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Research Article

Identification of mare colostrum proteins

Weronika Medeńska¹, Alicja Dratwa-Chałupnik ¹ , Małgorzata Ożgo ¹, Aleksandra Cichy¹, Ryszard Pikuła ², Janusz Bobik³

¹Department of Physiology, Cytobiology and Proteomics, West Pomeranian University of Technology in Szczecin, Klemensa Janickiego 29, 71-270 Szczecin, Poland

²Department of Monogastric Animal Sciences, Laboratory of Horse Breeding and Animalotherapy, West Pomeranian University of Technology in Szczecin, Klemensa Janickiego 33, 71-270 Szczecin, Poland

³Nowielice Stud Farm Sp. z o.o., Nowielice, 72-320 Trzebiatów, Poland

1. Acta Sci. Pol. Zootechnica, 19(4), 2020, 25‒32
2. DOI: 10.21005/asp.2020.19.4.03

Abstract. Colostrum is an essential feed of foals. It is a source of nutrients and functional proteins significant for foals’ growth and development. In the presented research using two-dimensional electrophoresis coupled via spectrometry mass MALDI-TOF in the mares’ colostrum (whey proteins fraction) were identified 24 proteins representing 15 different gene products. The identified proteins were involved in supporting foals’ immature immune systems and in the transport of various compounds. Further research of mares’ colostrum will allow determining more gene products. An in-depth analysis of mares’ milk will provide information about biochemical processes occurring in the mammary gland of the mare during the lactation period.

Keywords: proteomics, colostrum, whey, mare, foals

INTRODUCTION

Colostrum is a body fluid with high biological value, rich in nutrients and regulatory components, which underlies proper growth and development of newborns. The most important compounds of milk are proteins that are not the only the source of crucial amino acids, but are also, as functional factors, involved in various metabolic pathways and proteins which helps newborns to adapt to the new extra-uterus life environment.

Proteomics allows to study and determining the protein profile of body fluids. Highly specialized proteomics tools enable protein identification which we cannot detect using traditional biochemical methods.

Due to the significance of ruminants milk in the human diet, there is a lot of research on the proteome of this body fluid [Le et al. 2011Le, A., Barton, L.D., Sanders, J.T., Zhang, Q. (2011). Exploration of bovine milk proteome in colostral and mature whey using an ion-exchange approach. J. Proteome Res., 10, 692–704. https://doi.org/10.1021/pr100884z, Zhang et al. 2015aZhang, L., Boeren, S., Hageman, A.J., Hooijdonk, T., Vervoort, J., Hettinga, K. (2015a). Bovine milk proteome in the first 9 days: protein interactions in maturation of the immune and digestive system of the newborn. PLoS ONE, 10, e0116710. https://doi.org/10.1371/journal.pone.0116710, 2015b, Tacoma et al. 2016Tacoma, R., Fields, J., Ebenstein, D., Lam, Y.W., Greenwood, S.L. (2016). Characterization of the bovine milk proteome in early-lactation Holstein and Jersey breeds of dairy cows. J. Proteom., 130, 200–210. https://doi.org/10.1016/j.jprot.2015.09.024, Delosière et al. 2019Delosière, M., Pires, J., Bernard, L., Cassar-Malek, I., Bonnet, M. (2019). Milk proteome from in silico data aggregation allows the identification of putative biomarkers of negative energy balance in dairy cows. Sci. Rep., 9, 9718. https://doi.org/10.1038/s41598-019-46142-7].

