Praca oryginalna Original paper
Metalloproteases are enzymes from the peptidase group that contain metal ions, in most cases Zn2+, in
their active centres (22). A zinc ion is coordinated by two histidines, one chelating glutamate and one water molecule. The coordinated water is converted to the metal-bound hydroxide that is assisted by Glu, which is a general acid catalyst. The hydroxide then acts as the nucleophile to attack the peptide carbonyl carbon which is polarized with the assistance of Arg (1). Metalloproteases are subdivided into two groups: metalloexopeptidases (EC 3.4.17) and metaloendo-peptidases (EC 3.4.24) (3, 27).
Metalloproteases are important for both physio-logical and pathophysio-logical processes. They participate, among other things, in wound healing, cell migration and proliferation, the inflammatory response, as well as in apoptosis and angiogenesis. Metalloproteases in humans also participate in the etiopathogenesis of
a number of skin diseases, including psoriasis, lichen planus, acne rosacea, bladder diseases, scleroderma and crural ulceration. Wawrzycka et al. (28) have shown an important role of metalloproteinases in the etio-pathogenesis of common acne and in solar-induced skin senescence. These enzymes are present in clothes moth larvae Tineola bisselliella (Lepidoptera). The molecular weight of these digestive proteases is 24 kDa, at an optimal pH 9.4. Metalloproteases manifest digestive specificity towards carboxy-methyl A and B chains of insulin, as well as towards glukagon. Their specificity is similar to that of serine proteases and subtilisin, but they differ considerably from microbio-logical metalloproteases and from metalloproteases isolated from snake venom (8, 19, 27).
Metalloprotease concentration and activity rises in skin disorders, burns and cancers in mammals and under the influence of adverse environmental factors,
Body-surface metalloprotease activity
in Apis mellifera L. workers
relative to environmental pollution
ANETA STRACHECKA, GRZEGORZ BORSUK, JERZY PALEOLOG, KRZYSZTOF OLSZEWSKI, JACEK CHOBOTOW*, DOMINIKA SKOCZYLAS**
Department of Biological Basis of Animal Production, Faculty of Biology and Animal Breeding, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland
*Department of Zoology, Institute of Biology and Biochemistry, Faculty of Biology and Biotechnology, Akademicka 19, 20-033 Lublin, Poland
**Jean Monnet Chair of Economics of the European Integration, Faculty od Economics, Maria Curie-Sk³odowska University in Lublin, Pl. Marii Curie-Sk³odowskiej 5, 20-031 Lublin, Poland
Strachecka A., Borsuk G., Paleolog J., Olszewski K., Chobotow J., Skoczylas D.
Body-surface metalloprotease activity in Apis mellifera L. workers relative to environmental pollution
Summary
Metalloproteases are enzymes containing metallic ions, predominantly zinc ions, in their active sites. They are important for the maintenance of homeostasis and prevention of infections. The study was aimed at analysing the activity of metalloproteases on the cuticle of bees depending on environmental pollution and the develop-ment stage of the bees. The experidevelop-ments were conducted on bees from two apiaries: one located in a polluted environment (close to the trunk road no. 17) and the other situated in a clean area (Nowiny). Eggs, larvae and pupae, as well as one-day-old, two-week-old, three-week-old and four-week-old workers, were collected in spring, summer, autumn and winter. The following methods were employed: protein content testing by the Lowry method (modified by Schacterle-Pollack) and metalloprotease activity testing by the Lee and Lin method (using o-phenantroline). Protein concentration values were found to be higher in the eggs, larvae and pupae than in the workers in both environments. Metalloprotease activity was not observed in the workers from the clean environment, but it was found in those kept in the polluted area. An elevated metalloprotease activity in the latter environment was observed only in summer. Bee workers, which synthesise and secrete metalloproteases onto their body surface, can be regarded as bioindicators of environmental pollution.
e.g. pollution or diet in the case of humans (15, 18, 29). There-fore, it is extremely interesting to find out how the activity of these enzymes changes in bees influenced by environmental pol-lution. The aim of the study was to define the activity of metallo-proteases on the body surface of eggs, larvae, pupae and workers of different ages collected during spring, summer, autumn, and winter in polluted and unpollu-ted environments.
