In this study, three inhibitors were purified from the porcine liver by using a combination of chromato-graphic techniques, and identified to be the C-terminal truncated fragments with dif
Trang 1fragments on furin
Jinbo Han1, Ling Zhang2, Xiaoxia Shao2, Jiahao Shi1, and Chengwu Chi1,2
1 Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai, China
2 Institute of Protein Research, Tong-ji University, Shanghai, China
Following protein biosynthesis, the post-translational
modifications ultimately lead to the maturation of
bioactive molecules Within the secretory pathway,
these modifications include cleavage at specific sites by
endo- or exo-peptidase, amidation, glycosylation and
sulfonation, etc [1] Among these modifications, the
limited proteolysis of proproteins is a mechanism
widely used to regulate the activation of peptides and
proteins that play important roles in various biological
events from homeostasis to diseases Many inactive
precursors are cleaved at paired or multiple basic
amino acids by a family of proteolytic enzymes called
proprotein convertases (PCs) PCs are
calcium-depend-ent serine proteases whose catalytic domain shares
some homology with that of the bacterial subtilisin To
date, seven distinct PCs (furin, PC2, PC1⁄ PC3, PC4,
PACE4, PC5⁄ PC6 and LPC ⁄ PC7 ⁄ PC8 ⁄ SPC7) have
been identified in mammalian species [2]
Furin was the first identified mammalian PC and the most extensively studied member of the known seven PCs It is responsible for the activation of var-ious substrates ranging from the blood clotting fac-tors, serum proteins, growth facfac-tors, and hormone receptors to matrix metalloproteinases [3] Recently, some ion channels such as the epithelial sodium channel and the yeast chloride channel were also found to be processed by furin-like enzymes [4,5] In addition to endogenous proteins, many pathogens such as viral envelope glycoproteins and bacterial exotoxins are also activated by furin [6] Thus, furin
is an attractive target for therapeutic agents Many peptide- or protein-based inhibitors were designed, including the peptidyl inhibitor decanoyl-Arg-Val-Lys-Arg-CH2Cl, the bioengineered variant of a1-anti-trypsin Portland (a1-PDX) [7], polyarginines [8], Drosophila Serpin 4 [9,10], and the serpin-derived
Keywords
furin; gene expression; histone H1;
inhibitory activity; limited proteolysis;
peptide synthesis
Correspondence
C Chi, Shanghai Institute of Biochemistry
and Cell Biology, Chinese Academy of
Sciences, 320 Yue Yang Road,
Shanghai 200031, China
Fax: +86 21 54921011
Tel: +86 21 54921165
E-mail: chi@sunm.shcnc.ac.cn.
(Received 28 April 2006, revised 27 July
2006, accepted 4 August 2006)
doi:10.1111/j.1742-4658.2006.05451.x
Many physiologically important proproteins, pathogenic bacterial exo-toxins and viral envelope glycoproteins are activated by the proprotein con-vertase furin, which makes furin inhibitor a hot target for basic research and drug design Although synthetic and bioengineered inhibitors of furin have been well characterized, its endogenous inhibitor has not been directly purified from mammalian tissues to date In this study, three inhibitors were purified from the porcine liver by using a combination of chromato-graphic techniques, and identified to be the C-terminal truncated fragments with different sizes of histone H1.2 The gene of porcine histone H1.2 was cloned and sequenced, further confirming the determined sequences These three C-terminal fragments inhibited furin with Ki values around
2· 10)7mwhile the full-length histone H1.2 inhibited it with a lesser activ-ity, suggesting that the inhibitory activity relies on the C-terminal lysine-rich domain Though the inhibition was temporary, these inhibitors were specific, and the reactive site of one C-terminal fragment was identified
A 36 amino acid peptide around the reactive site was synthesized, which could still inhibit furin with a Kiof 5.2 · 10)7m
Abbreviation
PCs, proprotein convertases.
