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 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 Ki 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 LCI is a compactly folded globular protein, stable to a wide range of pH and denaturing conditions.
Reverter
D
D
Departament de Bioquímica i Biologia Molecular, Unitat de Ciències, and Institut de Biologia Fonamental, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
Vendrell
J
J
Canals
F
F
Horstmann
J
J
Avilés
F X
FX
Fritz
H
H
Sommerhoff
C P
CP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007865:Leeches / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; C116332:LCI protein, Hirudo medicinalis; D011506:Proteins; D012333:RNA, Messenger; D002268:Carboxypeptidases