Mimotope selection of blood group A antigen from a phage display 15-mer peptide library

Mimotope selection of blood group A antigen from a phage display 15-mer peptide library

TRIM-00851; No of Pages 4 Transplant Immunology xxx (2013) xxx–xxx Contents lists available at SciVerse ScienceDirect Transplant Immunology journal ...

411KB Sizes 0 Downloads 4 Views

TRIM-00851; No of Pages 4 Transplant Immunology xxx (2013) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Transplant Immunology journal homepage: www.elsevier.com/locate/trim

Mimotope selection of blood group A antigen from a phage display 15-mer peptide library Zhao Ming Tang a, Wei Chao Jiang a, Pan Pan Chang b, Min Fang a, Yu Rong a, Li Hua Hu a,⁎ a b

Department of Clinical Laboratory Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jiefang Da Dao 1277#, Wuhan 430022, China Department of Laboratory Medicine center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jiefang Da Dao 1277#, Wuhan 430022, China

a r t i c l e

i n f o

Article history: Received 28 May 2013 Received in revised form 14 June 2013 Accepted 18 June 2013 Available online xxxx Keywords: Blood group A antigen Epitopes Molecular mimicry Peptide library

a b s t r a c t We select the peptide mimics of blood group A antigen by a monoclonal anti-A from a phage display 15-mer peptide library. Monoclonal anti-A was used in biopanning a phage display 15-mer peptide library. After four rounds of panning, ELISA was carried out to confirm the positive phage clones. The exogenous DNAs of the positive phages were sequenced and the corresponding amino acid sequences were deduced. Both the synthesized peptide and the phage clones were used to bind to anti-A in competitive ELISA. Erythrocyte agglutination inhibition tests were carried out to determine the mimic ability of the free synthesized peptide to the natural blood group A antigen. Computer softwares were used to simulate the interaction between the peptide and anti-A. After four rounds of biopanning, the eluted phage reached an enrichment of approximately 1600 times. Thirty-seven phage clones were chosen randomly and amplified. There were eleven clones that interacted specifically with anti-A in ELISA. DNA sequencing of the inserted oligonucleotide revealed that nine clones present a same peptide — TRWLVYFSRPYLVAT (named TRW) and each of the other two clones presented a different peptide. The synthesized free peptide TRW could inhibit the interaction of both phage displayed peptide and group A red blood cell with anti-A in competitive ELISA and hemagglutination inhibition test. Both the peptide TRW and the natural group A antigen were docked into a same cavity of anti-A in a computer simulation assay. The results indicate that peptide TRW can mimic blood group A antigen. It may be used as a proxy of natural blood group A antigen in clinical application. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Carbohydrates and proteins are the most popular materials for antigen construction. Research on carbohydrate is more difficult than that on protein. An important reason is that a carbohydrate cannot be expressed directly by modern molecular biology technology. As a peptide can be expressed or synthesized easily, research on peptide mimics of carbohydrate antigens attracts more and more scientists [1]. Peptide is an important research field of life science. The organ or tissue specific peptides can be used to aid in constructing novel nanoparticles. Side effects can be decreased deeply when drugs are based on these kinds of nanoparticles [2–4]. Peptide based biosensors can increase the sensitivity and specificity [5–7]. Currently, scientists have achieved great success in ABO incompatible kidney transplantation. It is profit from using natural blood group A/B antigens to filter the anti-A/B antibodies before transplantation

⁎ Corresponding author. Tel.: +86 27 85726325. E-mail addresses: [email protected] (Z.M. Tang), [email protected] (L.H. Hu).

[8]. Natural blood group antigens are important to ensure success, but natural blood group A/B antigens are constructed by oligosaccharides which are difficult to synthesize and are very expensive. Actually, the shortage of natural blood group A/B antigen is a main barrier for researching on and extending ABO incompatibility organ transplantation [9]. To find substitutions for natural blood group A antigen, in our previous work, we have investigated the 12-mer peptide mimics of blood group A antigen [10]. In this study, we have identified 15-mer peptide mimics of group A antigen using the phage display technology platform. These peptides help us to enrich the database of proxy for blood group A antigen.

