A carboxypeptidase inhibitor from the medical leech Hirudo medicinalis - Isolation, sequence analysis, cDNA cloning, recombinant expression, and characterization

A novel metallocarboxypeptidase inhibitor was isolated from the medical lee ch Hirudo medicinalis, Amino acid sequence analysis provided a nearly compl ete primary structure. which was subsequently verified and completed by cDN A cloning using reverse transcriptase-polymerase chain reaction/rapid ampli fication of cDNA end techniques. The inhibitor, called LCI (leech carboxype ptidase inhibitor), is a cysteine-rich polypeptide composed of 66 amino aci d residues. It does not show sequence similarity to any other protein excep t at its C-terminal end. In this region, the inhibitor shares the amino aci d sequence -Thr-Cys-X-Pro-Tyr-Val-X with Solanacea carboxypeptidase inhibit ors, suggesting a similar mechanism of inhibition where the C-terminal tail of the inhibitor interacts with the active center of metallocarboxypeptida ses in a substrate-like manner. This hypothesis is supported by the hydroly tic release of the C-terminal glutamic acid residue of LCI after binding to the enzyme. Heterologous overexpression of LCI in Escherichia coli, either into the medium or as an intracellular thioredoxin fusion protein, yields a protein with full inhibitory activity, Both in the natural and recombinan t forms, LCI is a tightly binding, competitive inhibitor of different types of pancreatic-like carboxypeptidases, with equilibrium dissociation consta nts K-i of 0.2-0.4 x 10(-9) M for the complexes with the pancreatic enzymes A1, A2, and B and plasma carboxypeptidase B, Circular dichroism and nuclea r magnetic resonance spectroscopy analysis indicate that recombinant LCT is a compactly folded globular protein, stable to a wide range of pH and dena turing conditions.