Given the reduced sequence similarities and structural differences, no cross-reactivity of C2_2E12 for IL-1 and IL-6 seems to be reasonable. (GST-IL-1 and IL-6) and three unrelated proteins (GST, bovine serum albumin (BSA) and IlvC). Immunoblot assay results showed that C2_2E12 only reacted with IL-33 (Number 1C). It is interesting that C2_2E12 did not react with IL-1 and IL-6, since IL-33, IL-1, and IL-6 belong to the same subfamily. Human being IL-33 shows low sequence identities to IL-1 and IL-6 despite all three belonging to the same subfamily: 13.5% with IL-1 and 12.9% with IL-6, respectively (Number 1D). The structure of IL-1 (PDB ID: 1L2H) is similar to that of IL-33 (PDB ID: 4KC3) having a root mean square deviation (r.m.s.d.) of 1 1.93 ?, while the structure of IL-6 (PDB ID: 1ALU) is completely different from that of IL-33. Given the low sequence similarities and structural variations, no cross-reactivity of C2_2E12 for IL-1 and IL-6 seems to be sensible. The cross-reactivity results clearly Schisandrin B demonstrate that C2_2E12 specifically binds to IL-33. 2.2. Epitope Mapping To determine the epitope region in IL-33 for C2_2E12, a series of GST-IL-33112-270 N-terminal deletion mutants were constructed from the insertion of a stop codon at the end of each -helix or -strand of IL-33 based on the crystal structure of human being IL-33 (PDB ID: 4KC3) [23] (Number 2A). Immunoblot analysis exposed that residues 149C158 of the IL-33 comprised the epitope region, which corresponded to its receptor ST2 binding site in the crystal structure of the IL-33:ST2 complex (PDB ID: 4KC3). We found that the additional five scFv clones (C1_1E1, C2_1D5, C2_2A10, C2_2E1, C2_2E12, and C2_2H5) also acknowledged the same epitope region in the IL-33 (Number 2B). Alanine scanning mutagenesis was performed to determine the crucial residue(s) in the epitope region (Number 2C). Each residue in GST-IL-33149-158 was substituted to alanine by site-directed mutagenesis PCR. The effects of the IL-33 mutants were analyzed by immunoblots with C2_2E12 as the primary antibody. Alanine substitutions of L150 and K151 of IL-33 reduced the Schisandrin B binding with C2_2E12, rendering these the key residues in the epitope region. To obtain further insights on alanine scanning results in the molecular level, we performed molecular docking between IL-33 and C2_2E12 using the HADDOCK server with Schisandrin B restraints that only L150 and K151 residues of IL-33 and CDR residues of C2_2E12 should participate in interactions. Although alanine scanning data showed Schisandrin B that L150 of IL-33 is definitely a key residue of IL-33 and C2_2E12 binding, L150 seemed to not interact with any residue of C2_2E12. On the other hand, L150 seemed to possibly interact with the surrounding hydrophobic residues of the 149C158 epitope region of IL-33, and it also seemed to play an important role in keeping the shape of the loop (Number 2D). It seems that the L150A mutant inhibits the connection between IL-33 and C2_2E12 by local conformational changes of the loop. The docked structural model of IL-33:C2_2E12 suggested that K151 of IL-33 seemed Schisandrin B to interact electrostatically with the acidic pocket of C2_2E12 composed of D164, S166, Y168, A218, and Y230 (Number 2E). This structural analysis having a docked model between IL-33 and C2_2E12 helps that L150 and K151 residues of IL-33 are important for his or her binding to C2_2E12. Open in a separate window Number 2 FHF1 Epitope mapping of C2_2E12. (A) Immunoblot analysis of 14 GST-IL-33 deletion mutants to map the IL-33 epitope at.
Given the reduced sequence similarities and structural differences, no cross-reactivity of C2_2E12 for IL-1 and IL-6 seems to be reasonable
