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• W2139403670 abstract "Jerdostatin represents a novel RTS-containing short disintegrin cloned by reverse transcriptase-PCR from the venom gland mRNA of the Chinese Jerdons pit viper Trimeresurus jerdonii. The jerdostatins precursor cDNA contained a 333-bp open reading frame encoding a signal peptide, a pre-peptide, and a 43-amino acid disintegrin domain, whose amino acid sequence displayed 80% identity with that of the KTS-disintegrins obtustatin and viperistatin. The jerdostatin cDNA structure represents the first complete open reading frame of a short disintegrin and points to the emergence of jerdostatin from a short-coding gene. The different residues between jerdostatin and obtustatin/viperistatin are segregated within the integrin-recognition loop and the C-terminal tail. Native jerdostatin (r-jerdostatin-R21) and a R21K mutant (r-jerdostatin-K21) were produced in Escherichia coli. In each case, two conformers were isolated. One-dimensional 1H NMR showed that conformers 1 and 2 of r-jerdostatin-R21 represent, respectively, well folded and unfolded proteins. The two conformers of the wild-type and the R21K mutant inhibited the adhesion of α1-K562 cells to collagen IV with IC50 values of 180 and 703 nm, respectively. The IC50 values of conformers 2 of r-jerdostatin-R21 and r-jerdostatin-K21 were, respectively, 5.95 and 12.5 μm. Neither r-jerdostatin-R21 nor r-jerdostatin-K21 showed inhibitory activity toward other integrins, including αIIbβ3, αvβ3, α2β1, α5β1, α4β1, α6β1, and α9β1 up to a concentration of 24 μm. Although the RTS motif appears to be more potent than KTS inhibiting the α1β1 integrin, r-jerdostatin-R21 is less active than the KTS-disintegrins, strongly suggesting that substitutions outside the integrin-binding motif and/or C-terminal proteolytic processing are responsible for the decreased inhibitory activity. Jerdostatin represents a novel RTS-containing short disintegrin cloned by reverse transcriptase-PCR from the venom gland mRNA of the Chinese Jerdons pit viper Trimeresurus jerdonii. The jerdostatins precursor cDNA contained a 333-bp open reading frame encoding a signal peptide, a pre-peptide, and a 43-amino acid disintegrin domain, whose amino acid sequence displayed 80% identity with that of the KTS-disintegrins obtustatin and viperistatin. The jerdostatin cDNA structure represents the first complete open reading frame of a short disintegrin and points to the emergence of jerdostatin from a short-coding gene. The different residues between jerdostatin and obtustatin/viperistatin are segregated within the integrin-recognition loop and the C-terminal tail. Native jerdostatin (r-jerdostatin-R21) and a R21K mutant (r-jerdostatin-K21) were produced in Escherichia coli. In each case, two conformers were isolated. One-dimensional 1H NMR showed that conformers 1 and 2 of r-jerdostatin-R21 represent, respectively, well folded and unfolded proteins. The two conformers of the wild-type and the R21K mutant inhibited the adhesion of α1-K562 cells to collagen IV with IC50 values of 180 and 703 nm, respectively. The IC50 values of conformers 2 of r-jerdostatin-R21 and r-jerdostatin-K21 were, respectively, 5.95 and 12.5 μm. Neither r-jerdostatin-R21 nor r-jerdostatin-K21 showed inhibitory activity toward other integrins, including αIIbβ3, αvβ3, α2β1, α5β1, α4β1, α6β1, and α9β1 up to a concentration of 24 μm. Although the RTS motif appears to be more potent than KTS inhibiting the α1β1 integrin, r-jerdostatin-R21 is less active than the KTS-disintegrins, strongly suggesting that substitutions outside the integrin-binding motif and/or C-terminal proteolytic processing are responsible for the decreased inhibitory activity. The integrin family of cell adhesion proteins promotes the attachment and migration of cells on the surrounding extracellular matrix (1Ridley A.J. Schwartz M.A. Burridge K. Firtel R.A. Ginsberg M.H. Borisy G. Parsons J.T. Horwitz A.R. Science. 