Ukr.Biochem.J. 2016; Volume 88, Special Issue, pp. 79-86


Mapping of residues of fibrinogen cleaved by protease II of Bacillus thuringiensis var. israelensis IMV B-7465

E. M. Stohniy1, V. O. Chernyshenko1*, N. A. Nidialkova2, A. V. Rebriev1,
L. D. Varbanets2, V. E. Hadzhynova1, T. M. Chernyshenko1,
I. M. Kolesnikova1, E. V. Lugovskoy1

1Palladin Institute of Biochemistry, National Academy
of Sciences of Ukraine, Kyiv;
2Zabolotny Institute of Microbiology and Virology, National Academy
of Sciences of Ukraine, Kyiv;

The limited proteolysis of macromolecules allows obtaining the fragments that preserve the structure and functional properties of the whole molecule and could be used in the study of proteins structure and function. Proteases targeted to fibrinogen and fibrin are of interest as the tool for obtaining of functionally active fragments of fibrin(ogen) and for the direct defibrination in vivo. That is why the aim of the present work was to study the proteolytic action of Protease II (PII) purified from Bacillus thuringiensis var. israelensis IMV B-7465 on fibrinogen.
Hydrolysis products of fibrinogen by PII were analysed by SDS-PAGE under reducing conditions with further immunoprobing using the mouse monoclonal 1-5A (anti-Aα509-610) and ІІ-5С (anti-Aα20-78) antibodies. It was shown that PII cleaved preferentially the Aα-chain of fibrinogen splitting off the peptide with apparent molecular weight of 10 kDa that corresponded the C-terminal part of Aα-chain of fibrinogen molecule.
MALDI-TOF analysis of hydrolysis of fibrinogen was performed using a Voyager-DE. Results analyzed by Data Explorer allowed to detect the main peak occurring at mass/charge (M/Z) ratio of 11 441 Da. According to “Peptide Mass Calculator” this peptide corresponded to fragment Аα505-610 of fibrinogen molecule. The result showed that PII cleaves the peptide bond AαAsp-Thr-Ala504-Ser505.
Thus, PII can be used for the obtaining of unique fragments of fibrinogen molecule. As far as αC-domain contains numerous sites of fibrin intermolecular interactions we can consider PII as a prospective agent for their study and for the defibrination.

