Identification and Characterization of Peptides Binding AgEG1 from a Phage Display Library Chen Min1

Zhang Zhi-yi2*

1

College of Resources and Environment, Beijing Forestry University, Beijing 100083, P. R. China

2

College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, P. R. China

ABSTRACT

Endoglucanases are the main cellulolytic enzymes of Anoplophora glabripennis. Their high activities in cellulose

digestion as well as its good kinetic properties make it an attractive target for development of cellulase inhibitors. In this study, random peptide phage display technology was employed to identify peptides that bound the AgEG1, a member of endoglucanase isozymes. Phage clones with peptide LPPNPTK and XPP (X is residue T, L, A or H) motif frequently occurred in the selected phage population and showed a higher phage recovery than other clones. Peptide LPPNPTK was chemically synthesized and characterized for its binding activities to AgEG1. The synthetic peptide exhibited high specificity for AgEG1. The peptide LPPNPTK has the potential to be developed into inhibitors of the endoglucanase of A. glabripennis. KEY WORDS

larvae of Anoplophora glabripennis, random peptide phage display library, AgEG1, synthetic peptide

[Supported by the National Natural Science Foundation of China (Grant No. 39900116)]

1

Introduction

Larvae Anoplophora glabripennis Motsch (Coleoptera: Cerambycide: Lamiinae) are major borers of poplars, willows, elms, etc. and are distributed in most provinces of China (Xiao 1991). Conventional pest control approaches are less effective due to the borer’s biological characteristics such as a long generation time and living inside the stem of the host trees. There are considerable amounts of cellulases in the gut to digest cellulose from the food for these wood-feeding animals. Therefore, cellulase can serve as the target for developing and implementing new methods of control, such as investigations of cellulase inhibitors. However, studies on insect cellulases have mainly focused on the origin of the cellulose digestion (Kuker and Martin 1986, Kuker et al. 1988, Jiang et al. 1996) and few insect cellulases have been purified and characterized (Tokuda et al. 1997). It has been reported that enzymatic hydrolysis of cellulose requires synergistic action of endoglucanase (EC 3.2.1.4, EG), β-glucosidase (EC 3.2.1.21, BG) and exoglucanase (EC 3.2.1.91, CBH) (Yan 2000). We have previously reported that endoglucanase and β-glucosidase are major cellulolytic enzymes in the gut of A. glabripennis and endoglucanase has the highest activity as well as good kinetic properties such as a wide range of pH and temperature and high thermal stability for the enzymatic activity which makes it an attractive target for the development of cellulase inhibitors. In order to exploit inhibitors of endogluca*

Author for correspondence. E-mail: [email protected]

nase of A. glabripennis, we investigated the characteristics of endoglucanase isozymes and detected two isozymes of endoglucanase, denoted AgEG1 (also named IsoA, 26 kD) and AgEG2 (also called IsoB, 39 kD) by chemical coloration and isolated them by elution from the native gel (Chen et al. 2002). The AgEG1 sample is easier to prepare for its higher content in the crude extract than AgEG2. Therefore, in this study we attempted to identify peptides that specifically and tightly bound the AgEG1 target. Advances in powerful combinatorial technologies such as phage display library suggest new approaches for selecting protease inhibitors. A phage display peptide library is a mixture of filamentous phage with foreign peptides on their surface and the coding sequences for the peptides in the viral DNA. Surface display is accomplished by fusing the peptide coding sequence to a coat protein gene—either the minor coat protein cpⅢ or the major coat protein cpⅧ (Scott and Smith 1990). Each phage clone displays a single peptide, but a library as a whole may represent billions of peptides altogether. The vital advantage of surface exposure is that it allows phage display libraries with vast numbers of peptides to be easily surveyed for clones whose displayed peptides bind specifically to any given molecular target. A number of peptides that bind receptor molecules (Natalia et al. 2002) and enzyme inhibitors (Jespers et al. 1995, Xu et al. 1998, Li and Wang 2001) have been identified using phage display libraries. In this study, we identified peptides using a hepForestry Studies in China, 7(4): 1–4

2

tapeptide phage display library against AgEG1. One of the isolated peptides, LPPNPTK (p1) and the XPP motif frequently appeared in the selected phage population and exhibited higher phage recovery than other clones. The 7-amino acid p1 peptide was chemically synthesized for examination of its binding affinities and specificities. Results of this further analysis demonstrated that the synthetic peptide specifically bound the AgEG1. Therefore the p1 peptide has the potential to be used as a vehicle for delivery of the inhibiting agent to AgEG1 for the control purpose of A. glabripennis.

