The Flexible and Clustered Lysine Residues of Human Ribonuclease 7 Are Critical for Membrane Permeability and Antimicrobial Activity

Abstract

The ubiquitous ribonucleases (RNases) play important roles in RNA metabolism, angiogenesis, neurotoxicity, and antitumor or antimicrobial activity. Only the antimicrobial RNases possess high positively charged residues, although their mechanisms of action remain unclear. Here, we report on the role of cationic residues of human RNase7 (hRNase7) in its antimicrobial activity. It exerted antimicrobial activity against bacteria and yeast, even at 4 °C. The bacterial membrane became permeable to the DNA-binding dye SYTOX® Green in only a few minutes after bactericidal RNase treatment. NMR studies showed that the 22 positively charged residues (Lys18 and Arg4) are distributed into three clusters on the surface of hRNase7. The first cluster, K1,K3,K111,K112, was located at the flexible coil near the N terminus, whereas the other two, K32,K35 and K96,R97,K100, were located on rigid secondary structures. Mutagenesis studies showed that the flexible cluster K1,K3,K111,K112, rather than the catalytic residues His15, Lys38, and His123 or other clusters such as K32,K35 and K96,R97,K100, is critical for the bactericidal activity. We suggest that the hRNase7 binds to bacterial membrane and renders the membrane permeable through the flexible and clustered Lys residues K1,K3,K111,K112. The conformation of hRNase7 can be adapted for pore formation or disruption of bacterial membrane even at 4 °C. The ubiquitous ribonucleases (RNases) play important roles in RNA metabolism, angiogenesis, neurotoxicity, and antitumor or antimicrobial activity. Only the antimicrobial RNases possess high positively charged residues, although their mechanisms of action remain unclear. Here, we report on the role of cationic residues of human RNase7 (hRNase7) in its antimicrobial activity. It exerted antimicrobial activity against bacteria and yeast, even at 4 °C. The bacterial membrane became permeable to the DNA-binding dye SYTOX® Green in only a few minutes after bactericidal RNase treatment. NMR studies showed that the 22 positively charged residues (Lys18 and Arg4) are distributed into three clusters on the surface of hRNase7. The first cluster, K1,K3,K111,K112, was located at the flexible coil near the N terminus, whereas the other two, K32,K35 and K96,R97,K100, were located on rigid secondary structures. Mutagenesis studies showed that the flexible cluster K1,K3,K111,K112, rather than the catalytic residues His15, Lys38, and His123 or other clusters such as K32,K35 and K96,R97,K100, is critical for the bactericidal activity. We suggest that the hRNase7 binds to bacterial membrane and renders the membrane permeable through the flexible and clustered Lys residues K1,K3,K111,K112. The conformation of hRNase7 can be adapted for pore formation or disruption of bacterial membrane even at 4 °C. Ribonucleases (RNases) 3The abbreviations used are: RNase, ribonuclease; hRNase, human RNase; cfu, colony-forming unit(s); CD, circular dichroism; HSQC, heteronuclear single quantum correlation; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy. are found widely among living organisms, and they play an important role in the metabolism of RNA (1.D'Alessio G. Trends Cell Biol. 1993; 3: 106-109Abstract Full Text PDF PubMed Scopus (97) Google Scholar). Recently, novel biological functions other than the intrinsic ribonucleolytic activity have been demonstrated for several members of the bovine RNaseA superfamily. In the human RNase family, eosinophil-derived neurotoxin (RNase2) and eosinophil cationic protein (RNase3) have neurotoxic (2.Durack D.T. Ackerman S.J. Loegering D.A. Gleich G.J. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5165-5169Crossref PubMed Scopus (185) Google Scholar) as well as antiparasitic activity (3.McLaren D.J. McKean J.R. Olsson I. Venges P. Kay A.B. Parasite Immunol. 