2003; 302: 1704-1709Crossref PubMed Scopus (3870) Google Scholar, 2Springer T.A. Wang J. Adv. Protein Chem. 2004; 68: 29-63Crossref PubMed Scopus (138) Google Scholar). Through signals transduced upon integrin ligation by extracellular matrix proteins, several integrins play key roles in promoting angiogenesis and tumor metastasis (3Danen E.H. Curr. Pharm. Des. 2005; 11: 881-891Crossref PubMed Scopus (99) Google Scholar). However, although antagonists of several integrins (e.g. α5β1, αvβ3, and αvβ5, the primary targets of endostatin, an endogenous negative regulator of angiogenesis (4Rehn M. Veikkola T. Kukk-Valdre E. Nakamura H. Ilmonen M. Lombardo C.R. Pihlajaniemi T. Alitalo K. Vouri K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1024-1029Crossref PubMed Scopus (421) Google Scholar)) are now under evaluation in clinical trials to determine their potential as therapeutics for cancer and other diseases (5Jin H. Varner J. Br. J. Cancer. 2004; 90: 561-565Crossref PubMed Scopus (482) Google Scholar, 6Mistry A. Harbottle R. Hart S. Hodivala-Dilke K.M. Curr. Opin. Mol. Ther. 2004; 5: 603-610Google Scholar), the precise regulation and exact action of integrins is still unclear (7Hynes R.O. Nat. Med. 2002; 8: 918-921Crossref PubMed Scopus (488) Google Scholar, 8Hodivala-Dilke K.M. Reynolds A.R. Reynolds L.E. Cell Tissue Res. 2003; 314: 131-144Crossref PubMed Scopus (128) Google Scholar). Thus, the integrins α1β1 and α2β1 are highly up-regulated by vascular endothelial growth factor in cultured endothelial cells, resulting in an enhanced α1β1- and α2β1-dependent cell spreading on collagen and it has been reported that these integrins provide critical support for vascular endothelial growth factor signaling, endothelial cell migration, and tumor angiogenesis (9Senger D.R. Perruzzi C.A. Streit M. Koteliansky V.E. De Fougerolles A.R. Detmar M. Am. J. Pathol. 2002; 160: 195-204Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). The α1β1 and α2β1 integrins are highly expressed on the microvascular endothelial cells, and blocking of their adhesive properties by monoclonal antibodies (9Senger D.R. Perruzzi C.A. Streit M. Koteliansky V.E. De Fougerolles A.R. Detmar M. Am. J. Pathol. 2002; 160: 195-204Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 10Senger D.R. Claffey K.P. Benes J.E. Perruzzi C.A. Sergiou A.P. Detmar M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13612-13617Crossref PubMed Scopus (459) Google Scholar) or by the snake venom disintegrin obtustatin (11Marcinkiewicz C. Wainreb P.H. Calvete J.J. Kisiel D.G. Mousa S.A. Tuszynski G.P. Lobb R.R. Cancer Res. 2003; 63: 2020-2023PubMed Google Scholar) significantly reduced the vascular endothelial growth factor-driven neovascularization ratio and tumor growth in animal models. Moreover, null-mice lacking integrin α1β1 develop normally, but exhibit reduced vascularity of the skin (9Senger D.R. Perruzzi C.A. Streit M. Koteliansky V.E. De Fougerolles A.R. Detmar M. Am. J. Pathol. 2002; 160: 195-204Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar) and have reduced number and size of intratumoral capillaries (12Pozzi A. Moberg P.E. Miles L.A. Wagner S. Soloway P. Gardner H.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2202-2207Crossref PubMed Scopus (350) Google Scholar). Ongoing studies with α2 knock-out mice also suggest a critical role in angiogenesis for the α2β1 integrins (13Mercurio A.M. Am. J. Pathol. 