The proteomics approach have allowed identification of thousands of proteins of cow's milk [Delosière et al. 2019Delosière, M., Pires, J., Bernard, L., Cassar-Malek, I., Bonnet, M. (2019). Milk proteome from in silico data aggregation allows the identification of putative biomarkers of negative energy balance in dairy cows. Sci. Rep., 9, 9718. https://doi.org/10.1038/s41598-019-46142-7]. These included proteins associated with mammary gland development, milk components synthesis, and calf growth [Zhang et al. 2015aZhang, L., Boeren, S., Hageman, A.J., Hooijdonk, T., Vervoort, J., Hettinga, K. (2015a). Bovine milk proteome in the first 9 days: protein interactions in maturation of the immune and digestive system of the newborn. PLoS ONE, 10, e0116710. https://doi.org/10.1371/journal.pone.0116710]. Research shows differences in the protein profile between colostrum and milk [Zhang et al. 2015bZhang, L., Boeren, S., Hageman, J.A., Hooijdonk, T. van, Vervoort, J., Hettinga, K. (2015b). Perspective on calf and mammary gland development through changes in the bovine milk proteome over a complete lactation, J. Dairy Sci. 98, 5362–5373. https://doi.org/10.3168/jds.2015-9342]. Furthermore, proteomic studies demonstrated protein composition of milk fat globule membrane [Lu et al. 2016Lu, J., Liu, L., Pang, X., Zhang, S., Jia, Z., Ma, Ch., Zhao, L., Lv, J. (2016). Comparative proteomics of milk fat globule membrane in goat colostrum and mature milk. Food Chem., 209, 10–16. https://doi.org/10.1016/j.foodchem.2016.04.020, Wang et al. 2017Wang, X., Zhao, X., Huang, D., Pan, X., Qi ,Y. Yang, Y., Zhao, H., Cheng, G. (2017). Proteomic analysis and cross species comparison of casein fractions from the milk of dairy animals. Sci. Rep. 7, 43020. https://doi.org/10.1038/srep43020].

Compared to cow milk, mare milk has a higher content of whey protein fraction, which is albumin-type milk [Potočnik et al. 2011Potočnik, K., Gantner, V., Kuterovac, K., Cividini, A. (2011). Mare's milk: composition and protein fraction in comparison with different milk species. Mljekarstvo, 61, 107–113. Google Scholar]. It is widely known that whey proteins have a significant impact on infant development.

No research has been reported so far on colostrum whey low-content proteins in mares. Accordingly, this research was undertaken to study mare colostrum proteins, especially those involved in the adaptation of newborn foals to new environmental conditions. To archive this aim, we used two-dimensional electrophoresis with spectrometry mass MALDI-TOF.

MATERIAL AND METHODS

Research material and sample preparation

The experiment was carried out on colostrum collected from six mares, Polish noble half-breed in 12 hours after parturition. The animals came from horse stables in Nowielice and were kept in a stable-pasture system. Colostrum samples were centrifuged at 4℃ (4500 g, 30 minutes), and the lipid-rich cream layer was removed to receive skim milk (fat-free precipitant). The next step was to precipitate the casein with 30% acetic acid to adjust the pH to 4.6 and centrifuged at 4℃ (3380 g, 15 minutes). The obtained supernatant was collected and placed in new Eppendorf tubes. To each tube added acetone (–20℃) to extract proteins. The precipitant was diluted in lysis buffer containing: 5 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, 0.2% ampholytes pH 3–10, and 2 mM TBP. Protein concentration was measured using the Protein-Assay (Bio-Rad).

Two-dimensional electrophoresis and identification of the proteins

Colostrum samples containing 800 μg of proteins were applied on 24 cm, pH 4–7 linear IPG strips, and rehydrated. Rehydration was performed in two steps: passive rehydration (6 h, 0 V) and active rehydration (12 h, 50 V). First-dimensional isoelectric focusing performed using the following procedure: 50 V for 100 Vh, 250 V for 250 Vh, 500 V for 500 Vh, 1000 V for 1000 Vh, 2h in linearly increasing voltage from 1000 V to 5000 V, and subsequently 5000 V for 90000 Vh. After the focusing process, IPG strips were equilibrated according to Lepczyński et al. [2018]Lepczyński, A., Ożgo, M., Dratwa-Chałupnik, A., Robak, P., Pyć, A., Zaborski, D., Herosimczyk, A. (2018). An update on medium- and low-abundant blood plasma proteome of horse. Animal, 12, 76–87. https://doi.org/10.1017/S1751731117001409. The second step of the 2d electrophoresis was to separate proteins according to their molecular weight. Equilibrated IPG strips were placed on the top of 12% polyacrylamide gels. The migration of proteins was run at 40 V for 3.5 hours and 90 V for 14 hours. After migration, the mass spectrometry identification was performed according to Dratwa-Chałupnik et al. [2016]Dratwa-Chałupnik, A., Ożgo, M., Lepczyński, A., Herosimczyk A., Michałek, K. (2016). Excessive amount of lactose in the diet of two-week-old calves induces urinary protein chan ges. Arch. Anim. Breed. 59, 417–422. https://doi.org/10.5194/aab-59-417-2016 procedure.

