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

被引:76
作者
Reverter, D
Vendrell, J
Canals, F
Horstmann, J
Avilés, FX [1 ]
Fritz, H
Sommerhoff, CP
机构
[1] Univ Autonoma Barcelona, Dept Bioquim & Biol Mol, Unitat Ciencies, E-08193 Barcelona, Spain
[2] Univ Autonoma Barcelona, Inst Biol Fonamental, E-08193 Barcelona, Spain
[3] Univ Munich, Surg Clin, Dept Clin Chem & Clin Biochem, D-80336 Munich, Germany
关键词
D O I
10.1074/jbc.273.49.32927
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A novel metallocarboxypeptidase inhibitor was isolated from the medical leech Hirudo medicinalis, Amino acid sequence analysis provided a nearly complete primary structure. which was subsequently verified and completed by cDNA cloning using reverse transcriptase-polymerase chain reaction/rapid amplification of cDNA end techniques. The inhibitor, called LCI (leech carboxypeptidase inhibitor), is a cysteine-rich polypeptide composed of 66 amino acid residues. It does not show sequence similarity to any other protein except at its C-terminal end. In this region, the inhibitor shares the amino acid sequence -Thr-Cys-X-Pro-Tyr-Val-X with Solanacea carboxypeptidase inhibitors, suggesting a similar mechanism of inhibition where the C-terminal tail of the inhibitor interacts with the active center of metallocarboxypeptidases in a substrate-like manner. This hypothesis is supported by the hydrolytic 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 recombinant forms, LCI is a tightly binding, competitive inhibitor of different types of pancreatic-like carboxypeptidases, with equilibrium dissociation constants 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 nuclear magnetic resonance spectroscopy analysis indicate that recombinant LCT is a compactly folded globular protein, stable to a wide range of pH and denaturing conditions.
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页码:32927 / 32933
页数:7
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