The potent inhibitory activity of histone H1.2 C-terminal fragments on furin Jinbo Han1, Ling Zhang2, Xiaoxia Shao2, Jiahao Shi1, and Chengwu Chi1,2 Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai, China Institute of Protein Research, Tong-ji University, Shanghai, China 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 August 2006) doi:10.1111/j.1742-4658.2006.05451.x Many physiologically important proproteins, pathogenic bacterial exotoxins and viral envelope glycoproteins are activated by the proprotein convertase 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 chromatographic 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 · 10)7 m while the full-length histone H1.2 inhibited it with a lesser activity, suggesting that the inhibitory activity relies on the C-terminal lysinerich 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 Ki of 5.2 · 10)7 m 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-dependent 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 various substrates ranging from the blood clotting factors, serum proteins, growth factors, 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-ValLys-Arg-CH2Cl, the bioengineered variant of a1-antitrypsin Portland (a1-PDX) [7], polyarginines [8], Drosophila Serpin [9,10], and the serpin-derived Abbreviation PCs, proprotein convertases FEBS Journal 273 (2006) 4459–4469 ª 2006 The Authors Journal compilation ª 2006 FEBS 4459 Endogenous furin inhibitors from porcine liver J Han et al peptides, as well as the barley serine proteinase inhibitor 2-derived cyclic peptides [11] Some of these inhibitors are used to prevent the activation of bacterial toxin, the processing of envelope glycoprotein in viral replication and the metastasis of cancer [12– 14] The propeptide of furin itself [15,16], the interalpha-inhibitor protein [17] and human proteinase inhibitor [18] have been found to be potent furin inhibitors, and our earlier work identified a high positively charged protein, namely, nonhistone chromosomal 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-terminally truncated fragments generated from histone H1.2 with 126, 120 and 103 amino acid residues, respectively The activity assay showed that the fulllength histone H1.2 could also inhibit furin with a Ki value of 4.6 · 10)7 m The identification of lysine-rich histone H1.2 and its C-terminal fragments as inhibitors of furin will undoubtedly pave the way for the development 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 second, a variety of furin substrates are precisely processed in the liver, compelling the existence of furin inhibitor to modulate the enzyme activity The purification procedure is described in Fig 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 chromatography, 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 4460 Porcine liver TCA extraction Ammonium sulfate precipitation CM-52 (cation) column Phenyl-sepharose CL-4B Superdex 75 column HPLC Fig 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 precipitated 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 inhibitory activity against furin were purified a phenyl Sepharose CL-4B column, the highest inhibitory 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, indicating that they are derived from the same protein (Fig 3A) A database search revealed that the N-terminal 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 elucidate 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 predicted 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 FEBS Journal 273 (2006) 4459–4469 ª 2006 The Authors Journal compilation ª 2006 FEBS J Han et al Endogenous furin inhibitors from porcine liver A 0.80 Absorbance (214 nm) 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 Retention time (min) P3 B 0.90 P2 P4 Fig 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 mLỈmin)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 and 20–100% (v ⁄ v) acetonitrile in 0.1% (v ⁄ v) trifluoroacetic acid in 20–50 at a flow rate of mLỈmin)1 The peaks marked (P1-P6) were collected and the peaks marked by P2, P3 and P4 exhibited 