Isolation and Characterization of Mauritanicain, a Serine Protease from the Latex of Euphorbia mauritanica L.

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• Plant material
• Purification
• Proteolytic activity
• Protein concentration
• Molecular weight determination
• Determination of the protease family
• Behavior against pH changes
• Behavior against temperature
• Isoelectric focusing
• References

Abstract

A protease called Mauritanicain was isolated from the latex of Euphorbia mauritanica L. (Euphorbiaceae) by combining ion exchange chromatography, ultrafiltration, and gel filtration chromatography. It has a high proteolytic activity against casein. The activity was only inhibited by specific serine protease inhibitors, classifying it to the serine protease family. An optimal degradation of the substrate casein takes place at a temperature of 55–65 °C and a pH of 5.5–6.5, and is unstable at pH < 5 and pH > 9. The protease is stable at temperatures from 20–70 °C, whereby the activity decreases drastically to less than 20 % at 75 °C. SDS-PAGE and matrix-assisted laser desorption time-of-flight analysis yielded a molecular weight of 73 kDa; possibly, it is natively present as a non-covalently linked dimer of a higher molecular mass > 132 kDa. Without heat denaturation, a breakdown in fractions of 73 kDa and 52 kDa was observed in SDS-PAGE. Only in some properties it shows a similarity to other characterized proteases in the plant family Euphorbiaceae, such that Mauritanicain can be presented as a new isolated protease.

Introduction

Latex is an aqueous emulsion or a suspension found in laticifers of about 9 % of all angiosperms, spread over approximately 40 plant families [1]. Generally, it is a nontransparent white liquid, sometimes also being clear and/or colored [2]. The composition, thus, may vary accordingly, for example, the presence of alkaloids, cardenolides, furanocoumarins, phenols, proteins, terpenes, and amylum has been described. The proteins contained include proteases, oxidases, lectins, chitin-binding proteins, chitinases, lipases, glucosidases, and phosphatases. In certain cases, protease inhibitors may also be present [2]. After the slightest injury, the pressurized latex leaks from foliage, stems, fruits, or roots, followed by wound closure within a few minutes.

Proteases are widespread in the latex of different plant families. They belong mainly to the cysteine and serine protease families and were mainly isolated from the plant families Apocynaceae, Caricaceae, Euphorbiaceae, and Moraceae [3]. A protease belonging to the aspartic family has also been identified [4]. In this context, the proteases from Euphorbiaceae species have recently been reviewed [3], [5]. Compared to the amount of latex-containing species in these plant families, proteases could only be isolated and characterized from a few of them. The role of proteases in plant latex is not yet completely clarified. There is a known participation in the defense mechanism of the plant. They might be involved in the promotion of the coagulation of the exiting latex, which can, in addition to the wound closure, prevent a penetration of pathogens. The coagulation serves also as a defense against herbivores because it adheres to the clamps of insects. Besides toxic diterpene esters and proteins with chitinase and lysozyme activity, latex may contain some proteases that are also known to be toxic for predators and pathogens [1], [2], [3], [6], [7], [8], [9], [10].

Former investigations indicated a possible synergistic toxicity between proteases and phorbol esters in IL-6 release from monocytic cells while investigating a large number of plants (129 species) of the plant family Euphorbiaceae Juss. Therefore, there was a need to assay these for proteolytic activity [11], [12]. The screening of the latices showed that certain species had high proteolytic activities and would thus be interesting for further investigation [12]. An isolation and characterization of the specific latex proteases could therefore help to further elucidate the abovementioned mechanisms. Furthermore, it opens the possibility to investigate whether these proteases could be used industrially, for example, in the food, pharmaceutical, or cleaning agents industry. From the results of the screening and due to other factors such as location, availability, and amount of latex, the latex of Euphorbia mauritanica L. was chosen to isolate a protease, which is presented in this article.