2002; 161: 3-6Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 14DeClerck Y.A. Mercurio A.M. Stack M.S. Chapman H.A. Zutter M.M. Muschel R.J. Raz A. Matrisian L.M. Sloane B.F. Noel A. Hendrix M.J. Coussens L. Padarathsingh M. Am. J. Pathol. 2004; 164: 1131-1139Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Thus, inhibitors of the α1β1 and α2β1 integrins alone or in combination with antagonists of other integrins involved in angiogenesis may prove beneficial in the control of tumor neovascularization. α1β1 and α2β1 belong to the I-domain bearing subfamily of integrins, and specifically interact with collagen (15White D.J. Puranen S. Johnson M.S. Heino J. Int. J. Biochem. Cell Biol. 2004; 36: 1405-1410Crossref PubMed Scopus (145) Google Scholar). However, despite sharing large structural homology, these two integrins have distinct collagen binding preferences: α1β1 integrin is a very selective receptor of basement membrane type IV collagen, whereas α2β1 is highly specific for fibrillar collagen types I-III (16Dickeson S.K. Mathis N.L. Rahman M. Bergelson J.M. Santoro S.A. J. Biol. Chem. 1999; 274: 32182-32191Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 17Tulla M. Pentikäinen O.T. Viitasalo T. Käpylä J. Impola U. Nykvist P. Nissinen L. Johnson M.S. Heino J. J. Biol. Chem. 2001; 276: 48206-48212Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Substitution of the cytoplasmic domains of the α1 and α2 subunits in transfected human mammary epithelial cells revealed that the two integrins participate in different signal transduction pathways (18Klekotka P.A. Santoro S.A. Ho A. Dowdy S.F. Zutter M. Am. J. Pathol. 2001; 159: 983-992Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Noteworthy, the α1β1 and α2β1 integrins are the targets of snake venom toxins belonging to different protein families. C-type lectin-like proteins include selective and potent (i.e. EMS16 from Echis multisquamatus;IC50 = 6nm) inhibitors of α2β1 (19Wang R. Kini R.M. Chung M.C.M. Biochemistry. 1999; 38: 7584-7595Crossref PubMed Scopus (69) Google Scholar, 20Marcinkiewicz C. Lobb R.R. Marcinkiewicz M.M. Daniel J.L. Smith J.B. Dangelmaier C. Weinreb P.H. Beacham D.A. Niewiarowski S. Biochemistry. 2000; 39: 9859-9867Crossref PubMed Scopus (90) Google Scholar), whereas the only to date known snake venom proteins that specifically antagonize the function of the α1β1 integrin are the disintegrins obtustatin (IC50 = 2 nm) from the venom of Vipera lebetina obtusa (11Marcinkiewicz C. Wainreb P.H. Calvete J.J. Kisiel D.G. Mousa S.A. Tuszynski G.P. Lobb R.R. Cancer Res. 2003; 63: 2020-2023PubMed Google Scholar, 21Moreno-Murciano P. Daniel Monleón D. Calvete J.J. Celda B. Marcinkiewicz C. Protein Sci. 2003; 12: 366-371Crossref PubMed Scopus (45) Google Scholar), viperistatin (IC50 = 0.08 nm) from Vipera palestinae (22Kisiel D.G. Calvete J.J. Katzhendel J. Fertala A. Lazarovici P. Marcinkiewicz C. FEBS Lett. 2004; 577: 478-482Crossref PubMed Scopus (58) Google Scholar) and lebestatin (IC50 = 0.4 nm) from Macrovipera lebetina. 3M. El Ayeb, personal communication. The crystal structure of EMS16 in complex with the integrin α2 I-domain has provided insight into the structural basis of the integrin inhibitory specificity of this C-type lectin protein (23Horii K. Okuda D. Morita T. Mizuno H. J. Mol. Biol. 2004; 341: 519-527Crossref PubMed Scopus (50) Google Scholar). On the other hand, the primary α1β1 binding motif of obtustatin and viperistatin is a KTS tripeptide located in a lateral position of the mobile disintegrins active loop (24Moreno-Murciano M.P. Monleón D. Marcinkiewicz C. Calvete J.J. Celda B. J. Mol. Biol. 2003; 329: 135-145Crossref PubMed Scopus (46) Google Scholar), which displays concerted motions with the C-terminal region (25Monleón D. Moreno-Murciano M.P. Kovacs H. Marcinkiewicz C. Calvete J.J. Celda B. J. Biol. Chem. 