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Protein & Peptide Letters

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ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

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Research Article

Fusion Expression and Fibrinolytic Activity of rPA/SP-B

Author(s): Yi-Shan Tang, Xiao-Jun Zhang, Wan-Neng Wang*, Ting Wang, Wu-Long Cao, Qiu-Han Zhang and Fu Chen

Volume 28, Issue 9, 2021

Published on: 01 March, 2021

Page: [1033 - 1042] Pages: 10

DOI: 10.2174/0929866528666210301151302

Price: $65

Abstract

Background: Pulmonary surfactant dysfunction is an important pathological factor in acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF).

Objective: In this study, the characteristics of recombinant mature surfactant protein B (SP-B) and reteplase (rPA) fusion protein maintaining good pulmonary surface activity and rPA fibrinolytic activity in acute lung injury cell model were studied.

Methods: We studied the characteristics of SP-B fusion expression, cloned rPA gene and N-terminal rPA/C-terminal SP-B co-expression gene, and constructed them into eukaryotic expression vector pEZ-M03 to obtain recombinant plasmids pEZ-rPA and pEZ-rPA/SP-B. The recombinant plasmids was transfected into Chinese hamster ovary (CHO) K1 cells and the expression products were analyzed by Western Blot. Lipopolysaccharide (LPS) was used to induce CCL149 (an alveolar epithelial cell line) cell injury model. Fluorescence staining of rPA and rPA/SP-B was carried out with the enhanced green fluorescent protein (eGFP) that comes with pEZ-M03; the cell Raman spectroscopy technique was used to analyze the interaction between rPA/SP-B fusion protein and the phospholipid structure of cell membrane in CCL149 cells. The enzyme activity of rPA in the fusion protein was determined by fibrin-agarose plate method.

Results: The rPA/SP-B fusion protein was successfully expressed. In the CCL149 cell model of acute lung injury (ALI), the green fluorescence of rPA/SP-B is mainly distributed on the CCL149 cell membrane. The rPA/SP-B fusion protein can reduce the disorder of phospholipid molecules and reduce cell membrane damage. The enzyme activity of rPA/SP-B fusion protein was 3.42, and the fusion protein still had good enzyme activity.

Conclusion: The recombinant eukaryotic plasmid pEZ-rPA/SP-B is constructed and can be expressed in the eukaryotic system. Studies have shown that rPA/SP-B fusion protein maintains good SP-B lung surface activity and rPA enzyme activity in acute lung injury cell model.

Keywords: Reteplase, pulmonary surfactant, fusion protein, acute lung injury, acute respiratory distress syndrome (ARDS), pulmonary fibrosis (PF).

