Trypsin inhibitor SFTI-1, the smallest member of the family of Bowman–Birk inhibitors BBIs, has been found in sunflower seeds [1].. A monocyclic analogue of SFTI-1, containing only the di
Trang 1Trypsin inhibitor SFTI-1, the smallest member of the
family of Bowman–Birk inhibitors (BBIs), has been
found in sunflower seeds [1] This homodetic peptide
consists of 14 amino acid residues and its structure is
stabilized by a disulfide bridge (Fig 1) The reactive
site P1-P1¢ of this peptide is located between Lys5-Ser6
As a result of the high sequential and structural
homology of SFTI-1 with the binding loop of the
canonical inhibitors of the BBI family, SFTI-1 forms a
complex with the target enzyme in stoichiometric ratio
of 1 : 1 As reported by Marx et al [2], upon the
incu-bation with trypsin, the ratio of native SFTI-1 to its
acyclic permutant with hydrolyzed P1-P1¢ is
approxi-mately 9 : 1 As a result of its small size, very strong
trypsin inhibitory activity and circular backbone
scaf-fold (i.e well defined 3D structure, displayed b-hairpin
motive), SFTI-1 has attracted interest ever since its dis-covery Recent studies on SFTI-1 are summarized in three reviews [3–5] Because small proteinaceous inhibi-tors are of commercial interest, SFTI-1 soon became
an attractive template for the design of new protease inhibitors with potential use as therapeutic and agro-chemical agents
Subsequent to the discovery of SFTI-1, several stud-ies have shown that the presence of both cycles in the inhibitor molecule is not essential for its activity
A monocyclic analogue of SFTI-1, containing only the disulfide bridge, appeared to have trypsin inhibitory activity matching that of the wild-type SFTI-1 [6–8]
In addition, it displayed proteinase resistance similar
to that of the parent compound [9] Another analogue, containing only head-to-tail cyclization, ([Abu3,11]
splicing; serine proteinases; SFTI-1
Correspondence
A Łe˛gowska, Faculty of Chemistry,
University of Gdan´sk, Sobieskiego 18,
80-952 Gdan´sk, Poland
Fax: +48 5852 3472
Tel: +48 5852 3359
E-mail: legowska@chem.univ.gda.pl
(Received 4 February 2010, revised 10
March 2010, accepted 15 March 2010)
doi:10.1111/j.1742-4658.2010.07650.x
two molecules of trypsin inhibitor sunflower trypsin inhibitor 1 (SFTI-1) bound through a peptide bond The peptides in their reactive positions (5 and 19 of the peptide chain) contain two Lys ([KK]BiSFTI-1) and two Phe ([FF]BiSFTI-1) residues, along with a combination of the amino acid residues named thereafter [KF]BiSFTI-1 and [FK]BiSFTI-1 Association constants of the analogues determined with trypsin and chymotrypsin, respectively, indicated that they were potent inhibitors of cognate protein-ases An MS study of the associates revealed that incubation of the com-pounds with the proteinases resulted in cutting out a fragment of the peptide chain to restore the native monocyclic molecule of SFTI-1 or its analogue [Phe5]SFTI-1 This process, analogous to that of the DNA and protein splic-ing, can be referred to as ‘peptide splicing’
Abbreviations
BBI, Bowman–Birk inhibitor; ESI, electrospray ionization.