2003; 278: 45570-45576Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Now, we report the molecular cloning, primary structure, recombinant expression, and integrin inhibitory characteristics of two conformers of jerdostatin, an RTS-containing disintegrin from the Chinese Jerdon pit viper Trimeresurus jerdonii, and of a R21K mutant. In cell adhesion assays, the recombinant (r-) jerdostatin 4The abbreviations used are: r-jerdostatinrecombinant jerdostatinHPLChigh performance liquid chromatographyMALDI-TOFmatrix-assisted laser-desorption ionization time-of-flightMSmass spectrometry. conformers of both the wild-type (r-jerdostatin-R21) and the R21K mutant (r-jerdostatin-K21) selectively blocked, albeit with different potency, the adhesion of K562 cells expressing the integrin α1β1 to collagen IV. recombinant jerdostatin high performance liquid chromatography matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. Materials—Human vitronectin was purchased from Chemicon (Temecula, CA). Purified, human collagen IV was provided by Dr. A. Fertala (Thomas Jefferson University, Philadelphia, PA). Highly purified human fibrinogen was a gift from Dr. A. Budzynski (Temple University, Philadelphia, PA). Recombinant human VCAM-1/Ig was a generous gift of Dr. Roy R. Lobb (Biogen). Human fibronectin and laminin were purchased from Sigma. ExTaq® DNA polymerase, dNTP, and DNA marker were from TaKaRa Biotechnology Co., Ltd. (Dalian). PolyATtract® System 1000 kit and the Reverse Transcription System kit were from Promega Biotech. Cell Lines—K562 cells transfected with α1, α2, and α6 integrins were provided by Dr. M. Hemler (Dana Farber Cancer Institute, Boston, MA). JY cells expressing αvβ3 were a gift from Dr. Burakoff (Dana Farber Cancer Institute, Boston, MA). α9- and mock-transfected SW480 cells were generated as described (26Yokosaki Y. Palmer E.L. Prieto A.L. Crossin K.L. Bourdon M.A. Pytela R. Sheppard D. J. Biol. Chem. 1994; 269: 26691-26696Abstract Full Text PDF PubMed Google Scholar). K562 and Jurkat cell lines, which express α5β1 and α4β1 integrins, respectively, were purchased from ATCC (Manassas, VA). PCR Amplification of Jerdostatin cDNA—The T. jerdonii venom glands were collected from Yiliang, Yunnan, China. Isolation of mRNA and reverse transcription was conducted using the PolyATtract System 1000 kit and Reverse Transcription System kit, respectively, according to the manufacturer's protocols. DNA was amplified by PCR using total reverse transcriptase-PCR products as template. The forward primer, 5′-CCAAATCCAG(C/T)CTCCAAAATG-3′, and the reverse primer, 5′-TTCCA(G/T)CTCCATTGTTG(G/T)TTA-3′, were designed according to the highly conserved 5′- and 3′-noncoding regions of the cDNAs encoding for elegantin-2a from Trimeresurus elegans (GenBank® accession number AB059572), elegantin-1a from T. elegans (GenBank accession number AB059571), and HR2a from Trimeresurus flavoviridis (27Yamada D. Shin Y. Morita T. FEBS Lett. 1999; 451: 299-302Crossref PubMed Scopus (30) Google Scholar). The PCR amplification protocol included 35 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 2 min. The recovered PCR products were cloned into PMD18-T vector (TaKaRa), and then transformed into Escherichia coli strain JM109. The white transformants were screened by PCR and the positive clones were subjected to sequencing on an Applied Biosystems model 377 DNA sequencing system. Generation of r-Jerdostatin R21K Mutant—Site-directed mutagenesis was performed essentially as described in the QuikChange® site-directed mutagenesis kit of Stratagene (La Jolla, CA). To this end, plasmid pET-32a containing the wild-type jerdostatin sequence flanked by NcoI and XhoI restriction sites was used as the template in the PCR (denaturation