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[1]
Mathai, S.K.; Schwartz, D.A. Translational research in pulmonary fibrosis. Transl. Res., 2019, 209, 1-13.[http://dx.doi.org/10.1016/j.trsl.2019.02.001] [PMID: 30768925]
[2]
Schuliga, M.; Grainge, C.; Westall, G.; Knight, D. The fibrogenic actions of the coagulant and plasminogen activation systems in pulmonary fibrosis. Int. J. Biochem. Cell Biol., 2018, 97, 108-117.[http://dx.doi.org/10.1016/j.biocel.2018.02.016] [PMID: 29474926]
[3]
Han, S.; Mallampalli, R.K. The role of surfactant in lung disease and host defense against pulmonary infections. Ann. Am. Thorac. Soc., 2015, 12(5), 765-774.[http://dx.doi.org/10.1513/AnnalsATS.201411-507FR] [PMID: 25742123]
[4]
Liekkinen, J.; Enkavi, G.; Javanainen, M.; Olmeda, B.; Pérez-Gil, J.; Vattulainen, I. Pulmonary surfactant lipid reorganization induced by the adsorption of the oligomeric surfactant protein B complex. J. Mol. Biol., 2020, 432(10), 3251-3268.[http://dx.doi.org/10.1016/j.jmb.2020.02.028] [PMID: 32135191]
[5]
Williams, I.; Squires, T.M. Evolution and mechanics of mixed phospholipid fibrinogen monolayers. J. R. Soc. Interface, 2018, 15(141), 15.[http://dx.doi.org/10.1098/rsif.2017.0895] [PMID: 29618528]
[6]
Beck-Broichsitter, M. Biophysical activity of impaired lung surfactant upon exposure to polymer nanoparticles. Langmuir, 2016, 32(40), 10422-10429.[http://dx.doi.org/10.1021/acs.langmuir.6b02893] [PMID: 27641633]
[7]
Seeger, W.; Elssner, A.; Günther, A.; Krämer, H.J.; Kalinowski, H.O. Lung surfactant phospholipids associate with polymerizing fibrin: loss of surface activity. Am. J. Respir. Cell Mol. Biol., 1993, 9(2), 213-220.[http://dx.doi.org/10.1165/ajrcmb/9.2.213] [PMID: 8338688]
[8]
Günther, A.; Markart, P.; Kalinowski, M.; Ruppert, C.; Grimminger, F.; Seeger, W. Cleavage of surfactant-incorporating fibrin by different fibrinolytic agents. Kinetics of lysis and rescue of surface activity. Am. J. Respir. Cell Mol. Biol., 1999, 21(6), 738-745.[http://dx.doi.org/10.1165/ajrcmb.21.6.3514] [PMID: 10572071]
[9]
Schermuly, R.T.; Günther, A.; Ermert, M.; Ermert, L.; Ghofrani, H.A.; Weissmann, N.; Grimminger, F.; Seeger, W.; Walmrath, D. Conebulization of surfactant and urokinase restores gas exchange in perfused lungs with alveolar fibrin formation. Am. J. Physiol. Lung Cell. Mol. Physiol., 2001, 280(4), L792-L800.[http://dx.doi.org/10.1152/ajplung.2001.280.4.L792] [PMID: 11238021]
[10]
Mohammadi, E.; Seyedhosseini-Ghaheh, H.; Mahnam, K.; Jahanian-Najafabadi, A.; Mir Mohammad Sadeghi, H. Reteplase: structure, function, and production. Adv. Biomed. Res., 2019, 8, 19.[http://dx.doi.org/10.4103/abr.abr_169_18] [PMID: 31016177]
[11]
Walther, F.J.; Gordon, L.M.; Waring, A.J. Design of surfactant protein B peptide mimics based on the saposin fold for synthetic lung surfactants. Biomed. Hub, 2016, 1(3), 1.[http://dx.doi.org/10.1159/000451076] [PMID: 28503550]
[12]
Lampland, A.L.; Wolfson, M.R.; Mazela, J.; Henderson, C.; Gregory, T.J.; Meyers, P.; Plumm, B.; Worwa, C.; Mammel, M.C. Aerosolized KL4 surfactant improves short-term survival and gas exchange in spontaneously breathing newborn pigs with hydrochloric acid-induced acute lung injury. Pediatr. Pulmonol., 2014, 49(5), 482-489.[http://dx.doi.org/10.1002/ppul.22844] [PMID: 24039229]
[13]
Notter, R.H.; Wang, Z.; Walther, F.J. Activity and biophysical inhibition resistance of a novel synthetic lung surfactant containing Super-Mini-B DATK peptide. PeerJ, 2016, 4, e1528.[http://dx.doi.org/10.7717/peerj.1528] [PMID: 26793419]
[14]
Akinbi, H.T.; Breslin, J.S.; Ikegami, M.; Iwamoto, H.S.; Clark, J.C.; Whitsett, J.A.; Jobe, A.H.; Weaver, T.E. Rescue of SP-B knockout mice with a truncated SP-B proprotein. Function of the C-terminal propeptide. J. Biol. Chem., 1997, 272(15), 9640-9647.[http://dx.doi.org/10.1074/jbc.272.15.9640] [PMID: 9092492]
[15]