Trang 2peptides, as well as the barley serine proteinase
inhibitor 2-derived cyclic peptides [11] Some of these
inhibitors are used to prevent the activation of
bac-terial toxin, the processing of envelope glycoprotein
in viral replication and the metastasis of cancer [12–
14] The propeptide of furin itself [15,16], the
inter-alpha-inhibitor protein [17] and human proteinase
inhibitor 8 [18] have been found to be potent furin
inhibitors, and our earlier work identified a high
positively charged protein, namely, nonhistone
chro-mosomal protein HMG-17, from porcine kidney as a
suicide substrate inhibitor of a furin like enzyme
kexin [19] However, no other protein that possesses
furin inhibitory activity has been directly purified
from mammalian tissue
In this study, in contrast to constructing artificial
furin inhibitors, we purified three fractions from the
porcine liver using a combination of chromatographic
techniques They all possessed high inhibitory activity
against furin and have been identified to be the
C-ter-minally truncated fragments generated from histone
H1.2 with 126, 120 and 103 amino acid residues,
respectively The activity assay showed that the
full-length histone H1.2 could also inhibit furin with a Ki
value of 4.6· 10)7m The identification of lysine-rich
histone H1.2 and its C-terminal fragments as inhibitors
of furin will undoubtedly pave the way for the
devel-opment of therapeutically useful furin inhibitors and
for the mechanistic studies of the regulation of furin
activity in vivo
Results
Purification and identification of the endogenous
furin inhibitors from the porcine liver
Porcine liver was used as the raw material in the
search for the endogenous furin inhibitor for two
reasons: firstly, furin is expressed more abundantly
in the liver than in other tissues or organs; and
sec-ond, a variety of furin substrates are precisely
proc-essed in the liver, compelling the existence of furin
inhibitor to modulate the enzyme activity The
purifi-cation procedure is described in Fig 1 To avoid
possible proteolytic degradation, the fresh porcine
livers were immediately treated as an acetone powder
and extracted with 2.5% trichloroacetic acid After
centrifugation, the supernatant was precipitated with
two-step ammonium sulfate fractionation The active
portion was subjected to a cation exchange
chroma-tography, and the inhibitory activity was found in
the fraction eluted by 0.4 m NaCl (data not shown)
The active fraction was then pooled and loaded onto
a phenyl Sepharose CL-4B column, the highest inhibi-tory activity was found in the unbound fraction (data not shown) The unbound fraction was then further purified on a Superdex-75 column (Fig 2A) and then
on a Hamilton PRP-3 column (Fig 2B) Six fractions were finally obtained and assayed for their inhibitory activity Among them, the major fractions P2, P3 and P4 have a strong inhibitory activity against furin The homogeneity of the three fractions was detected by SDS⁄ PAGE, and their apparent sizes were 21, 24 and
25 kDa, respectively (Fig 2C)
The three purified proteins were sequenced by Edman degradation Unexpectedly, their N-terminal partial sequences were found to be overlapping, indi-cating that they are derived from the same protein (Fig 3A) A database search revealed that the N-ter-minal sequences of these three proteins matched the C-terminal sequence of human histone H1.2, except for a few sites which were not conserved between human and porcine histone H1.2 In order to eluci-date the whole protein sequence, we cloned the porcine histone H1.2 gene (Genebank Accession
#DQ060698) from the porcine genomic DNA, as described in the experimental procedures The predic-ted protein sequence of the porcine histone H1.2 was aligned with that of human histone H1.2, because,
as shown in Fig 3(B), they share 92% identity The
Porcine liver TCA extraction
Ammonium sulfate precipitation
CM-52 (cation) column
Phenyl-sepharose CL-4B
Superdex 75 column
HPLC
Fig 1 Diagram showing the purification procedure Fresh porcine livers were immediately treated as an acetone powder and extracted with 2.5% trichloroacetic acid The extraction was precipi-tated with a two-step ammonium sulfate fractionation and further separated on a CM-52 cation exchange chromatography, a phenyl Sepharose CL-4B column, a Superdex-75 column and a Hamilton PRP-3 column Finally, three fractions that possessed high inhibi-tory activity against furin were purified.
