Plasma proteome of buffaloes - kaivo.com.br · Leticia Gomes de Pontes 1, Nayara Rodrigues Vieira...

1600138 (1 of 6) Proteomics Clin. Appl. 11, 9–10, 2017, 1600138DOI 10.1002/prca.201600138

DATASET BRIEF

Plasma proteome of buffaloesLeticia Gomes de Pontes1, Nayara Rodrigues Vieira Cavassan1, Luciana Curtolo de Barros2,Rui Seabra Ferreira Junior1,2, Benedito Barraviera1,2 and Lucilene Delazari dos Santos1,2

1 Graduate Program in Tropical Diseases, Botucatu Medical School, Sao Paulo State University (UNESP), Botucatu,Sao Paulo, Brazil

2 Center for the Studies of Venoms and Venomous Animals (CEVAP), Sao Paulo State University (UNESP),Botucatu, Sao Paulo, Brazil

Received: October 4, 2016Revised: April 9, 2017

Accepted: April 25, 2017

The proteomic approach has aroused the interest of veterinary medicine researchers, especiallyregarding the production of biopharmaceuticals and diagnosis of diseases in farm animals.Water buffaloes have gained prominence in the world economy due to the quality of theirmilk, meat, and leather, in addition to being an important donor of blood components. Thiswork aimed to identify and characterize the proteins present in the blood plasma of Murrahbuffaloes (Bubalus bubalis) through 2D electrophoresis, in gel protein digestion followed bymass spectrometry technique and for albumin depletion, in solution protein digestion fol-lowed by shotgun analysis. Our results showed the identification of 112 protein spots and35 individual proteins, respectively. The abundant proteins were represented by albumin,fibrinogen-�, fibrinogen-�, fibrinogen-�, immunoglobulins in general, �-1-antiproteinase,�-1B-glycoprotein, �-2-HS-glycoprotein, �-macroglobulin, apolipoprotein A1, antithrombin-III, endopin 2B, fetuin-B, retinol-binding protein, serotransferrin, transthyretin and vitaminD-binding protein. Most of these proteins are related to the signaling pathways of the com-plement system and coagulation cascade. The results allowed a better understanding of theprotein composition of these blood components, thus promoting studies on animal health inthe search for molecular markers of zoonotic diseases in buffaloes.

Keywords:

2D-electrophoresis / Bubalus bubalis / Fibrin sealant / Mass spectrometry / Plasma

� Additional supporting information may be found in the online version of this article atthe publisher’s web-site

The search for candidate molecules using the toxins and bloodof large animals has reached its pinnacle in the last 20 years[1–5]. In addition, biotechnological research involving farmanimals has gained prominence in the pharmaceutical indus-try because these animals are more adequate experimentalmodels than rodents [6] and they can be used for the produc-tion of biopharmaceuticals [7,8]. Ruminants, especially cattle,buffaloes, goats, and sheep, are responsible for the global pro-duction of 30% of the meat and 100% of the milk available forhuman consumption [9, 10]. The meat and milk of Bubalusbubalis buffaloes present characteristics pleasant for the hu-man palate, such as a differential texture and flavor as wellas high amounts of fat, total proteins and mineral salts. Inaddition, water buffaloes have been used as donors of rawmaterial for the production of biopharmaceuticals [11–13].

Correspondence: Dr. Lucilene Delazari dos Santos, CEVAP-UNESP, Av. Jose Barbosa de Barros, 1780; Botucatu, SP, CEP18610-307, BrazilE-mail: [email protected]

A new heterologous fibrin sealant has been developedfrom the fibrinogen-rich cryoprecipitate of the blood ofBubalus bubalis buffalo, which is of the Murrah breed, anda thrombin like-enzyme from the venom of the snake Cro-talus durissus terrificus. This new sealant has coagulant, sealantand adhesive activities [11–14] as well as no risk of the trans-mission of human blood-borne infectious diseases. It hasbeen used in several clinical and biotechnological applica-tions, including as a biological glue in the removal of nasalskin tumors [15], in nerve implantation [16–18], as a biologi-cal scaffold for stem cells [19], and in the treatment of chronicvenous ulcers [13, 20].