RESULTS

The main aim of the study was the identification of mare’s colostrum proteins. To achieve this goal, we applied two-dimensional electrophoresis coupled via mass spectrometry MALDI-TOF. Among 250 spots, we identified 24 proteins, representing 15 different gene products (Table 1). Furthermore, 20 proteins were characteristic for equines (Equus caballus, Equus przewalskii, and Equus sinus). Proteins were separated in the 4–7 range, and molecular mass between 250–10 kDa (Fig. 1). For all identified proteins were determined theoretical and experimental molecular weight (Table 1). According to the Uniprot database, identified proteins were assigned their cell localization and function, as shown in Table 1. Most of the proteins were located in the extracellular region (secreted).

DISCUSSION

Proteomics tools are successfully used in the analysis of farm animals colostrum and milk proteome: cow [Zhang et al. 2015aZhang, L., Boeren, S., Hageman, A.J., Hooijdonk, T., Vervoort, J., Hettinga, K. (2015a). Bovine milk proteome in the first 9 days: protein interactions in maturation of the immune and digestive system of the newborn. PLoS ONE, 10, e0116710. https://doi.org/10.1371/journal.pone.0116710, Zhang et al. 2015bZhang, L., Boeren, S., Hageman, J.A., Hooijdonk, T. van, Vervoort, J., Hettinga, K. (2015b). Perspective on calf and mammary gland development through changes in the bovine milk proteome over a complete lactation, J. Dairy Sci. 98, 5362–5373. https://doi.org/10.3168/jds.2015-9342, Tacoma et al. 2016Tacoma, R., Fields, J., Ebenstein, D., Lam, Y.W., Greenwood, S.L. (2016). Characterization of the bovine milk proteome in early-lactation Holstein and Jersey breeds of dairy cows. J. Proteom., 130, 200–210. https://doi.org/10.1016/j.jprot.2015.09.024, Delosière et al. 2019Delosière, M., Pires, J., Bernard, L., Cassar-Malek, I., Bonnet, M. (2019). Milk proteome from in silico data aggregation allows the identification of putative biomarkers of negative energy balance in dairy cows. Sci. Rep., 9, 9718. https://doi.org/10.1038/s41598-019-46142-7], sheep [Cunsolo et al. 2017Cunsolo, V., Fasoli, E., Di Francesco, A., Saletti, R., Muccilli, V., Gallina, S., Righetti, P.G., Foti, S. (2017). Polyphemus, Odysseus and the ovine milk proteome. J. Proteom.,152, 58–74. https://doi.org/10.1016/j.jprot.2016.10.007], and goat [Cunsolo et al. 2015Cunsolo, V., Fasoli, E., Saletti, R., Muccilli, V., Gallina, S., Righetti, P.G., Foti, S. (2015). Zeus, Aesculapius, Amalthea and the proteome of goat milk. J. Proteom., 14, 69–82. https://doi.org/10.1016/j.jprot.2015.07.009, Cunsolo et al. 2017Cunsolo, V., Fasoli, E., Di Francesco, A., Saletti, R., Muccilli, V., Gallina, S., Righetti, P.G., Foti, S. (2017). Polyphemus, Odysseus and the ovine milk proteome. J. Proteom.,152, 58–74. https://doi.org/10.1016/j.jprot.2016.10.007]. So far, proteomic approaches have allowed knowing the ruminant’s colostrum and milk protein profile and capturing changes in protein profile during cow lactation [Zhang et al. 2015bZhang, L., Boeren, S., Hageman, J.A., Hooijdonk, T. van, Vervoort, J., Hettinga, K. (2015b). Perspective on calf and mammary gland development through changes in the bovine milk proteome over a complete lactation, J. Dairy Sci. 98, 5362–5373. https://doi.org/10.3168/jds.2015-9342].