1981; 3: 359-373Crossref PubMed Scopus (101) Google Scholar). Angiogenin (RNase5), which induces blood vessel formation, is also an RNase (4.Fett J.W. Strydom D.J. Lobb R.R. Alderman E.M. Bethune J.L. Riordan J.F. Vallee B.L. Biochemistry. 1985; 24: 5480-5486Crossref PubMed Scopus (845) Google Scholar). In the frog RNase family, most of the members have antitumor activity (5.Liao Y.D. Huang H.C. Leu Y.J. Wei C.W. Tang P.C. Wang S.C. Nucleic Acids Res. 2000; 28: 4097-4104Crossref PubMed Scopus (41) Google Scholar). For the above-mentioned functions, the catalytic activities of RNases are essential. Some members of RNase superfamily, such as human RNases 3, 5, and 7 (hRNase3, hRNase5, and hRNase7), mouse angiogenins 1 and 4 (mAng1 and mAng4), as well as chicken RNaseA2, also have antimicrobial activities (6.Zhang J. Dyer K.D. Rosenberg H.F. Nucleic Acids Res. 2003; 31: 602-607Crossref PubMed Scopus (86) Google Scholar, 7.Harder J. Schroder J.M. J. Biol. Chem. 2002; 277: 46779-46784Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 8.Hooper L.V. Stappenbeck T.S. Hong C.V. Gordon J.I. Nat. Immunol. 2003; 4: 269-273Crossref PubMed Scopus (739) Google Scholar, 9.Holloway D.E. Hares M.C. Shapiro R. Subramanian V. Acharya K.R. Protein Expression Purif. 2001; 22: 307-317Crossref PubMed Scopus (39) Google Scholar, 10.Nitto T. Dyer K.D. Czapiga M. Rosenberg H.F. J. Biol. Chem. 2006; 281: 25622-25634Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The mechanisms for these antibacterial activities remain unclear, although some distinct properties exist in these bactericidal RNases. First of all, the calculated isoelectric points of bactericidal RNases are higher than those of non-bactericidal RNases. Second, the net positive charges of the bactericidal RNases are higher than those of non-bactericidal RNases (Table 1). There is no evidence, however, that the high positive charge or high pI value is critical for the antimicrobial activity. In this study, we have presented evidence that one flexible Lys cluster K1,K3,K111,K112, rather than the catalytic residues His15, Lys38, His123 or other Lys clusters such as K32,K35, and K96,R97,K100,is critical for the bactericidal activity of hRNase7. The functional role of these Lys residues (K1,K3,K111,K112) on the bactericidal activity of hRNase7 will be discussed.TABLE 1Properties of selected RNases in bovine RNase A superfamilyRNaseFunctionCatalytic activityAmino acid no.LysArgAspGluNet chargespIdAccession codes are as follows: hRNase 2 (X16546), hRNase 3 (X16545), hRNase 7 (NM_032572), hRNase 5 (NM_001145), mAng (NM_007447), mAng-4 (AY219870), bRNase A (X07283), RC-RNase (AF039104), and RC-RNase 6 (AF242556)hRNase 2Antiviral, neurotoxic0.65aZhang, et al. (6)1344841+79.20hRNase 3Antimicrobial0.048aZhang, et al. (6)13311960+1410.72hRNase 7Antibacterial0.021aZhang, et al. (6)12818442+169.80hRNase 5Angiogenesis, antibacterialExtremely lowa,Zhang, et al. (6)bHolloway, et al. (9)12371364+109.73mAngAngiogenesis, antibacterial30% of hRNase 5bHolloway, et al. (9)12191174+99.54mAng-4AntibacterialUnknownbHolloway, et al. (9)120111077+79.20RNase ARNA catabolism1085.6cLiao, et al. (5)12410455+48.64RC-RNaseAntitumor964.4cLiao, et al. (5)1114612+79.20RC-RNase6Antitumor0.007cLiao, et al. (5)10515582+109.39a Zhang, et al. (6.Zhang J. Dyer K.D. Rosenberg H.F. Nucleic Acids Res. 2003; 31: 602-607Crossref PubMed Scopus (86) Google Scholar)b Holloway, et al. (9.Holloway D.E. Hares M.C. Shapiro R. Subramanian V. Acharya K.R. Protein Expression Purif. 