at 94 °C for 2 min, followed by 12 cycles of denaturation (30 s at 94 °C), annealing (60 s at 55 °C), and extension (12 min at 68 °C), and a final extension for 10 min at 68 °C) using the forward primer 5′-GGAACAACATGCTGGAAAACCAGTGTATCAAGTCATTACTGC-3′ and the reverse primer 5′-ACTTGATACACTGGTTTTCCAGCATGTTGTTCCTGCCGGC-3′ in which the Arg codon AGA has been substituted AAA (Lys) (in boldface). The mutant DNA was sequenced to confirm the absence of undesired mutations. Synthetic Peptides—Individual peptides and a library of peptides representing the entire integrin-recognition loop of obtustatin but differing in the residue at a single position (19CX1KTSLTSHYC29; CWX2TSLTSHYC; etc., where Xn is an equimolar mixture of all amino acids except cysteine) were prepared by manual simultaneous multiple peptide synthesis using standard N-(9-fluorenyl)methoxycarbonyl (Fmoc) chemistry as described (28Pastor M.T. De la Paz M.L. Lacroix E. Serrano L. Pérez-Payá E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 614-619Crossref PubMed Scopus (63) Google Scholar). The mixtures at positions “X” were incorporated by coupling a mixture of 19 L-amino acids (cysteine was omitted), with the relative ratio suitability adjusted to yield close to equimolar incorporation. The quality of the synthesized peptide mixtures was validated by mass spectrometry. Individual peptides were purified by preparative reverse phase-HPLC. Peptide identity was confirmed by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The following molar absorption coefficients (ϵ) at 280 nm were used for quantification of peptide mixtures (29Gill S.C. Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5073) Google Scholar): ϵ for the control peptide CWKTSLTSHYC = 5600 (Trp) + 1500 (Tyr) = 7100 m-1 cm-1; ϵ for the peptide library CX2KTSLTSHYC = 1500 + (1/19 × 5600) + (1/19 × 1500) = 1873.7 m-1 cm-1; for peptide libraries with X at positions 2-8, ϵ = 5600 + 1500 + (1/19 × 5600) + (1/19 × 1500) = 7473.7 m-1 cm-1; and ϵ for the peptide library CWKTSLTSHX9C = 5600 + (1/19 × 5600) + (1/19 × 1500) = 5973.7 m-1 cm-1. Purification of KTS-disintegrins—Obtustatin and viperistatin were purified from the venoms of V. lebetina obtusa and V. palestinae, respectively, using the previously described two-step reversed-phase HPLC (11Marcinkiewicz C. Wainreb P.H. Calvete J.J. Kisiel D.G. Mousa S.A. Tuszynski G.P. Lobb R.R. Cancer Res. 2003; 63: 2020-2023PubMed Google Scholar, 21Moreno-Murciano P. Daniel Monleón D. Calvete J.J. Celda B. Marcinkiewicz C. Protein Sci. 2003; 12: 366-371Crossref PubMed Scopus (45) Google Scholar, 22Kisiel D.G. Calvete J.J. Katzhendel J. Fertala A. Lazarovici P. Marcinkiewicz C. FEBS Lett. 2004; 577: 478-482Crossref PubMed Scopus (58) Google Scholar). The purity of the disintegrins was assessed by SDS-PAGE. The monoisotopic masses of the purified disintegrins were determined either by electrospray ionization mass spectrometry with a triple quadrupole-ion trap hybrid instrument (QTrap from Applied Biosystems) equipped with a nanospray source (Protana, Denmark) or by MALDI-TOF mass spectrometry (MS) using an Applied Biosystems DE-Pro spectrometer, operated in delayed extraction and reflector modes, and α-hydroxycinnamic acid saturated in 0.1% trifluoroacetic acid in 70% acetonitrile as the matrix. A tryptic peptide mixture of Cratylia floribunda seed lectin (SwissProt accession code P81517) prepared and previously characterized in our laboratory was used as a mass calibration standard (mass range 450-3300 Da). For determination of isotope-averaged molecular masses, the instrument was operated in the linear mode using 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) saturated in 70% acetonitrile and 0.1% trifluoroacetic acid as the