Crystal structure of Ara h 3, a major allergen in peanut (1)
Ara h 2 and Ara h 3 have been reported to have activities similar to bovine pancreatic trypsin inhibitor (BPTI) and a peanut trypsin inhibitor with high degree of homology to part of the N-terminal domain of Ara h 3 has been isolated. The peanut major allergen Ara h 3 is an 11S seed storage protein. The 11S protein has multiple isoforms in numerous species. Each isoform of the 11S globulin is generally encoded by a single gene that produces a precursor that is post-translationally cleaved by an asparaginyl endopeptidase after the formation of an interchain disulfide bond between the N-terminal and the C-terminal subunits.
There are at least 5 isoforms of Ara h 3, and 12 different Ara h 3 sequences were in the NCBI protein database, pos- sibly from different cultivars. The sequence identity between this Ara h 3 and arachin 6, Ara h 3.0101, and Ara h 4.0101 are 90%, 95%, and 93%, respectively.
The overall structure of Ara h 3 consists of an N-terminal domain and a C-terminal domain, each assumes a cupin fold and they are related by a pseudo-dyad axis. Three regions of the N-terminal domain (G119-Q138 and Q212-G259, and D311-N345) and the tail of C-terminal domain (S522-A530) were disordered.
The greatest differences between Ara h 3 and the soybean 11S glycinin (PDB:1OD5A) are seen in the loops and in the helical regions. Structure based alignment of the two sequences of the structurally ordered parts of Ara h 3 and soybean glycinin resulted in a sequence identity of 47.2%.
Similar to soybean glycinin A3B4, mature Ara h 3 is a hexamer formed by a head-to-head association of two trimers. One of the notable differences between the packing of the two structures is near the three-fold symmetric axis of the trimer. While there is a large cavity channel in the doughnut shaped soybean glycinin, there is no channel present in the Ara h 3 trimer.
The N-terminus of the C-terminal domain is located at the trimer–trimer interface and there was no space left for additional residues at its N-terminal, suggesting that the peptide linkage between the two domains would prevent the formation of an Ara h 3 hexamer.
Linear epitope 1 and 2 as well as the structured portion of epitope 3 was mapped on the final 1.73 Å monomeric structure of Ara h 3 and in the Ara h 3 homohexamer.
These 3 epitopes are partially exposed on the surface of the native allergen
L305, I307, L308, and P310 were determined to be the critical residues in epitope 3 (Rabjohn et al., 1999). The average SAS for L305, I307, and L308 is 12%. The SAS for P310 is 61%, but this value would have been lower if it were not the last locatable residue of the N-terminal domain of Ara h 3.
Native Ara h 3 is most likely not to be recognized by the IgE antibodies that recognized linear epitope 1 and 2 in the 8 patient sera. This may indicate that the allergen is degraded as a result of digestion and the linear epitopes are exposed to interaction with the immune system. Epitope 4 and part of epitope 3 are flexible in the native protein. IgE antibodies that rec- ognized these epitopes are likely to recognize Ara h 3 in its native state.