Trang 3three fragments P4, P3 and P2 start from 88th, 94th
and 111th residue of histone H1.2 with 126-,
120-and 103- amino acid residues, respectively It is
worth pointing out that both fragments P4 and P3
were generated by a proteolytic cleavage between the
Leu–Val peptide bond Obviously this bond was cleaved by the same protease, while the fragment P2 was most probably generated by furin itself or by a furin-like enzyme, as there are paired basic residues prior to the cleaved bond
A
B
C
0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00
Retention time (min)
Retention time (min)
P3
P4 P2
P6
0.00
0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00
5.00 10.00 15.00 20.00 25.00 30.00
Fig 2 Purification of three fractions with
furin inhibitory activity from porcine liver.
(A) The active fraction, separated from the
phenyl Sepharose CL-4B column, was
loaded onto a Superdex 75 column The
column was equilibrated and eluted with
20 mM sodium acetate ⁄ acetic acid buffer,
pH 5.4, at a flow rate of 0.5 mLÆmin)1 The
fractions with inhibitory activity marked by a
bar were pooled (B) The pooled fraction
from the Superdex 75 column was further
separated on a HPLC Hamilton PRP-3
column equilibrated with 0.1% (v ⁄ v)
trifluoroacetic acid, and the bound proteins
were eluted with a linear gradient of 0–20%
(v ⁄ v) acetonitrile in 0.1% (v ⁄ v) trifluoroacetic
acid in 0–20 min and 20–100% (v ⁄ v)
aceto-nitrile in 0.1% (v⁄ v) trifluoroacetic acid in
20–50 min at a flow rate of 1 mLÆmin)1 The
peaks marked (P1-P6) were collected and
the peaks marked by P2, P3 and P4
exhib-ited a high inhibitory activity against furin.
(C) Aliquots of the P2, P3 and P4 fractions
were subjected to electrophoresis on 15%
SDS ⁄ PAGE and visualized by silver staining.
Protein markers are indicated on the left.
Trang 4Temporary inhibition and identification of the
reactive site of the inhibitor
The inhibitory activity of the three fragments of
his-tone H1.2 against furin was assayed and analyzed
using Dixon’s plot to determine their inhibition
con-stants Ki Table 1 showed that the Ki values of three
H1.2 fragment inhibitors were around 2· 10)7m
Pro-longed incubation over half an hour caused a gradual
loss of inhibitory activity, suggesting that the
inhibi-tion was temporary
To identify the reactive site of the inhibitor, the P4
fragment was incubated with furin and the reaction
was stopped at the indicated times and analyzed on
SDS⁄ PAGE (Fig 4) The P4 fragment was gradually
degraded, yielding two smaller fragments within the
first 2 h (Fig 4A) Obviously, this cleaved site should
be the reactive site of the P4 fragment inhibitor The two degraded peptides were then separated on SDS⁄ PAGE, trans-blotted to the polyvinylidene difluo-ride membrane and sequenced separately (Fig 4B) According to the sequence of the P4 fragment inhib-itor, its reactive site was deciphered to be K91-A92 (Fig 4C) As known, in most cases, the preferential P1 residue for furin is arginine [20]; it is understood that the cleavage of the P4 fragment by furin is very slow, and was detected only after half an hour under our experimental conditions We believe that this cleavage does not affect the Kidetermination since all reactions for inhibitory activity analysis were finished in less than 10 min
Expression of full-length porcine histone H1.2 and its N- and C-terminal fragments and their inhibitory activity assay toward furin
In order to check whether the full-length histone H1.2
or its N-terminal fragment also exhibit an inhibitory activity against furin, and to obtain enough C-terminal fragment P4 (C-H1) for further study, they were expressed as His-tag fusion proteins The recombinant proteins were purified by metal affinity column and RP-HPLC, and analyzed by SDS⁄ PAGE and mass spectrometry (Fig 5) The molecular masses of the recombinant full-length histone H1.2 and its N- and C-terminal fragments determined by mass spectrometry
Fig 3 Identification of the P2, P3 and P4 fractions by N-terminal sequencing (A) The partial N-terminal sequences of the P2, P3 and P4 frac-tions determined by Edman degradation (B) Alignment of the protein sequences of histone H1.2 from Homo sapiens (human) and Sus scrofa (pig) Arrows indicated the starting residue of P2, P3, and P4, respectively The different residues between porcine and human his-tone H1.2 are marked with gray or black shadow for the less conserved or unconserved residues, respectively The underlined amino acids indicated the putative furin recognition site (Fig 5).