Material and methods
The research was performed at two apiaries. One of them was situated near a trunk road (no. 17) in an industrialized part of the Lublin region, Poland (polluted environment). The other was situated by a forest, away from major roads, in the village Nowiny, Lublin region (unpolluted environ-ment).
In two consecutive seasons, 2600 eggs, 1400 larvae, 1400 pupae and 5200 workers (tab. 1) were sampled from both apiaries. The biological material was collected from hives in winter (January), spring (May), summer (August) and autumn (October), and thereupon immediately frozen (8°C). Next, after being defrosted, samples for bio-chemical analyses were taken three times from each set of the biological material. Subsequently, the samples were placed on Miracloth and rinsed with distilled water, and
the polluted refuse was discarded. Afterwards, the samples were placed again in test-tubes and shaken for three minutes in distilled water (neutral and alkaline proteases) and then in a 1% detergent solution (Triton X-100, Serva; acidic pro-teases) in order to wash out body surface proteins. Next, the washings were frozen in Eppendorf test-tubes at 20°C. On defrosting, they were tested for the surface protein content by the Lowry method modified by Schacterle and Pollack (26). Next, the samples were assayed for metallo-protease activity according to the Lee and Lin method (17), taking advantage of the so-called diagnostic protease inhibitor: o-phenantroline, which is used in enzymology to determine the activity level of such proteins and to identify catalytic types of proteases.
The proteolytic activity was also assayed through PAGE electrophoresis with 1% gelatin as the initial protein
sub-f o e p y t e l p m a S e e b y e n o h n o s a e s e n o n i t n e m n o ri v n e d e t u ll o p n u / d e t u ll o P Totalsamples rfomtwo s n o s a e s o w t m o rf s t n e m n o ri v n e g n ir p S Summer Autumn Winter s g g E 250 250 150 *n.a.* 2600 e a v r a L 125 125 100 n.a. 1400 e a p u P 125 125 100 n.a. 1400 s r e k r o w d l o -y a d -1 100 100 100 n.a. 1200 s r e k r o w d l o -k e e w -2 100 100 100 n.a. 1200 s r e k r o w d l o -k e e w -3 100 100 100 n.a. 1200 s r e k r o w d l o -k e e w -4 100 100 100 100 1600
Tab. 1. Biological material: Sample database
Explanation: * non-analysed samples are due to the unavailability of specific forms at a given time
Tab. 2. Seasonal body-surface protein concentration measured in different developmental stages of honeybee workers from the clean and polluted habitats
t n e m n o ri v n E Samples Proteinconcenrtaiton(mg/m)l g n ir p S Summer Autumn Winter Totalmean n a e l C s g g e 0.523±0.002 0.626a±0.011 0.482a±0.010 *n.a.* 0.545±0.321 e a v r a l 0.021±0.009 0.598a±0.009 0.461a±0.006 n.a. 0.352±0.411 e a p u p 0.017±0.005 0.538a±0.008 0.205a±0.008 n.a. 0.254±0.248 s e n i g a m i d l o -y a d -1 0.072±0.009 0.112a±0.012 0.109a±0.017 n.a. 0.099±0.095 s e n i g a m i d l o -k e e w -2 0.059±0.011 0.109a±0.017 0.106a±0.016 n.a. 0.092±0.089 s e n i g a m i d l o -k e e w -3 0.051±0.010 0.089a±0.015 0.101a±0.013 n.a. 0.081±0.078 s e n i g a m i d l o -k e e w -4 0.132a±0.009 0.520a±0.007 0.211a±0.004 0.112a±0.003 0.289±0.231 n a e m l a r e v O 0.135±0.112 0.378±0.297 0.242±0.199 0.112±0.003 0.248±0.237 d e t u ll o P s g g e 0.521±0.012 0.563b±0.008 0.462b±0.008 n.a. 0.521±0.099 e a v r a l 0.024±0.013 0.831b±0.012 0.421b±0.012 n.a. 0.435±0.411 e a p u p 0.020±0.009 0.859b±0.014 0.235b±0.014 n.a. 0.375±0.352 s e n i g a m i d l o -y a d -1 0.076±0.014 0.459b±0.014 0.205b±0.016 n.a. 0.251±0.213 s e n i g a m i d l o -k e e w -2 0.062±0.012 0.204b±0.015 0.197b±0.009 n.a. 0.154±0.121 s e n i g a m i d l o -k e e w -3 0.053±0.013 0.185b±0.013 0.175b±0.009 n.a. 0.138±0.099 s e n i g a m i d l o -k e e w -4 0.038b±0.009 0.145b±0.009 0.132b±0.011 0.089b±0.012 0.105±0.871 n a e m l a r e v O 0.121±0.099 0.472±0.299 0.263±0.211 0.089±0.012 0.284±0.211
strate in a 10% gel. The electrophoreses were performed in the Laemmli (16) system modified for non-denaturing conditions. The zymographies were performed in a Mini-protean II apparatus (Bio-Rad, USA). A sample (100 µl) from each worker age was loaded in each lane. The gels were then incubated in appropriate buffers (0.1 M glycine HCl at pH 2.4; 0.1 M Tris HCl at pH 7; 0.1 M glycine NaOH at pH 11.4) at 37°C for 2 hours. The gels were stained according to the methods of Heussen and Dowdle (12) with 0.5% Coomassie Brilliant Blue R-250 and de--stained with 40% methanol and 10% acetic acid. The gels were analysed with the ImageMaster software.