The goal of this study was to identify and characterize theproteins present in the blood plasma from Bubalus bubalisof the Murrah breed since this information is not avail-able in the world literature. From this standardization using20 healthy animals, future proteomic approaches may re-veal biological markers of the main diseases that affect theseanimals (Supporting information, eMethods). All animalexperiments were approved by the Ethics Committee on An-imal Experimentation of Botucatu Medical School of Sao

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Proteomics Clin. Appl. 11, 9–10, 2017, 1600138 (2 of 6) 1600138

Clinical Relevance

The proteomic approach has aroused the interestof veterinary medicine researchers, especially re-garding the production of biopharmaceuticals anddiagnosis of diseases in farm animals. Water buf-faloes have gained prominence in the world econ-omy due to the quality of their milk, meat and leather,in addition to being an important donor of bloodcomponents. A new heterologous fibrin sealant hasbeen developed from the fibrinogen-rich cryopre-cipitate of the blood of Bubalus bubalis buffalo. Ithas been used in several clinical and biotechnologi-cal applications, including as biological glue in the

removal of nasal skin tumors, in nerve implanta-tion, as a biological scaffold for stem cells and inthe treatment of chronic venous ulcers. However,this unprecedented description was extremely valu-able because it provided a better understanding ofthe physiology of plasma of buffaloes, thus pro-moting studies in this area of animal health. Theidentification of molecular markers of zoonotic dis-eases in buffaloes through clinical proteomic re-search strategies will assist the development ofmore specific diagnostic platforms for this farmanimal.

Paulo State University (UNESP), under process numberCEEA 970-2012, and in accordance with the ethical principlesadopted by the Brazilian College of Animal Experimentation(COBEA).

The plasma pool used was prepared using blood col-lected from the jugular vein of the animals and it was sub-jected to 2D electrophoresis assays. For this purpose, 7-cmstrips, isoelectric point (pI) 4–7, and 10% polyacrylamidegels (w/v) were used under denaturing as well as reduc-ing conditions. The analyses of the proteomic maps ob-tained with the software Platinum v.7 showed that the plasmaprotein profile contain proteins with molecular masses be-tween 110 and 20 kDa and isoelectric points between 4.5and 7 (Fig. 1). One hundred twenty-nine protein spotswere found in the 2D protein profile, in which were ex-cised and hydrolyzed in the presence of the enzyme trypsin.

The tryptic digests were subjected to mass spectrometric anal-ysis carried out on a Nano-LC-ESI-MS/MS system. From129 proteins found in the blood plasma, 112 were identi-fied and validated statistically through the bioinformatics toolMascot (Matrix Science) (Supporting information, eTable 1).The most proteins identified include: albumin, fibrinogen-�, fibrinogen-�, fibrinogen-�, immunoglobulins in general,�-1-antiproteinase, �-1B-glycoprotein, �-2-HS-glycoprotein,�-macroglobulin, apolipoprotein A1, antithrombin-III, en-dopin 2B, fetuin-B, retinol-binding protein, serotransferrin,transthyretin, and vitamin D-binding protein.

Additionally, the plasma from Bubalus bubalis wassubmitted to albumin depletion assays by using an affinitychromatography column (Agilent Technologies). The ligandfraction was submitted to in solution protein digestionprotocol followed by mass spectrometry and 35 individual

Figure 1. Protein profile of the blood plasmaof Murrah buffaloes (Bubalus bubalis) obtainedby 2D electrophoresis. One hundred twenty-nineprotein spots were found by the software ImageMaster 2D Platinum v7.05 (GE Healthcare, Pitts-burgh, USA).


