[Note]
Proteomic Assessment of Dung Beetle, Copris tripartitus Immune Response
Hwa-Jin Suh, Hea-Son Bang, Seong-Ryul Kim, Eun-Young Yun, Kwan-Ho Park, Bo Ram Kang, Iksoo Kim1, Jae-Pil Jeon2 andJae-Sam Hwang*
National Academy of Agricultural Science, RDA, Suwon 441-100, South Korea
1College of Agriculture & Life science, Chonnam National University, Gwangju 500-757, South Korea
2KoreaNational Institute of Health, Seoul 136-713, South Korea (Received 06 November 2008; Accepted 02 December 2008)
Dung beetle larvae at the 3rd instar were injected with lipopolysaccaride and inducible proteins were exam- ined within a pI level of 3-10 and a size level by pro- teomics, including 1-D SDS PAGE analysis and antibacterial assay. The immune infected larvae extracts provided seven protein bands in one-dimen- sional electrophoresis and its antibacterial activity also checked. Hemolymph protein from immune infected larvae of the dung beetle were separated by two- dimensional gel electrophoresis and compared with those from native larvae. In 2-D gel electrophoresis, we detected 63 immune infected unique and 32 up-regu- lated proteins, and 36 proteins that were down-regu- lated or not present in treated gel. Ten protein spots from unique proteins and those presented as different level of abundance in infected and native larvae were specially expressed. These differentially expressed pro- teins were proposed to be involved in the defense mechanism against microorganism.
Key words:Copris tripartitus, Proteomics, Antimicrobial protein, Hemolysis, Two-dimensional electrophoresis
Introduction
Living organisms are exposed daily to microbial infec- tions and pathogens. In order to defend themselves against the unclean environment, they have developed potent
defensive mechanism that is a part of innate and adaptive immunity (Bulet et al., 2004). AMPs play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals (Koczulla and Bals, 2003; Lehrer et al., 1993;
Zasloff, 2002). There has been particular interest in these peptides as a new class of antibiotics with potential clin- ical value. In addition, they are known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses (Boman et al., 1991).
Previous works in insect AMPs are classified into four major types by structure and size: (i) peptides with an a- helical conformation (insect cecropins, marginins, etc.), (ii) cyclic and open-ended cyclic peptides with pairs of cysteine residues (defensins, protegrin, etc.), (iii) peptides with an over-representation of some amino acids (proline rich, glycine rich, histidine rich, etc.), and (iv) lysozymes.
In recent years, a series of novel AMPs have been dis- covered as processed forms of large proteins. Despite the extreme diversity in their primary and secondary struc- ture, all natural AMPs have the in vitro particularity to affect a large number of microorganisms (bacteria, fungi, yeast, virus, etc.) with identical or complimentary activity spectra.
Insects have developed an efficient host defense against microorganism. Numerous data highlight similarities between defense responses of insects and innate immunity of mammals. We are interested in understanding the biol- ogy of dung beetle Copris tripartitus in order to seek new antibacterial peptides. Here we present results from pro- teomic comparison of immune infected and native sam- ple. We attempted 2-dimensional electrophoresis (2-DE) using hemolymph. In this study, we focused on how we have approached to seek a new AMPs candidate protein from this insect larvae and the character of its antibacterial
Industrial Entomology
*To whom the correspondence addressed
National Academy of Agricultural Science, RDA, Suwon 441- 100, South Korea. Tel: +82-31-290-8573; Fax: +82-31-290- 8543; E-mail: hwangjs@rda.go.kr
activity.
Materials and Methods
Animals and Immunization
Dung beetle, Copris tripartitus Waterhouse was collected in the field of the Jeju Island, South Korea, and cultured in a room kept at 25oC (Bang et al., 2003). Third-instar lar- vae were cooled on ice and injected with lipopolysaccar- ide (LPS, 2µg) suspended in a saline buffer. The larvae were kept at 25oC for 24 hrs. To eliminate the intestines, they were grinded in liquid nitrogen with care.
Electrophoresis and Image analysis
SDS-PAGE was performed on 12.5% acrylamide gels system (Laemmli, 1970) and 2-DE was performed in a horizontal apparatus (IPGphor and Hoefer 600 SE; Amer- sham Biosciences, Uppsala, Sweden). For the analytical gels, 1 mg of protein was applied onto immobilized pH gradient (IPG) strips (24 cm, pH 3~10) containing Immo- bilines pH 3~10, thiourea, CHAPS, and urea, according to the method of Rabilloud et al. After isoelectric focus- ing, the strips were applied to the top of SDS-PAGE gel (9
~17% gradient), and the proteins were separated accord- ing to their molecular mass. The SDS-PAGE gels were stained using Coomassie Brilliant Blue G250 to detect proteins.
