A Fluorogenic Peptide Containing the Processing Site of Human SARS Corona Virus S‐Protein: Kinetic Evaluation and NMR Structure Elucidation
Identifieur interne : 000102 ( Istex/Curation ); précédent : 000101; suivant : 000103A Fluorogenic Peptide Containing the Processing Site of Human SARS Corona Virus S‐Protein: Kinetic Evaluation and NMR Structure Elucidation
Auteurs : Ajoy Basak ; Abhijit Mitra [États-Unis] ; Sarmistha Basak ; Carolyn Pasko ; Michel Chrétien ; Pamela Seaton [États-Unis]Source :
- ChemBioChem [ 1439-4227 ] ; 2007-06-18.
English descriptors
- Teeft :
- Active enzyme, Amide, Amide protons, Amino, Amino acid analysis, Amino acids, Analytical column, Authentic samples, Basak, Biochem, Bruker biospin, Chembiochem, Cleavage, Cleavage site, Cleavage sites, Comparative study, Crucial role, Crude digests, Data show, Digestion, Enzyme activity, Experimental section, Fluorogenic, Fluorogenic peptide, Furin, Furin cleavage, Furin inhibitors, Further studies, Glutamic acid, Glycoprotein, Gmbh, Hsars coronavirus cleavage, Hsars coronavirus cleavage figure, Human sars corona virus, Identical conditions, Identical levels, Inhibitor, Kcat, Kgaa, Kinetic parameters, Mass spectrometry, Model peptide, Peak intensity, Peptide, Peptide synthesis, Proteolytic maturation, Proton, Random structures, Roesy, Roesy correlations, Roesy spectra, Sars, Sars infection, Secondary structure, Seidah, Sheet content, Sheet structure, Soluble, Soluble forms, Soluble pc5a, Spike, Spike protein, Tocsy patterns, Various concentrations, Verlag, Verlag gmbh, Weinheim, Weinheim chembiochem.
Abstract
Human severe acute respiratory syndrome coronavirus (hSARS‐CoV) is the causative agent for SARS infection. Its surface glycoprotein (spike protein) is considered to be one of the prime targets for SARS therapeutics and intervention because its proteolytic maturation by a host protease is crucial for host–virus fusion. Using intramolecularly quenched fluorogenic (IQF) peptides based on hSARS‐CoV spike protein (Abz‐755Glu‐Gln‐Asp‐Arg‐Asn‐Thr‐Arg‐Glu‐Val‐Phe‐Ala‐Gln766‐Tyx‐NH2) and in vitro studies, we show that besides furin, other PCs, like PC5 and PC7, might also be involved in this cleavage event. Through kinetic measurements with recombinant PCs, we observed that the peptide was cleaved efficiently by both furin and PC5, but very poorly by PC7. The cleavage could be blocked by a PC‐inhibitor, α1‐PDX, in a dose‐dependent manner. Circular dichroism spectra indicated that this peptide possesses a high degree of sheet structure. Following cleavage by furin, the sheet content increased, possibly at the expense of turn and random structures. 1H NMR spectra from 2D COSY and ROESY experiments under physiological buffer and pH conditions indicated that this peptide possesses a structure with a turn at its C‐terminal segment, close to the cleavage site. The data suggest that the cleavable peptide bond is located within the most exposed domain; this is supported by the nearby turn structure. Several strong to weak NMR ROESY correlations were detected, and a 3D structure of the spike IQF peptide that contains the crucial cleavage site R761↓E has been proposed.
Cut and fuse. A key event in SARS infection is the proteolytic cleavage of viral spike protein by host proteases. By using intramolecularly quenched fluorogenic (IQF) peptides based on hSARS‐CoV spike protein and in vitro studies, we show that in addition to furin, proprotein convertases PC5 and PC7 cleave the model IQF peptide. Cleavage could be blocked by the PC inhibitor, α1‐PDX, in a dose‐dependent manner. The 3D structure of the model peptide was investigated by circular dichromism and 2D 1H NMR spectroscopy.
