Array-Based Rational Design of Short Peptide Probe-Derived from an

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Array-based rational design of short peptide probe derived from an anti-TNT monoclonal antibody Mina Okochi, Masaki Muto, Kentaro Yanai, Masayoshi Tanaka, Takeshi Onodera, Jin Wang, Hiroshi Ueda, and Kiyoshi Toko ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.7b00035 • Publication Date (Web): 28 Aug 2017 Downloaded from http://pubs.acs.org on September 1, 2017

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ACS Combinatorial Science

Array-based rational design of short peptide probe derived from an anti-TNT monoclonal antibody Mina Okochia,b *, Masaki Mutoa,b, Kentaro Yanaia, Masayoshi Tanakaa,b, Takeshi Onoderab,c, Jin Wangb,c, Hiroshi Uedad, Kiyoshi Tokob,c,e a

Department of Chemical Science and Engineering, School of Materials and Chemical

Technology, Tokyo Institute of Technology, 2-12-1, O-okayama, Meguro-ku, Tokyo 152-8552, Japan b

JST, ImPACT, Sanban-cho 5, Chiyoda-ku, Tokyo 102-0075, Japan

c

Research and Development Center for Taste and Odor Sensing, Kyushu University, Fukuoka,

744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan d

Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of

Technology, R1-18, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503, Japan e

Graduate School of Information Science and Electrical Engineering, Kyushu University, 744

Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan

KEYWORDS: 2,4,6-Trinitrotoluene, Peptide probe, Anti-TNT antibody, Complementarity determining region

1 Environment ACS Paragon Plus


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ABSTRACT:

Complementarity-determining regions (CDRs) are sites on the variable chains of antibodies responsible for binding to specific antigens. In this study, a short peptide probe for recognition of 2,4,6-trinitrotoluene (TNT), was identified by testing sequences derived from the CDRs of an anti-TNT monoclonal antibody. The major TNT-binding site in this antibody was identified in the heavy chain CDR3 by antigen docking simulation and confirmed by an immunoassay using a spot-synthesis based peptide array comprising amino acid sequences of six CDRs in the variable region. A peptide derived from heavy chain CDR3 (RGYSSFIYWF) bound to TNT with a dissociation constant of 1.3 µM measured by surface plasmon resonance. Substitution of selected amino acids with basic residues increased TNT binding while substitution with acidic amino acids decreased affinity, an isoleucine to arginine change showed the greatest improvement of 1.8-fold. The ability to create simple peptide binders of volatile organic compounds from sequence information provided by the immune system in the creation of an immune response will be beneficial for sensor developments in the future.


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INTRODUCTION: Antibodies are among the most useful biomolecules for biosensor development

1,2

.

Complementarity-determining regions (CDRs) in the variable regions of antibodies are regarded as paratopes, which are the sites of specific antigen binding 3. The relationships between amino acid sequences and tertiary structures within the typical loops of each CDR segment generally confer the target molecular-recognition property of the antibody. Small antibody-mimicking molecules, single-chain variable fragment (scFv)

4,5

, and nanobodies

6

containing the paratopes

of constructed CDR loops have been fabricated through genetic engineering techniques to reduce production costs, engineering processes, and immunogenicity. The use of standalone peptides created from the sequences of antibody CDRs has also attracted attention as a way to use a minimum recognition unit of the antibody, mostly against large protein targets

7,8

. For example, synthetic peptides derived from the CDR sequences against the

gp120 envelope glycoprotein of human immunodeficiency virus type 1, and against the F glycoprotein of human respiratory syncytial virus, showed virus-binding function and neutralization properties.7–10 However, to our knowledge, no such extraction of simple peptide sequences from antibodies has been demonstrated for the binding of small molecules. Volatile organic compounds (VOC) are important analytes for a wide range of applications such as noninvasive diagnosis by breath analysis, environmental pollutant monitoring, and explosive detection

11–13

. Several VOC sensors that use affinity biomolecular probes such as antibodies,

olfactory receptors, and peptides have been reported

14–16

. A summary of previously developed

ligands for 2,4,6-trinitrotoluene (TNT) is shown in Table S1. Among these target-recognition biological molecular probes, short peptides have several advantages, including high stability,



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simplicity of library development from a combination series of 20 natural amino acids, chemical synthesis root, and ease of quality control

17

. The use of trinitrotoluene and dinitrotoluene-

recognition peptide probes have been applied for development of selective sensors using field effect transistor, electrochemiluminescence, and quartz crystal microbalance

16,18,19

. The

detection of VOCs such as alcohols and amines have been also demonstrated in gas sensor arrays by using peptide probes screened from olfactory receptor proteins

20,21

. These VOCs binding

peptides have been identified from in silico-based study of olfactory receptor proteins

20,21

and

from the screening by phage display 22–24, however no peptide probes have been identified from anti-VOC antibodies so far. In this study, the design of VOC recognition peptide probes from a monoclonal antibody was demonstrated by the sequence analysis of the CDRs of the developed monoclonal antibody for TNT25 combined with peptide array technology. This simple method should be applicable to other VOCs, which are small enough to be recognized by contiguous peptide sequences of antibody binders. This may help accelerate the development of practical sensors for these important analytes.