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 Absorbance (214 nm) 0.80 0.70 0.60 0.50 P6 0.40 0.30 P5 P1 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 Retention time (min) C three fragments P4, P3 and P2 start from 88th, 94th and 111th residue of histone H1.2 with 126-, 120and 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 FEBS Journal 273 (2006) 4459–4469 ª 2006 The Authors Journal compilation ª 2006 FEBS 4461 Endogenous furin inhibitors from porcine liver J Han et al Fig Identification of the P2, P3 and P4 fractions by N-terminal sequencing (A) The partial N-terminal sequences of the P2, P3 and P4 fractions 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 histone 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) Temporary inhibition and identification of the reactive site of the inhibitor The inhibitory activity of the three fragments of histone H1.2 against furin was assayed and analyzed using Dixon’s plot to determine their inhibition constants Ki Table showed that the Ki values of three H1.2 fragment inhibitors were around · 10)7 m Prolonged incubation over half an hour caused a gradual loss of inhibitory activity, suggesting that the inhibition 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 Table Inhibition constants of the three purified C-terminal fragments 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 procedures The results obtained were used to compute the Ki values Each value represents the mean ± SD determined from three independent experiments Inhibitor Inhibition constant Ki (M) P2 P3 P4 2.4 ± 0.08 · 10)7 2.3 ± 0.17 · 10)7 2.1 ± 0.18 · 10)7 4462 first 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 difluoride membrane and sequenced separately (Fig 4B) According to the sequence of the P4 fragment inhibitor, 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 Ki determination since all reactions for inhibitory activity analysis were finished in less than 10 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 FEBS Journal 273 (2006) 4459–4469 ª 2006 The Authors Journal compilation ª 2006 FEBS J Han et al Endogenous furin inhibitors from porcine liver A kDa 97 66 43 B kDa 97 66 43 31 31 20 14 20 14 15 30 VSKGTLVQ AAKPKKAA 30 60 120 (min) 60 (min) C P4 10 20 30 40 50 VSKGTLVQTK GTGASGSFKL NKKAATGETK PKAKKSGAAK PKKSAGAAKK 60 70 80 90 100 PKKATGSATP KKAAKKTPKK AKKPAAAAVT KKVAKSPKKA KAAKPKKAAK 110 120 SAAKAVKPKA AKPKVAKPKK AAPKKK Reactive site Fig Determination of the reactive site of the P4 fragment inhibitor (A) The degradation of the P4 fragment incubated with furin at different time at 37 °C in 100 mM Hepes buffer, pH 7.5, mm CaCl2, 0.5% Triton X-100 and mm 2-mercaptoethanol At the indicated time, the reaction was immediately terminated by boiling the sample at 100 °C for min, then separated on 15% SDS ⁄ PAGE and stained with Coomassie brilliant blue Protein markers are indicated on the left (B) The cleaved fragments were separated and transferred to the polyvinylidene 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 matched 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 fragments 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)7 m, 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 fulllength histone H1.2 and its C-terminal fragment is specific, 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 KVAKSPKKAKAAKPKKAAKSAAKAVKPK) with 36- amino acid residues was synthesized The determined molecular weight of the purified peptide was consistent with the theoretical one (Fig 5B) Moreover, this synthetic 36- amino acid peptide was still a potent furin inhibitor with a Ki value only about twofold 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 fragments were also examined by CD spectroscopy (Fig 6) As previously reported [23], histone H1.2 and its fragments not have a clear secondary structure Their secondary structures were found to be dominated 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, 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 FEBS Journal 273 (2006) 4459–4469 ª 2006 The Authors Journal compilation ª 2006 FEBS 4463 Endogenous furin