Statistical calculations were carried out with the SAS software (25). The verification of statistical differences between the experimental factors was performed by two-way ANOVA.
Results and discussion The eggs, larvae and pupae had higher protein concentrations than the imagines in the polluted and un-polluted environments. The cuticle protein concentration was medium and similar in both environments in spring. The highest concentration was observed in the summer time independently of the pollution levels (tab. 2). Later, the concentration de-creased in autumn and was the lowest in the winter time. In the cases of eggs and 4-week-old workers, the protein concentrations were higher in the unpolluted environment than in the polluted one. As regards the larvae, pupae and 1-, 2- and 3-week-old workers, the situation was inverse.
The proteins were almost comple-tely used up over winter. This was confirmed especially by the analysis of the samples from the beginning of
the vegetative period. The winter protein concentra-tion amounted to 0.112 mg/ml in the mature forms of the honey bee workers from the clean environment and 0.089 mg/ml in those from the polluted habitat.
Only at a neutral pH (7.0) and only in the polluted environment were the worker cuticle metalloproteases active (tab. 3). The activities were the highest for each developmental stage in the summer time. Particularly high activities of these enzymes were observed in the older imagines at that time. The distribution pattern of the activity of the analysed metalloproteases was similar for the spring and autumn periods. In the case
Tab. 3. Seasonal body-surface metalloprotease activity measured in different developmental stages of honeybee workers from the polluted habitat (#)
s e l p m a S Metalloproteaseacitvtiy(U/mg)inpH=7.0 g n ir p S Summer Autumn Winter Totalmean s g g E 0.034±0.013 1.123±0.018 0.023±0.011 *n.a.* 0.391±0.362 e a v r a L 0.043±0.018 1.143±0.012 0.026±0.017 n.a. 0.405±0.345 e a p u P 0.051±0.016 1.154±0.013 0.031±0.015 n.a. 0.413±0.368 s e n i g a m i d l o -y a d -1 0.032±0.021 0.871±0.016 0.028±0.017 n.a. 0.311±0.287 s e n i g a m i d l o -k e e w -2 0.028±0.019 2.683±0.015 0.024±0.014 n.a. 0.912±0.787 s e n i g a m i d l o -k e e w -3 0.023±0.020 3.352±0.014 0.016±0.011 n.a. 1.131±1.099 s e n i g a m i d l o -k e e w -4 0.016±0.021 2.769±0.015 0.012±0.013 0.006±0.009 0.933±0.881 n a e m l a r e v O 0.033±0.019 1.871±1.152 0.022±0.017 0.006±0.009
Explanations: # metalloprotease activities were not observed in the clean habitat and their values were zero. Therefore, they were not included in the table; * as in tab. 1.
Tab. 4. PAGE zymography of the cuticle-metalloprotease activity in A. mellifera workers from the polluted habitat
of the winter period, the metallopro-teolytic activities (in workers only) were the lowest.