Keywords: , , , ,


  1. Hoge R., Pelzer A., Rosenau F., Wilhelm S. Weapons of a pathogen: Proteases and their role in virulence of Pseudomonas aeruginosa.  In Current Research, Technology and Education Topics in Applied  Microbiology and Microbial Biotechnology, Ed by A. Medes-Vilas, 2010.  Pharmatex. P. 383-395.
  2. Khan NA, Jarroll EL, Panjwani N, Cao Z, Paget TA. Proteases as markers for differentiation of pathogenic and nonpathogenic species of Acanthamoeba. J Clin Microbiol. 2000 Aug;38(8):2858-61. PubMedPubMedCentral
  3. Matseliukh OV, Nidialkova NA, Varbanets LD, Andreeva NO, Shepelevych VV, Zelena PP, Yumyna JM. Ability of microorganisms from different ecological niches to hydrolyze the insoluble proteins. Mikrobiol Zh. 2015 May-Jun;77(3):16-22. Ukrainian. PubMed
  4. Peng Y, Yang X, Zhang Y. Microbial fibrinolytic enzymes: an overview of source, production, properties, and thrombolytic activity in vivo. Appl Microbiol Biotechnol. 2005 Nov;69(2):126-32. PubMed, CrossRef
  5. Lakshmi Bhargavi P, Prakasham RS. A fibrinolytic, alkaline and thermostable metalloprotease from the newly isolated Serratia sp RSPB11. Int J Biol Macromol. 2013 Oct;61:479-86. PubMed, CrossRef
  6. Fricke B, Parchmann O, Kruse K, Rücknagel P, Schierhorn A, Menge S. Characterization and purification of an outer membrane metalloproteinase from Pseudomonas aeruginosa with fibrinogenolytic activity. Biochim Biophys Acta. 1999 Aug 30;1454(3):236-50. PubMed
  7. Majumdar S, Sarmah B, Gogoi D, Banerjee S, Ghosh SS, Banerjee S, Chattopadhyay P, Mukherjee AK. Characterization, mechanism of anticoagulant action, and assessment of therapeutic potential of a fibrinolytic serine protease (Brevithrombolase) purified from Brevibacillus brevis strain FF02B. Biochimie. 2014 Aug;103:50-60. PubMed, CrossRef
  8. Mukherjee AK, Rai SK, Thakur R, Chattopadhyay P, Kar SK. Bafibrinase: A non-toxic, non-hemorrhagic, direct-acting fibrinolytic serine protease from Bacillus sp. strain AS-S20-I exhibits in vivo anticoagulant activity and thrombolytic potency. Biochimie. 2012 Jun;94(6):1300-8.  PubMed, CrossRef
  9. Majumdar S, Dutta S, Das T, Chattopadhyay P, Mukherjee AK. Antiplatelet and antithrombotic activity of a fibrin(ogen)olytic protease from Bacillus cereus strain FF01. Int J Biol Macromol. 2015 Aug;79:477-89.  PubMed, CrossRef
  10. Park JY, Park JE, Park JW, Yoon SM, Lee JS.  Purification and characterization of a novel alkaline serine protease secreted by Vibrio metschnikovii. Int J Mol Med. 2012 Feb;29(2):263-8.  PubMed, CrossRef
  11. Imamura T, Nitta H, Wada Y, Kobayashi H, Okamoto K. Impaired plasma clottability induction through fibrinogen degradation by ASP, a serine protease released from Aeromonas sobria. FEMS Microbiol Lett. 2008 Jul;284(1):35-42.  PubMed, PubMedCentral, CrossRef
  12. Reshma CV, Zuhara KF. Response surface methodology based optimization of a new isolate Bacillus pumilus ZR LS S2 with fibrinolytic activity.  Br Biotechnol J. 2015; 6(2): 51-61. CrossRef
  13. Ahn MJ, Ku HJ, Lee SH, Lee JH. Characterization of a Novel Fibrinolytic Enzyme, BsfA, from Bacillus subtilis ZA400 in Kimchi Reveals Its Pertinence to Thrombosis Treatment. J Microbiol Biotechnol. 2015 Dec 28;25(12):2090-9. PubMed, CrossRef
  14. Matselyukh ОV, Nidialkova NA, Varbanets LD. Purification and physicochemical properties of Bacillus thuringiensis ІМВ В-7324 peptidase with elastolytic and fibrinolytic activity. Ukr Biokhim Zhurn. 2012 Nov-Dec;84(6):25-36. Ukrainian.  PubMed
  15. Varetskaia TV. Preparation of a fibrin monomer and studies on some of its properties. Ukr Biokhim Zhurn. 1965;37(2):194-206. Ukrainian.  PubMed
  16. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680-5.  PubMedCrossRef
  17. Chapman J. R. Mass Spectrometry of Proteins and Peptides. Humana Press, 2000, 538. CrossRef
  18. Gershkovich AA, Kibirev VK. Chromogenic and fluorogenic peptide substrates of proteolytic enzymes. Bioorg Khim. 1988 Nov;14(11):1461-88. Russian. PubMed
  19. Nіdialkova NA, Varbanets LD. A bacterial strain Bacillus thuringiensis var. israelensis – producer of extracellular fibrinolytic peptidase. UA Patent. (In Ukrainian).
  20. Hadzhynova VE, Kolesnikova IM, Pozniak TA, Kostiuchenko OP.  Monoclonal antibody I-5A against AlphaC-region of fibrin(ogen) molecule and their practical use. In: “Hot Topics on Biochemistry and Biotechnology – 2015”. Kyiv, 2015. Sanchenko.  P. 63.
  21. Pozniak TA, Kolesnikova IN, Litvinova LM, Kostiuchenko OP, Urvant LP, Khadzhynova VE, Lugovskoi EV, Komisarenko SV.  Monoclonal antibodies specific to the E-region of human fibrin(ogen). Report Natl Acad Sci Ukraine. 2014;4:162-167. (In Ukrainian).