2. Objective We hypothesized that some short peptides could mimic blood group A carbohydrate antigen and may be used as a substitute in clinical practice. The aim of this study is to get this kind of peptides from biopanning a phage display 15-mer random peptide library and a series of validation experiments.

0966-3274/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.trim.2013.06.001

Please cite this article as: Tang ZM, et al, Mimotope selection of blood group A antigen from a phage display 15-mer peptide library, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.06.001

2

Z.M. Tang et al. / Transplant Immunology xxx (2013) xxx–xxx

Table 1 Enrichment of the ratios of output to input for four rounds of biopanning. Cycle

Input (pfu)

I II III IV

1.0 2.0 1.6 1.2

× × × ×

1012 1012 1012 1012

Output (pfu) 6.0 2.0 6.0 1.2

× × × ×

102 103 105 106

Output/input 6.0 1.0 3.8 1.0

× × × ×

10 10 10 10

−10 −7 −7

(0.5 mg/mL in 0.1 M NaHCO3) for 2 h at 4 °C. Phages were added to the wells and incubated for 1 h at 37 °C. The primary phage library was used as negative control. The wells were washed six times with TBST (0.1% v/v Tween-20). The bound phages were detected by anti-phage-HRP antibody and enzyme substrate (TMB and H2O2). Each experiment was repeated three times.

−6

3.4. DNA sequencing 3. Materials and methods

Single strand DNAs of phages were purified by NaI method. The exogenous DNAs were sequenced in Sangon Biotech (Shanghai, China). The primer sequence was as follows: TGAATTTTCTGTATGAGG.

3.1. Materials Monoclonal anti-blood group A (Clone Number: 9A) was purchased from Abcam Inc. (Cambridge, USA). The horse radish peroxidase (HRP) labeled antibody against phage was obtained from Sigma Inc. The phage display 15-mer peptide library and the host strain E. coli K91 were a gift from Professor George P. Smith (Division of Biological Science, University of Missouri). The microcolumn gel cards were products of DiaMed AG (Cressier, Switzerland). Anti-A and Anti-B used in the hemagglutination tests were plasma from blood group B and blood group A volunteers of our laboratory. The other reagents were bought from Sangon Biotech (Shanghai, China). The peptide was synthesized by ShineGene Company (Shanghai, China). 3.2. Biopanning of the phage display 15-mer peptide library The panning procedure was referred to Adda et al. [11] with some modifications. Anti-A (1 μg/well in 150 μL 0.1 M NaHCO3, pH 8.6) was coated in one well of a microplate (Greiner bio-one, German). The well was incubated at 4 °C under moist condition. Blocking buffer (0.5 mg/ml BSA or 0.3% gelatin in 0.1 M NaCO3 buffer) was used to block the well for 2 h at 4 °C. After washing six times with TBST, 10 μL phage library suspended in 100 μL TBST was added into the well and incubated for 1 h at 37 °C. Unbound phages were discarded and wells were washed 10 times by TBST (the concentration of Tween-20 was 0.1% in the first round and 0.5% in other rounds of panning). The bound phages were eluted by 0.1 M Glycine–HCl buffer (pH 2.2). Tris–HCl (pH 9.1) was used to neutralize the Glycine–HCl buffer. The procedures for phage amplification and tittering were the same as previously described [11]. Four rounds of panning were carried out. One uncoated well was used as negative control.

