A spatial similarity of stereochemical environments formed by amino acid residues defines a common epitope of two non-homologous proteins

It is critical for development of high-quality antibodies in research and diagnostics to predict accurately their cross-reactivities with “off-target” molecules, which potentially induce false results. Herein, we report a good example of such a cross-reactivity for an off-target due to a stereochemical environment of epitopes, which does not simply depend on amino acid sequences. We found that significant subpopulation of a polyclonal peptide antibody against Bcnt (Bucentaur) (anti-BCNT-C antibody) cross-reacted with a completely different protein, glutamine synthetase (GS), and identified four amino acids, GYFE, in its C-terminal region as the core amino acids for the cross-reaction. Consistent with this finding, the anti-BCNT-C antibody strongly recognized endogenously and exogenously expressed GS in tissues and cultured cells by Western blotting and immunohistochemistry. Furthermore, we elucidated that the cross-reaction is caused by a spatial similarity between the stereochemical environments formed by amino acid residues, including the GYFE of GS and the GYIE of Bcnt, rather than by their primary sequences. These results suggest it is critical to comprehensively analyze antibody interactions with target molecules including off-targets with special attention to the physicochemical environments of epitope-paratope interfaces to decrease the risk of false interpretations of results using antibodies in science and clinical applications.

4 purity. Two and six Hartley guinea pigs (5)(6) week-old males) were immunized at different times by subcutaneous injection of 0.2 and 0.25 mg antigen peptide, respectively, which was coupled to keyhole limpet hemocyanin through the cysteine residue, supplemented with Freund's complete adjuvant. Typically, two booster injections with Freund's incomplete adjuvant were carried out on days 14 and 28. Whole blood was collected from each animal on day 38-52 and sera were prepared. All these processes were outsourced to Takara Bio.
Two other anti-Bcnt/Cfdp1 Abs, designated anti-Bcnt-Cter Ab and anti-mBcnt-N Ab, were similarly generated in each three guinea pigs using a peptide of 10 amino acids, RDLRLSKMKP-COOH, located at the C-terminus of BCNT with an additional cysteine at the N-terminus and a peptide of 16 amino acids, acetyl-GEEQAEKTKGKRRKAQ, located at the N-terminal region of mouse Bcnt with an additional cysteine at the C-terminus, respectively. These peptides were synthesized by AnyGen. Immunization of animals and collecting whole blood were also outsourced to Takara Bio. The anti-BCNT-C Ab was affinity-purified from each serum of guinea pigs using a HiTrap NHS-activated HP column (GE Healthcare) coupled with the antigen peptide. The anti-Bcnt-Cter Abs and the anti-mBcnt-N Abs were purified from each serum by using SulfoLink Coupling Resine (Thermo Fisher Scientific) coupled with each antigen peptide through the additional cysteine.

Construction of expression vectors for deletion and substitution mutants of mGS
Expression vectors for deletion and substitution mutants of mGS were constructed from pCold II DNA plasmid carrying mGS according to a protocol of PrimeSTAR mutagenesis basal kit (Takara Bio) using PrimeSTAR GXL DNA polymerase or KAPA HiFi HotStart DNA polymerase (KAPA Biosystems). PCRs were performed using 0.3 μM of each set of mutagenesis primers (Table S3-2) under either of following two conditions: 1) initial denaturation for 3 min at 95℃ and 35 cycles of denaturation for 20 sec at 98 ℃, annealing and extension for 3 min at 72℃ when using KAPA HiFi HotStart DNA polymerase for a A4(AAGYFEAA) mutant, or 2) initial denaturation for 3 min at 95℃ and 35 cycles of denaturation for 10 sec at 98℃, annealing for 15 sec at 60℃, and extension for 2 min at 68℃ when using PrimeSTAR GXL DNA polymerase for other mutants. These PCR products were used for transformation of E. coli XL1-Blue after confirmation of their amplicon size by 0.8% TAE-agarose gel electrophoresis and purification by Wizard SV Gel and PCR Clean-Up System (Promega). All constructs obtained were expressed in BL21 (DE3) cells (Delphi Genetics S.A.) after confirmation of their ORF sequences and the promoter region using the designed primers (Table S3-3) and BigDye Terminator V3.1 Cycle Sequencing Kit (Thermo Fisher Scientific). PCR products were also inserted into one of the following three kinds of mammalian expression vectors by using restriction enzymes BglII or BamHI for 3' end and XhoI for 5' end: BsrGI-MCS-pcDNA3.1 (Accession No.

Preparation of an anti-BCNT-C peptide Ab-linked agarose
Anti-BCNT-C Ab-affinity agarose was prepared according to a protocol of Abcam (www.abcam.com/technical) by mixing 400 μg anti-BCNT-C Ab with 250 μL agarose (packed beads of Protein A/G PLUS-Agarose, Santa Crutz Biotech) and by coupling with dimethyl pimelimidate (Sigma-Aldrich). For preparation of a control agarose, the affinity agarose resin described above was boiled in Hepes buffer (HB; Hepes-NaOH pH 7.5) for 5 min, and an unrelated calmoldulin-agarose (Sigma-Aldrich) was also used.

