单环刺螠重组血红蛋白酶解组分hemocidins的初步抗菌活性分析
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摘要
目的:研究重组血红蛋白酶解片段抗菌活性,以发现活性强的抗微生物多肽。方法:本研究分别采用糜蛋白酶、胃蛋白酶、胰蛋白酶和木瓜蛋白酶4种酶水解单环刺螠重组血红蛋白(Uu Hb-F-I),然后通过超滤将酶解产物分成不同分子量段组分(> 10k,3～10k,<3k),并琼脂扩散法初步测定各组分抗菌活性。结果:血红蛋白源的各酶解组分对所用菌种有不同程度的抗菌作用,其中胃蛋白酶和胰蛋白酶水解组分对阳性菌的抑制作用强于阴性菌,而糜蛋白酶和木瓜蛋白酶水解组分(> 10k)对阴性菌的抑制作用强于阳性菌;此外,4种酶水解后的各组分对真菌均无抑制作用。另外,研究结果发现抗菌活性随着肽链的长度缩小而降低,抗菌活性强的多肽片段可能位于酶解组分(> 10k,3～10k)中。结论:血红蛋白酶解片段具有抗菌活性,且抗菌活性强的是相对较长的多肽,进一步水解可能导致活性丧失。本研究结果有助选择具有较好抗菌性的酶解多肽片段,为研发新型抗微生物多肽提供思路。
Objective: To detect the antimicrobial activities of the hydrolyzed fragments of recombinant hemoglobin from Urechisunicinctus( r Uu Hb-F-I) in order to find new antimicrobial peptides. Methods: The r Uu HbF-I which was expressed according to our previous work,was hydrolyzed by four kinds of enzymes including chymotrypsin,pepsin,trypsin and papain. And then the enzymolysis products were divided into different molecular fragments( > 10 k,3 ～ 10 k,< 3 k) through ultrafiltration. The antimicrobial activities of those peptide fragments were determined by the method of agar diffusion assay. Results: The hydrolyzed fragments by pepsin or trypsin had more potent activities against Gram-positive bacteria than against Gram-negative bacteria,while the fragments hydrolyzed by chymotrypsin or papain( > 10 k) had more potent activities against Gram-negative bacteria than against the positive strains,and all of the fragments had almost no effect on fungus. In addition,the fragments larger than 10 k and 3 ～ 10 k had more potent antimicrobial activity than those with molecular weights of < 3 k.Conclusion: The hydrolyzed fragments of hemoglobin have different antimicrobial activities. And the peptides with higher molecular weight have better antimicrobial activity than the lower ones,indicating that further hydrolysis maylead to loss of activity. The results may help to select a highly potent antibacterial peptide and provide inspiration for development of new antimicrobial peptides.
Objective: To detect the antimicrobial activities of the hydrolyzed fragments of recombinant hemoglobin from Urechisunicinctus( r Uu Hb-F-I) in order to find new antimicrobial peptides. Methods: The r Uu HbF-I which was expressed according to our previous work,was hydrolyzed by four kinds of enzymes including chymotrypsin,pepsin,trypsin and papain. And then the enzymolysis products were divided into different molecular fragments( > 10 k,3 ～ 10 k,< 3 k) through ultrafiltration. The antimicrobial activities of those peptide fragments were determined by the method of agar diffusion assay. Results: The hydrolyzed fragments by pepsin or trypsin had more potent activities against Gram-positive bacteria than against Gram-negative bacteria,while the fragments hydrolyzed by chymotrypsin or papain( > 10 k) had more potent activities against Gram-negative bacteria than against the positive strains,and all of the fragments had almost no effect on fungus. In addition,the fragments larger than 10 k and 3 ～ 10 k had more potent antimicrobial activity than those with molecular weights of < 3 k.Conclusion: The hydrolyzed fragments of hemoglobin have different antimicrobial activities. And the peptides with higher molecular weight have better antimicrobial activity than the lower ones,indicating that further hydrolysis maylead to loss of activity. The results may help to select a highly potent antibacterial peptide and provide inspiration for development of new antimicrobial peptides.
引文
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[2] IVANOV VT,KARELIN AA,PHILIPPOVA MM,et al. Hemoglobin as a source of endogenous bioactive peptides:the concept of tissue-specific peptide pool[J]. Biopolymers,1997,43(2):171-188.