The electrophorograms (tab. 4) show high- and low-molecular metal-loproteases observed in all develop-mental stages only in the polluted habitat. These protease bands were not observed in the clean-environ-ment samples. In each developmen-tal stage the medevelopmen-talloprotease mole-cular weight was about 66 kDa. The highest number of bands in the elec-trophorograms was observed for the eggs, larvae and pupae. The bands were very vivid and broad in the case of high-molecular proteases in all seasons. The spring larvae and spring and autumn pupae (tab. 4 A, C) also had low-molecular proteases with molecular weights ranging from 25 kDa to 28 kDa. In the mature bees, the bands were far less pronounced. The spring and summer samples (tab. 4 A, B) contained proteases with the molecular weight of about 43 kDa. Additionally, the imagines also had low-molecular proteases (about 26 kDa) in spring.
These results demonstrate that metalloprotease activity varied, abo-ve all, according to the degree of pol-lution of the respective environment. It also varied with developmental phases and seasons of the year. The research vindicated a well-known claim that bees are environmental bioindicators. If a bee colony lives in a polluted environment, the plant material used by the bees and the air they breathe are also contaminated. As a result, some of the pollution accumulates in their systems as well (10, 13, 24). This can cause disequi-librium in the homeostasis of the honeybees, involving the destabilisa-tion of the immune system which in-cludes anatomical and physiological body surface structures and the pro-teolytic system along with metallo-proteases. Most probably, these en-zymes are synthesised on the body surface of bees owing to the effect of environmental pollution factors, the same as in the case of humans (15).
The highest enzymatic activity was observed in the summer period in the polluted environment alone. This
C. Autumn Tab. 4. B. Summer
Explanations: Rf ranges of the protein migration path (0-40 high-molecular; 41-60 medium-molecular; 61-100 low molecular), OD width of the bands, MW molecular standard
pattern probably results from the position and nume-rous functions of workers in a bee colony. During the vegetative season (in summer) mature workers are the most exposed to pathogen infection (2, 9), and it was in them that high values of metalloprotease activity were observed. Considering insect body-surface im-munity, a parallel role may be played by metallopro-teases in the control of microbial biofilms in ants (5, 6). Such speculations would be supported by the detection of specific metalloproteases (only o-phenan-troline-inhibited) and their inhibitors and by the ana-lysis of typical interactions between them (11, 14, 23, 30). The body-surface proteolytic system may be in-volved in pheromone metabolism (4, 7), as indicated by high metalloprotease activities in the worker ima-gines from summer. In the case of the winter period, a very low body-surface protein concentration and metalloprotease activity were observed. This situation may be related to the low temperature of the environ-ment and the changing metabolism in bees in this season (they accumulate excrements in their rectums) (20, 21, 31).
Metalloproteases are a protease group which is very important for combating diseases and infections on the body surface of honey bee workers. Workers, which synthesise and secrete metalloproteases on their body surface, can be treated as bioindicators of environ-mental pollution.
References
1.Atwood J., Steed J.: Encyclopedia of supramolecular chemistry. Teylor and Francis, New York 2004, 1632-1636.
2.Bania J., Polanowski A.: Bioinsecticides and insect defense mechanisms. Post. Biochem. 1999, 45, 143-150.
3.Billingsley P.: Blood digestion in the mosquito, Anopheles stephensi Liston (Diptera: Culicidae): Partial characterization and post-feeding activity of midgut aminopeptidases. Arch. Insect Biochem. Physiol. 1999, 15, 116-146. 4.Cornette R., Farine J., Quennedey B., Riviere S., Brossut R.: Molecular characterization of Lma-p45, a new epiculticular surface protein in the cock-roach Leucophaea maderae (Dictyoptera, oxyhaloine). Insect Biochem. Mol. Biol. 2002, 32, 1635-1642.
5.Currie C.: A community of ants, fungi and bacteria: a multilateral approach to studying symbiosis. Annu. Rev. Microbiol. 2001, 55, 357-380. 6.Currie C., Scott J., Summerbell R., Malloch D.: Fungus-growing ants use
antibiotic-producing bacteria to control garden parasites. Nature 1999, 398, 701-704.
7.Evans J., Aronstein K., Chen Y., Hetru C., Imler J., Jiang H., Kanost M., Thompson G., Zou Z., Hultmark D.: Immune pathways and defence mecha-nisms in honey bees Apis mellifera. Insect Mol. Biol. 2006, 15, 645-656. 8.Fox J., Serrano S.: Structural considerations of the snake venom
metallopro-teinases, key members of the M12 reprolysin family of metalloproteinases. Toxicon 2005, 45, 969-985.