Table 1 Inhibition constants of the three purified C-terminal
frag-ments P2, P3 and P4 of histone H1.2 against furin The rate of
hydrolysis of pyrArg-Thr-Lys-Arg-7-amino-4-methylcoumarin (MCA)
by furin was determined in the presence of various concentrations
of the different proteins, as described in the Experimental
proce-dures The results obtained were used to compute the K i values.
Each value represents the mean ± SD determined from three
inde-pendent experiments.
Trang 5matched their calculated ones very well (Fig 5), but
the apparent molecular weights of the proteins on
SDS⁄ PAGE were about 10 kDa larger A similar result
has been reported by Konishi et al [21] The aberrant
behavior of recombinant and native histone H1
frag-ments on SDS⁄ PAGE (Figs 2,4 and 5) are most likely
caused by their net basic charges, as basic proteins
migrate slower and acidic proteins migrate faster than
neutral proteins with the same molecular weight on
SDS⁄ PAGE [22]
The inhibitory assay of these recombinant proteins
showed that the full-length histone H1.2 also inhibited
furin with a Ki value of 4.6· 10)7m, comparable
to the C-terminal fragment P4 (Table 2) But the basic
N-terminal 87- amino acid fragment of histone H1.2
(N-H1) and another highly basic protein cytochrome c
hardly inhibited furin, with the Ki values being several
orders of magnitude higher This huge difference
strongly indicates that the inhibitory activity of
full-length histone H1.2 and its C-terminal fragment is
spe-cific, and not a general property of positively charged
proteins This is in accordance with the existence of a
specific active site in P4 (Fig 4)
Synthesis of a smaller peptide inhibitor
As the three naturally occurring fragments P2, P3 and
P4 have similar Ki values against furin, some N- and
C-terminal residues may not be necessary for the
inhibitory activity In order to know whether a smaller
fragment around the observed cleavage site is still active, an appropriately sized peptide (PAAATVTK
36- amino acid residues was synthesized The deter-mined molecular weight of the purified peptide was consistent with the theoretical one (Fig 5B) More-over, this synthetic 36- amino acid peptide was still a potent furin inhibitor with a Ki value only about two-fold higher than that of P2, P3 and P4 (Table 2) Based on these results, it was possible to further design
a smaller potent furin inhibitor
The secondary structure determination of histone H1 and its fragments
The secondary structures of histone H1 and its frag-ments were also examined by CD spectroscopy (Fig 6) As previously reported [23], histone H1.2 and its fragments do not have a clear secondary structure Their secondary structures were found to be domin-ated by a random coil (negative peak at 196–193 nm), and the contents of the a-helix (estimated from the ellipticity value at 222 nm [24]) are only 1.6, 1 and 0.5% for full-length H1, P4 and the 36- amino acid peptide, respectively
Discussion
Due to the physiological importance of furin substrates, furin is a hot target for functional and mechanistic
A
C
B
kDa
97
66
43
31
20
14
0
VSKGTLVQTK
PKKATGSATP
SAAKAVKPKA AKPKVAKPKK AAPKKK
KKAAKKTPKK AKKPAAAAVT KKVAKSPKKA KAAKPKKAAK GTGASGSFKL NKKAATGETK PKAKKSGAAK PKKSAGAAKK
15 30 60 120
kDa
97
66
43
31
20
30 60 (min)
50 100
90
80
Reactive site
70
60
110 120
40
30
20
10 P4
AAKPKKAA
(min)
Fig 4 Determination of the reactive site of the P4 fragment inhibitor (A) The degradation of the P4 fragment incubated with furin at differ-ent time at 37 C in 100 mM Hepes buffer, pH 7.5, 1 mm CaCl2, 0.5% Triton X-100 and 1 mm 2-mercaptoethanol At the indicated time, the reaction was immediately terminated by boiling the sample at 100 C for 5 min, then separated on 15% SDS ⁄ PAGE and stained with Coo-massie brilliant blue Protein markers are indicated on the left (B) The cleaved fragments were separated and transferred to the polyvinylid-ene difluoride membrane The membrane was stained with ponceaus, and the bands were cut out for sequencing Arrows showed the partial N-terminal sequences of two cleaved fragments, respectively (C) The reactive site within the P4 fragment is shown by the arrow.