RESULTS AND DISCUSSION: Identification of amino acid sequences of variable regions in the anti-TNT antibody We made use here of hybridoma cells producing the known monoclonal rat anti-TNT antibody QM2014-0130, provided at the Material Management Center of Kyusyu University.25 cDNAs coding for different regions of the heavy and light chains (360 bp and 321 bp, respectively) were specifically amplified from total RNA extracts using previously reported primer sets


26

. The

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target amino acid sequences of CDRs in the heavy and light chains were identified using IMGT/V-QUEST

software

to

be

GYSITSHY

(HCDR1),

ISYSGST

(HCDR2),

ARGYSSFIYWFFDF (HCDR3), QDIGNY (LCDR1), SAT (LCDR2), and LQHYSAPYT (LCDR3) (Fig. 1a). These sequences are rich in aromatic amino acids tryptophan, tyrosine, phenylalanine, and histidine, which are capable of π-π stacking with the planar surface of TNT 22,27

. Using this sequence information, the TNT-binding site within the CDR was then simulated

by antigen-docking simulation

28

using Autodock software. This was aided by the use of a 3D

structure of the anti-methamphetamine scFv (PDB ID: 3gm0, which exhibited 67.8% amino acid sequence similarity by SWISS-MODEL) as a homology model 29. The best TNT docking model is shown in Fig. 1b; its score of −5.52 kcal/mol is comparable to that obtained for tetrodotoxin, a small-molecular-weight neurotoxin, docked onto anti-tetrodotoxin scFv 30. Based on this in silico analysis, the binding site of TNT was suggested to be comprised of regions of the heavy and light chains of CDR3 and the light chain of CDR1. The highlight image shows that W and Y of HCDR3, H of LCDR3, and Y of LCDR1 are predicted to be close to TNT, suggesting the importance of these aromatic amino acids.



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(a) Heavy chain HCDR2 HCDR1 EVQLQESGPGLVKPSQSLSLTCSVTGYSITSHYWAWIRKFPGNKMEWMGYISYSGST HCDR3 GYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARGYSSFIYWFFDFWGPGTMVTVSS Light chain LCDR1 LCDR2 DIQMTQSPASLSASLEEIVTITCQASQDIGNYLSWYQQKLGKSPQLLIHSAT LCDR3 SLADGVPSRFSASRSGTQYSLKINRLQVEDTGIYYCLQHYSAPYTFGAGTKLELK

(b) HCDR2

HCDR1 HCDR3 LCDR1 LCDR3

TNT

Y

R

TNT

S

LCDR2

T

G

F

H Y

D

F

Q L

S

Y LCDR3

F

Y W F

Side-view

Y

HCDR3

LCDR1

N

Vertical-view focused on CDRs

Fig. 1 Amino acid sequences of the anti-TNT monoclonal antibody variable region and the simulation analysis of binding between the identified scFv and TNT. (a) Amino acid sequence of variable region in heavy chain and light chain from anti-TNT antibody. The CDR regions were predicted by IMGT/V-QUEST program v3.4.0 (www.imgt.org/IMGT_vquest/share/textes) and indicated with underline and colored fonts. (b) TNT docking model simulated by AutoDock4.2.6 within the identified scFv is shown 28. The homology model of scFv was generated using the SWISS-MODEL website in conformity to template with the highest sequence identities from the database 31. The CDR regions in the heavy and light chains are shown in red and orange, respectively. In vertical-view focused on CDRs, atoms within 10 Å radius of TNT were selected from heavy chain and light chain CDR amino acid residues.


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Screening of TNT-binding peptides from CDRs To identify TNT-binding peptides from the above analysis, a peptide array of sequences derived from these CDR regions was synthesized along with two different positive control peptides, previously identified by phage display.22,32 To assist in the detection of binding, the target molecule was displayed on keyhole limpet hemocyanin (KLH) by reaction of the protein with 2,4,6-trinitrobenzenesulfonate.33 This resulted in the attachment of approximately 150-380 2,4,6trinitrophenyl groups to lysine side chains of the protein (designated TNP-KLH) while allowing the use of a FITC-labeled anti-KLH antibody for detection. As shown in Fig. 2a, the peptides HCDR1 and especially HCDR3 (see Figure 1) arrayed on a paper support were bound by TNP-KLH, while little binding was observed for HCDR2, and LCDR1-3. The phage display-derived peptides (WHRTPSTLWGVI; WHWQRPLMPVSI) 22,32 were also active in this assay, and WHRTPSTLWGVI bound more higher than the other. The specific nature of the peptide recognition of the TNP group was illustrated by three observations: negative control peptides (AAAAAA and GGGGGG) showed little binding to TNP-KLH, the interaction of arrayed HCDR3 was 14-fold greater with TNP-KLH than with KLH alone (Supplementary Fig. 1), and the interaction with HCDR3 with TNP-KLH showed a well-defined dose-response curve (Fig. 2c).