2001; 22: 307-317Crossref PubMed Scopus (39) Google Scholar)c Liao, et al. (5.Liao Y.D. Huang H.C. Leu Y.J. Wei C.W. Tang P.C. Wang S.C. Nucleic Acids Res. 2000; 28: 4097-4104Crossref PubMed Scopus (41) Google Scholar)d Accession codes are as follows: hRNase 2 (X16546), hRNase 3 (X16545), hRNase 7 (NM_032572), hRNase 5 (NM_001145), mAng (NM_007447), mAng-4 (AY219870), bRNase A (X07283), RC-RNase (AF039104), and RC-RNase 6 (AF242556) Open table in a new tab Preparation and Enzymatic Assay of RNases−The coding region of the human RNase7 (hRNase7) gene was obtained from the genomic DNA of HeLa cells by PCR. The NdeI and BamHI restriction sites were hung on the 5′ and 3′ end of the gene fragment, respectively, and subcloned into the T7 RNA polymerase-driven expression vector pET22b (Novagen) (11.Hsu C.H. Liao Y.D. Pan Y.R. Chen L.W. Wu S.H. Leu Y.J. Chen C. J. Mol. Biol. 2003; 326: 1189-1201Crossref PubMed Scopus (17) Google Scholar). Site-directed mutagenesis was made by PCR as previously described (12.Huang H.C. Wang S.C. Leu Y.J. Lu S.C. Liao Y.D. J. Biol. Chem. 1998; 273: 6395-6401Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The uniformly 15N- or 13C-labeled hRNase7 with an extra Met at the N terminus was produced in Escherichia coli BL21(DE3). The recombinant hRNase7 from inclusion bodies were refolded and purified as previously described (11.Hsu C.H. Liao Y.D. Pan Y.R. Chen L.W. Wu S.H. Leu Y.J. Chen C. J. Mol. Biol. 2003; 326: 1189-1201Crossref PubMed Scopus (17) Google Scholar). The ribonucleolytic activity of recombinant hRNase7 was analyzed by the zymogram assay on RNA-casting PAGE as previously described (13.Liao Y.D. Wang J.J. Eur. J. Biochem. 1994; 222: 215-220Crossref PubMed Scopus (30) Google Scholar). Antimicrobial Activity Assay−The bacteria were cultured in Nutrient broth and plated on Nutrient agar (Difco 0001) for Pseudomonas aeruginosa Migula (American Type Culture Collection (ATCC) 27853), tryptic soy broth/agar (Difco 0369) for Staphylococcus aureus subspecies Aureus Rosenbach (ATCC 6538P), and Luria-Bertani broth/agar for E. coli DH5α. The yeast Candida albicans (Robin) Berkhout (ATCC 14053) was cultured and plated in/on yeast malt broth/agar, and Pichia pastoris X-33 was cultured and plated in/on yeast extract-peptone-dextrose broth/agar. The microbes were grown overnight, washed, and diluted 1:100 in 10 mm sodium phosphate, pH 7.4. 45 μl of the microbes (5–10 × 104 colony-forming units (cfu)) was mixed with various concentrations of RNase/oligopeptide (5 μl), which was dissolved in 20 mm Hepes, pH 7.4, 50 mm NaCl, and incubated at 37 °C for 3 h. Serial dilution of each RNase-treated bacteria/yeast was prepared and plated for the determination of the remaining cfu (14.Rosenberg H.F. Dyer K.D. J. Biol. Chem. 1995; 270 (Correction (1995) J. Biol. Chem.270, 30234): 21539-21544Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Binding of RNase to Bacteria−Small aliquots of RNases (2 μg in 5 μl) were incubated with 45 μl(5 × 106 cfu) of sodium phosphate-washed P. aeruginosa at 37 °C for 30 min. The RNases bound onto bacteria were spun down, washed five times with 10 mm sodium phosphate, and analyzed by SDS-PAGE and silver staining. Assays of the Permeability of Bacterial Membrane−The overnight culture of P. aeruginosa (107 cfu) was washed and resuspended in 100 μl of water and incubated with 1 μm SYTOX® Green (Molecular Probes) in a dark 96-well plate for 15 min in the dark. After the addition of RNase, the increase of fluorescence due to the binding of the dye to the intracellular DNA was measured in the same microplate reader using 485- and 520-nm filters for excitation and emission wavelengths, respectively (15.Nekhotiaeva N. Elmquist A. Rajarao G.K. Hallbrink M. Langel U. Good L. FASEB J. 2004; 18: 394-396Crossref PubMed Scopus (122) Google Scholar). Nomenclature of Mutated hRNase7 and Oligopeptide−Some of the hRNase7 mutants are designated as follows: K1A (substitution of Lys1 by Ala); H15A (substitution of His15 by Ala); K32N/K35Q (substitution of Lys32 by Asn and Lys35 by Gln); K96A/R97A/K100T (substitution of