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DOI: 10.1002/cbic.200700007
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Ajoy Basak<affiliation><mods:affiliation>Hormone, Growth, and Development Program, Regional Protein Chemistry Center, Ottawa Health Research Institute, University of Ottawa, 725 Parkdale Ave., Ottawa, ON K1Y 4E9, Canada, Fax: (+1) 613‐761‐4355</mods:affiliation><wicri:noCountry code="subField">(+1) 613‐761‐4355</wicri:noCountry></affiliation><affiliation><mods:affiliation>Hormone, Growth, and Development Program, Regional Protein Chemistry Center, Ottawa Health Research Institute, University of Ottawa, 725 Parkdale Ave., Ottawa, ON K1Y 4E9, Canada, Fax: (+1) 613‐761‐4355</mods:affiliation><wicri:noCountry code="subField">(+1) 613‐761‐4355</wicri:noCountry></affiliation><affiliation><mods:affiliation>Hormone, Growth, and Development Program, Regional Protein Chemistry Center, Ottawa Health Research Institute, University of Ottawa, 725 Parkdale Ave., Ottawa, ON K1Y 4E9, Canada, Fax: (+1) 613‐761‐4355</mods:affiliation><wicri:noCountry code="subField">(+1) 613‐761‐4355</wicri:noCountry></affiliation><affiliation><mods:affiliation>Hormone, Growth, and Development Program, Regional Protein Chemistry Center, Ottawa Health Research Institute, University of Ottawa, 725 Parkdale Ave., Ottawa, ON K1Y 4E9, Canada, Fax: (+1) 613‐761‐4355</mods:affiliation><wicri:noCountry code="subField">(+1) 613‐761‐4355</wicri:noCountry></affiliation>Le document en format XML
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first="Ajoy" last="Basak">Ajoy Basak</name><affiliation><mods:affiliation>Hormone, Growth, and Development Program, Regional Protein Chemistry Center, Ottawa Health Research Institute, University of Ottawa, 725 Parkdale Ave., Ottawa, ON K1Y 4E9, Canada, Fax: (+1) 613‐761‐4355</mods:affiliation><wicri:noCountry code="subField">(+1) 613‐761‐4355</wicri:noCountry></affiliation></author><author><name sortKey="Mitra, Abhijit" sort="Mitra, Abhijit" uniqKey="Mitra A" first="Abhijit" last="Mitra">Abhijit Mitra</name><affiliation wicri:level="1"><mods:affiliation>Department of Chemistry and Biochemistry, Manhattan College/College of Mount Saint Vincent, Riverdale, NY 10471, USA</mods:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Chemistry and Biochemistry, Manhattan College/College of Mount Saint Vincent, Riverdale, NY 10471</wicri:regionArea></affiliation></author><author><name sortKey="Basak, Sarmistha" sort="Basak, Sarmistha" uniqKey="Basak S" first="Sarmistha" last="Basak">Sarmistha Basak</name><affiliation><mods:affiliation>Hormone, Growth, and Development Program, Regional Protein Chemistry Center, Ottawa Health Research Institute, University of Ottawa, 725 Parkdale Ave., Ottawa, ON K1Y 4E9, Canada, Fax: (+1) 613‐761‐4355</mods:affiliation><wicri:noCountry code="subField">(+1) 613‐761‐4355</wicri:noCountry></affiliation></author><author><name sortKey="Pasko, Carolyn" sort="Pasko, Carolyn" uniqKey="Pasko C" first="Carolyn" last="Pasko">Carolyn Pasko</name><affiliation><mods:affiliation>Hormone, Growth, and Development Program, Regional Protein Chemistry Center, Ottawa Health Research Institute, University of Ottawa, 725 Parkdale Ave., Ottawa, ON K1Y 4E9, Canada, Fax: (+1) 613‐761‐4355</mods:affiliation><wicri:noCountry code="subField">(+1) 613‐761‐4355</wicri:noCountry></affiliation></author><author><name sortKey="Chretien, Michel" sort="Chretien, Michel" uniqKey="Chretien M" first="Michel" last="Chrétien">Michel Chrétien</name><affiliation><mods:affiliation>Hormone, Growth, and Development Program, Regional Protein Chemistry Center, Ottawa Health Research Institute, University of Ottawa, 725 Parkdale Ave., Ottawa, ON K1Y 4E9, Canada, Fax: (+1) 613‐761‐4355</mods:affiliation><wicri:noCountry code="subField">(+1) 613‐761‐4355</wicri:noCountry></affiliation></author><author><name sortKey="Seaton, Pamela" sort="Seaton, Pamela" uniqKey="Seaton P" first="Pamela" last="Seaton">Pamela Seaton</name><affiliation wicri:level="1"><mods:affiliation>Department of Chemistry and Biochemistry, University of North Carolina, Wilmington, NC 28403, USA</mods:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Chemistry and Biochemistry, University of North Carolina, Wilmington, NC 28403</wicri:regionArea></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">ChemBioChem</title><title level="j" type="alt">CHEMBIOCHEM</title><idno type="ISSN">1439-4227</idno><idno type="eISSN">1439-7633</idno><imprint><biblScope unit="vol">8</biblScope><biblScope