inhibitors from porcine liver J Han et al A B Max 2.9e8cps Mass reconstruction of + EMS: 1.030 to 3.088 from Sample 30 (kkak_p1) of 040812wiff (Turbo 3570.0 2.9e8 2.8e8 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 3552.0 3028.0 3046.0 3126.0 3000 3100 3242.0 3313.0 3409.0 3200 3300 3441.0 3400 3640.0 3667.0 3739.0 3836.0 3922.0 3976.0 3498.0 3500 3600 Mass, amu 3700 3800 Table Inhibition constants of various recombinant proteins and the synthetic peptide against furin Inhibitor Inhibition constant Ki (M) C-H1 Full length histone H1 N-H1 Cytochrome c 36- amino acid peptide 3.1 4.6 7.8 3.3 5.2 ± ± ± ± ± 0.12 0.02 0.06 0.21 0.16 · · · · · 10)7 10)7 10)5 10)5 10)7 studies from basic-research and clinical-application viewpoints Many efforts have been made to develop peptidyl, nonpeptidyl and protein-derived furin inhibitors 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 4464 3900 4000 Fig SDS ⁄ PAGE and mass spectrometry analysis of the recombinant full-length histone H1.2 and its N- and C-terminal fragments (A) The recombinant full-length histone H1.2 and its N- and C-terminal fragments were expressed in E coli and purified by TALON superflow metal affinity column and RP-HPLC (see Experimental procedures) The purified recombinant proteins 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 in vitro (Ki ¼ 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 glycoprotein of HIV type 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- FEBS Journal 273 (2006) 4459–4469 ª 2006 The Authors Journal compilation ª 2006 FEBS J Han et al Endogenous furin inhibitors from porcine liver 20000 Fig The conformation of full-length H1, C-H1 and the 36-amino acid peptide measured by far-UV CD spectra in 10 mM phosphate buffer, pH 7.0 at 20 °C ——, - - - and ỈỈỈỈỈỈ indicate the full-length H1, C-H1 and the 36- amino acid peptide, respectively Molar ellipticity 10000 Proteins or peptide Percentages of alpha-helix % H1 1.6 C-H1 36 aa peptide 0.5 –10000 –20000 –30000 190 200 itor 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 · 10)7 m, comparable with that of polyarginines such as L6R (hexa-l-arginine) (Ki ¼ 1.14 · 10)7 m) [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 (Ki ¼ 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 temporary, 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 (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 210 220 230 240 250 Wavelength (nm) 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 various 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 facilitating its transport to the cell surface or to other subcellular compartments These suggest that the inhibition of furin by histone H1 fragments may be physiologically 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 damageinduced apoptosis [21], to selectively inhibit the activation of calmodulin-dependent enzymes [45], and to be FEBS Journal 273 (2006) 4459–4469 ª 2006 The Authors Journal compilation ª 2006 FEBS 4465 Endogenous furin inhibitors from porcine liver J Han et al the 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 fragmentation factor, DFF40 ⁄ CAD) and of stimulating the DNA cleavage [47], and this current work indicates another potential function of the multifunctional protein 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 pyrArgThr-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 recombinant mouse furin was a generous gift from I Lindberg (Louisiana 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 m ammonium sulfate and applied onto a phenyl Sepharose CL-4B column pre-equilibrated with m ammonium sulfate in buffer A The unbound fraction from 4466 the column was collected, dialyzed with water and lyophilized 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 mLỈmin)1 in 0–20 min, and of 20–100% acetonitrile in 0.1% (v ⁄ v) trifluoroacetic acid at a flow rate of mLỈmin)1 in 20–50 The elute was monitored at 214 nm, collected and assayed for furin inhibitory activity Enzyme activity assay The fluorogenic MCA substrate, pyrArg-Thr-Lys-ArgMCA, was used for the furin activity assay as previously