Results and Discussion

The protease in the latex of E. mauritanica L. was purified in three steps. First, the polymerized fraction (rubber content) was eliminated by freezing for 24 h, centrifugation (18 626 × g), and filtration (0.2 µm cellulose acetate). In the second step, an ion exchange chromatography with a stepwise increase of NaCl led to a separation of the proteins in the latex. Simultaneous to the protein concentration, the proteolytic activity was measured ([Fig. 1]). The first peak maxima (fraction 3 and 6) elutes without NaCl in the buffer. Fractions 16–30 were eluted with 0.05 M NaCl and fractions 31–42 with 0.1 M NaCl in phosphate buffer, resulting, in each case, to a peak in fractions 17 and 32. After increasing the salt concentration to 0.15 M in fraction 43–57, a peak with a high protein concentration and proteolytic activity was obtained (fraction 44). The elution with 0.2 M NaCl of fractions 58–67, with 0.4 M NaCl of fractions 67–82, and, finally, with 1 M NaCl of fractions 83–90 led to four more peaks, with three of them having proteolytic activity. A SDS-PAGE of the supernatant of the crude extract and the fractions 3, 6, 17, 32, 44, 58, 68, 72, and 83 was conducted, where the separation could be monitored ([Fig. 2]). The transition is often fluid and several fractions contain proteins of the same size, but in varying amounts.

Fraction 44, with the highest protein concentration and proteolytic activity, was separated in a third step with a gel filtration column. The separation resulted in three peaks ([Fig. 3 A]), which could be collected. Peak 1 showed a high proteolytic activity, where the largest protein of fraction 44 is responsible for the activity. Following [Fig. 2], this has a mass in the range between 66–97 kDa. Fraction 44 was then filtered through a membrane with a 50 kDa molecular weight cutoff to eliminate all smaller proteins and to concentrate the protein of interest. A gel filtration of this concentrate resulted in one main peak and a second smaller peak ([Fig. 3 B]). The main peak was collected between the retention times 14.4–17.4 min and again subjected to gel filtration ([Fig. 3 C]). Therefore, and also due to the further results shown below, the fraction which resulted from the mentioned retention interval was considered a pure fraction of a protein and this protein was named Mauritanicain. Other fractions turned out not to be interesting for further study in this work.

A separation on gel electrophoresis showed the results of the purification ([Fig. 4]). The molecular weight was determined by SDS-PAGE ([Fig. 4 B]) and with matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) analysis ([Fig. 5]). The mass spectrum showed a peak of high intensity at 73 kDa and the SDS-PAGE, under reducing and denaturing conditions, showed an intensive band at the same mass. In addition, other bands of lesser intensity can be reported in the SDS-PAGE, which might arise, for example, through self-digestion. Therefore, an electrophoresis was performed under a native condition, which showed only a single band ([Fig. 4 A]). However, BSA, which was applied on the same gel, suggested that the molecular weight must be > 132 kDa. BSA showed two bands in the gel, which are probably attributed to its monomer and dimer form. Therefore, Mauritanicain could also be a dimer. But it should be noted that the Native-PAGE is not a method to determine a molecular mass. By renouncing SDS, heat denaturation, and β-mercaptoethanol, the running behavior of proteins is, in addition to the molecular weight, influenced by the charge and the folding of each protein through, e.g., disulfide and hydrogen bridges as well as hydrophobic and ionic interactions. Another explanation for the single band of higher molecular weight could be that it is a complex of several subunits or degradation products, which are held together by non-covalent secondary binding forces. To check this possibility, an SDS-PAGE was carried out in a next step in which the samples were treated differently. Alternately, the addition of β-mercaptoethanol and the heat denaturation were omitted ([Fig. 4 B]). A protein of several subunits, which are held together by disulfide bonds, is therefore not likely because the addition of β-mercaptoethanol has no significant impact on the number of bands. However, there is a difference between a treatment with or without heat denaturation. Without heating for 5 min at 95 °C, two intense bands can be seen at about 73 kDa and 52 kDa. On the other hand, with heating, only the band with a molecular mass of 73 kDa appears. However, it must also be considered that without the heat denaturation, the folding of the protein has an influence on the gel running behavior and, thus, also on the molecular weight of the samples, the latter being difficult to determine. One possible explanation for the occurrence of two bands could be that the band at 52 kDa could be a degradation product of the protein at 73 kDa, which is formed by instability to SDS. This product could then be self-digested under the heat treatment. Corresponding time-dependent studies need to be performed to confirm this possibility. Finally, it canʼt be clarified clearly why a second intense band in the gel is visible without heat denaturation, but there is some evidence that Mauritanicain is a protease with a molecular mass of 73 kDa, which is possibly present in native form as a non-covalently linked dimer of a higher molecular mass.

Mauritanicain is significantly reduced in its proteolytic activity only by the inhibitors aprotinin and AEBSF-HCl [4-(2-Aminoethyl)benzenesulfonylfluoride] ([Fig. 6]). Consequently, we may classify, on basis of these results, Mauritanicain as a serine protease. So far, most of the isolated latex proteases belong to the serine and cysteine protease class. The cysteine proteases isolated from latex have almost exclusively molecular weights from 20 to 30 kDa, whereas the isolated serine proteases have mainly a mass between 60–80 kDa [3], [13].