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Fig. 2 Evaluation of TNT-binding activities, using the CDRs fragment-derived peptide array. (a) Image of peptide array after binding assay with TNP-KLH. Each peptide spot was comprised of 6 CDR peptides identified from the anti-TNT antibody with two previously reported TNT binding peptides (1, WHRTPSTLWGVI; 2, WHWQRPLMPVSI)

22,32

and two

negative control peptide (1, AAAAAA; 2, GGGGGG). The black spot derived from the FITCtagged anti-KLH antibody indicates the binding activity between the peptide and TNP-KLH. (b) Quantitative analysis of binding activity between each peptide and TNP-KLH. Binding intensity was evaluated from fluorescent intensity derived from the FITC-tagged anti-KLH antibody. (c) Dose-response curve of TNP-KLH binding with the HCDR3 peptide (ARGYSSFIYWFFDF).


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TNT binding assay using surface plasmon resonance (SPR) Binding of the HCDR3 peptide and TNT was directly evaluated by SPR. The HCDR3 peptide was immobilized on the sensor chip and response of various TNT concentrations was analyzed. According to the increase in TNT concentration, the positive signal gradually increased and the binding constant was 1.305 µM (Fig. 3). Also, we assayed TNT binding to the HCDR3 peptide by the competitive assay using the mixture of TNT and TNT analogue (TNP-KLH) as the analytes. The sensor responses decreased when equivalence amount and excess amount of TNT was added, suggesting that TNT competitively inhibit the binding between TNP-KLH and HCDR3 peptide (Fig.4). Thus, the selected HCDR3 peptide showed binding property to TNT. The binding constant was significantly higher than the peptide array-based analysis results using TNT analogue, TNP-KLH. Since the assay condition is different, the value of binding constants are not directly comparable, however the difference might be occurred by the avidity effect of multiple TNP in a TNP-KLH. Also, binding analysis with KLH and TNP-KLH was also confirmed as the control SPR experiment. The SPR response of TNP-KLH shows interaction to the HCDR3 peptide while KLH alone did not show binding response (Supplementary Fig.2). It was shown that the HCDR3 peptide has the ability to directly bind to TNT.



16 14 12

Response (RU)

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10 8 6 4 2 0 -2 0

1

2

3

4

5

6

7

8

9

10

TNT concentration (µM) Fig. 3 Affinity measurements between TNT and the HCDR3 peptide by surface plasmon resonance spectroscopy. The TNT responses of the HCDR3 (ARGYSSFIYWFFDFC, circle) and control (AAAAC; square) peptide immobilized CM5 sensor chip was obtained. The SPR response of TNT as the analyte was obtained at flow rate of 30 µl/min with contact and dissociation time of 180 s.


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TNP-KLH TNP-KLH:TNT=1:1 TNP-KLH:TNT=1:1000

20

Response (RU)

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15 10 5 0

-100

0 -5

100

200

300

400

500

600

Time (s)

Fig. 4 SPR sensorgrams of TNP-KLH with competitive TNT in interaction with the HCDR3 (ARGYSSFIYWFFDFC) peptide. The HCDR3 peptide was immobilized on the Biacore CM5 sensor chip and TNP-KLH (500 nM for TNP) or the mixture of TNP-KLH and TNT (500 nM for 1:1 (blue line) and 5 µM for 1:1000 of TNP-KLH:TNT (gray line)) were respectively injected as the analyte.



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Truncation and amino acid substitution analyses of TNT-binding peptide To specify the key amino acid sequences involved in TNT-binding within HCDR3 [ARGYSSFIYWFFDF, amino acids (aa) 1–14], truncated peptide derivatives were designed and evaluated by the peptide array (Fig. 5). Five peptide derivatives including ARGYSSFIYWFF (aa1–12), RGYSSFIYWFFD (aa2–13), ARGYSSFIYWF (aa1–11), RGYSSFIYWFF (aa2–12), and RGYSSFIYWF (aa2–11) were found to have clearly higher binding intensity than the original HCDR3 peptide with statistical p-value

Array-Based Rational Design of Short Peptide Probe-Derived from an

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