Trang 6studies from basic-research and clinical-application
viewpoints Many efforts have been made to develop
peptidyl, nonpeptidyl and protein-derived furin
inhibi-tors The two most widely used inhibitors are the
peptide inhibitor decanoyl-Arg-Val-Lys-Arg-CH2Cl and
the bioengineered variant of a1-antitrypsin Portland
(a1-PDX), the latter of which is highly selective for furin
in vitro(Ki¼ 0.6 nm) and has been used to prevent the replication of pathogenic viruses, and the activation of bacterial exotoxin and cancer metastasis [6] Some protein-derived inhibitors were obtained by engineering other protease inhibitors, such as eglin C mutants [25–27], turkey ovomucoid third domain [28] and
a2-macroglobulin [29], based on the consensus substrate recognition sequence of furin Recently, polyarginine
or polyarginine-containing peptides [30] were found
to be able to inhibit furin in vitro and, as a result, they were also able to inhibit the maturation of the glyco-protein of HIV type 1 gp160, and to prevent the Pseudomonas aeruginosa exotoxin A activation in vivo [13,14]
Based on the principle that wherever a protease exists, its counterpart inhibitor can also be found, we embarked on the search for an endogenous furin
inhib-A
B
Mass reconstruction of + EMS: 1.030 to 3.088 min from Sample 30 (kkak_p1) of 040812wiff (Turbo Max 2.9e8cps.
3570.0 2.9e8
2.6e8
2.4e8
2.2e8
2.0e8
1.8e8
1.6e8
1.4e8
1.2e8
1.0e8
8.0e7
6.0e7
4.0e7
2.0e7
3000 3100 3200 3300 3400 3500
Mass, amu
3600 3700 3800 3900 4000
3976.0 3922.0 3836.0 3739.0 3667.0 3640.0 3552.0 3498.0 3441.0 3409.0 3313.0 3242.0 3126.0
3046.0
3028.0
Fig 5 SDS ⁄ PAGE and mass spectrometry analysis of the recombinant full-length his-tone H1.2 and its N- and C-terminal frag-ments (A) The recombinant full-length histone H1.2 and its N- and C-terminal frag-ments were expressed in E coli and puri-fied by TALON superflow metal affinity column and RP-HPLC (see Experimental procedures) The purified recombinant pro-teins were examined on SDS ⁄ PAGE (left panel) The determined molecular masses and the calculated ones of the recombinant proteins were aligned in the table (B) The sequence of the synthetic 36- amino acid peptide around the reactive site (underlined) and the mass spectrometry of its molecular mass.
Table 2 Inhibition constants of various recombinant proteins and
the synthetic peptide against furin.