described [25] To determine the inhibitory activity, different amounts of the sample were preincubated with a fixed amount of enzyme (2 · 10)3 units) at 37 °C in 100 mm Hepes buffer, pH 7.5, containing mm CaCl2 for min, the residual enzyme activity was then measured The final substrate concentration for all assays was lm The fluorescence 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¢ and 5¢-GGTGGCTCTGAAAAGAGCC(G ⁄ T)TTTG-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-terminal fragment with 87- amino acid residues (N-H1) and FEBS Journal 273 (2006) 4459–4469 ª 2006 The Authors Journal compilation ª 2006 FEBS J Han et al C-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 lgỈmL)1 kanamycin were induced with isopropylthiogalactoside when OD600 reached 0.8 The harvested cells were lysed by sonicating The recombinant proteins with His-tag were purified by TALON superflow metal affinity column (BD Clontech) according to the manufacturer’s instructions The eluted fraction 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 The purified recombinant proteins were lyophilized for inhibitory activity assay Endogenous furin inhibitors from porcine liver CD spectroscopy Samples for CD spectroscopy were at a final concentration of 200 lgỈmL)1 in 10 mm phosphate buffer, pH 7.0 Spectra were obtained on a Jasco J-715 spectrometer in mm of cells at 20 °C The results were analyzed with standard analysis 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 content ẳ 100ẵh222 =39 500 2:57=nị 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 References 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 concentrations 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 equilibrium inhibition constant, Ki, as previously described [25] Peptide synthesis The 36- amino acid peptide (PAAATVTKKVAKSP KKAKAAKPKKAAKSAAKAVKPK) derived from histone H1.2 around the identified cleavage site was synthesized 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 h at room temperature The product was extracted by 0.1% trifluoroacetic acid containing 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 lyophilized 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 mLỈmin)1 The peptide was eluted by a two-step gradient system: 0–12% buffer B (70% acetonitrile, 0.08% trifluoroacetic acid) in 10 and 12–45% buffer B in 10–45 The purified peptide was checked by the ABI API2000 Q-trap mass spectroscope Han KK & Martinage A (1992) Post-translational chemical modification(s) of proteins Int J Biochem 24, 19–28 Nakayama K (1997) Furin: a mammalian subtilisin ⁄ Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins Biochem J 327, 625–635 Rockwell NC, Krysan DJ, Komiyama T & Fuller RS (2002) Precursor processing by kex2 ⁄ furin proteases Chem Rev 102, 4525–4548 Wachter A & Schwappach B (2005) The yeast CLC chloride channel is proteolytically processed by the furin-like protease Kex2p in the first extracellular loop FEBS Lett 579, 1149–1153 Hughey RP, Bruns JB, Kinlough CL, Harkleroad KL, Tong Q, Carattino MD, Johnson JP, Stockand JD & Kleyman TR (2004) Epithelial sodium channels are activated by furin-dependent proteolysis J Biol Chem 279, 18111–18114 Thomas G (2002) Furin at the cutting edge: from protein traffic to embryogenesis and disease Nat Rev Mol Cell Biol 3, 753–766 Jean F, Stella K, Thomas L, Liu G, Xiang Y, Reason AJ & Thomas G (1998) alpha1-Antitrypsin Portland, a bioengineered serpin highly selective for furin: application as an antipathogenic agent Proc Natl Acad Sci 95, 7293–7298 Cameron A, Appel J, Houghten RA & Lindberg I (2000) Polyarginines are potent furin inhibitors J Biol Chem 275, 36741–36749 FEBS Journal 273 (2006) 4459–4469 ª 2006 The Authors Journal compilation ª 2006 FEBS 4467 Endogenous furin inhibitors from porcine liver J Han et al Oley M, Letzel MC & Ragg H (2004) Inhibition of furin by serpin Spn4A from Drosophila melanogaster FEBS Lett 577, 165–169 10 Osterwalder T, Kuhnen A, Leiserson WM, Kim YS & Keshishian H (2004) Drosophila serpin functions as a neuroserpin-like inhibitor of subtilisin-like proprotein convertases J Neurosci 