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[11] WICHEREK L,MAK P,KLIMEK M,et al. The length of menstrual bleeding and the risk of urogenital infections in the context of the activity of hemoglobin-derived microbicidal peptides[J].Eur J Obstet Gynecol Reprod Biol,2007,131(1):97-100.
[12] SAHA D,KOLI S,PATGAONKAR M,et al. Expression of hemoglobin-αandβsubunits in human vaginal epithelial cells and their functional significance[J]. PloS One,2017,12(2):e0171084.
[13] CARVALHO LAC,REMUZGO C,PEREZ KR,et al. Hb40-61a:novel analogues help expanding the knowledge on chemistry,properties and candidacidal action of this bovineα-hemoglobin-derived peptide[J]. Biochim Biophys Acta,2015,1848(12):3140-3149.
[14] CARVALHO LAC,MACHINI MT. Hemocidins derived from hemoglobin:structures,properties and perspectives[J]. Química Nova,2012,36(7):1021-1029.
[15] CATIAU L,TRAISNEL J,DELVAL-DUBOIS V,et al. Minimal antimicrobial peptidic sequence from hemoglobin alpha-chain:KYR[J]. Peptides,2011,32(4):633-638.
[16]杨洁芳,刘会平,张宇,等.菌酶协同制备大豆肽的工艺优化及其抗氧化特性的研究[J].食品工业科技,2013,34(13):245-249.
[17] NIU R,CHEN X. Full-length cDNA,prokaryotic expression,and antimicrobial activity of UuHb-F-I from Urechisunicinctus[J]. Bio Med Res Inter,2016,2:1-8.
[18] ULLAL AJ,LITAKER RW,NOGA EJ. Antimicrobial peptides derived from hemoglobin are expressed in epithelium of channel catfish(Ictalurus punctatus,Rafinesque)[J]. Devel Compar Immunol,2008,32(11):1301-1312.
[19] BOMAN HG,AGERBERTH B,BOMAN A. Mechanisms of action on Escherichia coli of cecropin P1 and PR-39,two antibacterial peptides from pig intestine[J]. Infect Immun,1993,61(7):2978-2984.
[20] OLIWIA B,MARI RK,NATSLI W,et al. Secreted aspartic peptidases of Candida albicans liberate bactericidal hemocidins from human hemoglobin[J]. Peptides,2013,48(10):49-58.
[21] PELLEGRINI A,THOMAS U,BRAMAZ N,et al. Identification and isolation of a bactericidal domain in chicken egg white lysozyme[J]. J Appl Microbiol,1997,82(3):372-378.
[22] ZUCHT HD,RAIDA M,ADERMANN K,et al. Casocidin-I:a casein-αs2 derived peptide exhibits antibacterial activity[J].Febs Letters,1995,372(2-3):185-188.
[23] SCHALLY AV,HUANG WY,REDDING TW,et al. Isolation,structural elucidation and synthesis of a tetradecapeptide with in vitro ACTH-releasing activity corresponding to residues 33-46 of the alpha-chain of porcine hemoglobin[J]. Biochem Biophys Res Commun,1978,82(2):582-588.
[24] RIBEIRO NM,TONIOLO EF,CASTRO LM,et al. AGH is a new hemoglobin alpha-chain fragment with antinociceptive activity[J]. Peptides,2013,48(5):10-20.
[25] GOMES I,DALE CS,CASTEN K,et al. Hemoglobin-derived peptides as novel type of bioactive signaling molecules[J]. AAPS J,2010,12(4):658-669.
[2] IVANOV VT,KARELIN AA,PHILIPPOVA MM,et al. Hemoglobin as a source of endogenous bioactive peptides:the concept of tissue-specific peptide pool[J]. Biopolymers,1997,43(2):171-188.
[3] PETROV RV,MIKHAILOVA AA,FONINA LA. Bone marrow immunoregulatory peptides(myelopeptides):isolation,structure and functional activity[J]. Biopolymers,1997,43(2):139-146.
[4] MAK P,WOJCIK K,SILBERRING J,et al. Antimicrobial peptides derived from heme-containing proteins:hemocidins[J].Antonie Van Leeuwenhoek,2000,77(3):197-207.
[5]陈翔,牛荣丽.一类新型血红素蛋白源抗菌肽:hemocidins[J].中国新药杂志,2014,23(13):1536-1539.
[6] SHIN JR,LIM KJ,CHO JH,et al. Display of multimeric antimicrobial peptides on the escherichia coli cell surface and its application as whole-cell antibiotics[J]. PloS One,2013,8(3):e58997.