Trang 7itor from the porcine liver where furin is relatively
abundant Unexpectedly, three C-terminal fragments
of histone H1.2 with different sizes were found to be
potent inhibitors of furin with Ki values around
2· 10)7 m, comparable with that of polyarginines
such as L6R (hexa-l-arginine) (Ki¼ 1.14 · 10)7m) [8]
The structure of the catalytic domain of mouse furin
revealed that the active site of furin resides in an
extended substrate-binding groove that is lined with
many negatively charged residues [31] Previous works
of peptide inhibitor indicated that positively charged
residues are preferred for being a furin inhibitor
[30,32] In our purified fragments P2, P3 and P4, the
multiple positively charged Lys should contribute to
the potency of inhibition However, compared with the
peptide inhibitor nona-l-arginine (Ki¼ 4.2 nm), which
produced hexa- and heptamers when cleaved by furin
[8], the inhibition of the histone H1 P4 fragment is
specific to the cleavage site being K178-A179 (the
sequence number of histone H1 is shown in Fig 4)
The inhibition by histone H1 fragments is
tempor-ary, as the incubation with furin over half an hour
resulted in digestion at the specific active site (Fig 4)
However, the temporary inhibition of histone H1
against furin is understandable, as furin is involved in
many subtly regulated physiological events, for which
the permanent inhibition is not desirable A similar
case has been reported on 7B2, an endogenous PC2
inhibitor [33] The neuroendocrine protein 7B2
contains two domains, a 21-kDa chaperon domain
required for the maturation of prohormone convertase
2 (PC2) and a C-terminal peptide capable of inhibiting
PC2 at nanomolar concentration [34] When the 7B2
C-terminal peptide was incubated with PC2, a smaller
peptide (CT peptide 1–18 containing Lys-Lys at the
C-terminus) was generated, and its inhibitory activity
was lost when incubated with carboxypeptidase E to
remove the last two Lys residues [35]
As histone H1 forms little secondary structure in
solution (Fig 6), the specific conformation may not be
necessary for the inhibitory activity These indicate
that furin recognizes specific sequence around KKAKflA in the histone fragments, which explains why the three fragments, P2, P3 and P4, have similar inhibitory activity, as well as the full-length histone H1 and the synthetic 36- amino acid peptide around the cleavage site It would be interesting to further identify the minimum sequence around the cleavage site required for inhibition
Furin is predominantly located at the trans-Golgi network and cell surface in vivo [36], whereas histone H1 normally binds to the linker DNA of chromosome
in nucleus However, some studies have shown that nuclear proteins could be located on the surface of var-ious cells, including intestinal microvilli, monocytes and lymphocytes [37,38] Histone fragments released from epithelia were shown to have strong antimicrobial activities [39–41] During the cell apoptosis induced by virus or bacteria, histones are released and bind to the negatively charged surfaces of neighboring viable cells [42] In addition, it is interesting that the N-terminals
of both fragments P3 and P4 were Val, generated from the cleavage of Leu-Val bond by an unknown protease
It has been indicated that an endopeptidase in the DNase I-containing extract from the bovine pancreas was able to cleave human H1 into two fragments of
8 and 14 kDa [43] We speculate that there might be
a specific protease in the porcine liver to cleave histone H1 at Leu-Val site into smaller fragments, thus facilita-ting its transport to the cell surface or to other subcel-lular compartments These suggest that the inhibition
of furin by histone H1 fragments may be physiologi-cally relevant, which remains to be clarified
It is well known that, besides the housekeeping role of chromosomal condensation, histone H1 has some other biological functions, such as the regulation
of gene expression and the stimulation of myoblast proliferation [44] Moreover, histone H1.2 was found
to be a signal molecule that triggers the release of cytochrome c from mitochondria in the DNA damage-induced apoptosis [21], to selectively inhibit the activa-tion of calmodulin-dependent enzymes [45], and to be
20000
10000
–10000
–20000 –30000
Wavelength (nm)
Proteins or peptide
H1
C-H1
36 aa peptide
1.6
1 0.5
Percentages of alpha-helix %
0
Fig 6 The conformation of full-length H1,
C-H1 and the 36-amino acid peptide
meas-ured by far-UV CD spectra in 10 mM
phos-phate buffer, pH 7.0 at 20 C ——,
-and ÆÆÆÆÆÆ indicate the full-length H1, C-H1 -and
the 36- amino acid peptide, respectively.