matrix. The mass calibration standard consisted of a mixture of the following proteins, whose isotope-averaged molecular mass in daltons are given in parentheses: bovine insulin (5,734.6), E. coli thioredoxin (11,674.5), and horse apomyoglobin (16,952.6). Protein concentration was determined with the bicinchoninic acid (BCA) protein quantification kit (Pierce) with bovine serum albumin as a standard, or by amino acid analysis (after hydrolysis in 6 n HCl for 24 h at 110 °C in air-evacuated and sealed ampoules) using a Biochrom (Amersham Biosciences) amino acid analyzer. Cloning and Production of Recombinant r-Jerdostatin-Thioredoxin-His6 Fusion Proteins—The jerdostatin cDNA coding for wild-type and R21K fragments were amplified by PCR using primers synthesized by Sigma-Genosys (Haverhill, UK). The forward primer was 5′-CGTGCCATGGATTGTACAACTGGACCATG-3′, which contained a NcoI restriction site (underlined) and the sequence coding for the first six residues of the protein. The reverse primer was 5′-GCCTCGAGTATTAGCCATTCCCGGGATAAC-3′, which includes a restriction site for XhoI (underlined), a stop codon (in italics and bold), and the last six C-terminal residues of jerdostatin. The PCR protocol included denaturation at 94 °C for 2 min, followed by 40 cycles of denaturation (10 s at 94 °C), annealing (15 s at 55 °C), and extension (20 s at 72 °C), and a final extension for 7 min at 72 °C. The amplified fragments were purified using the Perfect Pre Gel Clean Up kit (Eppendorf, Hamburg, Germany) and cloned in a pGEM-T vector (Promega, Madison, WI). E. coli DH5α cells (Novagen, Madison, WI) were transformed by electroporation using an Eppendorf 2510 electroporator following the manufacturer's instructions. Positive clones, selected by growing the transformed cells in Luria broth (LB) medium containing 100 μg/ml ampicilin, were confirmed by PCR amplification using the above primers, and the PCR-amplified fragments were sequenced (using an Applied Biosystems model 377 DNA sequencer) to check the correctness of the sequences of the wild-type and the R21K jerdostatins open reading frame. To construct an expression vector of jerdostatin-thioredoxin-His6 wild-type and mutated fusion proteins the pGEM-T-jerdostatin plasmid and a pET32a vector (Novagen) were digested with NcoI and XhoI for 12 h at 37 °C and the 132-bp jerdostatin fragments and the pET32a vector were purified after agarose gel electrophoresis with the Eppendorf Perfect Pre Gel Clean Up kit. The jerdostatin fragments and the open pET32a vector were ligated with T4 DNA ligase (Invitrogen) overnight at 13 °C. These constructs were used to transform electrocompetent E. coli DH5α cells. The plasmidic DNAs from positive clones were used to transform (by electroporation) E. coli Origami® B cells (Novagen). Another pool of cells were transformed with mock pET32a plasmid and used as negative control for the recombinant expression of jerdostatin-thioredoxin fusion protein. Recombinant Expression of Jerdostatin-Thioredoxin-His6 Fusion Proteins—Positive E. coli Origami B clones, shown by PCR to contain the jerdostatin-thioredoxin fusion constructs, wild-type or R21K mutant, were grown overnight at 37 °C in LB medium containing 100 μg/ml of ampicillin, 33 μg/ml of kanamycin, and 12 μg/ml of tetracyclin, followed by a 1:10 (v/v) dilution in the same medium. For the induction of the expression of the recombinant fusion proteins, isopropyl β-d-thiogalactosidase was added to a final concentration of 1 mm, and the cell suspensions were incubation for another 7 h at 37°C. Thereafter, the cells were pelleted by centrifugation, resuspended in the same volume of 20 mm sodium phosphate, 150 mm NaCl, pH 7.4, washed three times with the same buffer, and resuspended in 100 ml/liter of