Digitized images of the stained gels were analyzed using the 2-DE gel analysis program Image Master Plat- inum 5 (Amersham Pharmacia Biotech, Uppasla, Swe- den). A comparison report of the qualitative and quan- titative differences between the samples for each data set of was generated.
In-gel digestion and MALDI-TOF Mass spectrometry Differentially expressed protein spots were excised from the gels, cut into smaller pieces, and digested with trypsin (Promega, Madison, WI, USA) (Shevchenko et al., 1996).
For MALDI-TOF MS analysis, tryptic peptides extracts for each spot were pooled, lyophilized, and stored at −20oC.
Digested peptide samples were dissolved in 0.1% for- mic acid, and concentrated and cleaned in a C18 column.
Peptides were separated by application of the gradient condition (0~90% 0.5M ammonium acetate, 0.1% formic acid). Clean fractions were collected and directly onto a MALDI plate by a Probot spotting robot. Matrix solution (CHCA containing 10 mM ammonium citrate in 60%
ACN) was mixed with column eluent at a flow rate of 1µl/min. The dried droplets were analyzed using a 4700 proteomics Analyzer (Applied Biosystems, MA, USA) in MS and automated MS/MS mode. All samples were irra-
diated at 355 nm using a Nd:YAG laser. A source accel- eration voltage of 25 kV was used with two-stage re- flection in MS mode. In the MS/MS experiment, collision energy was set to 1 kV. Data were searched using MAS- COT (Matrix Science, London, UK) as the search engine with the MASCOT database.
Assay of antibacterial activity and Hemolysis
Bacteria were grown to mid-log phase in 10 ml of full- strength(3%, w/v) trypticase soy broth (TSB) (BD Bio- sciences). The microorganisms were washed once with PBS, and then 2×104 bacterial colony-forming units (CFU) were added to 10 ml of the underlay-agarosegel (0.03% TSB, 1% low electroendosmosis-type agarose, 1 mM sodium citrate, and 9 mM sodium phosphate (pH 7.4). The underlay was poured into a100×15 mm dish. After agarose solidification, 3 mm-diameterwells were punched, and 5µl of test peptide was addedto each well. Plates were incubated at 37oC for 1 h toallow dif- fusion of the peptides. The underlay gel was then covered with 10 ml of molten overlay (6% TSB and 1% low elec- troendosmosis-typeagarose in distilled H2O). The anti- microbial activity of a peptidewas visualized as a zone of clearing around each well afterincubating 18~24 hrs at 37oC (Lehrer et al., 1991).
Erythrocytes freshly prepared from mouse blood were suspended in PBS to an 8% suspension and then rinsed three times, and resuspended in PBS. Red blood cells (RBC) of the suspension (4% in PBS, v/v) were plated in 96-well microtiter plates, after which an equal volume of the peptide solution dissolved in PBS was added. Plates were incubated for 2 hrs at 37oC and then centrifuged at 1000 g for 5 min. Aliquots (100µl) of the supernatant were then transferred to a new 96-well microtiter plates, where released hemoglobin was monitored using micro- plate ELISA reader (Molecular Devices) by measuring the absorbance at 405 nm. The degree of hemolysis was assessed by hemoglobin content in the supernatant of the incubated samples. Zero and total hemolysis were deter- mined in PBS and 0.1% Triton X-100, respectively.
Results and Discussion
SDS-PAGE analysis of larvae extract and Antibac- terial activity
One-dimensional SDS-PAGE analysis of larvae extracts between native and infected larvae extract is shown in Fig. 1. In lane 4, protein band located in closed circle is caused by infection of LPS. Even though the precise mechanism of action of these proteins is not fully under- stood, the proteins are expected to exert their biological
function by enhancing the protecting action of pathogenic cells. Thus, these were analogized that they seem to have an antibacterial activity. To further understand the protein,
we performed radial diffusion assay (RDA). Antibacterial activities of larvae extracts were examined against E. coli,
Staphylococcus and Streptococcus. The activity of growth inhibition was determined by RDA methods (Fig. 2).
Although not evident against bacteria, the infected larvae extracts showed some effect considering the size of the inhibition zone compared with native. Cytotoxicity of the hemolymph against mammalian cells was studied by mea- suring hemoglobin release at 405 nm from 4% suspen- sions of fresh mouse red blood cells. Erythrocytes lysis induced by adding melittin that is well-known cytotoxic peptide resulted in a sharp increase, whereas addition of hemolymph did not increased with the increase in the quantity. A slight increasing was observed at the quantity above 50µl addition (data not shown).