Trang 8the intestinal protein receptor for 987P fimbriae of
enterotoxigenic Escherichia coli [46] The C-terminal
domain of histone H1 was reported to be capable of
binding to an apoptotic nuclease (a DNA
fragmenta-tion factor, DFF40⁄ CAD) and of stimulating the
DNA cleavage [47], and this current work indicates
another potential function of the multifunctional
pro-tein histone H1
In summary, this study has shown for the first time
that poly-lysine protein histone H1 and its C-terminal
fragments are potent furin inhibitors In contrast to
other synthetic furin inhibitors, histone H1 and its
C-terminal fragments are endogenous proteins and
should exhibit little toxicity if used clinically Our results
give a new indications for understanding the regulation
of furin activity in vivo, as well as for developing novel
tools to inhibit furin-mediated pathogenic processes
Experimental procedures
Materials
Phenyl-Sepharose 4B and Superdex 75 column were from
Amersham Pharmacia (Uppsala, Sweden), Hamilton PRP-3
column from Hamilton Co (Reno, NV, USA) TALON
superflow metal affinity resin was from Clontech (Mountain
View, CA, USA) The fluorogenic substrate
pyrArg-Thr-Lys-Arg-7-amino-4-methylcoumarin (MCA) was from
Bachem Bioscience (San Diego, CA, USA), cytochrome c
from Sigma (St Louis, MO, USA) The purified
recombin-ant mouse furin was a generous gift from I Lindberg
(Lou-isiana State University, New Orleans, LA, USA)
Purification of furin inhibitors
The porcine liver was excised and homogenized with five
volumes of cold acetone previously kept in)20 C freezer
About 100 g of acetone powder was obtained per kilogram
of liver The 300 g of acetone powder was extracted with
10 volumes of 2.5% trichloroacetic acid overnight After
centrifugation, the supernatant was subjected to stepwise
precipition with 0.5 and 0.9 saturated ammonium sulfate
The pellet of the 0.9 saturated ammonium sulfate portion
was dissolved in a small volume of distilled water and was
dialyzed with 20 mm sodium acetate⁄ acetic acid buffer
(pH 4.5) The dialyzed sample was loaded onto a CM-52
column pre-equilibrated with 20 mm sodium acetate⁄ acetic
acid buffer, pH 4.5 (buffer A), washed with three column
volumes of buffer A and eluted stepwise The fraction
eluted with buffer A containing 0.4 m NaCl was found to
have furin inhibitory activity This fraction was adjusted
to 2 m ammonium sulfate and applied onto a phenyl
Sepharose CL-4B column pre-equilibrated with 2 m
ammonium sulfate in buffer A The unbound fraction from
the column was collected, dialyzed with water and lyophi-lized The lyophilized fraction was then dissolved in 250 lL buffer A, loaded onto a Superdex 75 column equilibrated with buffer A The fraction with a furin inhibitor activity from the gel filtration column was further loaded onto a Hamilton PRP-3 (150· 4.1 mm) column equilibrated with 0.1% trifluoroacetic acid on a Waters 510 HPLC pump and 2487 dual absorbance detector (Milford, MA, USA) The bound proteins were eluted with a linear gradient of 0–20% acetonitrile in 0.1% (v⁄ v) trifluoroacetic acid at a flow rate of 1 mLÆmin)1 in 0–20 min, and of 20–100% acetonitrile in 0.1% (v⁄ v) trifluoroacetic acid at a flow rate
of 1 mLÆmin)1 in 20–50 min The elute was monitored at
214 nm, collected and assayed for furin inhibitory activity
Enzyme activity assay
The fluorogenic MCA substrate, pyrArg-Thr-Lys-Arg-MCA, was used for the furin activity assay as previously described [25] To determine the inhibitory activity, differ-ent amounts of the sample were preincubated with a fixed amount of enzyme (2· 10)3 units) at 37C in 100 mm Hepes buffer, pH 7.5, containing 1 mm CaCl2 for 5 min, the residual enzyme activity was then measured The final substrate concentration for all assays was 1 lm The fluor-escence of the released MCA was measured on-line with a Hitachi F-2500 spectrofluorimeter using an excitation and
an emission wavelength of 370 nm (slit width, 10 nm) and
460 nm (slit width, 10 nm), respectively
N-terminal sequencing
Amino acid sequencing was performed on a Perkin-Elmer Applied Biosystems 494 pulsed-liquid phase protein sequencer [Procise, PE Applied Biosystems (Foster City,