  22. Duncan EA, Brown MS, Goldstein JL, Sakai J. Cleavage site for sterol-regulated protease localized to a leu-Ser bond in the lumenal loop of sterol regulatory element-binding protein-2. J Biol Chem. 1997 May 9;272(19):12778-85. PubMedCrossRef
  23. Feder J, Schuck JM. Studies on the Bacillus subtilis neutral-protease- and Bacillus thermoproteolyticus thermolysin-catalyzed hydrolysis of dipeptide substrates. Biochemistry. 1970 Jul 7;9(14):2784-91. PubMed, CrossRef
  24. Hung SH, Hedstrom L. Converting trypsin to elastase: substitution of the S1 site and adjacent loops reconstitutes esterase specificity but not amidase activity. Protein Eng. 1998 Aug;11(8):669-73. PubMed, CrossRef
  25. Weisel JW, Medved L. The structure and function of the alpha C domains of fibrinogen. Ann N Y Acad Sci. 2001;936:312-27.  PubMed, CrossRef
  26. Tsurupa G, Hantgan RR, Burton RA, Pechik I, Tjandra N, Medved L. Structure, stability, and interaction of the fibrin(ogen) alphaC-domains. Biochemistry. 2009 Dec 29;48(51):12191-201. PubMedPubMedCentral, CrossRef
  27. Collet JP, Moen JL, Veklich YI, Gorkun OV, Lord ST, Montalescot G, Weisel JW. The alphaC domains of fibrinogen affect the structure of the fibrin clot, its physical properties, and its susceptibility to fibrinolysis. Blood. 2005 Dec 1;106(12):3824-30. PubMed, PubMedCentral, CrossRef
  28. Gorkun OV, Veklich YI, Medved LV, Henschen AH, Weisel JW. Role of the alpha C domains of fibrin in clot formation. Biochemistry. 1994 Jun 7;33(22):6986-97.  PubMed
  29. Hawiger J, Kloczewiak M, Bednarek MA, Timmons S. Platelet receptor recognition domains on the alpha chain of human fibrinogen: structure-function analysis. Biochemistry. 1989 Apr 4;28(7):2909-14.  PubMed, CrossRef
  30. Yakovlev S, Mikhailenko I, Tsurupa G, Belkin AM, Medved L. Polymerisation of fibrin αC-domains promotes endothelial cell migration and proliferation. Thromb Haemost. 2014 Dec;112(6):1244-51. PubMed, PubMedCentral, CrossRef
  31. Sato H, Weisel JW. Polymerization of fibrinogen-derived fragment X and subsequent rearrangement of fibers. Thromb Res. 1990 May 1;58(3):205-12. PubMed, CrossRef
  32. Gorkun OV, Henschen-Edman AH, Ping LF, Lord ST. Analysis of A alpha 251 fibrinogen: the alpha C domain has a role in polymerization, albeit more subtle than anticipated from the analogous proteolytic fragment X. Biochemistry. 1998 Nov 3;37(44):15434-41. PubMed, CrossRef
  33. Cottrell BA, Doolittle RF. The amino acid sequence of a 27-residue peptide released from the alpha-chain carboxy-terminus during the plasmic digestion of human fibrinogen. Biochem Biophys Res Commun. 1976 Aug 9;71(3):754-61. PubMed, CrossRef
  34. Mihalyi E. Kinetics and molecular mechanism of the proteolytic fragmentation of fibrinogen. Ann N Y Acad Sci. 1983 Jun 27;408:60-70.  PubMed, CrossRef
  35. Harty DW, Farahani RM, Simonian MR, Hunter L, Hunter N. Streptococcus gordonii FSS2 Challisin affects fibrin clot formation by digestion of the αC region and cleavage of the N -terminal region of the Bβ chains of fibrinogen. Thromb Haemost. 2012 Aug;108(2):236-46.  PubMed, CrossRef
  36. Lee SY, Kim JS, Kim JE, Sapkota K, Shen MH, Kim S, Chun HS, Yoo JC, Choi HS, Kim MK, Kim SJ. Purification and characterization of fibrinolytic enzyme from cultured mycelia of Armillaria mellea. Protein Expr Purif. 2005 Sep;43(1):10-7.  PubMedCrossRef
  37. Gomes MS, Naves de Souza DL, Guimarães DO, Lopes DS, Mamede CC, Gimenes SN, Achê DC, Rodrigues RS, Yoneyama KA, Borges MH, de Oliveira F, Rodrigues VM. Biochemical and functional characterization of Bothropoidin: the first haemorrhagic metalloproteinase from Bothrops pauloensis snake venom. J Biochem. 2015 Mar;157(3):137-49. PubMedCrossRef
  38. Chernyshenko VO. Limited proteolysis of fibrinogen by fibrinogenase from Echis multisquamatis venom. Protein J. 2015 Apr;34(2):130-4.  PubMed, CrossRef
  39. Hiller O, Lichte A, Oberpichler A, Kocourek A, Tschesche H. Matrix metalloproteinases collagenase-2, macrophage elastase, collagenase-3, and membrane type 1-matrix metalloproteinase impair clotting by degradation of fibrinogen and factor XII. J Biol Chem. 2000 Oct 20;275(42):33008-13.  PubMedCrossRef
  40. Kirschbaum NE, Budzynski AZ. A unique proteolytic fragment of human fibrinogen containing the A alpha COOH-terminal domain of the native molecule. J Biol Chem. 1990 Aug 15;265(23):13669-76.  PubMed

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