A strong influence on the proteolytic activity of Mauritanicain against pH changes was observed while applying BODIPY FL casein. The optimum pH value for a reaction at 37 °C is in the range of pH 5.5–6.5 ([Fig. 7]). The degradation of the substrate decreases to less than 40 % at pH levels below pH 5 and above pH 9 with respect to the optimum range.

Compared to standard proteins, an isoelectric focusing (IEF) of Mauritanicain led to an isoelectric point of about 6.5 ([Fig. 8]).

At different temperatures, proteolytic activity could be determined from the beginning of the measurement at 20 °C up to 80 °C ([Fig. 9]). Above 70 °C, however, the activity decreases drastically. At 75 °C, only less than 20 % residual activity was present. The optimal temperature for activity towards casein was between 55–65 °C. Storage for 20 min at various temperatures does not affect the proteolytic activity in the range of 20 to 70 °C. The influence of storage temperatures > 70 °C results in a strong activity reduction as well.

Many of the isolated proteases from the family Euphorbiaceae have a molecular weight in the range of 60–80 kDa and either consist of several subunits, or are unstable under the conditions of SDS-PAGE [3], [14]. Cotinifolin, for example, seems to consist of several subunits because the mass spectrum shows a molecular weight of 80 kDa, whereas in the SDS-PAGE, only bands of 25–30 kDa were detected [14]. Euphorbain p, which has a very similar molecular weight (74 kDa) to Mauritanicain, shows, in the SDS-PAGE, one main band of 35 kDa and two other bands of 20 kDa and 15 kDa. The authors also propose a protein with several subunits [15]. Most similar in this property to Mauritanicain seems to be the isolated proteases Euphorbain la₁, lc, and y₃ from Euphorbia lactea Haw. However, other properties like pH optimum and pI differ from it [16]. Therefore, Mauritanicain can be announced as a newly isolated and characterized protein. With its proteolytic activity being retained under higher temperatures and a proteolytic activity of over 40 % in the pH range of 4–9, Mauritanicain opens a broad field of application possibilities. Further studies will include, for example, the influence of the enzyme in blood coagulation (hemostasis) and the dissolution of blood clots (fibrinolysis).

Materials and Methods

Plant material

The plant latex was collected from E. mauritanica L. in the Botanical Garden Berlin, Freie Universität Berlin (accession number: 125–08–74–80).

Purification

To obtain the latex, the plant was scratched at various points using a clean scalpel. The fresh latex was collected in plastic tubes prefilled with phosphate buffer (10 mM, pH 7.0) until the solution showed no light transmittance. The tubes were shaken, centrifuged (18 626 × g) at 4 °C for 30 min, and frozen for 24 h. After thawing and recentrifugation, 10 mL of the clear supernatant was used for further purification. It was filtered through a 0.2-µm cellulose acetate syringe filter and was loaded onto a DEAE (diethylaminoethyl) ion exchange column (AcroSep DEAE Ceramic HyperD F, Pall Life Sciences) equilibrated with 10 mM phosphate buffer at pH 7.0. Fractions of 1.5 mL volume were collected during elution with a stepwise increase of NaCl from 0 to 1 M for different time intervals ([Fig. 1]) and a flow rate of 1.5 mL/min. Fractions with high proteolytic activity were analyzed for purity by SDS-PAGE. After ultrafiltration (Vivaspin 500, 50 MWCO PES, Sartorius Stedim) of the most active fraction, 50 µL were loaded onto a gel filtration column (Discovery BIO GFC 150 30 cm × 4.6 mm I. D. 5 µm 150 Å column, Supelco, Sigma-Aldrich) with a flow rate of 0.15 mL/min using the same buffer. Peaks were detected at 210 nm and also analyzed by SDS- and Native-PAGE.

Proteolytic activity

All samples were analyzed for proteolytic activity with the EnzChek Protease Assay (Invitrogen). Twenty-five µL of a sample were put onto a 96-well black microtiter plate and 100 µL of the substrate BODIPY FL casein was added, shortly shaken, and incubated for 1 h at 37 °C. The change in fluorescence was measured by exciting at 485 nm and noting the emission at 535 nm using a microtiter plate fluorescence reader (Infinite F200, Tecan) against a negative control (buffer + substrate) [17].