Trang 2SFTI-1) displayed trypsin inhibitory activity that was
only 2.5-fold lower than the wild-type inhibitor [6] As
reported by Korsinczky et al [8,9], solution structures
of such monocyclic SFTI-1 analogues are remarkably
similar to the solution and the crystal structures of the
wild-type SFTI-1 A higher structural flexibility of
[Abu3,11]SFTI-1 compared to that of SFTI-1 is
com-patible with its lower activity and higher hydrolysis
rate In the wild-type of SFTI-1, a substrate specificity
P1 position [1] is occupied by Lys residues For this
reason, SFTI-1 and its monocyclic analogues with
Lys5 were demonstrated to be strong trypsin inhibitors
[6], whereas their chymotrypsin inhibitory activity was
three orders of magnitude lower when association
con-stants (Ka) with appropriate serine proteinases were
used as a measure of their strength [7]
Monosubstitu-tion of Lys5 by Phe reversed the SFTI-1 specificity
Thus, [Phe5]SFTI-1 did not inhibit trypsin but
exhib-ited strong chymotrypsin inhibitory activity with
Ka= 2.0· 109m)1[10]
When designing compounds of commercial
impor-tance, the aim is to reduce their size and simplify the
original structure of a naturally occurring compound
(e.g protein) Bearing in mind the potential
applica-tions of BBIs, and considering the results presented
above, we designed four SFTI-1 analogues based on
the double-sequence of the wild-type inhibitor The
primary structures of dimeric SFTI-1 analogues are
shown in Fig 1 In all of the compounds, two
sequences are bound by a peptide bond formed
between the C-terminal a-carboxyl group of Asp in the
first molecule and the N-terminal a-amino group of
Gly in the second one To form one disulfide bond
only, two Cys residues located in the middle of the
peptide chain (positions 11 and 17) were replaced by
their structural counterparts of a-aminobutyric acid (Abu) residues, whereas the remaining two formed a disulfide bond The dimeric SFTI-1 analogues differ at positions 5 and 19 Our synthesized analogues, [KK]BiSFTI-1 (5) and [FF]BiSFTI-1 (6), as well as [KF]BiSFTI-1 (7) and [FK]BiSFTI-1 (8), contain Lys and Phe in positions 5 and 19, in addition to combina-tions of both amino acid residues Our intention was
to design low-molecular compounds containing two reactive sites, with the second one located between positions 19 and 20 We assumed that these analogues would be able to form complexes with trypsin or chy-motrypsin with a stoichiometry of 2 : 1 (analogues of
6 and 7), whereas the two remaining analogues would inhibit both trypsin and chymotrypsin simultaneously and independently Jaulent and Leatherbarrow [11] reported the synthesis and kinetic studies of a bicyclic and bifunctional proteinase peptidic inhibitor consist-ing of 16 amino acids The inhibitor was designed by combining two binding loops of BBI As postulated by Jaulent and Leatherbarrow [11], the size of the inhibi-tor was incompatible with the simultaneous binding of trypsin and chymotrypsin We predicted that the size
of 28 amino acid residues peptides might be sufficient
to accommodate both enzyme molecules
Results and Discussion
As indicated in Table 1, all four dimeric SFTI-1 per-mutants, with the exception of 8, incubated with tryp-sin, are potent inhibitors The Ka values for the compounds are approximately one order of magnitude lower than those for their monomeric counterparts Surprisingly, [FK]BiSFTI-1 (8) did not block trypsin activity This enzyme regained its activity (within
A
B
Fig 1 Chemical structures of (A) SFTI-1 and (B) synthesized analogues [KK]BiSFTI-1 (5), [FF]BiSFTI-1 (6), [KF]BiSFTI-1 (7) and [FK]BiSFTI-1 (8).
