Human recombinant membrane-bound aminopeptidase P: production of a soluble form and characterization using novel, internally quenched fluorescent substrates

Abstract

APP (aminopeptidase P) has the unique ability to cleave the N-terminal amino acid residue from peptides exhibiting a proline at P(1)'. Despite its putative involvement in the processing of bioactive peptides, among them the kinins, little is known about the physiological roles of both human forms of APP. The purpose of the present study is first to engineer and characterize a secreted form of hmAPP (human membrane-bound APP). Our biochemical analysis has shown that the expressed glycosylated protein is fully functional, and exhibits enzymic parameters similar to those described previously for mAPP purified from porcine or bovine lungs or expressed from a porcine clone. This soluble form of hmAPP cross-reacts with a polyclonal antiserum raised against a 469-amino-acid hmAPP fragment produced in Escherichia coli. Secondly, we synthesized three internally quenched fluorescent peptide substrates that exhibit a similar affinity for the enzyme than its natural substrates, the kinins, and a higher affinity compared with the tripeptide Arg-Pro-Pro used until now for the quantification of APP in biological samples. These new substrates represent a helpful analytical tool for rapid and reliable screening of patients susceptible to adverse reactions associated with angiotensin-converting enzyme inhibitors or novel vasopeptidase (mixed angiotensin-converting enzyme/neprilysin) inhibitors.

FE(II) Is the Native Cofactor for Escherichia coli Methionine Aminopeptidase

“…E. coli prolidase was reported as a Co(II) enzyme (55), while human prolidase is believed to be a Mn(II) enzyme (56). Although E. coli aminopeptidase P (57, 58) and human cytosolic homolog (59) were characterized as Mn(II) enzymes, both human and porcine membrane-bound aminopeptidase P were described as Zn(II)-containing enzymes (60,61). Several bacterial nitrile hydratases have been characterized, and they contain either cobalt or ferric ions, although they have similar sequences and share the same metal ligand residues (45,62).…”

The Kallikrein-Kinin System: Current and Future Pharmacological Targets

“…A secreted form of mAPP has recently been produced and characterized in vitro in a transfected HEK-293 cell line. This protein exhibits a molecular mass of 85 kDa (101).…”

The effect of electronegativity and angiotensin-converting enzyme inhibition on the kinin-forming capacity of polyacrylonitrile dialysis membranes

“…The plasma samples were screened for APP and ACE activities according to methods described previously [12]. The results are expressed in units/mL (mmol of substrate hydrolyzed/mL/min).…”