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 Ca2+ ion each) was described for Allergen M, analogous to carp parvalbumin pI 4.25. The suggested structure 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 Allergen M was assigned by: 1. 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.(ABSTRACT TRUNCATED AT 400 WORDS)