Trang 35 min) after incubation with 8, thus suggesting that 8
behaved as a substrate rather than an inhibitor At the
same time, this shows that the peptide bond between
Lys19 and Ser20 (the reactive site) and the Arg16
-Abu17 bond are both rapidly hydrolyzed by the
enzyme (Fig 2) and the hydrolysis products are
quickly released from the enzyme’s substrate pocket It
is interesting to note that, when compounds 7 and 8
were preincubated with one enzyme each and then
their inhibitory activities were checked against another
enzyme, they displayed inhibitory activity that was at
least one order of magnitude higher On the basis of
these results, it can be assumed that compounds 7
and 8 inhibit two proteinases independently and
simultaneously We also found that each of the
used chromogenic substrates was specific for one of
the experimental proteinases and remained intact in
the presence of the other enzyme Consequently, the
hypothesis that the inhibitory activity of the
permu-tants in the presence of both enzymes could have been
caused by experimental conditions can be ruled out
Indeed, we conducted the experiments under the
condi-tions recently described by Jaulent and Leatherbarrow
[11], who reported synthesis and kinetic studies on a
bicyclic and bifunctional proteinase peptidic inhibitor
consisting of 16 amino acids The inhibitor (BiKF)
was designed by combining two building loops of
BBI and was able to inhibit both trypsin and
chymo-trypsin independently but not simultaneously This
means that, after preincubation with one enzyme, it
completely lost its inhibitory activity against the other
one As claimed by Jaulent and Leatherbarrow [11],
results obtained in these experiments were not convinc-ing and are not discussed here
One of the methods of choice for studying noncova-lent complexes formed by proteins is MS with electro-spray ionization (ESI) An in-depth analysis of complexes formed between bovine pancreatic trypsin inhibitor and target proteinases was provided by Nesatyy [12], who also emphasized that a correlation between the solution and gas phase binding of the complexes was not straightforward There was a not-oceable difference in the strength of the complexes formed in the aqueous and gas phase, whereas their stoichiometry was preserved
Figures 3 and 4 represent MS spectra of trypsin and chymotrypsin along with those recorded after their incubation with [KK]BiSFTI-1 (5) and [FF]BiSFTI-1 (6), respectively The ESI spectra of bovine b-trypsin (Fig 3A) exhibited two charge states of +9 and +10 The molecular mass of the enzyme calculated from the first peak was 23 322 Da, whereas the other peak cor-responded to a trypsin molecule with a trapped cal-cium ion After incubation of 5 with trypsin, among the four peaks seen in the MS spectrum, those with
m⁄ z 2333.2753 and 2592.4874 were assigned to free trypsin, whereas the remaining two with m⁄ z 2486.4686 and 2762.4797 revealed the appearance of a
1 : 1 complex of trypsin with monocyclic SFTI-1 (Fig 3B) Essentially, an identical peak pattern was seen with an increasing incubation time of up to 20 h (data not shown) The MS spectrum of bovine a-chy-motrypsin (Fig 4A) produced charge states of +10 and +11 The monoisotopic molecular mass (calculated using the SNAP procedure in the Bruker Data Analysis program; Bruker Daltonics, Bremen, Germany) of the proteinase derived from those peaks was 25 225 Da
In the spectrum of a 1 : 1 mixture of chymotrypsin and [FF]BiSFTI-1 (6) incubated for 30 min (Fig 4B), two peaks were seen with charge states of +10 and +11, both representing a 1 : 1 complex between
7 [KF]BiSFTI-1 (2.6 ± 0.2) · 10 (8.7 ± 0.2) · 10
(1.2 ± 0.2) · 10 10
(Ch) (5.3 ± 0.2) · 10 9
(T)
8 [FK]BiSFTI-1 ND (3.0 ± 0.4) · 10 8
(1.3 ± 0.3) · 10 9 (T)
a With the exception of wild-type SFTI-1, all inhibitors are monocyclic with
one disulfide bridge only or a head-to-tail cyclization (compound 3).
Trang 4chymotrypsin and monocyclic (disulfide bridge only)
[Phe5]SFTI-1 It is worth emphasizing that the
con-ditions for the enzyme–inhibitor incubation used in
the MS study differed from those applied for the determination of Ka To detect the complexes, we had
to exchange the buffer for a more volatile one (an
A
B
Fig 2 MS spectra and results of HPLC analysis of (A) [FK]BiSFTI-1 (8) and (B) a mixture of b-trypsin and [FK]BiSFTI-1: peak 2, analogue 8 without tripeptide Abu-Thr-Lys; peak 3, analogue 8 with cleaved
Abu-Thr-Lys and Gly-Arg fragments.
