A rapid fluorometric assay for the proteolytic activity of SKI‐1/S1P based on the surface glycoprotein of the hemorrhagic fever Lassa virus

FEBS Letters

Chrétien, Michel²  Basak, Ajoy²  Seidah, Nabil G.¹ 

[1] Biochemical Neuroendocrinology Laboratory, Clinical Research Institute of Montreal, 110 Pine Ave. West, Montreal, QC, Canada H2W 1R7;Regional Protein Chemistry Center, Diseases of Ageing Unit, Ottawa Health Research Institute, Loeb Building, Ottawa Hospital, Civic Campus, 725 Parkdale Ave., Ottawa, ON, Canada K1Y 4E9

关键词: Subtilisin kexin isozyme 1;    Site 1 protease;    Lassa virus glycoprotein;    Intramolecularly quenched fluorogenic substrate;    Enzyme assay;    Kinetic study;    V max (app)/K m (app);    Abz;    2-amino benzoic acid;    Tyx;    3-nitro tyrosine [Tyr(3-NO2)];    SKI-1;    subtilisin kexin isozyme-1;    S1P;    site 1 protease;    PC;    proprotein convertase;    Fmoc;    fluorenylmethyloxycarbonyl;    RP-HPLC;    reverse-phase high performance liquid chromatography;    GP-C;    glycoprotein C of Lassa virus;    MALDI-tof MS;    matrix-assisted laser desorption ionization time of flight mass spectroscopy;    R t;    retention time;    m/z;    mass/charge;    TFA;    trifluoroacetic acid;   

DOI  :  10.1016/S0014-5793(02)02394-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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-²⁵¹Asp-Ile-Tyr-Ile-Ser-Arg-Arg-Leu-Leu↓Gly-Thr-Phe-Thr²⁶³-3-NitroTyr-Ala-CONH₂], 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.

【 授权许可】

Unknown