unit="issue">9</biblScope><biblScope unit="page" from="1029">1029</biblScope><biblScope unit="page" to="1037">1037</biblScope><biblScope unit="page-count">9</biblScope><publisher>WILEY‐VCH Verlag</publisher><pubPlace>Weinheim</pubPlace><date type="published" when="2007-06-18">2007-06-18</date></imprint><idno type="ISSN">1439-4227</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1439-4227</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Active enzyme</term><term>Amide</term><term>Amide protons</term><term>Amino</term><term>Amino acid analysis</term><term>Amino acids</term><term>Analytical column</term><term>Authentic samples</term><term>Basak</term><term>Biochem</term><term>Bruker biospin</term><term>Chembiochem</term><term>Cleavage</term><term>Cleavage site</term><term>Cleavage sites</term><term>Comparative study</term><term>Crucial role</term><term>Crude digests</term><term>Data show</term><term>Digestion</term><term>Enzyme activity</term><term>Experimental section</term><term>Fluorogenic</term><term>Fluorogenic peptide</term><term>Furin</term><term>Furin cleavage</term><term>Furin inhibitors</term><term>Further studies</term><term>Glutamic acid</term><term>Glycoprotein</term><term>Gmbh</term><term>Hsars coronavirus cleavage</term><term>Hsars coronavirus cleavage figure</term><term>Human sars corona virus</term><term>Identical conditions</term><term>Identical levels</term><term>Inhibitor</term><term>Kcat</term><term>Kgaa</term><term>Kinetic parameters</term><term>Mass spectrometry</term><term>Model peptide</term><term>Peak intensity</term><term>Peptide</term><term>Peptide synthesis</term><term>Proteolytic maturation</term><term>Proton</term><term>Random structures</term><term>Roesy</term><term>Roesy correlations</term><term>Roesy spectra</term><term>Sars</term><term>Sars infection</term><term>Secondary structure</term><term>Seidah</term><term>Sheet content</term><term>Sheet structure</term><term>Soluble</term><term>Soluble forms</term><term>Soluble pc5a</term><term>Spike</term><term>Spike protein</term><term>Tocsy patterns</term><term>Various concentrations</term><term>Verlag</term><term>Verlag gmbh</term><term>Weinheim</term><term>Weinheim chembiochem</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Human severe acute respiratory syndrome coronavirus (hSARS‐CoV) is the causative agent for SARS infection. Its surface glycoprotein (spike protein) is considered to be one of the prime targets for SARS therapeutics and intervention because its proteolytic maturation by a host protease is crucial for host–virus fusion. Using intramolecularly quenched fluorogenic (IQF) peptides based on hSARS‐CoV spike protein (Abz‐755Glu‐Gln‐Asp‐Arg‐Asn‐Thr‐Arg‐Glu‐Val‐Phe‐Ala‐Gln766‐Tyx‐NH2) and in vitro studies, we show that besides furin, other PCs, like PC5 and PC7, might also be involved in this cleavage event. Through kinetic measurements with recombinant PCs, we observed that the peptide was cleaved efficiently by both furin and PC5, but very poorly by PC7. The cleavage could be blocked by a PC‐inhibitor, α1‐PDX, in a dose‐dependent manner. Circular dichroism spectra indicated that this peptide possesses a high degree of sheet structure. Following cleavage by furin, the sheet content increased, possibly at the expense of turn and random structures. 1H NMR spectra from 2D COSY and ROESY experiments under physiological buffer and pH conditions indicated that this peptide possesses a structure with a turn at its C‐terminal segment, close to the cleavage site. The data suggest that the cleavable peptide bond is located within the most exposed domain; this is supported by the nearby turn structure. Several strong to weak NMR ROESY correlations were detected, and a 3D structure of the spike IQF peptide that contains the crucial cleavage site R761↓E has been proposed.</div><div type="abstract" xml:lang="en">Cut and fuse. A key event in SARS infection is the proteolytic cleavage of viral spike protein by host proteases. By using intramolecularly quenched fluorogenic (IQF) peptides based on hSARS‐CoV spike protein and in vitro studies, we show that in addition to furin, proprotein convertases PC5 and PC7 cleave the model IQF peptide. Cleavage could be blocked by the PC inhibitor, α1‐PDX, in a dose‐dependent manner. The 3D structure of the model peptide was investigated by circular dichromism and 2D 1H NMR spectroscopy.</div></front></TEI></record>Pour manipuler ce document sous Unix (Dilib)
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