Recombinant expression, purification, and kinetic and inhibitor characterisation of human site-1-protease
(2007) In Protein Expression and Purification 51(2). p.308-319- Abstract
Human site-1-protease (S1P, MEROPS S08.8063), also widely known as subtilisin/kexin isozyme 1 (SKI-1), is a membrane bound subtilisin-related serine protease, that belongs to a group of nine mammalian proprotein convertases. Among these proteases, S1P displays unique substrate specificity, by showing preferred cleavage after non-basic amino acids. S1P plays a key role in a proteolytic pathway that controls the cholesterol content of membranes, cells and blood. S1P also participates in the activation of viral coat glycoproteins of the lassa virus, the lympocytic choriomeningitis virus and the crimean congo hemorrhagic fever virus. We expressed recombinant human S1P using the baculovirus expression vector system and characterized the... (More)
Human site-1-protease (S1P, MEROPS S08.8063), also widely known as subtilisin/kexin isozyme 1 (SKI-1), is a membrane bound subtilisin-related serine protease, that belongs to a group of nine mammalian proprotein convertases. Among these proteases, S1P displays unique substrate specificity, by showing preferred cleavage after non-basic amino acids. S1P plays a key role in a proteolytic pathway that controls the cholesterol content of membranes, cells and blood. S1P also participates in the activation of viral coat glycoproteins of the lassa virus, the lympocytic choriomeningitis virus and the crimean congo hemorrhagic fever virus. We expressed recombinant human S1P using the baculovirus expression vector system and characterized the highly purified enzyme. Featuring a new chromogenic substrate (Acetyl-Arg-Arg-Leu-Leu-p-nitroanilide) we show that the enzymatic activity of S1P is not calcium dependent, but can be modulated by a variety of mono- and divalent cations. S1P displayed pronounced positive cooperativity with a substrate derived from the viral coat glycoprotein of the lassa virus. The screening of a limited number of protease inhibitors showed that S1P was not inhibited by specific inhibitors of other proprotein convertases or by Pefabloc SC (4-(2-aminoethyl) benzene sulphonyl fluoride, AEBSF). We found 3,4-dichloroisocoumarin (DCI) to be a potent slow binding inhibitor of human S1P, with a K(iapp) = 6.8 microM, thus representing a new small molecule inhibitor of S1P. These findings show that S1P differs significantly from other proprotein convertases with respect to kinetics, co-factor requirement and inhibition.
(Less)
- author
- Bodvard, Kristofer ; Mohlin, Johanna and Knecht, Wolfgang LU
- publishing date
- 2007-02
- type
- Contribution to journal
- publication status
- published
- keywords
- Amino Acid Sequence, Animals, Buffers, Capsid Proteins/metabolism, Cations, Divalent/pharmacology, Edetic Acid/pharmacology, Egtazic Acid/pharmacology, Electrophoresis, Polyacrylamide Gel, Glycosylation, Humans, Kinetics, Lassa virus, Molecular Sequence Data, Proprotein Convertases/antagonists & inhibitors, Recombinant Proteins/antagonists & inhibitors, Sequence Alignment, Serine Endopeptidases/genetics, Spodoptera, Substrate Specificity
- in
- Protein Expression and Purification
- volume
- 51
- issue
- 2
- pages
- 12 pages
- publisher
- Academic Press
- external identifiers
-
- scopus:33751525056
- pmid:16973377
- ISSN
- 1046-5928
- DOI
- 10.1016/j.pep.2006.07.015
- language
- English
- LU publication?
- no
- id
- bc91c6e7-bde8-43f0-91d3-663d961dabbd
- date added to LUP
- 2020-07-22 14:12:28
- date last changed
- 2024-01-02 15:00:47
@article{bc91c6e7-bde8-43f0-91d3-663d961dabbd,
abstract = {{<p>Human site-1-protease (S1P, MEROPS S08.8063), also widely known as subtilisin/kexin isozyme 1 (SKI-1), is a membrane bound subtilisin-related serine protease, that belongs to a group of nine mammalian proprotein convertases. Among these proteases, S1P displays unique substrate specificity, by showing preferred cleavage after non-basic amino acids. S1P plays a key role in a proteolytic pathway that controls the cholesterol content of membranes, cells and blood. S1P also participates in the activation of viral coat glycoproteins of the lassa virus, the lympocytic choriomeningitis virus and the crimean congo hemorrhagic fever virus. We expressed recombinant human S1P using the baculovirus expression vector system and characterized the highly purified enzyme. Featuring a new chromogenic substrate (Acetyl-Arg-Arg-Leu-Leu-p-nitroanilide) we show that the enzymatic activity of S1P is not calcium dependent, but can be modulated by a variety of mono- and divalent cations. S1P displayed pronounced positive cooperativity with a substrate derived from the viral coat glycoprotein of the lassa virus. The screening of a limited number of protease inhibitors showed that S1P was not inhibited by specific inhibitors of other proprotein convertases or by Pefabloc SC (4-(2-aminoethyl) benzene sulphonyl fluoride, AEBSF). We found 3,4-dichloroisocoumarin (DCI) to be a potent slow binding inhibitor of human S1P, with a K(iapp) = 6.8 microM, thus representing a new small molecule inhibitor of S1P. These findings show that S1P differs significantly from other proprotein convertases with respect to kinetics, co-factor requirement and inhibition.</p>}},
author = {{Bodvard, Kristofer and Mohlin, Johanna and Knecht, Wolfgang}},
issn = {{1046-5928}},
keywords = {{Amino Acid Sequence; Animals; Buffers; Capsid Proteins/metabolism; Cations, Divalent/pharmacology; Edetic Acid/pharmacology; Egtazic Acid/pharmacology; Electrophoresis, Polyacrylamide Gel; Glycosylation; Humans; Kinetics; Lassa virus; Molecular Sequence Data; Proprotein Convertases/antagonists & inhibitors; Recombinant Proteins/antagonists & inhibitors; Sequence Alignment; Serine Endopeptidases/genetics; Spodoptera; Substrate Specificity}},
language = {{eng}},
number = {{2}},
pages = {{308--319}},
publisher = {{Academic Press}},
series = {{Protein Expression and Purification}},
title = {{Recombinant expression, purification, and kinetic and inhibitor characterisation of human site-1-protease}},
url = {{http://dx.doi.org/10.1016/j.pep.2006.07.015}},
doi = {{10.1016/j.pep.2006.07.015}},
volume = {{51}},
year = {{2007}},
}