9.Gliñski Z., Kostro K., Luft-Deptu³a D.: Apian diseases. PWRiL, Warszawa 2006, 37-73.
10.Gromisz Z.: Apian protection against contamination. PWRiL, Warszawa 1990, 37-55.
11.Harrison C., Dowdle E.: Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymeri-zed substrates. J. Apic. Res. 1980, 23, 61-63.
12.Heussen C., Dowdle E.: Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and co-polymeri-zed substrates. Anal. Biochem. 1980, 102, 196-202.
13.Hoffel I.: Schwermetallen in Bienen und Bienenprodukten. Apidologie 1985, 16, 6-197.
14.Imamura N., Ishikawa T., Takeda K., Fukami H., Konno A., Nishida R.: The relationship between a leaf-rolling moth (Dactylioglypha tonica) and fungi covering the cocoon. Biosci. Biotechnol. Biochem. 2001, 65, 1965-1969.
15.Jorma E. Fräki, Väinö K. Hopsu-Havu: Human skin proteases. Arch. Derm. Forsch. 1972, 243, 153-163.
16.Laemmli U.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680-685.
17.Lee T., Lin Y.: Trypsin inhibitor and trypsin-like protease activity in air- or submergence-grown rice (Oryza sativa L.) coleoptiles. Plant Sci. 1995, 106, 43-54.
18.North M.: Comparative biochemistry of the proteinases of eukaryotic micro-organisms. Microbiol. Rev. 1982, 46, 308-340.
19.Pavlukova E., Belozersky M., Dunaevsky Y.: Extracellular proteolityc enzy-mes of filamentous fungi. Biochemistry (Moscow) 1998, 63, 1059-1089. 20.Pliszczyñski M., Che³miñski M., Bizoñ K.: Hemocytic immune parameters of
the wintering workers of the honey bee Apis mellifera L. (Apidae). Ann. Univ. Mariae Curie-Sk³odowska 2006, 20, 157-172.
21.Pliszczyñski M., Luft-Deptu³a D., Bizoñ K.: Monitoring the resistance of win-tering honeybee workers, Apis mellifera L. (Apidae) based on the protective activity test. Ann. Univ. Mariae Curie-Sk³odowska 2006, 21, 173-178. 22.Ray J., Stetler-Stevenson W.: The role of matrix metalloproteases and their
inhibitors in tumour invasion, metastasis and angiogenesis. Eur. Respir. J. 1994, 7, 2062-2072.
23.Rohlfs M.: Clash of kingdoms or why Drosophila larvae positively respond to fungal competitors. Front. Zool. 2005, 2, 1-7.
24.Roman A.: Bees and bee products as environmental pollution bioindicators in areas influenced by copper (LGOM) and cement and lime (Opole) indu-stries. Zesz. Nauk. AR, Wroc³aw 1997, 323, 175-195.
25.SAS Institute (2002-2003) SAS/STAT Users Guide release 9.13, Cary, NC, Statistical Analysis System Institute, license 86636.
26.Schacterle G., Pollack R.: Simplified method for quantitative assay of small amounts of protein in biological material. Anal. Biochem. 1973, 51, 654--655.
27.Walter R., Clèlia F.: Insect digestive enzymes: properties, compartmentaliza-tion and funccompartmentaliza-tion. Comp. Biochem. Physiol. 1994, 109B, 1-62.
28.Wawrzycka-Kaflik A., Pe³ka M., Broniarczyk-Dy³a G.: Metalloproteinases of the extracellular matrix and their tissue inhibitors biochemical profile and clinical importance. Dermatologia Estetyczna 2007, 51, 10-16.
29.Zeeuwen P.: Epidermal differentiation: The role of proteases and their inhibitors. Eurpean J of Cell Biol. 2004, 83, 761-773.
30.Zhang N., Suh S., Blackwell M.: Microorganisms in the gut of beetles: evidence from molecular cloning. J. Invertebrate Pathol. 2003, 84, 226-233. 31.¯ó³towska K., Fr¹czek R., Lipiñski Z.: Hydrolases of developing worker brood and newly emerged worker of Apis mellifera ceranica. J. Apic. Sci. 2011, 55, 27-35.
Corresponding author: Aneta Strachecka PhD, Akademicka 13, 20-950 Lublin; e-mail: aneta.strachecka@up.lublin.pl