[7] HARTMANN R,MEISEL H. Food-derived peptides with biological activity:from research to food applications[J]. Curr Opin Biotech,2007,18(2):163-169.
[8] ZHANG DL,GUAN RZ,HUANG WS,et al. Isolation and characterization of a novel antibacterial peptide derived from hemoglobin alpha in the liver of Japanese eel,anguilla japonica[J]. Fish Shellfish Immunol,2012,35(3):625-631.
[9] SRIHONGTHONG S,PAKDEESUWAN A,DADUANG S,et al.Complete amino acid sequence of globin chains and biological activity of fragmented crocodile hemoglobin(crocodylussiamensis)[J]. Protein J,2012,31(6):466-476.
[10] BELMONTE R,CRUZ CE,PIRES JR,et al. Purification and characterization of Hb 98-114:a novel hemoglobin-derived antimicrobial peptide from the midgut of rhipicephalus(boophilus)microplus[J]. Peptides,2012,37(1):120-127.
[11] WICHEREK L,MAK P,KLIMEK M,et al. The length of menstrual bleeding and the risk of urogenital infections in the context of the activity of hemoglobin-derived microbicidal peptides[J].Eur J Obstet Gynecol Reprod Biol,2007,131(1):97-100.
[12] SAHA D,KOLI S,PATGAONKAR M,et al. Expression of hemoglobin-αandβsubunits in human vaginal epithelial cells and their functional significance[J]. PloS One,2017,12(2):e0171084.
[13] CARVALHO LAC,REMUZGO C,PEREZ KR,et al. Hb40-61a:novel analogues help expanding the knowledge on chemistry,properties and candidacidal action of this bovineα-hemoglobin-derived peptide[J]. Biochim Biophys Acta,2015,1848(12):3140-3149.
[14] CARVALHO LAC,MACHINI MT. Hemocidins derived from hemoglobin:structures,properties and perspectives[J]. Química Nova,2012,36(7):1021-1029.
[15] CATIAU L,TRAISNEL J,DELVAL-DUBOIS V,et al. Minimal antimicrobial peptidic sequence from hemoglobin alpha-chain:KYR[J]. Peptides,2011,32(4):633-638.
[16]杨洁芳,刘会平,张宇,等.菌酶协同制备大豆肽的工艺优化及其抗氧化特性的研究[J].食品工业科技,2013,34(13):245-249.
[17] NIU R,CHEN X. Full-length cDNA,prokaryotic expression,and antimicrobial activity of UuHb-F-I from Urechisunicinctus[J]. Bio Med Res Inter,2016,2:1-8.
[18] ULLAL AJ,LITAKER RW,NOGA EJ. Antimicrobial peptides derived from hemoglobin are expressed in epithelium of channel catfish(Ictalurus punctatus,Rafinesque)[J]. Devel Compar Immunol,2008,32(11):1301-1312.
[19] BOMAN HG,AGERBERTH B,BOMAN A. Mechanisms of action on Escherichia coli of cecropin P1 and PR-39,two antibacterial peptides from pig intestine[J]. Infect Immun,1993,61(7):2978-2984.
[20] OLIWIA B,MARI RK,NATSLI W,et al. Secreted aspartic peptidases of Candida albicans liberate bactericidal hemocidins from human hemoglobin[J]. Peptides,2013,48(10):49-58.
[21] PELLEGRINI A,THOMAS U,BRAMAZ N,et al. Identification and isolation of a bactericidal domain in chicken egg white lysozyme[J]. J Appl Microbiol,1997,82(3):372-378.
[22] ZUCHT HD,RAIDA M,ADERMANN K,et al. Casocidin-I:a casein-αs2 derived peptide exhibits antibacterial activity[J].Febs Letters,1995,372(2-3):185-188.
[23] SCHALLY AV,HUANG WY,REDDING TW,et al. Isolation,structural elucidation and synthesis of a tetradecapeptide with in vitro ACTH-releasing activity corresponding to residues 33-46 of the alpha-chain of porcine hemoglobin[J]. Biochem Biophys Res Commun,1978,82(2):582-588.
[24] RIBEIRO NM,TONIOLO EF,CASTRO LM,et al. AGH is a new hemoglobin alpha-chain fragment with antinociceptive activity[J]. Peptides,2013,48(5):10-20.
[25] GOMES I,DALE CS,CASTEN K,et al. Hemoglobin-derived peptides as novel type of bioactive signaling molecules[J]. AAPS J,2010,12(4):658-669.