A rapid fluorometric assay for the proteolytic activity of SKI-1/S1P based on the surface glycoprotein of the hemorrhagic fever Lassa virus.
The subtilase subtilisin kexin isozyme-1 (SKI-1)/site 1 protease (S1P), has been implicated in the processing of Lassa virus glycoprotein C (GP-C) precursor into GP1 and GP2 that are responsible for viral fusion with the host cell membrane. Here, we studied in vitro the kinetics of this cleavage by hSKI-1 using an intramolecularly quenched fluorogenic (IQF) peptide, Q-GPC(251-263) [Abz-(251)Asp-Ile-Tyr-Ile-Ser-Arg-Arg-Leu-Leu/Gly-Thr-Phe-Thr(263)-3-NitroTyr-Ala-CONH(2)], containing the identified site. The measured V(max (app))/K(m (app)) was compared to those for other IQF SKI-substrates. Q-GPC(251-263) is cleaved 10-fold more efficiently than the previously known best SKI-substrate, Q-hproSKI(134-142). This study confirmed the role of SKI-1 in GP-C processing and provides a novel, rapid and efficient enzymatic assay of SKI-1.
Basak
Ajoy
A
Regional Protein Chemistry Center, Diseases of Ageing Unit, Ottawa Health Research Institute, Loeb Building, 725 Parkdale Ave., Ottawa, Ontario, Canada K1Y 4E9. abasak@ohri.ca
Chrétien
Michel
M
Seidah
Nabil G
NG
D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry / Q000379:methods*; D007700:Kinetics; D007836:Lassa virus / Q000737:chemistry*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D043484:Proprotein Convertases; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000032:analysis* / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014760:Viral Fusion Proteins / Q000737:chemistry; D015678:Viral Structural Proteins / Q000737:chemistry*
D005456:Fluorescent Dyes; D010455:Peptides; D014760:Viral Fusion Proteins; D015678:Viral Structural Proteins; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; C115575:membrane-bound transcription factor peptidase, site 1