Trang 52000 2100 2200 2300 2400 2500 2600 2700 2800 2900 m/z
0
1
2
Fig 3 MS spectra of (A) bovine b-trypsin and (B) a mixture of b-trypsin and [KK]BiSFTI-1 (5).
2294.149111+
2523.520110+
2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 m/z
0.0
0.2
0.4
0.6
0.8
1.0
2435.0841 11+
2678.6160 10+
0.0
0.5
1.0
1.5
2.0
[Phe ]SFTI-1- chymotrypsin complex
5
Fig 4 MS spectra of (A) bovine a-chymotrypsin and (B) a mixture of a-chymotrypsin and [FF]BiSFTI-1 (6).
Trang 6ammonium formate buffer, pH 5.8) In a separate
experiment, we demonstrated that, under the conditions
used for the MS study, the peptides exhibited their full
inhibitory activity In a similar way, we studied
interac-tions of the remaining inhibitors, [KF]BiSFTI-1 (7) and
[FK]BiSFTI-1 (8), by using ESI-MS Compound 7
incubated with trypsin generated a peak corresponding
to a noncovalent complex of SFTI-1 with trypsin
However, its formation was significantly slower
com-pared to that of a mixture of 5 with trypsin In a
mix-ture of 8 with trypsin, only trace amounts of a complex
of [Phe5]SFTI-1 with the enzyme were found after 1 h
of incubation Compound 8 incubated with
chymotryp-sin formed a noncovalent complex, although the
reac-tion was definitely slower than that with compound 6
In a mixture of 7 with chymotrypsin, no noncovalent
complex was found after 1 h of incubation On the
other hand, incubation of both compounds 7 and 8 in
a mixture of trypsin and chymotrypsin resulted in the
immediate high-yield formation of noncovalent
com-plexes, SFTI-1–trypsin and [Phe5
]SFTI-1–chymotryp-sin, respectively Figures 5 and 6 show the results of
the MS analyses of those mixtures
These results clearly indicate that all dimeric ana-logues undergo proteolysis when incubated with target enzymes In all cases, P1-P1¢ reactive sites are located between positions 5⁄ 6 and 19 ⁄ 20 to release fragments with Ser6 and Lys19 at their N- and C-termini, respec-tively The cleavage is followed by resynthesis of the peptide bond between Lys5 and Ser20 to pro-duce monocyclic SFTI-1 or its [Phe5]SFTI-1 analogue Figure 7 shows the splicing of the permutants medi-ated by target enzymes These results are compatible with the inhibitory activity of the peptides (Table 1), with all of them being potent inhibitors of the target enzymes
Proteolytic susceptibility of the inhibitors was found
to be in excellent agreement with their inhibitory activ-ity In all cases where the dimeric species were less pro-teolytically resistant than their monocyclic reference compounds (i.e SFTI-1 and [Phe5]SFTI-1), their inhib-itory activities, expressed in terms of Ka, were one order of magnitude lower On the other hand, in all cases where the reference inhibitors were formed after proteolysis, the Ka values matched those determined for the reference monomers
10+
2333.2814
10+
2486.3689
10+
2523.4314
9+
2592.4929
0
2
4
6
8
Trypsin
Chymotrypsin
SFTI-1-trypsin complex
Fig 5 MS spectrum of a mixture a-chymotrypsin, b-trypsin and [KF]BiSFTI-1 (7).
Trang 7Despite our expectations, the peptides did not
form 2 : 1 complexes with the enzymes, nor did they
simultaneously and independently (7 and 8) inhibit
experimental proteinases; instead, they undergo
prote-olysis The enzymatic process involving proteolytic
cleavage, combined with resynthesis of the peptide bond, is an intriguing finding It may serve as a model for the in vivo formation of cyclic peptides by enzy-matic processing of their precursors generated by stan-dard translation Some bioactive cyclic peptides
Fig 7 Splicing of the double-sequence SFTI-1 analogues mediated by target enzymes.