Lys96 by Ala, Arg97 by Ala, and Lys100 by Thr); K111Q/K112Q (substitution of Lys111 by Gln and Lys112 by Gln); Δ1KPKG4 (deletion of N-terminal Met and Lys1-Pro2-Lys3-Gly4 residues from the recombinant hRNase7; and (R3S1)3, (12-mer oligopeptide containing the triple-repeated Arg-Arg-Arg-Ser sequence). Circular Dichroism (CD) Experiments−CD experiments were carried out using an Aviv CD 202 spectrometer (Lakewood, NJ) using a 1-mm path length cuvette with 20 μm hRNase7 in 20 mm sodium phosphate. The CD spectra at different temperatures and pH values were recorded from 190 to 260 nm using a wavelength step of 0.5 nm. Equilibrium thermal denaturing experiments were performed using protein samples dissolved in 20 mm phosphate buffer, pH 3.5, by measuring the change of molar ellipticity at 201 nm. Data were collected as a function of temperature with a scan rate of 2 °C/min and allowing 3 min to reach equilibrium over the range of 4–95 °C. Equilibrium unfolding induced by guanidine HCl was monitored by CD as described previously (11.Hsu C.H. Liao Y.D. Pan Y.R. Chen L.W. Wu S.H. Leu Y.J. Chen C. J. Mol. Biol. 2003; 326: 1189-1201Crossref PubMed Scopus (17) Google Scholar). The curves were fitted and analyzed using SigmaPlot version 8.02 (SPSS Inc.). NMR Spectroscopy−All NMR experiments were performed on Bruker AVANCE 600 and 800 spectrometers equipped with triple (1H, 13C, and 15N) resonance probes, including a shielded z-gradient. The RNase samples (0.6 mm in 0.35 ml) were prepared in 50 mm phosphate buffer in 90% H2O/10% D2Oor 99.9% D2O at pH 3.5, 310 K and kept in a Shigemi NMR tube. All heteronuclear NMR experiments were carried out as described previously (16.Clore G.M. Gronenborn A.M. Methods Enzymol. 1994; 239: 349-363Crossref PubMed Scopus (253) Google Scholar). Sequence-specific assignment of the backbone atoms of hRNase7 was achieved by the independent connectivity analysis of CBCA(CO)NH, HNCACB, HNCO, HN(CA)CO, and C(CO)NH. The 1H resonances were assigned using TOCSY-HSQC, HAHB(CO)NH, and HCCH-TOCSY. Combined information from two-dimensional 1H-15N HSQC and three-dimensional NOESY-HSQC experiments yielded for resonances of the Asn and Gln resonances were assigned using two-dimensional HSQC, NOESY, and was used in the and to the All spectra were using the S. G. J. A. J. 1995; PubMed Scopus Google Scholar) and analyzed using version Methods Mol. Biol. 2004; Google Scholar). NMR and of information was by the G. A. J. PubMed Scopus Google Scholar). The information was obtained from D2O monitored by two-dimensional 1H-15N HSQC The nuclear Overhauser effect from 1H-15N NOESY-HSQC and spectra were assigned by the of T. P. J. Mol. Biol. 2002; PubMed Scopus Google Scholar) and NMR were calculated on by using the The 15 that the total no and no were The of the backbone of the were by the of the which the from the by using R. J.M. J. PubMed Scopus Google Scholar) and R. M. J. Mol. PubMed Scopus Google Scholar). Data Accession of recombinant hRNase7 at pH and 310 K have been in the The 15 with the of have been in the Protein Data Antimicrobial Activity of RNase hRNase7 was μm for in cfu with that of buffer only against bacteria cfu P. than (5 whereas the RNases RNaseA and and other RNases were bactericidal at The positively charged oligopeptide (R3S1)3, however, bactericidal activity as that of hRNase7 A and The antimicrobial of hRNase7 was also and the yeast P. pastoris X-33 μm for in cfu with that of buffer was the most among of the microbes and in the P. aeruginosa whereas the S. aureus and E. coli bacteria and yeast C. albicans 20 were and Binding and Permeability to Bacterial Membrane−The cfu of bacteria was in only a few minutes after RNase addition The bactericidal RNases and were bound to the P. aeruginosa 2 and In the non-bactericidal bovine RNaseA and RC-RNase to the bacteria 5 and The membrane of