate proteoglycans
- Source :
- The international journal of biochemistrycell biology. 40(12)
- Publication Year :
- 2008
-
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 (Ki=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.
- Subjects :
- Metallopeptidase
CHO Cells
Biochemistry
Chromatography, Affinity
Substrate Specificity
Glycosaminoglycan
chemistry.chemical_compound
Cricetulus
Affinity chromatography
Cricetinae
Fluorescence Resonance Energy Transfer
Animals
Chondroitin sulfate
Chemistry
Heparin
Endoplasmic reticulum
Circular Dichroism
Hydrolysis
PHEX
Proteins
Cell Biology
Heparan sulfate
Molecular biology
Recombinant Proteins
Metalloproteases
Proteoglycans
Heparitin Sulfate
Chromatography column
Heparan Sulfate Proteoglycans
Protein Binding
Subjects
Details
- ISSN :
- 13572725
- Volume :
- 40
- Issue :
- 12
- Database :
- OpenAIRE
- Journal :
- The international journal of biochemistrycell biology
- Accession number :
- edsair.doi.dedup.....c95bcbe7ac99a07beff5e3685fc18a74