2 Materials and methods 2.1 Phage display peptide library The heptapeptide phage display library (Ph.D-7Tm Phage display peptide library Kit, #E8100S) was purchased from New England Biolabs (NEB). The E. coli ER2738 host strain and the sequencing primer 5′-CCCTCATAGTTAGCGTAACG-3′ came along with the kit. 2.2 Insects The larvae of A. glabripennis were collected from trunks of infected poplar trees in the suburb of Tianjin City, northern China, and maintained indoor by their natural food. 2.3 Purification of the AgEG1 The enzyme extract was prepared from the gut of A. glabripennis larvae and the AgEG1 was located by chemical coloration on the carboxymethylcellulose polyacrymide gel (CMC-gel) and then eluted from native polyacrylamide gel slices according to the methods described in our previous study (Chen et al. 2002). 2.4 Biopanning Heptapeptide phage display library was used for screening in the biopanning procedure. The selection was performed according to the manufacturer’s protocol (available on http://www.neb.com) with minor modifications. Briefly, wells of the microtiter plates were coated with the target (100 µg·mL–1) in 0.1 mol·L–1 NaHCO3 (pH 8.6) and then filled with blocking buffer (0.1 mol·L–1 NaHCO3, pH 8.6, 5 mg·mL–1 BSA, 0.02% NaN3). The phage display library was incubated first with native polyacrylamide gel slices in TBS (50 mmol·L–1 Tris-HCl, pH 7.5, 150 mmol·L–1 NaCl) to remove phage clones binding to polyacrylamide gel, then incubated with coated wells for biopanning. The plates were washed with TBST (TBS + 0.1% (V/V) Tween-20) to remove unbound phage, and bound phage was eluted using elution buffer (0.2 mol·L–1 Glycine-HCl, pH 2.2). The increased recovery,

Forestry Studies in China, Vol.7, No.4, 2005

defined as the ratio of bound phage (yield) to input phage, served as an intrinsic indicator of successful biopanning. After each round of screening, except for the last, the elution of the bound phage was amplified and used as the input for the next round. Individual clones were characterized by DNA sequencing after the third round of selection. 2.5 Binding activities of the selected phage clones Individual phage clones from the third round of screening were isolated randomly, amplified respectively and used as the input for another round of selection to examine their binding activities with AgEG1. Phage recovery reflects the binding activities of the selected phage clones 2.6 Peptide sequence analysis and peptide synthesis Individual phage isolates were sequenced on the ABI Prism 310 using BigDye Terminator V3.0 Ready reaction cycle sequencing kit (from ABI Inc.). The sequence similarity searches were performed using the BLAST program provided on website http://ncbi. nlm.nig.gov/BLAST/. The peptide with consensus sequence was chemically synthesized and biotinylated by Beijing Sai Bai Sheng (SBS) Company. 2.7 Dot blot analysis of binding activity of the p1 peptide Purified AgEG1 and bovine serum albumin (BSA) were immobilized on a nylon membrane at various quantities (2–0.1 µg). After washing for 1–5 min with TBS, the membrane was blocked with 1% blocking reagent (Roche Molecular Biochemicals, IN) in TBS for 1 h at room temperature, washed with TBS and then incubated with synthetic peptide (1 µg·mL–1) in a blocking buffer with a gentle agitation for 2 h at 4°C. Afterwards the membrane was washed with TBS and incubated with alkaline phosphatease-labeled streptavidin (SA-AP) for 1 h at room temperature. A color reaction was performed using 5-bromo-4-choro-3indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) solution (Roche Molecular Biochemicals, IN) in the dark at room temperature.

3 Results 3.1 Purification and characterization of the AgEG1 The AgEG1 was detected by chemical coloration on the CMC-gel and then purified from native polyacrylamide gel slices as described previously (Chen et al. 2002). The identity of the AgEG1 was confirmed by endoglucanase activity analysis using 1% (W/V) carboxymethylcellulose (CMC, from Sigma) as substrate. The purified protein yielded a single band by

Chen Min et al.: Identification and Characterization of Peptides Binding AgEG1 from a Phage Display Library

sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of ~26 kD (Fig. 1). The isolated AgEG1 was used in the following experiments.

