Partial alanine scan of mast cell degranulating peptide (MCD): Importance of the histidine- and arinine residues

The influence of the two histidine and two arginine residues of mast cell degranulating peptide (MCD) in activity and binding was studied by replacing these amino acids in the MCD sequence with L-alanine. Their histamine releasing activity was determined on rat peritoneal mast cells. Their binding affinity to the FcERIα binding subunit of the human mast cell receptor protein, was carried out using fluorescence polarization. The histamine assay showed that replacement of His¹³ by Ala occurred without loss of activity compared with the activity of MCD. Alanine substitutions for Arg⁷ and His⁸ resulted in an approximately 40-fold increase, and for Arg¹⁶ in a 14-fold increase in histamine-releasing activity of MCD. The binding affinities of the analogs were tested by competitive displacement of bound fluorescent MCD peptide from the FcERIα binding protein of the mast cell receptor by the Ala analogs using fluorescence polarization. The analogs Ala⁸ (for His) and Ala¹⁶ (for Arg) showed the same binding affinities as MCD, whereas analog Ala⁷ (for Arg) and analog Ala¹³ (for His) showed slightly better binding affinity than the parent compound. This study showed that the introduction of alanine residues in these positions resulted in MCD agonists of diverse potency. These findings will be useful in further MCD structure-activity studies.