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Figure 2. Classification of proteins present in the blood plasmaof Bubalus bubalis buffaloes according to their participation ina (A) biological function, (B) molecular function, and (C) cellularcomponents. The classification of proteins was analyzed by theSTRING software according to the annotations deposited in theGene Ontology database.

proteins in 33 clusters were identified by MASCOT (MatrixScience) (Supporting information, eTable 2). This procedurewas realized to remove the excess of albumin in this sampleand permitted to visualize proteins that were not identifiedin the 2D-SDS-PAGE approach as such serotransferrin,complement C3, apolipoprotein A-II, complement factor B,Inter-alpha-trypsin inhibitor heavy chain H4, Kininogen-1,beta-2-glycoprotein 1, prothrombin, protein HP-20 homolog,complement factor H, histidine-rich glycoprotein, pigment

epithelium-derived fator, hemopexin, pregnancy-zoneprotein, complement C4, ceruloplasmin and clusterin.

At the end, a total of 35 different types of proteins wereidentified in the blood plasma from Bubalus bubalis by usingtwo different proteomic strategies. It was observed that manyprotein spots revealed to be the same protein. This fact wasalready expected due to genetic and epigenetic events [21]and post-translational modifications that contribute to proteinheterogeneity.



Figure 3. (A) Interaction network among proteins identified in the blood plasma of Murrah buffaloes (Bubalus bubalis). The strongerassociations are represented by thicker lines. (B) In red are the proteins that participated in the complement system and blood coagulationcascade, one of the signaling pathways of the immune system. The protein network was analyzed using STRING software, and theassociations between the twelve proteins of this signaling pathway were detected using the Kyoto Encyclopedia at Genes and Genomes(KEGG) tool. FGA, fibrinogen-�; FGB, fibrinogen-�; FGG, fibrinogen-�; A2M, �-2-macroglobulin; SERPINC1, antithrombin-III; SERPINA1CLU,antithrombin-III; F2 Prothrombin Activation peptide fragment; KNG1, kininogen-1; C3, complemente C3; CFB, complemente fator B; CFH,complemente fator H precursor; C4A, complement C4 precursor.

Once the most abundant proteins in the blood plasmawere identified, they were analyzed in regards to their bio-logical function, their molecular function and cell location.For this purpose, the annotations deposited in the Gene On-tology database were consulted, and the results are shownin Fig. 2. Most of the proteins are related to the signalingpathway of the complement system, blood coagulation cas-cade, ion and molecule transport, homeostasis, inflammatoryprocesses, neoplasms, organism defense, protease inhibitors,and antioxidant and anti-inflammatory effects. The protein–protein interaction network was also obtained to better un-derstand the relationship between the proteins present in theblood plasma of buffaloes (Fig. 3).

It was possible to observe a huge overlap between Bubalusbubalis and Bos taurus datasets with some studies in the lit-erature [22,23]. Fibrinogen, albumin, serpins, apolipoproteinA1, retinol binding protein, �-1-antitrypsin, fetuins and im-munoglobulins in general identified in the blood plasma ofBubalus bubalis had already been identified in previous stud-ies on the proteomic characterization of blood plasma andserum of equines and bovines [24–26]. However, there aretwo particular features of the protein profiles of the bloodproducts of these farm animals that are worth noting. First,the protein haptoglobin was not identified in the plasma ofBubalus bubalis, although it was present in the equine andbovine protein profiles previously cited. It is known that hap-toglobin is an important protein marker of acute-phase re-action in buffaloes, showing increased concentrations in theserum of these animals during inflammatory processes [27].

We suggest that haptoglobin was not detected due to it be-ing a minor protein in these blood products. In addition, thisfinding suggests that the animals studied were healthy.

The second particularity is the presence of much moreforms of fibrinogen (fibrinogen-� [18 spots], fibrinogen-�[13 spots], fibrinogen-� [6 spots]) in the blood plasma ofbuffaloes when compared to the two-dimensional proteinprofiles of farm and domestic animals plasma and serum[24–26]. This peculiarity of the heterogeneity of fibrinogenwas already expected [21], but the amount of these forms cor-roborated the findings of Thomazini-Santos et al. [14]. In thisstudy, the plasma of Bubalus bubalis showed a fibrinogen con-centration above that observed in humans, cattle, horses, andsheep, although the coagulation time of the plasma of buf-faloes was similar to the values obtained in human samples[14]. This evidence supported the creation of a new biodrugusing the cryoprecipitate of these animals to development ofheterologous fibrin sealant without the use of human biolog-ical insumes [11–13].