Ruppert, C.; Mahavadi, P.; Wygrecka, M.; Weaver, T.E.; Magdolen, V.; Idell, S.; Preissner, K.T.; Seeger, W.; Günther, A.; Markart, P. Recombinant production of a hybrid plasminogen activator composed of surfactant protein B and low-molecular-weight urokinase. Thromb. Haemost., 2008, 100(6), 1185-1192.[http://dx.doi.org/10.1160/TH08-03-0142] [PMID: 19132247]
[16]
Ruppert, C.; Schmidt, R.; Grimminger, F.; Suzuki, Y.; Seeger, W.; Lehr, C.M.; Günther, A. Chemical coupling of a monoclonal antisurfactant protein-B antibody to human urokinase for targeting surfactant-incorporating alveolar fibrin. Bioconjug. Chem., 2002, 13(4), 804-811.[http://dx.doi.org/10.1021/bc0255081] [PMID: 12121136]
[17]
Lippi, G.; Mattiuzzi, C.; Favaloro, E.J. Novel and emerging therapies: thrombus-targeted fibrinolysis. Semin. Thromb. Hemost., 2013, 39(1), 48-58.[PMID: 23034825]
[18]
Wang, X.; Xu, Z.; Tian, Z.; Zhang, X.; Xu, D.; Li, Q.; Zhang, J.; Wang, T. The EF-1α promoter maintains high-level transgene expression from episomal vectors in transfected CHO-K1 cells. J. Cell. Mol. Med., 2017, 21(11), 3044-3054.[http://dx.doi.org/10.1111/jcmm.13216] [PMID: 28557288]
[19]
Azarkan, M.; González, M.M.; Esposito, R.C.; Errasti, M.E. Stem bromelain proteolytic machinery: study of the effects of its components on fibrin (ogen) and blood coagulation. Protein Pept. Lett., 2020, 27(11), 1159-1170.
[20]
Gao, X.H.; Zhang, S.D.; Wang, L.T.; Yu, L.; Zhao, X.L.; Ni, H.Y.; Wang, Y.Q.; Wang, J.D.; Shan, C.H.; Fu, Y.J. Morus alba L. Anti-inflammatory effects of neochlorogenic acid extract from mulberry leaf (Morus alba L.) against LPS-stimulated inflammatory response through mediating the AMPK/Nrf2 signaling pathway in A549 cells. Molecules, 2020, 25(6), 1385.[http://dx.doi.org/10.3390/molecules25061385] [PMID: 32197466]
[21]
Ryan, M.A.; Qi, X.; Serrano, A.G.; Ikegami, M.; Perez-Gil, J.; Johansson, J.; Weaver, T.E. Mapping and analysis of the lytic and fusogenic domains of surfactant protein B. Biochemistry, 2005, 44(3), 861-872.[http://dx.doi.org/10.1021/bi0485575] [PMID: 15654742]
[22]
Olmeda, B.; García-Álvarez, B.; Gómez, M.J.; Martínez-Calle, M.; Cruz, A.; Pérez-Gil, J. A model for the structure and mechanism of action of pulmonary surfactant protein B. FASEB J., 2015, 29(10), 4236-4247.[http://dx.doi.org/10.1096/fj.15-273458] [PMID: 26089319]
[23]
Zhang, X.J.; Wang, W.N.; Zhou, J.H.; Liu, Q.; Gu, S.L.; Zhou, D.J.; Qiu, J.; Dai, W.; Yuan, D.F.; Liu, D.W. Prokaryotic expression and purification of mature rat pulmonary surfactant protein B. Progress In Veterinary Medicine, 2016, 37(4), 18-23.
[24]
Calkovska, A.; Mokra, D.; Calkovsky, V. Lung surfactant alterations in pulmonary thromboembolism. Eur. J. Med. Res., 2009, 38-41.[http://dx.doi.org/10.1186/2047-783X-14-S4-38]
[25]
Eckmann, D.M.; Diamond, S.L. Surfactants attenuate gas embolism-induced thrombin production. Anesthesiology, 2004, 100(1), 77-84.[http://dx.doi.org/10.1097/00000542-200401000-00015] [PMID: 14695727]
[26]
Wang, W.N.; Zhou, J.H.; Wang, P.; Zhang, X.J. The localization of SP-B and influences of lipopolysaccharide on it. Eur. Rev. Med. Pharmacol. Sci., 2016, 20(11), 2338-2345.[PMID: 27338059]
[27]
Günther, A.; Mosavi, P.; Heinemann, S.; Ruppert, C.; Muth, H.; Markart, P.; Grimminger, F.; Walmrath, D.; Temmesfeld-Wollbrück, B.; Seeger, W. Alveolar fibrin formation caused by enhanced procoagulant and depressed fibrinolytic capacities in severe pneumonia. Comparison with the acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med., 2000, 161(2 Pt 1), 454-462.[http://dx.doi.org/10.1164/ajrccm.161.2.9712038] [PMID: 10673185]
[28]
Nogee, L.M.; de Mello, D.E.; Dehner, L.P.; Colten, H.R. Brief report: deficiency of pulmonary surfactant protein B in congenital alveolar proteinosis. N. Engl. J. Med., 1993, 328(6), 406-410.[http://dx.doi.org/10.1056/NEJM199302113280606] [PMID: 8421459]

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