24, 5482–5491 11 Villemure M, Fournier A, Gauthier D, Rabah N, Wilkes BC & Lazure C (2003) Barley serine proteinase inhibitor 2-derived cyclic peptides as potent and selective inhibitors of convertases PC1 ⁄ and furin Biochemistry 42, 9659–9668 12 Bassi DE, Lopez De Cicco R, Mahloogi H, Zucker S, Thomas G & Klein-Szanto AJ (2001) Furin inhibition results in absent or decreased invasiveness and tumorigenicity of human cancer cells Proc Natl Acad Sci USA 98, 10326–10331 13 Kibler KV, Miyazato A, Yedavalli VS, Dayton AI, Jacobs BL, Dapolito G, Kim SJ & Jeang KT (2004) Polyarginine inhibits gp160 processing by furin and suppresses productive human immunodeficiency virus type infection J Biol Chem 279, 49055–49063 14 Sarac MS, Cameron A & Lindberg I (2002) The furin inhibitor hexa-d-arginine blocks the activation of Pseudomonas aeruginosa exotoxin A in vivo Infect Immun 70, 7136–7139 15 Basak A & Lazure C (2003) Synthetic peptides derived from the prosegments of proprotein convertase ⁄ and furin are potent inhibitors of both enzymes Biochem J 373, 231–239 16 Bissonnette L, Charest G, Longpre JM, Lavigne P & Leduc R (2004) Identification of furin pro-region determinants involved in folding and activation Biochem J 379, 757–763 17 Opal SM, Artenstein AW, Cristofaro PA, Jhung JW, Palardy JE, Parejo NA & Lim YP (2005) Inter-alphainhibitor proteins are endogenous furin inhibitors and provide protection against experimental anthrax intoxication Infect Immun 73, 5101–5105 18 Dahlen JR, Jean F, Thomas G, Foster DC & Kisiel W (1998) Inhibition of soluble recombinant furin by human proteinase inhibitor J Biol Chem 273, 1851– 1854 19 Fei H, Li Y, Wang LX, Luo MJ, Ling MH & Chi CW (2000) Nonhistone protein purified from porcine kidney acts as a suicide substrate inhibitor on furin-like enzyme Acta Pharmacol Sin 21, 265–270 20 Duckert P, Brunak S & Blom N (2004) Prediction of proprotein convertase cleavage sites Protein Eng Des Sel 17, 107–112 21 Konishi A, Shimizu S, Hirota J, Takao T, Fan Y, Matsuoka Y, Zhang L, Yoneda Y, Fujii Y, Skoultchi AI et al (2003) Involvement of histone H1.2 in apoptosis induced by DNA double-strand breaks Cell 114, 673–688 4468 22 Weber K, Pringle JR & Osborn M (1972) Measurement of molecular weights by electrophoresis on SDS-acrylamide gel Methods Enzymol 26, 3–27 23 Roque A, Iloro I, Ponte I, Arrondo JR & Suau P (2005) DNA-induced secondary structure of the carboxyl-terminal domain of histone H1 J Biol Chem 280, 32141–32147 24 Chen YH, Yang JT & Chau KH (1974) Determination of the helix and b form of proteins in aqueous solution by circular dichroism Biochemistry 13, 3350–3359 25 Liu ZX, Fei H & Chi CW (2004) Two engineered eglin c mutants potently and selectively inhibiting kexin or furin FEBS Lett 556, 116–120 26 Komiyama T, VanderLugt B, Fugere M, Day R, Kaufman RJ & Fuller RS (2003) Optimization of protease–inhibitor interactions by randomizing adventitious contacts Proc Natl Acad Sci USA 100, 8205– 8210 27 Komiyama T & Fuller RS (2000) Engineered eglin c variants inhibit yeast and human proprotein processing proteases, Kex2 and furin Biochemistry 39, 15156– 15165 28 Lu W, Zhang W, Molloy SS, Thomas G, Ryan K, Chiang Y, Anderson S & Laskowski M Jr (1993) Arg15-Lys17-Arg18 turkey ovomucoid third domain inhibits human furin J Biol Chem 268, 14583–14585 29 Van Rompaey L, Ayoubi T, Van De Ven W & Marynen P (1997) Inhibition of intracellular proteolytic processing of soluble proproteins by an engineered alpha 2-macroglobulin containing a furin recognition sequence in the bait region Biochem J 326, 507–514 30 Kacprzak MM, Peinado JR, Than ME, Appel J, Henrich S, Lipkind G, Houghten RA, Bode W & Lindberg I (2004) Inhibition of furin by polyarginine-containing peptides: nanomolar inhibition by nona-d-arginine J Biol Chem 279, 36788–36794 31 Henrich S, Cameron A, Bourenkov GP, Kiefersauer R, Huber R, Lindberg I, Bode W & Than ME (2003) The crystal structure of the proprotein processing proteinase furin explains its stringent specificity Nat Struct Biol 10, 520–526 32 Apletalina E, Appel J, Lamango NS, Houghten RA & Lindberg I (1998) Identification