Whey is a source of many regulating components, including bioactive peptides, antioxidants, and immunomodulating factors [Tai et al. 2016Tai, C.S., Chen, Y.Y., Chen, W.L. (2016). β-lactoglobulin influences human immunity and promotes cell proliferation. BioMed Res. Int. 7123587. https://doi.org/10.1155/2016/7123587]. Beta-lactoglobulin (LGB1) is the main whey protein of mare milk. Presented research showed the presence of beta-lactoglobulin in mare colostrum collected in 12 h after birth (Table 1). The percentage content of LGB1 in equine milk is about 36%, whereas in ruminants between 20% in cow milk and 77% of all whey proteins in ovine [Potočnik et al. 2011Potočnik, K., Gantner, V., Kuterovac, K., Cividini, A. (2011). Mare's milk: composition and protein fraction in comparison with different milk species. Mljekarstvo, 61, 107–113. Google Scholar]. LGB1 is a multifunctional protein. LGB1 can bind fatty acids and vitamins [Le Maux et al. 2014Le Maux, S., Bouhallab, S., Giblin, L., Brodkorb, A., Croguennec, T. (2014). Bovine β-lactoglobulin/ fatty acid complexes: binding, structural, and biological properties. Dairy Sci. Technol., 94, 409–426. https://doi.org/10.1007/s13594-014-0160-y]. It is a retinol carrier [Król et al. 2008Król, J., Litwińczuk, A., Zarajczyk, A., Liwtińczuk, Z. (2008). Alpha-lactalbumin and beta-lactoglobulin as bioactive compounds of the milk proteins. Med. Weter., 64, 1375–1378. Google Scholar]. Research by Tai et al. [2016]Tai, C.S., Chen, Y.Y., Chen, W.L. (2016). β-lactoglobulin influences human immunity and promotes cell proliferation. BioMed Res. Int. 7123587. https://doi.org/10.1155/2016/7123587 showed that LGB1 stimulates cell proliferation and growth. Moreover, β-lactoglobulin affects the secretion of proinflammatory cytokines and regulates the Th1/Th2 ratio [Tai et al. 2016Tai, C.S., Chen, Y.Y., Chen, W.L. (2016). β-lactoglobulin influences human immunity and promotes cell proliferation. BioMed Res. Int. 7123587. https://doi.org/10.1155/2016/7123587].

After birth, the foal immune system is not fully mature and requires delivery of immunomodulating proteins. Presented research showed the presence of immunoregulating proteins such as immunoglobulin mu heavy chain (IGHM) and immunoglobulin gamma 5 heavy chain (IGHG5), immunoglobulin gamma 4 heavy chain (IGHG4), nuclear antigen Sp-100-like, complement C3 alpha chain-like, the interleukin-24 precursor in mare colostrum (Table 1).

Intake of colostrum by the infant influences the correct development of innate immunity. Newborn foals are born with trace amounts of antibodies, because the epitheliochorial placenta prevents the passage of immunoglobulin from mother to fetus. In colostrum collected 12 hours after parturition, the following proteins have been identified: immunoglobulin mu heavy chain (IGHM); immunoglobulin gamma 5 heavy chain (IGHG5), and immunoglobulin gamma 4 heavy chain (IGHG4) (Table 1). The immunoglobulins delivered with colostrum protect the newborn foal against environmental pathogens. Results obtained by Markiewicz-Kęszycka et al. [2013]Markiewicz-Kęszycka, M., Wójtowski, J., Kuczyńska, B., Puppel, K., Czyżak-Runowska, G., Bognicka, E., Strzałkowska, N., Jaźwik, A., Krzyżewski, J. (2013). Chemical composition and whey protein fraction of late lactation mares' milk. Int. Dairy J., 31, 62–64. https://doi.org/10.1016/j.idairyj.2013.02.006 indicate that mare milk contains high amounts of immunoglobulins, 15.8% of the total whey proteins. Furthermore, in equine colostrum nuclear antigen Sp-100-like protein has been identified. This protein was located in the nucleus which is an antigen-stimulated by interferon.