Protein concentration

The protein concentration was determined with the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific) on a 96-well transparent microtiter plate. Two hundred µL of BCA reagent were added to a 25-µL sample, shaken for 30 s, and incubated 30 min at 37 °C. The absorbance was measured at 560 nm (Infinite F200, Tecan) and the polynomial regression of a BSA solution (20–2000 µg/mL) served as the standard [18].

Molecular weight determination

The molecular weight was determined by two methods, initially by SDS-PAGE under reducing conditions while applying a broad range molecular weight standard (Bio-Rad). The logarithms of the molecular weights were applied to the retention factors. A second determination was carried out with MALDI-TOF-MS analysis with an AUTOFLEX-III LRF200-CID equipped with a Smartbeam laser 200 (Bruker Daltonik). Two µL of the sample were mixed with 2 µL of matrix solution (7.6 mg 2,5-dihydroxyacetophenone dissolved in 375 µL of ethanol and 125 µL of a solution containing 18 mg/mL diammonium hydrogen citrate). Of this mixture, 0.5 µL was applied onto a steel target for measuring. The instrument was internally calibrated using the signals of the positive [M + H]⁺ mono isotopic ions of a protein I calibration standard (Bruker Daltonik).

Determination of the protease family

To determinate whether the protease belongs to the aspartic, cysteine, metallo, or serine protease family, the residual activity after the addition of specific inhibitors for each protease type was measured [Aprotinin from bovine lung, Pepstatin A from microbial source, Phosphoramidon (Sigma Aldrich); E-64 (Interchim); EDTA (Carl Roth); Pefabloc SC/AEBSF (Roche)]. To 25 µL of the samples, 50 µL of inhibitor were added and incubated for 30 min at room temperature, followed by the addition of the substrate for activity measurement. The residual activity was compared to samples with the addition of buffer instead of inhibitor. As a positive control, a corresponding reference protease was used (Papain from Carica papaya L. latex, Pepsin (EC.3.4.23.1) from porcine stomach mucosa, Thermolysin from Bacillus thermoproteolyticus ROKKO (Sigma Aldrich); Trypsin from porcine pancreas (Carl Roth)).

Behavior against pH changes

The optimum pH was determined with the following buffer systems: Glycine/HCl (2–3.5), acetic acid/sodium acetate (4–5.5), NaH₂PO₄/Na₂HPO₄ (6–8.5), NaHCO₃/Na₂CO₃ (9–11), NaH₂PO₄/NaOH (11.5–12.5). Twenty-five µL of a sample were incubated 30 min at 37 °C with 200 µL of a buffer solution. After the addition of 75 µL of the substrate BODIPY FL casein and incubation for 1 h, the change in fluorescence was measured. The actual pH during the reaction was determined from the reaction mixture of the three components directly in the reaction vessel.

Behavior against temperature

To determine the temperature activity maximum, 50 µL of sample (pH 7.0) and 100 µL BODIPY FL casein were kept separately for 10 min at a specific temperature. After that, the solutions were combined and incubated again for 10 min, followed by measuring the change in fluorescence. The maximum activity was the reference point for the other values. To determine the stability against temperature, 50 µL of sample were kept for 20 min at different temperatures. After cooling down to room temperature, 100 µL of BODIPY FL casein were added and after incubation for 1 h at 37 °C, the change in fluorescence was measured.

Isoelectric focusing

For isoelectric point analyses, a pH gradient gel was used. The method and evaluation of the results were carried out in accordance with instructions from the application guide for precast gels and the instructions for using the standard from Bio-Rad. The samples were diluted with sample buffer (50 % Glycerol). The anode buffer consisted of 7 mM phosphoric acid and the inner chamber was filled with 20 mM lysine and 20 mM arginine buffer. The electrophoresis was conducted with different constant voltages at 4–8 °C (100 V, 60 min; 250 V, 60 min; 500 V, 30 min). After the run, the gel was fixed in 3.5 % 5-sulfosalicylic acid, 10 % trichloracetic acid (TCA), and 30 % methanol for 1 h, followed by incubation four times in a solution of 12 % TCA and 30 % methanol for at least 2 h. The protein bands were visualized by silver staining [19]. The buffers and the standard (pI range: 4.45–9.6) were from Bio-Rad.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

Thanks to Prof. T. Borsch, Mr. T. Dürbye, and colleagues of the Botanical Garden Berlin for providing the plant material and M.Sc. T. Buchholz for helping with the MALDI-TOF-MS analysis.