P. aeruginosa became permeable to the DNA-binding dye SYTOX® Green in only a few minutes after the addition of μm hRNase7 or to the bacteria (107 cfu in 100 the non-bactericidal bovine RNaseA and RC-RNase no effect the same which was in with the antimicrobial assay and The bactericidal activity of hRNase7 was only at 4 °C μm for in cfu with that of μm at 37 whereas that of a bactericidal oligopeptide from bovine was suggest that the bactericidal activity of hRNase7 is whereas that of is effect of temperature on the bactericidal activity of hRNase7 and The antimicrobial activities of hRNase7 and against P. aeruginosa at 37 °C and 4 °C were and of the for the antimicrobial activity of the of the recombinant protein was analyzed by CD spectra and NMR The important are and the conformation of hRNase7 was over the range of The value of hRNase7 was °C. The was with a value at and values that hRNase7 is a protein in to other RNaseA of two-dimensional 1H-15N HSQC of the hRNase7 was obtained in which For the determination of a of were collected for these were were were and 4 were (Table The 15 of the total were to the of NMR were with and and no for and no for of each with the yielded an of for the backbone atoms and for the atoms in residues of the using that of the residues in the most and of the residues in in the (Table on the of 15 for human RNase range range × for the of in the atoms atoms atoms atoms in most in in in Open table in a new tab of hRNase7. NMR of hRNase7. the of 15 NMR is The are in in and other secondary are in the of the NMR that the total is The hRNase7 is of three and surface of hRNase7 is as a with positively and charged residues in and of charged residues, Lys in in in and in of 15 NMR of hRNase7 are as a The was on the of the backbone atoms in the and of only one is The charged residues that were for antimicrobial activity studies are with The of hRNase7 an which was of three and for the RNaseA superfamily. The was by and of the were located in the and positively charged residues were distributed into three clusters The first was of and the and Lys38, and the and the other the charged residues were distributed over hRNase7 for Activity of cationic residues were in bactericidal RNases as demonstrated in we to the role of the RNA catalytic activity or cationic in the bactericidal activity. First of all, we found that the catalytic mutants of hRNase7 and the same of antimicrobial activity as the hRNase7 A and and The that RNA catalytic activity is for the antibacterial activity of activities of recombinant hRNase7 and catalytic and or mutants of positively charged Δ1KPKG4 the of N-terminal Met and residues from the recombinant of the N-terminal Met and residues from recombinant hRNase7 the bactericidal activity the RNA catalytic activities and and analysis showed that the exerted bactericidal activity than the K1A and and the of the K111Q/K112Q residues in the same cationic cluster the bactericidal activity to a than N-terminal In in other positively charged clusters and the catalytic the bactericidal activity and and that the flexible as well as residues in the first cationic cluster are critical for the bactericidal activity. of and Mutated The of was to that of as was no in the of two-dimensional and three-dimensional and HSQC NMR spectra residues and although antimicrobial activity than hRNase7 The of bactericidal RNases and hRNase7 are to those of RNaseA with three and these only the bactericidal RNases positively charged residues on the the non-bactericidal RNases (Table 1). The of the cationic residues Lys and is also found in most antimicrobial possess that are of clustered cationic and residues on each of the although they have and different secondary PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus Google Scholar). The cationic residues their with the charged on the and