0.0
0.2
0.4
Fig 6 MS spectrum of a mixture b-trypsin, a-chymotrypsin and [FK]BiSFTI-1 (8).
Trang 8comprising proteinogenic l-amino acids (e.g the
pep-tides produced by Caryophyllaceae plants) are likely to
be formed by this mechanism, which can be named
‘peptide splicing’
Materials and methods
Peptide synthesis
All peptides were synthesized manually by the solid-phase
method using standard Fmoc chemistry on 2-chlorotrityl
chloride resin (substitution of Cl 1.46 meqÆg)1)
(Calbio-chem-Novabiochem AG, La¨ufelfingen, Switzerland)
apply-ing a previously described procedure [10] Durapply-ing the last
step, disulfide bridge formation was performed using 0.1 m
solution of I2 in MeOH as described previously [13] All
synthetic steps were monitored by HPLC analysis using an
RP Kromasil-100, C8, 5 lm column (4.6· 250 mm)
(Knauer, Berlin, Germany) The solvent systems were 0.1%
trifluoroacetic acid (A) and 80% acetonitrile in A (B) A
linear gradient of 20–80% B for 30 min was employed with
a flow rate of 1 mlÆmin)1, monitored at 226 nm Finally, all
peptides were purified on a semi-preparative HPLC column
RP Kromasil-100, C8, 5 lm column (8· 250 mm) (Knauer)
using the same solvent system as above A linear gradient
of 20–80% B for 30 min was employed with a flow rate of
2.5 mlÆmin)1, monitored at 226 nm To confirm the
correct-ness of molecular weights of the peptides, MS analysis was
carried out on a MALDI MS (Biflex III MALDI-TOF
spectrometer; Bruker Daltonics) using a-CCA matrix
Determination of association constants
The association constants were measured using a method
developed in the laboratory of Laskowski et al [14,15] The
procedure was described in detail previously [10]
The measurements were carried out at initial enzyme
con-centrations over the ranges 5.1–5.8 nm and 2.8–7.2 nm for
trypsin and chymotrypsin, respectively To determine the
Kavalues for [KF]BiSFTI-1 (7) and [FK]BiSFTI-1 (8) with
trypsin in the presence of chymotrypsin, a two-fold molar
excess over the inhibitor of the second enzyme was added
to each of the experimental cuvettes, followed 5 min later
by the addition of appropriate volumes of the trypsin and
substrate solutions The chymotrypsin inhibitory activity in
the presence of trypsin was determined by the same
proce-dure, using a reverse order of enzyme addition for
preincu-bation
Proteolytic susceptibility assays
Dimeric analogues of SFTI-1 were incubated in a 100 mm
Tris-HCl buffer (pH 8.3) containing 20 mm CaCl2 and
0.005% Triton X-100, using catalytic amounts of bovine
b-trypsin or bovine a-chymotrypsin (1 mol%) [11] The incubation was carried out at room temperature and aliquots of the mixture were taken out periodically and submitted to RP-HPLC analysis
Analysis of enzyme–inhibitor complexes using MS
A 1.4· 10)5m solution containing a proteolytic enzyme (trypsin or chymotrypsin) and inhibitor (1.7· 10)5m) in a
20 mm aqueous ammonium formate buffer (pH 5.8) was incubated for predetermined periods of 0.5, 1 and 20 h After incubation, the mixture was analysed directly by
ESI-MS spectrometry The experiments were performed using
an FT-MS instrument (Apex-Ultra 7T; Bruker Daltonic) equipped with a dual ESI-MADI Apollo source (Agilent Technologies Inc., Santa Clara, CA, USA) The samples were infused at a flow rate of 2 llÆmin)1 The potential between the spray needle and the orifice was set at 4.5 kV Capillary temperature was 200C, and N2 was used as a nebulizing gas
Acknowledgements
This work was supported by the Ministry of Science and Higher Education (grant no 2889⁄ H03 ⁄ 2008 ⁄ 34)
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