of inhibitors of prohormone convertases and using a peptide combinatorial library J Biol Chem 273, 26589–26595 33 Martens GJ, Braks JA, Eib DW, Zhou Y & Lindberg I (1994) The neuroendocrine polypeptide 7B2 is an endogenous inhibitor of prohormone convertase PC2 Proc Natl Acad Sci USA 91, 5784–5787 34 van Horssen AM, van den Hurk WH, Bailyes EM, Hutton JC, Martens GJ & Lindberg I (1995) Identification of the region within the neuroendocrine polypeptide 7B2 responsible for the inhibition of prohormone convertase PC2 J Biol Chem 270, 14292– 14296 FEBS Journal 273 (2006) 4459–4469 ª 2006 The Authors Journal compilation ª 2006 FEBS J Han et al 35 Zhu X, Rouille Y, Lamango NS, Steiner DF & Lindberg I (1996) Internal cleavage of the inhibitory 7B2 carboxyl-terminal peptide by PC2: a potential mechanism for its inactivation Proc Natl Acad Sci USA 93, 4919–4924 36 Molly SS, Thomas L, Vanslyke JK, Stenberg PE & Thomas G (1994) Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface EMBO J 13, 18–33 37 Mecheri S, Dannecker G, Dennig D, Poncet P & Hoffmann MK (1993) Anti-histone autoantibodies react specifically with the B cell surface Mol Immunol 30, 549–557 38 Watson K, Edwards RJ, Shaunak S, Parmelee DC, Sarraf C, Gooderham NJ & Davies DS (1995) Extranuclear location of histones in activated human peripheral blood lymphocytes and cultured T-cells Biochem Pharmacol 50, 299–309 39 Fernandes JM, Molle G, Kemp GD & Smith VJ (2004) Isolation and characterisation of oncorhyncin II, a histone H1-derived antimicrobial peptide from skin secretions of rainbow trout, Oncorhynchus mykiss Dev Comp Immunol 28, 127–138 40 Richards RC, O’Neil DB, Thibault P & Ewart KV (2001) Histone H1: an antimicrobial protein of Atlantic salmon (Salmo salar) Biochem Biophys Res Commun 284, 549–555 41 Rose FR, Bailey K, Keyte JW, Chan WC, Greenwood D & Mahida YR (1998) Potential role of epithelial cellderived histone H1 proteins in innate antimicrobial defense in the human gastrointestinal tract Infect Immun 66, 3255–3263 42 Watson K, Gooderham NJ, Davies DS & Edwards RJ (1999) Nucleosomes bind to cell surface proteoglycans J Biol Chem 274, 21707–21713 Endogenous furin inhibitors from porcine liver 43 Kanai Y (2003) The role of non-chromosomal histones in the host defense system Microbiol Immunol 47, 553–556 44 Henriquez JP, Casar JC, Fuentealba L, Carey DJ & Brandan E (2002) Extracellular matrix histone H1 binds to perlecan, is present in regenerating skeletal muscle and stimulates myoblast proliferation J Cell Sci 115, 2041–2051 45 Rasmussen C & Garen C (1993) Activation of calmodulin-dependent enzymes can be selectively inhibited by histone H1 J Biol Chem 268, 23788–23791 46 Zhu G, Chen H, Choi BK, Del Piero F & Schifferli DM (2005) Histone H1 proteins act as receptors for the 987P fimbriae of enterotoxigenic Escherichia coli J Biol Chem 280, 23057–23065 47 Widlak P, Kalinowska M, Parseghian MH, Lu X, Hansen JC & Garrard WT (2005) The histone H1 C-terminal domain binds to the apoptotic nuclease, DNA fragmentation factor (DFF40 ⁄ CAD) and stimulates DNA cleavage Biochemistry 44, 7871–7878 Supplementary material The following supplementary material is available online: Fig S1 Dixon’s plots (1 ⁄ V against [I]) of the three purified C-terminal fragments P2, P3 and P4 of histone H1.2, the recombinant full-length histone H1.2, its N- and C-terminal fragments, cytochrome c and the 36-amino acid peptide around the reactive site This material is available as part of the online article from http://www.blackwell-synergy.com FEBS Journal 273 (2006) 4459–4469 ª 2006 The Authors Journal compilation ª 2006 FEBS 4469 ... Temporary inhibition and identification of the reactive site of the inhibitor The inhibitory activity of the three fragments of histone H1.2 against furin was assayed and analyzed using Dixon’s plot to... inhibit furin with a Ki value of 4.6 · 10)7 m The identification of lysine-rich histone H1.2 and its C-terminal fragments as inhibitors of furin will undoubtedly pave the way for the development of therapeutically... 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]