Preparation of protein extracts of cow, mouse and rat organs
Dissected pieces of a bovine brain (2-year-old female) and mouse brains (mixtures of 6 months to 1 year old C57Bl/6J mice) were frozen in liquid N 2 and stocked at -80℃ until use.
A piece of frozen tissues was crushed in a metal cylinder containing liquid N 2 , and the resulted fine grains were homogenized in Hepes buffered saline (HBS; 10 mM Hepes-NaOH (pH 7.5), 150 mM NaCl) supplemented with protease inhibitor cocktail (Nacalai Tesque) and phosphatase inhibitors (10 mM NaF, 1 mM sodium vanadate, 12.5 mM β-glycerophosphate) with 40 strokes using a Dounce homogenizer. The crude extracts were centrifuged at 20,000 x g for 15 min at 4℃ (Hitachi PA50, rotor AG-6512C) and the supernatant (S-20) was collected. Extracts of rat olfactory bulb [1] and skeletal muscle of FOXO1-transgenic mice and wild littermates [2] were prepared as previously reported.

Immunoprecipitation of the 43 kDa protein with the anti-BCNT-C Ab from bovine brain extracts in RIPA buffer or those boiled once in SDS buffer
Bovine brain S-100 extract [3] was mixed with an equal volume of two-fold concentrated RIPA buffer (final concentration: 10 mM Hepes-NaOH (pH 7.4), 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 5 mM Ethylenediaminetetraacetic acid (EDTA), 2.5 mM Ethyleneglycoltetraacetic acid (EGTA)) supplemented with inhibitors of proteinases and phosphatases, and the mixture was sonicated by a Bioruptor in an ice-water bath (15 x 10 s pulses at 10 s intervals), followed by centrifugation at 28,000 x g for 10 min.
Twenty μL Staphylococcus aureus suspension (IgG-Sorb, The Enzyme Center, binding capacity of 1.9 mg IgG per mL of reconstructed solution) was added to 1 mL extract, and the mixture was incubated for 30 min using a rotary shaker and centrifuged at 12,000 x g for and boiled for 4 min. Then Nonidet P-40 (hereinafter called "NP40") was added to the solution followed by adding HBS making a final concentration of both NP40 and SDS to 0.1%, respectively [4]. After treatment with IgG-Sorb, immunoprecipitation was carried out exactly the same as in RIPA buffer.

Isolation of the 43 kDa protein from bovine brain extracts
S-20 extracts of bovine brain ( and subjected to immunoprecipitation as described above. The protein-bound agarose were washed three times with HBS containing 0.1% NP40 twice with HB, then eluted with HB containing 10 μM antigen peptides (50 μL). Elution was repeated three times and the eluates were pooled and subjected to acetone precipitation followed by SDS-PAGE.

Immunoprecipitation of the 43 kDa protein from extracts of rat olfactory bulb
Extracts of rat olfactory bulb (see, above) stored in 0.25 M sucrose at -80℃ were thawed and concentrated by 60% AS precipitation to remove sucrose and resolved once in HBS, and then subjected to acetone precipitation. Boiling of the extracts in SDS, dilution with HBS containing 0.1% NP40, and immunoprecipitation were exactly the same as described in immunoprecipitation from bovine brain extracts.

Competitive experiment with peptides in Western blot analysis
Extracts of E. coli expressing His-tagged GS were subjected to Western blot analysis (100 ng/lane). The anti-BCNT-C Ab (2 nM) was preincubated with 5 μM antigen peptide or each GS peptide (P1-P4 described in Fig. 4B, which were synthesized at JPT Peptide Technologies and dissolved in DMSO) in blocking reagent (0.2% casein in TBT) at room temperature for 1 h. Western blotting was carried out using each preincubated Ab. antibody or with anti-GFAP antibody similarly as described above. The immunoreactivity of these two antibodies was visualized using Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch). Finally, TO-PRO-3 (1:500, Molecular Probes) was used for nuclear staining. Immunostained sections were examined by a confocal laser scanning microscopy (Olympus FV1000).

Isolation of an anti-BCNT-C Ab subpopulation specific to GS
Antibody molecules that specifically react with GS were isolated from the anti-BCNT-C Ab essentially according to a previously reported method [6]. In brief, extracts of E. coli μL/50 μL). This procedure was repeated three times, and the collected solution was adjusted to TBS composition by adding 10-fold concentrated TBS for stock.     Nucleotide sequences of PCR primers used for amplification of mGS and mBcnt cDNA (Table S3-1), construction of deletion or substitution mutants of mGS (Table S3-2), and DNA sequencing (Table S3- (lanes 1, 4, 7). Note that these positive bands were completely abolished in the presence of the BCNT-C peptide, regardless of its terminal forms.

Ab.
Western blotting was conducted with the anti-BCNT-C Ab using extracts of bovine brain     Fig. S5A, was identified as GS with a score of 99 and an expect value of 3e-008 using Mascot (see also Supplementary Table S1).     The asterisk indicates non-specific band.