Primary structure and properties of the cathepsin G chymotrypsin inhibitorfrom the larval hemolymph of Apis mellifera

A member of the Ascaris inhibitor family exhibiting anti-cathepsin G and an ti-chymotrypsin activity was purified from the larval hemolymph of the hone y bee (Apis mellifera). Three forms of the inhibitor, designated AMCI 1-3, were isolated using gel filtration and anion-exchange chromatographies foll owed by reverse-phase HPLC. The amino-acid analyses indicated that AMCI-1 a nd AMCI-2 have an identical composition whereas AMCI-3 is shorter by two re sidues (Thr, Arg). All three forms contain as many as 10 cysteine residues and lack tryptophan, tyrosine, and histidine. The sequence of the isoinhibi tors showed that the major form (AMCI-1) consisting of 56 amino-acid residu es was a single-chain Protein of molecular mass 5972 Da, whereas the other two forms were two-chain proteins with a very high residue identity. The AM CI-2 appeared to be derived from AMCI-1, as a result of the Lys24-Thr25 pep tide bond splitting, while AMCI-3 was truncated at its N-terminus by the di peptide Thr25-Arg26. The association constants for the binding of bovine al pha-chymotrypsin to all purified forms of the inhibitor were high and nearl y identical, ranging from 4.8 x 10(10) M-1 for AMCI-1 to 2.7 x 10(9) M-1 fo r AMCI-3. The sensitivity of cathepsin G to inhibition by each inhibitor wa s different. Only the association constant for the interaction of this enzy me with AMCI-1 was high (2 x 10(8) M-1) whereas those for AMCI-2 and AMCI-3 were significantly lower, and appeared to be 3.7 x 10(7) M-1 and 4.5 x 10( 6) M-1, respectively The reactive site of the inhibitor, as identified by c athepsin G degradation and chemical modification, was found to be Lit Met30 -Gln31. A search in the Protein Sequence Swiss-Prot databank revealed a sig nificant degree of identity (44%) between the primary structure of AMCI and the trypsin isoinhibitor from Ascaris sp (ATI). On the basis of the cysteine residues alignment, the position of the reacti ve site as well as some sequence homology, the cathepsin G/chymotrypsin inh ibitor from larval hemolymph of the honey bee may be considered to be a mem ber of the Ascar is inhibitor family.