FIGURE 1

Isolated AgEG1 analyzed by SDS-PAGE

Lane A: AgEG1; Lane M: Protein MW marker

3.2 Isolation of peptide sequences that bind AgEG1 A random 7 amino acid peptide phage display library was selected against the AgEG1 to identify its affinity peptides. The library was based on the M13 phage vector and the displayed heptapeptides were expressed at the N-terminus of the minor coat protein pⅢ. Three rounds of selection were employed to enrich phage population for virions that displayed peptides with affinity to AgEG1 (Table 1). TABLE 1

Phage recovery during the screening

Rounds of screening

Target molecule

Input (pfu)

Output (pfu)

Recovery* (%)

Ⅰ

AgEG1

2×1011

3×104

1.5×10–5

Ⅱ

AgEG1

3×10

11

5

6.7×10–5

Ⅲ

AgEG1

2×1011

2×106

1.0×10–3

2×10

3

the propagation properties of the particular clone. TABLE 2

DNA and amino acid sequence of

the isolated peptides Clone DNA sequence a1 a2 a3 a4 a5 a6 a7 a8 a9 a10 a11 a12

5′-GATCCGGGGGCTACTCCTCCT-3′ 5′-ACGCTTACGCCGCCGCCTCTG-3′ 5′-CCGCAG TCTTCGCCTCCTCAT-3′ 5′-ACTAATCGTTTGCATCCTCCG-3′ 5′-ACTCGTCTGCCGCCTCTGTCG-3′ 5′-TTGCCGCCTAATCCGACGAAG-3′ 5′-TTGCCGCCTAATCCGACGAAG-3′ 5′-TTGCCTTTGTCGGGTAATGAT-3′ 5′-GCTCCTCCGGGTCGTCCTACG-3′ 5′-GCGAAGGCTCTGCCTCATACG-3′ 5′-GCTCCTCGTCATAATACGCAG-3′ 5′-GCTGCGCCGCTGCATAATAAG-3′

Amino acid sequence DPGATPP TLTPPPL PQSSPPH TNRLHPP TRLPPLS LPPNPTK LPPNPTK LPLSGND APPGRPT AKALPHT APRHNTQ AAPLHNK

3.3 Binding activities of the selected phage clones Selected phage clones were amplified and screened with immobilized AgEG1 to verify their binding abilities by using the same input of the library as control. Data from this experiment are presented in Fig. 2. The phage clones with the peptide LPPNPTK (a6 and a7) exhibited the highest affinity by phage recovery of 4×10–2–5×10–2 and the clones with the XPP motif (a1–a5, a9) also showed different binding activities by recovery of 3.5×10–3–2×10–2, while the others was 10–4–10–6 by recovery. This supported the idea that the enriched phage clones had affinity to AgEG1 and suggested that the LPP motif played a critical role in the interaction. Further investigation of the p1 peptide was pursued due to the high presentation in selection phage population and high binding activities.

*

Recovery (%) = output/input ×100

The phage recovery increased 67 times after three rounds of biopanning, which indicated that the screening was successful. Twelve phage clones (a1–a12) from the screening were selected randomly and DNA from the isolated clones was sequenced. The sequences of peptides exposed on the surface of these phages were identified (Table 2). Analysis of these sequences revealed 2 out of 12 phage clones encoding the same LPPNPTK peptides (p1) and the XPP (X is residue T, L, A or H) motif also appeared in the other 6 phage clones. Within these clones we found several nucleotide sequences with different bases in the third position of codons but the same amino acid sequence. This supports the fact that the peptide was selected due to the peptide-protein interaction and not due to

FIGURE 2

Phage recovery of the selected clones screened against AgEG1

3.4

Binding activities of p1 peptide To characterize further the identified peptide, it was important to verify that the peptide is capable of binding AgEG1 in the absence of phage particle. Therefore we had the p1 peptide chemically synthesized and dot blot analysis was performed to test the binding activities of the p1 peptide to AgEG1 with BSA as a control. AgEG1 and BSA were spotted on a nylon membrane at various amounts and exposed to

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Forestry Studies in China, Vol.7, No.4, 2005

the biotinlylated p1 peptide. The p1 peptide bound only AgEG1 with significant dose-dependent effect and did not react with BSA (Fig. 3). This provides direct evidence that the binding between the target and the phage clones are caused by the fused peptide itself rather than the coat protein or specific structure carried by the phage and also demonstrates that the P1 peptide is specifically bound to the AgEG1.

conclusion, the peptide LPPNPTK has the potential to be developed as inhibitors of the endoglucanase of A. glabripennis.