initial cell culture of 20 mm sodium phosphate, 250 mm NaCl, 10 mm imidazole, pH 7.4. The cells were lyzed by sonication (15 cycles of 15 s sonication followed by 1 min resting) in an ice bath. The lysates were centrifuged at 10,000 × g for 30 min at 4 °C, and the soluble and the insoluble fractions were analyzed by SDS-15% polyacrylamide gel electrophoresis. Purification of Recombinant Jerdostatin Molecules—The jerdostatin-thioredoxin-His6 fusion proteins, wild-type and R21K mutant, were purified from the soluble fraction of positive E. coli Origami clone, the lysate was purified by affinity chromatography using an ÄKTA Basic chromatograph equipped with a 5-ml HisTrap HP column (Amersham Biosciences) equilibrated in 20 mm sodium phosphate, 250 mm NaCl, 10 mm imidazole, pH 7.4, buffer. After absorbance at 280 nm of the flow-through fraction reached baseline, the bound material was eluted at a flow rate of 1.5 ml/min with a linear gradient of 10-500 mm imidazole for 60 min. The purified protein fractions (checked by SDS-PAGE) were pooled, dialyzed against 50 mm Tris/HCl, pH 7.4, and digested with 0.25 units of enterokinase (Invitrogen) per mg of recombinant protein. The reaction mixture was freed from enterokinase by chromatography on a 0.5-ml column of agarose-trypsin inhibitor (Sigma) equilibrated and eluted with 50 mm Tris-HCl, pH 7.4. Jerdostatin was separated from thioredoxin-His6 by chromatography of the agarose-trypsin-inhibitor non-bound fraction on a HisTrap column (as above), and the nonbound and retarded fractions, both containing jerdostatin, were further purified by reverse-phase HPLC followed by size-exclusion chromatography using an ÄKTA Basic chromatograph equipped with a Superdex Peptide column (Amersham Biosciences) eluted with phosphate-buffered saline buffer at a flow rate of 0.3 ml/min. The purity of the isolated proteins was assessed by SDS-PAGE, reverse-phase HPLC, N-terminal sequence analysis (using an Applied Biosystems Procise instrument), and MALDI-TOF mass spectrometry as described above for the KTS-disintegrins, and nanoelectrospray ionization mass spectrometry using a QTrap instrument (Applied Biosystems) equipped with a nanoelectrospray source (Proxeon, Denmark). Protein concentration of purified recombinant jerdostatin was determined spectrophotometrically using an ϵ at 280 nm of 10,677 m-1 cm-1 calculated by amino acid analysis as above. In-gel Tryptic Digestion and Mass Fingerprinting—The recombinant expression of the jerdostatin-thioredoxin-His6 fusion proteins and the purification of r-jerdostatin molecules were monitored by SDS-PAGE and mass fingerprinting. To this end, SDS-PAGE separated polypeptides were subjected to automated digestion with sequencing grade bovine pancreatic trypsin (Roche) at a final concentration of 20 ng/μlof 50 mm ammonium bicarbonate, pH 8.3, using a ProGest digestor (Genomic Solutions) following the manufacturer's instructions. Digestions were done with prior reduction with dithiothreitol (10 mm for 15 min at 65 °C) and carbamidomethylation with iodoacetamide (50 mm for 60 min at room temperature). The tryptic peptide mixtures were freed from reagents using a C18 Zip-Tip pipette tip (Millipore) activated with 70% acetonitrile and equilibrated in 0.1% trifluoroacetic acid. Following protein adsorption and washing with 0.1% trifluoroacetic acid, the proteins were eluted with 3 μl of 70% acetonitrile and 0.1% trifluoroacetic acid. For mass fingerprinting analysis, 0.85 μl of the digests were spotted onto a MALDI-TOF sample holder, mixed with an equal volume of a saturated solution of α-cyano-4-hydroxycinnamic acid (Sigma) in 50% acetonitrile containing 0.1% trifluoroacetic acid, dried, and analyzed with an Applied Biosystems Voyager-DE Pro