The complement system is a primary line of protection against pathogens, a significant element of innate immunity [Alcorlo et al. 2013Alcorlo, M., Tortajada, A., Rodriguez, S., Lorca, O. (2013). Structural basis for the stabilization of the complement alternative pathway C3 convertase by properdin. PNAS, 110, 13504–13509. https://doi.org/10.1073/pnas.1309618110]. Among the identified mare colostrum proteins was the protein participating in the activation of the complement system (all three pathways) complement C3 alpha chain-like (Table 1). The main role of the complement system is to create a membrane attack complex, which allows lysis of bacteria cells [Alcorlo et al. 2013Alcorlo, M., Tortajada, A., Rodriguez, S., Lorca, O. (2013). Structural basis for the stabilization of the complement alternative pathway C3 convertase by properdin. PNAS, 110, 13504–13509. https://doi.org/10.1073/pnas.1309618110].

Another identified protein in mare colostrum was the interleukin-24 precursor (Table 1). This protein belongs to the IL-10 cytokine family. IL-24 is produced by immune cells, including myeloid cells and lymphoid cells. Interleukin-24 is the immunoregulating cytokine [Persaud et al. 2016Persaud, L., Jesus, D. D., Brannigan, O., Richiez-Paredes, M., Human, J., Alvarado, G., Riker, L., Mendez, G., Dejoie, J., Sauane, M. (2016). Mechanism of Action and Application of Interleukin 24 in Immunotherapy. Int. J. Mol. Sci., 17, 869. https://doi.org/10.3390/ijms17060869].

All the above-mentioned proteins were connected with regulating foals’ immune response. After birth, foals are exposed to environmental pathogens. Except for immunomodulating proteins, along with colostrum are deliver protease inhibitors which have to protect proteins against degradation in the newborn digestive tract.

The presence of protease inhibitors in the study mare colostrum has been demonstrated, including alpha–1-antitrypsin, inter-alpha-trypsin inhibitor (ITIH4), and fetuin-b (FETUB) (Table 1). Research carried out by Zhang et al. [2015a] showed the presence of alpha-1-antitrypsin and ITIH4 in cow’s milk. According to the authors, the observed decrease of trypsin and immunoglobulin inhibitor concentration with subsequent lactation days indicates the protective role of this inhibitor against proteolytic degradation of immunoglobulin. Fetuin-B has an endopeptidase inhibitor activity and metalloendopeptidase inhibitor activity. Nissen et al. [2012]Nissen, A., Bendixen, E., Ingresrtsten, K.L., Røntved, C.M. (2012). In-deph analysis of low abundant proteins in bovine colostrum using different fractionation. Proteomics, 12, 2866–2878. https://doi.org/10.1002/pmic.201200231 using two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) showed fetuin-B in cow milk.

The presence of protease inhibitors in mare colostrum indicates their protective role against protein degradation, especially the immunomodulating proteins. Regulatory proteins transfer along with colostrum is possible owing to the “leaky intestinal barrier”. The physiological transfer of antibodies is possible up to 24–36 hours after birth, subsequently the enterocyte cell membrane is sealed. The Inhibiting of protein proteolysis allows their absorption in unchanged form.