References Chen M, Lu M Z, Zhang Z Y. 2002. Characteristics of cellulases from Anoplophora glabripennis Motsch (Coleoptera: Cerambucidae). Forestry Studies in China. 4(2): 43–47 Jespers L S, Messens J H, Dekeyser A, Eeckhout D, Van den Brande I, Gansemans Y G, Lauwereys M J, Vlasuk G P, Stanssens P E. 1995. Surface expression and ligand-based selection of cDNAs fused to filamentous phage gene Ⅵ. Bio-Technolog. 13: 378–382

FIGURE 3

Dot blot analysis of the p1 peptide against AgEG1 and BSA

4 Discussion Endoglucanases are the major cellulolyric enzymes for larvae beetles (Yin et al. 1996, Chen et al. 2002), thus it can serve as the target for the development of the cellulase inhibitors. However, up to date there have been few reports on inhibitors of animal cellulases. Based on our previous study on the purification and characterization of the endoglucanase of A. glabripennis, in the current investigation we used a phage display library to search for potential inhibitors of the endoglucanases. By screening against AgEG1, a member of endoglucanase isozymes of A. glabripennis, we identified that the LPPNPTK peptide enriched in the selection had a high affinity to AgEG1. Remarkably, the XPP motif appeared frequently in the peptides of the selected phage clones, which is consistent with the result of our previous results of in vivo biopanning against the gut of the living larvae A. glabripennis. Based on these facts we suggested that the XPP motif serves as a critical element in binding to AgEG1. Further dot blot analysis confirmed that the synthesized LPPNPTK peptide specifically bound to AgEG1. Attempting to test the inhibitory activities of the identified peptide, we measured the enzymatic activities of the AgEG1 in the absence and in the presence of the p1 peptide, and the analysis indicated that the peptide p1 was not able to inhibit enzymatic activities of protein AgEG1 (data not shown). This might be interpreted to mean that the peptide did not interact with the critical site of the molecular structure, such as the substrate binding regions or the active center for the enzymatic function. However, the p1 peptide can be used as a lead by means of delivery and enrichment of some inhibitory agents of AgEG1 to the target organs to strengthen its inhibiting ability. In

Jiang S N, Yin Y P, Wang Z K. 1996. Studies on the cellulases,s resource in some species of longicorn borers (Coleoptera: Ceranbycidae) (in Chinese with English abstract). Scientia Silvae Sinicae. 32(5): 441–446 Kukor J J, Cowan D P, Martin M M. 1988. The role of ingested fungal enzymes in cellulose digestion in the larvae of cerambycid beetles. Physiol Zool. 61(4): 364–371 Kukor J J, Martin M M. 1986. The transformation of Saperda calcarata (Coleoptera: Cerambucidae) into cellulose digester through the inclusion of fungal enzymes in its diet. Oecologia. 71: 138–141 Li J D, Wang K Y. 2001. Selection of α-glucosidase inhibitors from a phage display library. Acta Biochinica et Biophysica Sinica. 33(5): 513–518 Natalia G K, Vladislav V G, Ning X C, Ravichandra K, Thomas P Q. 2002. Identification and characterization of peptides that bind human ErB-2 selected from a bacteriophage. Journal of Proteim Chemistry. 21(4): 287–296 Scott J K, Smith G P. 1990. Searching for peptide ligangs with an epitope library. Science. 249: 386–390 Tokuda G, Watanabe H, Matsumoto T, Noda H. 1997. Cellulose digestion in the wood-eating higher termite, Nasutitermes takasagoensis (Shiraki): distribution of cellulases and properties of endo-b-1,4-glucanase. Zool Sci. 14: 83–93 Xiao G R. 1991. Forest Insects of China, 2nd edn (in Chinese). Beijing: China Forestry Publishing House. 455–457 Xu Z P, Duan Z J, Chen H, Chen C Z, Li B L. 1998. Selection of myristoyltransferase ingibitor phages from phage display random peptides library. Acta Biochinica et Biophysica Sinica. 30(2): 154–158 Yan B X. 2000. Substrate specificity of cellulases. Chinese Bulletin of Life Science. 12(2): 86–88 Yin Y P, Cheng J Q, Jiang S N. 1996. Studies on the kinetic properties of cellulase in Aprina gernari Hope (Coleoptera: Ceranbycidae) (in Chinese with English abstract). Scientia Silvae Sinicae. 32(5): 454–459 (Received July 15, 2005

Accepted September 5, 2005)