the residues their into membrane 2003; 24: PubMed Scopus Google Scholar). In this study, we have found that three clusters of cationic residues are located on the surface of hRNase7. The first cluster of residues, the and clusters and Only cationic residues in the first cluster, K1,K3,K111,K112, are critical for the bactericidal whereas the other K32,K35, and K96,R97,K100, are and The of that the of the and Lys112 residues in the first cluster are flexible than those of cationic residues in other In we also found that no in the first cluster of hRNase7 near the residues by NMR studies and no at each N-terminal region of bactericidal RNases by the of acid among the RNaseA members is that of cationic residues an is critical for the bactericidal activity of members in the RNase have antimicrobial the for antimicrobial activity were different in human and RNase7 (6.Zhang J. Dyer K.D. Rosenberg H.F. Nucleic Acids Res. 2003; 31: 602-607Crossref PubMed Scopus (86) Google Scholar, 7.Harder J. Schroder J.M. J. Biol. Chem. 2002; 277: 46779-46784Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 8.Hooper L.V. Stappenbeck T.S. Hong C.V. Gordon J.I. Nat. Immunol. 2003; 4: 269-273Crossref PubMed Scopus (739) Google Scholar, 9.Holloway D.E. Hares M.C. Shapiro R. Subramanian V. Acharya K.R. Protein Expression Purif. 2001; 22: 307-317Crossref PubMed Scopus (39) Google Scholar, 10.Nitto T. Dyer K.D. Czapiga M. Rosenberg H.F. J. Biol. Chem. 2006; 281: 25622-25634Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The of cationic residues of and from that of hRNase7 (Lys18 and the cationic residues and and residues and of which are to be for membrane binding and on the secondary E. E. Rosenberg H.F. Biochemistry. 2003; PubMed Scopus Google whereas the cationic residues K1,K3,K111,K112, of hRNase7 on the flexible coil and at the N-terminal to chicken RNaseA2, the residues in and are critical for the bactericidal the of the T. Dyer K.D. Czapiga M. Rosenberg H.F. J. Biol. Chem. 2006; 281: 25622-25634Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In addition to the of the of cationic residues, we were also in the of for RNases on the antimicrobial activity. that the bactericidal activity of RNase was with its backbone for the values hRNase7 and bactericidal and are and respectively, whereas those bactericidal hRNase7 and non-bactericidal and bovine RNaseA are and that the of the RNase backbone is critical for the antimicrobial activity. The hRNase7 was in bactericidal activity at 4 whereas the a oligopeptide from bovine was C.H. Chen C. Huang Wu S.H. Nucleic Acids Res. PubMed Scopus Google Scholar). The bactericidal RNases hRNase7 and from were bound to whereas the non-bactericidal RNases were suggest that some on the bacteria for the binding of hRNase7 and the action of hRNase7 is J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. 1998; PubMed Scopus Google Scholar). The bacterial membrane became permeable to the DNA binding dye SYTOX® Green a few minutes after the addition of hRNase7 that the increase of membrane is an important step for the bactericidal activity of hRNase7. we suggest that the hRNase7 to the charged of the bacterial membrane through the flexible and cationic The hRNase7 change its into the bacterial membrane through the and the disruption of we also that several which are of on the bacterial surface for the of the of and The of be by hRNase7 binding through the cationic residues and The of and of bacterial membrane even at The antibacterial mechanisms of these positively charged by disruption of bacterial membrane is different from those for which are of DNA RNA or protein to the bactericidal activity of the to be a new for bacterial the of The of hRNase7 gene expression or the of a oligopeptide from the hRNase7 be in the for We for critical of the for the and Wang for the of various hRNase7