Competitive ELISA was performed as ELISA described above except that 1010 pfu phage particles were added to the anti-A coated wells with the presence of decreasing amounts of synthetic peptide. Each experiment was repeated again. 3.6. Hemagglutination inhibition test Anti-A and the synthetic peptide were incubated for 30 min at 37 °C. Twenty microliters of the mixture and ten microliters of washed group A erythrocytes (2%) were added to the microcolumn gel cards. After incubation for 30 min at 37 °C, the gel cards were centrifuged at 85 g for 10 min and then the results were recorded by camera. The hemagglutination inhibition tests with anti-B, group B erythrocytes and the synthetic peptide were performed as specific control. 3.7. Molecular docking between the peptide and anti-A The web server, 3D-JIGSAW (http://bmm.cancerresearchuk.org/ ~3djigsaw/), was used to construct the 3D structure of the peptide. A 3D structure data of anti-A was retrieved from PDB database (No. 1JV5). Autodock software [12] was used to simulate the interaction between the peptide and anti-A. Lamarckian genetic algorithm, semi-flexible docking and semi-empirical free energy algorithm were used to estimate the minimum total energy of the complex structure. The natural blood group A antigen drawn from a complex structure (PDB No. 2vng) was also docked into anti-A. 4. Results

3.3. Phage ELISA Thirty-seven phage clones were randomly chosen from the tittering plate after the fourth round of panning. The clones were amplified in 1 mL volume. The supernatants were retained for subsequent use. Anti-A (200 ng/mL in 0.1 M NaHCO3) were coated in 96-wells plates at 4 °C overnight. The wells were blocked by BSA 1.6 1.4

A450nm/630nm

3.5. Competitive ELISA

4.1. Biopanning and the specific phages enrichment Four rounds of biopanning were carried out, anti-A monoclonal antibody was used as target molecule to biopanning the phage display 15-mer peptide library. BSA and gelatin were used alternately as blocking reagent. This operation may reduce the enrichment of nonspecific phage clones. The output phages of every round of elution were tittered. The ratios of output to input were calculated. As the biopanning rounds continue, the ratios increased gradually. The enrichment achieved about 1600 times (Table 1). There was no obvious increase of the output/input ratio in the negative control. The ELISA result also showed that the amplified elutes' affinity for anti-A were greater than that of the primary library phages (Fig. 1).

1.2 4.2. Phage clones' affinity for anti-A in ELISA experiment

1

Thirty-seven phage clones were randomly chosen from the tittering plate after four rounds of biopanning. Supernatant of the amplified phage clones were used to determine the affinity for anti-A by ELISA method. The primary library was diluted 100-fold and used as negative control. Those clones having absorbance values over three times higher than that of the negative control were considered as positive clones. There were eleven phage clones that satisfied this condition and the positive rate was 27.5% (Fig. 2).

0.8 0.6 0.4 0.2 0

Primary library

Round 1

Round 2

Round 3

Round 4

Fig. 1. ELISA result showing the phage's affinity for anti-A. The primary library given on the x-axis means the unpanninged phage display 15-mer peptide library.

4.3. DNA sequencing and the derivation of corresponding peptides Eleven positive phage clones' exogenous DNAs were sequenced and the corresponding peptides were deduced. Among these eleven phage clones, nine clones

Please cite this article as: Tang ZM, et al, Mimotope selection of blood group A antigen from a phage display 15-mer peptide library, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.06.001

Z.M. Tang et al. / Transplant Immunology xxx (2013) xxx–xxx

3

1.2

A450nm/630nm

1 0.8 0.6 0.4

0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

0.2

Fig. 2. ELISA results showing the phage clones' affinity for anti-A. 1–37 given on the x-axis mean thirty-seven phage clones and 38 means the primary 15-mer library which was used as negative control.

displayed the same peptide — TRWLVYFSRPYLVAT (phage clone 8/31/32/22/5/14/17/ 27/36, we named it TRW), the other two phage (clone 15 and clone 19) clones displayed two different peptides respectively. TRW was chosen for chemical synthesis.

4.4. Synthetic peptide inhibited binding of phage clones to anti-A To investigate if the free peptide still retains the anti-A-binding ability of the phage displayed peptide, peptide TRW was synthesized. Peptide TRW can inhibit the binding between its corresponding phage (clone 18) and anti-A in a concentration dependent manner. It also inhibited the binding of phage clones (15/19) to anti-A (Fig. 3).