MALDI-TOF mass spectrometer, operated in delayed extraction and reflector modes, as above. The peptide mass fingerprint obtained from each electrophoretic band was compared with the expected proteolytic digest of the fusion protein using the program PAWS. 5Proteometrics, available at prowl.rockefeller.edu. Collision-induced Dissociation by Tandem Mass Spectrometry—For peptide sequencing, the protein digest mixture was subjected to electrospray ionization tandem mass spectrometric (MS/MS) analysis using a QTrap mass spectrometer (Applied Biosystems) equipped with a nanoelectrospray source (Protana, Denmark). Doubly charged ions selected after Enhanced Resolution MS analysis were fragmented using the Enhanced Product Ion with the Q0 trapping option at 250 atomic mass units/s across the entire mass range. For MS/MS experiments, Q1 was operated at unit resolution, the Q1 to Q2 collision energy was set to 35 eV, the Q3 entry barrier was 8 V, the linear ion trap Q3 fill time was 250 ms, and the scan rate in Q3 was 1000 atomic mass units/s. Collision-induced dissociation spectra were interpreted manually or using the on-line form of the MASCOT program. 6www.matrixscience.com. Quantitation of Free Cysteine Residues and Disulfide Bonds—For quantitation of free cysteine residues and disulfide bonds (30Juárez P. Sanz L. Calvete J.J. Proteomics. 2004; 4: 327-338Crossref PubMed Scopus (102) Google Scholar), the purified proteins dissolved in 10 μl of 50 mm HEPES, pH 9.0, 5 m guanidine hydrochloride containing 1 mm EDTA) were heat denatured at 85 °C for 15 min, allowed to cool at room temperature, and incubated with either 10 mm iodoacetamide for 1 h at room temperature, or with 10 mm 1,4-dithioerythritol (Sigma) for 15 min at 80 °C, followed by addition of iodoacetamide at 25 mm final concentration and incubation for 1 h at room temperature. Carbamidomethylated proteins were freed from reagents using a C18 Zip-Tip pipette tip (Millipore) after activation with 70% acetonitrile and equilibration in 0.1% trifluoroacetic acid. Following protein adsorption and washing with 0.1% trifluoroacetic acid, the PE-proteins were eluted onto the MALDI-TOF plate with 1 μl of 70% acetonitrile and 0.1% trifluoroacetic acid and subjected to mass spectrometric analysis as above. The number of free cysteine residues (NSH) was determined using Equation 1, NSH=(M1A-MNAT)/57.05(Eq. 1) where MIA is the mass of the denatured but nonreduced protein incubated in the presence of iodoacetamide; MNAT is the mass of the native, HPLC-isolated protein; and 57.05 is the mass increment because of the carbamidomethylation of one thiol group. The number of total cysteine residues (NCys) can be calculated from Equation 2, NCys=(MCM-MNAT)/57.05(Eq. 2) where MCM is the mass (in Da) of the reduced and carbamidomethylated protein. Finally, the number of disulfide bonds NS-S can be calculated from Equation 3. NS-S=(Ncys-NSH)/2(Eq. 3) Cell Adhesion Studies—Adhesion studies of cultured cells labeled with 5-chloromethyl fluorescein diacetate were performed essentially as described (31Marcinkiewicz C. Calvete J.J. Marcinkiewicz M.M. Raida M. Lobb R.R. Senadhi V.-K. Huang Z. Niewiarowski S. J. Biol. Chem. 1999; 274: 12468-12473Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 32Marcinkiewicz C. Senadhi V.K. McLane M.A. Niewiarowski S. Blood. 1997; 90: 1565-1575Crossref PubMed Google Scholar). For inhibition studies, increasing concentrations of disintegrins were incu" @default.
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• W2139403670 title "cDNA Cloning and Functional Expression of Jerdostatin, a Novel RTS-disintegrin from Trimeresurus jerdonii and a Specific Antagonist of the α1β1 Integrin" @default.
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