CA, USA)] with an on-line 785 A PTH-amino acid analyzer
Gene cloning of the porcine histone H1.2
As there is no intron in the genes of histones, the genomic DNA from porcine liver was used as a template
to clone the gene of histone H1.2 The human and murine histone H1 cDNA sequences from the gene database were referred to design a pair of PCR primers
as follows: 5¢-ATGTCCGAGAC(C ⁄ T)GCTCC(T ⁄ C)GC-3¢
The PCR products were ligated into the pGEM-T Easy vector (Promega, Madison, WI, USA) and sequenced
Expression and purification of full-length histone H1.2 and its N- and C-terminal fragments
The genes of the full- length histone H1.2 (F-H1), its N-ter-minal fragment with 87- amino acid residues (N-H1) and
Trang 9C-terminal fragment of 126-amino acid residues (C-H1)
were cloned through the flanking NcoI and XhoI restriction
sites into the expression vector pET28a The sequences of
the constructions were verified by DNA sequencing The
primer pairs for the cloning were as follows: F-h1:
5¢-ccatgggcatgtccgagactgctcctgc-3¢, 5¢-ctcgagcttctttttgggtgca
gcctt-3¢; n-h1 : 5¢-ccatgggcatgtccgagactgctcctgc-3¢, 5¢-ctcgagc
aggctcttgagacccagct-3¢; c-h1 : 5¢-ccatgggcgtgagcaagggcacctt
g-3¢, 5¢-ctcgagcttctttttgggtgcagcctt-3¢
The expression vectors were transformed into E coli strain
BL21 Cells grown in LB medium containing 10 lgÆmL)1
kanamycin were induced with isopropylthiogalactoside when
OD600reached 0.8 The harvested cells were lysed by
soni-cating The recombinant proteins with His-tag were purified
by TALON superflow metal affinity column (BD Clontech)
according to the manufacturer’s instructions The eluted
frac-tion was further purified by RP-HPLC on Hamilton PRP-3
(150· 4.1 mm) column with gradient elution from 100%
buffer A (0.1% trifluoroacetic acid) to 100% buffer B (70%
acetonitrile with 0.1% trifluoroacetic acid) in 50 min The
purified recombinant proteins were lyophilized for inhibitory
activity assay
Measurement of the kinetic parameter Ki
The Ki values of inhibitors against furin were determined
by Dixon’s plot (1⁄ V against I) using two different
concen-trations of substrate pyrArg-Thr-Lys-Arg-MCA (1.5 lm,
and 3.0 lm) Data from three measurements were averaged
and graphically analyzed with equation to obtain the
equi-librium inhibition constant, Ki, as previously described [25]
Peptide synthesis
KKAKAAKPKKAAKSAAKAVKPK) derived from
his-tone H1.2 around the identified cleavage site was
synthes-ized using ABI 433 peptide synthesizer starting from
Fmoc-LysBoc-Wang resin The protected amino acids are:
Fmoc-Thr (tBu), Fmoc-Ser (tBu), Fmoc-Lys (Boc) The
resin was cleaved by trifluoroacetic acid containing 8%
p-cresol and 0.2% H2O for 1 h at room temperature The
product was extracted by 0.1% trifluoroacetic acid
contain-ing 20% acetonitrile The extract was then lyophilized and
purified on a Sephadex G-15 column, equilibrated with
0.1% trifluoroacetic acid The eluted fraction was
lyo-philized and further purified on a RP-HPLC Zorbax C8
column (9.4· 250 mm) equilibrated with buffer A
(0.1% trifluoroacetic acid) at a flow rate of 2 mLÆmin)1
The peptide was eluted by a two-step gradient system:
0–12% buffer B (70% acetonitrile, 0.08% trifluoroacetic
acid) in 10 min and 12–45% buffer B in 10–45 min The
purified peptide was checked by the ABI API2000 Q-trap
mass spectroscope
CD spectroscopy
Samples for CD spectroscopy were at a final concentration
of 200 lgÆmL)1in 10 mm phosphate buffer, pH 7.0 Spectra were obtained on a Jasco J-715 spectrometer in 1 mm of cells at 20C The results were analyzed with standard ana-lysis software (jasco) and expressed as mean residue molar ellipticity (h) The helical content was estimated from the ellipticity value at 222 nm (h222), according to the empirical equation of Chen et al [24]:
%helical content¼ 100½h222=39 500 ð1 2:57=nÞ where n is the number of residues per helix
Acknowledgements
We would like to thank Dr I Lindberg (Louisiana State University) for the purified recombinant mouse furin We also would like to appreciate Dr C Wang for discussion
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