4.5. Peptide TRW inhibited group A erythrocyte binding to anti-A The hemagglutination inhibition tests were performed to determine the mimic ability of the free peptide TRW to natural group A antigen. Peptide TRW inhibited the binding of group A erythrocyte to anti-A. A peptide with the same amino acid composition as peptide TRW but with a randomized sequence (PTARVTYWFVSRLYL) had no effect on inhibition (Fig. 4). It suggested that the primary sequence, and not simply the amino acid, appeared to be critical for the binding to the group A antigen-binding site of anti-A. Hemagglutination inhibition tests based on group B erythrocyte and anti-B were carried out. The results suggested that peptide TRW could not inhibit the agglutination of group B erythrocyte induced by anti-B (Fig. 4). Peptide TRW could not inhibit the agglutination of group O erythrocyte by anti-H antibody either (see supplementary data).

5. Discussion The synthetic blood group A carbohydrate antigen was used to immunoabsorb the recipient's anti-A preoperatively. This kind of perioperative operation is a very important way to prevent the hyperacute rejection during ABO incompatible organ transplantation. Because the synthesis of carbohydrate is very difficult, development of carbohydrate-mimetic peptides is very meaningful. In this study, we identified that peptide TRW can mimic natural blood group A carbohydrate antigen. Many developments have been achieved since the establishment of phage display technology. This wonderful technology has been used in panning peptide ligands of many materials including organic and inorganic matter. Calculating the ratio of output to input phages is a useful index for monitoring the panning procedure. The ratio should increase gradually as the panning continues. Previous researches have shown that the ratio may increase dozens of

4.6. Docking between peptide TRW and anti-A Autodock software was used to analysis the possible interaction between the peptide TRW and anti-A. After the 3D structure construction of the peptide by the web server mentioned above, peptide TRW was docked into the group A antigen-binding groove of anti-A (Fig. 5). The amino acids (threonine, tryptophan and tyrosine) at the N terminal of the peptide were closely docked into this groove and may be the most important amino acids for peptide mimic. The calculated minimum free energy for the peptide-anti-A complex was −6.5 kcal/mol, while this value for natural group A-anti-A complex was −6.1 kcal/mol.

1

2

7

Fig. 3. Synthetic peptide TRW inhibited the binding of phage displayed peptide to anti-A. The x-axis shows the dilution ratio of the synthetic peptide, the original concentration was 0.5 g/L. Scattered peptide sequence: PTARVTYWFVSRLYL.

3

8

9

4

5

6

10

Fig. 4. Peptide TRW inhibited the hemagglutination of group A erythrocyte induced by anti-A. Microcolumns labeled 1–6 are based on group A erythrocyte, anti-A and peptide inhibitor reaction system. Microcolumns labeled 1, 2 with the peptide TRW (5 μg/column), labeled 3, 4 with scattered peptide (5 μg/column) used as negative control, labeled 5, 6 with no peptide. Microcolumns labeled 7–10 were based on group B erythrocyte, anti-B and peptide inhibitor reaction system. Microcolumns labeled 7, 8 with the peptide TRW (5 μg/column), labeled 9,10 with no peptide.

Please cite this article as: Tang ZM, et al, Mimotope selection of blood group A antigen from a phage display 15-mer peptide library, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.06.001

4

Z.M. Tang et al. / Transplant Immunology xxx (2013) xxx–xxx

Fig. 5. Docking profile of anti-A. A: Peptide TRW docked into anti-A. B: Natural group A antigen docked into anti-A.

