Immunochemical Analysis of Cod Fish Allergen M: Locations of the Immunoglobulin Binding Sites as Demonstrated by the Native and Synthetic Peptides

The major allergen of codfish (Allergen M) is a muscle protein belonging to the family of calcium binding parvalbumins. The primary structure of the molecule was established and the molecular weight was estimated from the sequence data to be 12,328. Allergen M consists of 113 amino acid residues and one residue of glucose. A molecular arrangement of three domains (AB, CD and EF—the latter two bind one Ca 2+ ion each) was described for Allergen M, analogous to carp parvalbumin pI 4.25. The suggested strucutre was based on the extensive intramolecular amino acid homologies and the immunochemical cross‐reactivities of the intact molecule and the two major isolated fragments. The immunological structure of Allergen M was studied by: 1. Modification of certain amino acids residues and study of the reactivity of the modified derivatives. 2. Examination of the immunochemical reactivity of a large number of overlapping peptides obtained by limited and selective tryptic hydrolyses. 3. Solid phase peptide synthesis (SPPS) of segments selected in regard to the reactivity of pre‐examined native peptides. The immunological reactivity of the derivatives of Allertgen M was assigned by: I, Rocket line immunoelectrophoresis and quantitative precipitation using rabbit anti‐Allergen M in precipitating antibody‐mediated reactions and, 2. RAST/RAST‐inhibition and PK test/PK‐test inhibition using sera from individuals allergic to codfish in IgE‐mediated reactions. The modification of Tyr‐30 and Arg‐75 in isolated and purified peptides indicated that the former was part of a reactive site whereas the latter did not contribute to the activity. Masking of Arg‐residue or unchelating the two calcium ions from the native Allergen M, with the resulting perturbation of the tertiary structure, decreased the allergenicity by approximately 25%. Two major fragments of Allergen M were produced and purified; TM1 (residues 1–75) comprising domains AB and CD, and TM2 (residues 76–113) covering domain EF. Both were immunologically reactive; TM1 showing intermediate reactivity between Allergen M and TM2. A high degree of immunological cross reactivity was evident between TM1 and TM2. The finding was in concert with the high intramolecular amino acid homologies of Allergen M, and suggested that the reactive sites were repetitively distributed along the polypeptide chain. The immunological reactivity of several long‐sequence overlapping peptides obtained by limited and selective trypsin hydrolysis of Allergen M was studied. The immunologically reactive sites were accordingly assigned to the following regions of the chain: 1. Residues 33–44 on the junction between the AB and CD domains. 2. Residues 65–74 on the corresponding junction between the CD and EF domains. 3. Residues 88–96 on the calcium binding loop of the EF domain. These results were cross‐examined by SPPS of these segments. A synthetic peptide with the sequence 49–64 of Allergen M, located in the calcium binding loop of the CD domain confirmed the immunological reactivity of this region. Peptide 49–64 incorporates two homologous tetrapeptides (Asp‐Glu‐Asp‐Lys and Asp‐Glu‐Leu‐Lys) interspaced by six amino acid residues. Changing the residues of the spacer arm had no influence on the reactivities. The conclusion was that the tetrapeptides attached at a distance of six amino‐acids spacer‐arm formed a binding locus for Ig‐molecule. The same tetrapeptides are repeated in the sequence 51–64. A synthetic peptide of this sequence was also immuno‐logically reactive. An octapeptide comprising one single tetrapeptide lacked the immunochemical reactivity. Similarly, solid phase synthesis of peptide 88–103 of the calcium binding loop of the EF‐domain rendered an immunochemically reactive peptide. A high degree of homology between this peptide and the peptide 51–64 is evident although the former is devoid of the characteristic tetrapeptide. The synthesis of the N‐terminal domain peptide 13–32 (AB‐domain) was accomplished. The synthetic peptide was found to bind IgE antibodies from cod‐allergic individuals' sera in direct PK‐test. At identical concentration, a ratio of 1:6 for in vitro reactivity of the peptide relative to that of intact Allergen M was obtained. The peptide could also react with rabbit anti‐Allergen M antibodies in rocket immunoelectrophoresis. The ability of the peptide to give positive direct PK‐reaction and a rocket precipitate in immunoelectrophoresis preclude the achievement of a haptenic determinant. The synthetic peptides of Allergen M are currently utilized in trials on the regulation of IgE synthesis in experimental animals.