Differential expression of proteins
To examine the differential expression of proteins in hemolymph from infected dung beetle larvae and native controls, proteomic analysis was performed using high- resolution 2-DE. Fig. 3 is a representative mater gel image showing the separation of proteins from native beetle hemolymph. A comparison of the location and volumes of each spot showed that the majority of hemolymph pro- teins remained virtually unchanged. Several tens of total spots in Coomassie Blue-stained samples showed two- fold or higher differences between the two samples. These include 63 unique and 32 upregulated, and 36 downreg- ulated protein spots. Only proteins that showed relative abundance judged by the Coomassie Blue-staining and those showed at least the four-fold difference between native and infected were selected. Among them 10 from infected larvae are indicated in Fig. 3B. These differen- tially specifically expressed proteins were identified by MASCOT search engine.
As shown in Table 1, differentially expressed proteins from infected larvae were identified based on the Droso-
phila protein database. Spot no. 1235 was acidic and had a relatively small molecular mass. However, it was iden-
Fig. 1.SDS-PAGE analysis of larvae extract. Crude extracted protein was analyzed on 9~17% gradient SDS-PAGE and stained with Coomassie Blue: lane 1, native extract pellet; lane 2, native extract supernatant; lane 3, infected extract pellet;
lane 4, infected extract supernatant; and M, molecular weight.
Closed triangle (◀) indicate differential expressed protein.
Fig. 2.Antibacterial activity between native and infected lar- vae extracts against Esherichia coli, Staphylococcus aureus and Streptococcuspyogenes using radial diffusion assay.
Fig. 3.Two-dimensional electrophoresis of hemolymph proteins from native (A) and infected larvae (B) of C. tripartitus. Protein were separated by IEF in the first dimension, pH 3~ 10, then by size in the second dimension and stained with Coomassie Blue.
Arrows were specially expressed proteins.
tified as unknowns by database search. Spot no. 1238 and 1508 were slightly acidic, while others were basic. The database reported that spot no. 677 is GTP-binding nuclear protein RAN, spot no. 1206 has signal transducer activity, and spot no. 1238 has ATP-dependent RNA heli- case activity. Because the values of sequence coverage were relatively small in this case, we cannot exclude the possibility that spot no. 1235, 1238, 1508 are novel polypeptides. Spot no. 1178, 1235, and 1508 were iden- tified as unknowns by database search and suggested to be novel polypeptides induced by injection of LPS. Further- more, if these differentially specifically expressed pro- teins were made by amino acid synthesis or protein over expression, it was fairly interesting and valuable.
To identify diverse polypeptides rapidly and to monitor changes in them, the proteomics by peptide mass finger- printing method using 2-D gel electrophoresis, in-gel tryptic digestion, MALDI-TOF MS and database search- ing are one of the most powerful tools. The appeal of a proteomics analysis is that it is one step closer to func- tionality than a study based on mRNA. While mRNA based studies are enormously useful, one cannot be cer- tain that the transcripts are actually translated unless the work is complemented with protein studies (Gygi et al., 1999; Renaut et al., 2006). By working at the protein level it is also possible to detect multiple forms of a protein that
may be the consequence of families of closely related pro- teins or post-translational modifications (Li et al., 2007).
Proteomic knowledge of insect innate immunity is still limited in comparison with genomic knowledge of it. Fur- ther proteomic work is in progress in our laboratory.
In conclusion, a comparison of the result suggests that many of the proteins present in the hemolymph may be synthesized after immune stimulation, and implied that these proteins are related to immune defense mechanism against microorganism. The aforementioned methods to find out antibacterial proteins are fairly valuable.
Acknowledgements
This study was supported by Post Doctoral Course Pro- gram of National Academy of Agricultural Science, Rural Development Administration, Republic of Korea.
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Table 1.Identification of differentially expressed proteins base on the Drosophila protein database (Hemolymph) Spot no. Accession no. Molecular function Matched
peptide no.MW(kDa)
/pl Coverage
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677 AAF30287 GTP-binding nuclear protein RAN 7 24.9/7.63 40 4.7
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1206 NP_729679 signal transducer activity 12 120.1/9.04 15 5.5
1235 EAL26422 unknown 7 17.1/4.59 34 4.4
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1508 XP_001347662 unknown 12 62.6/5.38 33 -
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1627 EAL31926 unknown 5 33.1/9.80 20 2.2
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