A protein–protein interaction network has been proposedfor a better understanding of the relationship among themost abundant proteins identified in the blood plasma ofbuffaloes. One hundred fifty interactions were revealed, withmost of them having a synergistic effect on the activationof the complement system and blood coagulation cascade(Fig. 3B). However, some proteins showed no type of asso-ciation, likely due to them being involved in inflammatoryprocesses [28] and immune defense [24]. This study ofprotein–protein interactions emphasized the main presence


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of fibrinogen molecules that, when associated with thethrombin-like enzyme of Crotalus durissus terrificus snakevenom, lead to the formation of a stable fibrin network, thusallowing its use as a hemostatic adhesive or sealant [11–13].

The serum and/or plasma of farm animals such ascattle, pigs, horses, chickens, and sheep has been thetarget of many proteomic studies due to the need to main-tain animal welfare and monitor the transmission of infec-tious disease to humans [29]. In this context, the clinical pro-teomic approach has indicated that proteins are differentlyexpressed in the blood products of these animals affected byimportant zoonotic diseases [30]. Proteins such as fibrino-gen, albumin, �-1B-glycoprotein, antithrombin-III, and �-1-antiprotease have already been revealed as candidate proteinsfor molecular markers of mastitis in cattle and sheep [28,31],whereas, the �-2-HS-glycoprotein and vitamin D-binding pro-tein were differently expressed in sheep, pigs, and cattle af-flicted by bronchopulmonary diseases [32]. The Supportinginformation, eTable 3 shows several proteins present in theblood products of cattle, sheep and horses shown to be poten-tial molecular indicators of several infectious diseases. There-fore, it is believed that some of these proteins can also act asmolecular markers for the main zoonotic diseases affectingbuffaloes.

Finally, this pioneering work revealed the protein consti-tution of the blood plasma of clinically healthy Bubalus bubalisbuffaloes. Despite the efforts towards genetic sequencing ofBubalus bubalis, the protein sequences of the entire genomeof these animals are still not available. This unprecedenteddescription for Bubalus bubalis is extremely valuable becauseit provided a better understanding of the physiology of thisblood component of buffaloes, thus promoting studies in thisarea of animal health. The identification of molecular mark-ers of zoonotic diseases through clinical proteomic researchstrategies will assist the development of more specific diag-nostic platforms for a quality control routine in the productionof biopharmaceuticals and of the animal sanity.

The 2D-SDS-PAGE and MS/MS data have been deposited inthe Word-2DPAGE repository (http://world-2dpage.expasy.org/repository/0087/viewer) with the dataset identifier 0087.

This study was supported, in part, by a grant from the Foun-dation for Research Support of the State of Sao Paulo, (FAPESP)Proc. No 2014/13299-7 (LDS) and Proc. No. 2012/08101-8 (RS-FJr); CNPq (National Counsel of Technological and Scientific De-velopment) Proc. No. 458919/2014-4 (LDS) and 563582/2010-3 (BB); and CAPES (Coordination for the Improvement ofHigher Education Personnel) AUX-PE Toxinology Proc. No.23038.006285/2011-21 and PNPD No. 23038.008557/2010(BB). RSFJr is CNPq fellow researcher (310395/2014-3).Special thanks are also extended to the Center for the Study ofVenoms and Venomous Animals (CEVAP) of Sao Paulo StateUniversity (UNESP) for enabling the publication of this paper.Appreciation is also extended to Prof. Dr. Mario Sergio Palmaand Msc. Anally Ribeiro da Silva Menegasso for enabling the lab-oratorial infrastructure for the beginning of the development of this

work. We acknowledge the Mass Spectrometry Laboratory at theBrazilian Biosciences National Laboratory, CNPEM, Campinas,SP, Brazil for its support with the mass spectrometric analysis.

The authors have declared no conflict of interest.

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