times, and even thousand times [13–15]. In this study, after four rounds of panning, the ratio increased one thousand six hundred times. It is consistent with a previous report [11]. In order to determine if the free peptide still has the ability to bind to the target, we performed competitive ELISA. Free peptide can inhibit the binding between anti-A and phage displayed peptide as the ELISA result shows. This result suggested that the free peptide TRW could bind to anti-A. Peptide TRW was confirmed to mimic the natural blood group A antigen as the hemagglutination competitive test shows. In our previous work, we have investigated the 12-mer peptide mimetics of blood group A antigen [10]. As most carbohydrate antigens are based on conformational epitope, and it is easier for longer peptides to form a conformational structure, we performed this study to identify a 15-mer peptide which can mimic blood group A antigen. We finally found peptide TRW which can competitively bind to anti-A. There were eight polar amino acids in peptide TRW (T1,R2,Y6,S8,R9,P10,Y11,T15). Polar amino acids favor the formation of an antigen determinant because they are hydrophilic and easily present on the surface of an antigen molecule. Among these eight polar amino acid, there were three hydroxy amino acids (T1,S8,T15). These amino acids could tighten the interaction between peptide TRW and anti-A by hydrogen bonds. Tyrosine which occurred two times in peptide TRW was also a very popular amino acid in an antigen determinant. In conclusion, the peptide TRW which was selected from a phage display 15-mer peptide library is a mimic of blood group A antigen. This peptide may be a functional mimic of group A antigen. The detailed interaction, specificity and exact affinity should be further investigated. As the synthesis of peptides is easier than that of carbohydrates, peptide TRW may be used as a substitute for blood group A carbohydrate antigen in clinical applications. Acknowledgment This work was supported by the National Natural Science Foundation of China (NSFC), Grant No. 30972820.

Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.trim.2013.06.001. References [1] Fukuda MN. Peptide-displaying phage technology in glycobiology. Glycobiology 2012;22:318–25. [2] Hatakeyama S, Sugihara K, Shibata TK, Nakayama J, Akama TO, Tamura N, et al. Targeted drug delivery to tumor vasculature by a carbohydrate mimetic peptide. Proc Natl Acad Sci U S A 2011;108:19587–92. [3] Gray BP, Li S, Brown KC. From phage display to nanoparticle delivery: functionalizing liposomes with multivalent peptides improves targeting to a cancer biomarker. Bioconjug Chem 2013;24:85–96. [4] Hubbell JA, Chemistry Chilkoti A. Nanomaterials for drug delivery. Science 2012;337:303–5. [5] Wu J, Park JP, Dooley K, Cropek DM, West AC, Banta S. Rapid development of new protein biosensors utilizing peptides obtained via phage display. PLoS One 2011;6:e24948. [6] Cui Y, Kim SN, Naik RR, McAlpine MC. Biomimetic peptide nanosensors. Acc Chem Res 2012;45:696–704. [7] Xiao M, Hong Z, Sun L, Wu Y, Zhang N, Liu Y, et al. TMTP1, a novel tumor-homing peptide, specifically targets hematological malignancies and their metastases. J Huazhong Univ Sci Technolog Med Sci 2011;31:608–13. [8] Genberg H, Kumlien G, Wennberg L, Berg U, Tydén G. ABO-incompatible kidney transplantation using antigen-specific immunoadsorption and rituximab: a 3-year follow-up. Transplantation 2008;85:1745–54. [9] Tyden G. The European experience. Transplantation 2007;84:S2–3. [10] Tang Z, Wang L, Hu L, Li Y, Cui T, Xiong J, et al. Identification and characterization of peptide mimics of blood group A antigen. J Huazhong Univ Sci Technolog Med Sci 2008;28:222–6. [11] Adda CG, Tilley L, Anders RF, Foley M. Isolation of peptides that mimic epitopes on a malarial antigen from random peptide libraries displayed on phage. Infect Immun 1999;67:4679–88. [12] Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 2009;30:2785–91. [13] Li Y, Ning YS, Wang YD, Luo J, Wang W, Dong WQ, et al. Production of mouse monoclonal antibodies against Helicobacter pylori catalase and mapping the antigenic epitope by phage display library. Vaccine 2008;26:1263–9. [14] Houimel M, Dellagi K. Peptide mimotopes of rabies virus glycoprotein with immunogenic activity. Vaccine 2009;27:4648–55. [15] Owens GP, Shearer AJ, Yu X, Ritchie AM, Keays KM, Bennett JL, et al. Screening random peptide libraries with subacute sclerosing panencephalitis brain-derived recombinant antibodies identifies multiple epitopes in the C-terminal region of the measles virus nucleocapsid protein. J Virol 2006;80:12121–30.

Please cite this article as: Tang ZM, et al, Mimotope selection of blood group A antigen from a phage display 15-mer peptide library, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.06.001