• References

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Correspondence

• References

• 1 Konno K. Plant latex and other exudates as plant defense systems: roles of various defense chemicals and proteins contained therein. Phytochemistry 2011; 72: 1510-1530
  CrossrefPubMedSearch in Google Scholar
• 2 Agrawal AA, Konno K. Latex: a model for understanding mechanisms, ecology, and evolution of plant defense against herbivory. Annu Rev Ecol Syst 2009; 40: 311-331
  CrossrefPubMedSearch in Google Scholar
• 3 Domsalla A, Melzig MF. Occurrence and properties of proteases in plant latices. Planta Med 2008; 74: 699-711
  Thieme ConnectPubMedSearch in Google Scholar
• 4 Devaraj KB, Gowda LR, Prakash V. An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.). Phytochemistry 2008; 69: 647-655
  CrossrefPubMedSearch in Google Scholar
• 5 Flemmig M. Investigations on the proteolytic Activity of Plant Latices and their Influence on Hemostasis and Fibrinolysis [Dissertation]. Berlin: Freie Universität; 2015
  PubMedSearch in Google Scholar
• 6 Martin MN. The latex of Hevea brasiliensis contains high levels of both chitinases and chitinases/lysozymes. Plant Physiol 1991; 95: 469-476
  CrossrefPubMedSearch in Google Scholar
• 7 Sytwala S, Gunther F, Melzig MF. Lysozyme- and chitinase activity in latex bearing plants of genus Euphorbia – A contribution to plant defense mechanism. Plant Physiol Biochem 2015; 95: 35-40
  CrossrefPubMedSearch in Google Scholar
• 8 Van der Hoorn RA, Jones JD. The plant proteolytic machinery and its role in defence. Curr Opin Plant Biol 2004; 7: 400-407
  CrossrefPubMedSearch in Google Scholar
• 9 Siritapetawee J, Thammasirirak S, Samosornsuk W. Antimicrobial activity of a 48-kDa protease (AMP48) from Artocarpus heterophyllus latex. Eur Rev Med Pharmacol Sci 2012; 16: 132-137
  PubMedSearch in Google Scholar
• 10 Goel G, Makkar HP, Francis G, Becker K. Phorbol esters: structure, biological activity, and toxicity in animals. Int J Toxicol 2007; 26: 279-288
  CrossrefPubMedSearch in Google Scholar
• 11 Domsalla A, Melzig MF. Enhancement of protease-induced IL-6 release in monocytic U-937 cells by phorbol-12-myristate-13-acetate. Inflamm Res 2012; 61: 1125-1129
  CrossrefPubMedSearch in Google Scholar
• 12 Domsalla A. Investigations of Plant Latices with Regard to proteolytic Activity and the Influence on the Interleukin-6 Secretion in monocytic Cells [Dissertation]. Berlin: Freie Universität; 2012
  PubMedSearch in Google Scholar
• 13 Badgujar SB, Mahajan RT. Peptide mass fingerprinting and N-terminal amino acid sequencing of glycosylated cysteine protease of Euphorbia nivulia Buch.-Ham. J Amino Acids 2013; 2013: 569527
  PubMedSearch in Google Scholar
• 14 Kumar R, Singh KA, Tomar R, Jagannadham MV. Biochemical and spectroscopic characterization of a novel metalloprotease, cotinifolin from an antiviral plant shrub: Euphorbia cotinifolia . Plant Physiol Biochem 2011; 49: 721-728
  CrossrefPubMedSearch in Google Scholar
• 15 Lynn KR, Clevette-Radford NA. Euphorbain p, a serine protease from Euphorbia pulcherrima . Phytochemistry 1984; 23: 682-683
  CrossrefPubMedSearch in Google Scholar
• 16 Lynn KR, Clevette-Radford NA. Isolation and characterization of proteases from Euphorbia lactea and Euphorbia lactea cristata . Phytochemistry 1986; 25: 807-810
  CrossrefPubMedSearch in Google Scholar
• 17 Jones LJ, Upson RH, Haugland RP, Panchuk-Voloshina N, Zhou M. Quenched BODIPY dye-labeled casein substrates for the assay of protease activity by direct fluorescence measurement. Anal Biochem 1997; 251: 144-152
  CrossrefPubMedSearch in Google Scholar
• 18 Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem 1985; 150: 76-85
  CrossrefPubMedSearch in Google Scholar
• 19 Merril CR, Goldman D, Sedman SA, Ebert MH. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 1981; 211: 1437-1438
  CrossrefPubMedSearch in Google Scholar