Moreover, in the presented research identified in mare colostrum proteins were involved in binding and transport of compounds, such as serum albumin and vitamin D-binding protein, proline-rich and Gla domain 4 (transmembrane) isoform 3 (Table 2). Vitamin D-binding protein (GC) is a vitamin D transporting protein. GC has the availability of binding albumin [Chun 2012Chun, R.F. (2012). New perspectives on the vitamin D binding protein. Cell Biochem. Funct., 30, 445–56. https://doi.org/10.1002/cbf.2835]. The concentration of vitamin D in mare colostrum is 4.93 μg · L^–1 [Pieszka et al. 2016Pieszka, M., Łuczyński, J., Zamachowska, M., Augustyn, R., Długosz, B., Hędrzak, M. (2016). Is mare milk an appropriate food for people? – A review. Ann. Anim. Sci, 16, 33–51. https://doi.org/10.1515/aoas-2015-0041]. Delivery with colostrum vitamin D is essential for the correct growth and development of foals.

Proline-rich and Gla domain 4 (transmembrane) isoform 3 (PRRG4) is a protein belonging to the PRRG family [Yazicioglu et al. 2013Yazicioglu, M.N., Monaldini, L., Chu, K., Khazi, F.R., Murphy, S.L., Huang, H., Margaritis, P., High, K.A. (2013). Cellular localization and characterization of cytosolic binding partners for Gla domain-containing proteins PRRG4 and PRRG2. J. Biol. Chem. 288, 25908–25914. https://doi.org/10.1074/jbc.M113.484683]. According to the Uniprot database, the PRRG4 can bind calcium ions. Calcium is involved in many processes, including activation of some enzymes, and is a primary building block of bone mass. Calcium with vitamin D shows synergetic action, vitamin D increases the bioavailability of calcium. The content of calcium in mare milk is 93 mg · ml^–1 [Claeys et al. 2014Claeys, W.L., Varraes, C., Cardoen, S., De Block, J., Huyghebaert, A., Raes, K. (2014). Consumption of raw or heated milk from different species: an evaluation of the nutritional and potential health benefits. Food Control., 42, 188–201. https://doi.org/10.1016/j.foodcont.2014.01.045].

Among identified proteins in mares' colostrum was rho GTPase-activating protein 39, which has GTPase activity properties (Table 1). This protein belongs to the Rho GTPases family. Rho GTPases family participate in many cellular processes, including in migration, vesicular transport and cytokinesis [Hodge and Ridley 2016Hodge, R., Ridley, A. (2016). Regulating Rho GTPases and their regulators. Nat. Rev. Mol. Cell Biol., 17, 496–510. https://doi.org/10.1038/nrm.2016.67]. This protein is associated with mammary gland maturation.

Mammary gland duct morphogenesis is a complex process, during which cell has to proliferate and migrate to the fat pads where are differentiate into luminal and myoepithelial cell compartments. Studies showed that over-expression of Rho GTPases proteins in mammary gland tissue in the postnatal period results in higher branching and delay elongating of the milk ducts and disorganization of ductal trees [Vargo-Gogola et al. 2006Vargo-Gogola, T., Heckman, B.M., Gunther, E.J., Chodosh, L.A., Rosen, J.M. (2006). P190-B Rho GTPase-activating protein overexpression disrupts ductal morphogenesis and induces hyperplastic lesions in the developing mammary gland. Mol. Endcrinol., 20, 1391–1405. https://doi.org/10.1210/me.2005-0426]. Vesicle transport is a pathway of main milk components, among other proteins and lactose. Participating Rho GTPases proteins in vesicle transport may indicate their role in compounds secretion into milk.

CONCLUSION

To sum up, using two-dimensional electrophoresis with mass spectrometry MALDI-TOF allowed the identification of mare colostrum whey proteins, essential for biochemical process regulation. Most of the identified proteins were associated with supporting an immature foals’ immunological system. Further research of the mare colostrum proteome will enable determining important proteins that respond to the correct development of foals and milk components synthesis. Identification of low-abundant mare colostrum proteins and indicate in which metabolic pathways are involved may expand knowledge about biochemical processes occurring in the mammary gland and indicate proteins involved in foal growth and development.

ACKNOWLEDGEMENTS

The research was financed from funds for young researchers (project number 7377).

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