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J Biol Chem, Vol. 275, Issue 4, 2349-2358, January 28, 2000
From the Laboratories of Biochemical and enzymatic characterization of the
novel human subtilase hSKI-1 was carried out in various cell lines.
Within the endoplasmic reticulum of LoVo cells, proSKI-1 is converted to SKI-1 by processing of its prosegment into 26-, 24-, 14-, 10-, and
8-kDa products, some of which remain tightly associated with the
enzyme. N-terminal sequencing and mass spectrometric analysis were used
to map the cleavage sites of the most abundant fragments, which were
confirmed by synthetic peptide processing. To characterize its in
vitro enzymatic properties, we generated a secreted form of
SKI-1. Our data demonstrate that SKI-1 is a
Ca2+-dependent proteinase exhibiting optimal
cleavage at pH 6.5. We present evidence that SKI-1 processes peptides
mimicking the cleavage sites of the SKI-1 prosegment, pro-brain-derived
neurotrophic factor, and the sterol regulatory element-binding protein
SREBP-2. Among the candidate peptides encompassing sections of the
SKI-1 prosegment, the RSLK137- and
RRLL186-containing peptides were
best cleaved by this enzyme. Mutagenesis of the latter peptide allowed
us to develop an efficiently processed SKI-1 substrate and to assess
the importance of several P and P' residues. Finally, we demonstrate
that, in vitro, recombinant prosegments of SKI-1 inhibit
its activity with apparent inhibitor constants of 100-200
nM.
Biosynthesis and Enzymatic Characterization of Human SKI-1/S1P
and the Processing of Its Inhibitory Prosegment*
§,,
,,, and
Biochemical and
¶ Molecular Neuroendocrinology and ** Structure and Metabolism of
Neuropeptides, Protein Engineering Network of Centres of Excellence,
Clinical Research Institute of Montreal, University of Montreal,
Montreal, Quebec H2W 1R7 and the Protein Chemistry Center, Loeb
Health Research Institute at the Ottawa Hospital and University of
Ottawa, Ottawa, Ontario K1Y 4K9, Canada
*
This work was supported in part by Medical Research Council
(MRC) Group Grant MGC-11474 (to N. G. S. and M. C.), by
MRC Operating Grants MT-NGS (to N. G. S.) and MT-14766 (to
C. L.), and by the Government of Canada's Network of Centers of
Excellence program supported by the MRC and National Sciences and
Engineering Research Council of Canada through Protein Engineering
Network of Centers of Excellence.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Clinical Research
Inst. of Montreal, 110 Pine Ave. W., Montreal, Quebec H2W 1R7, Canada.
Tel.: 514-987-5609; Fax: 514-987-5542; E-mail:
seidahn@ircm.qc.ca.
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