Characterization of Recombinant Human Adipocyte-Derived Leucine Aminopeptidase Expressed in Chinese Hamster Ovary Cells

Abstract

Adipocyte-derived leucine aminopeptidase (A-LAP) is a recently identified novel member of the M1 family of zinc-metallopeptidases. Transfection of the A-LAP cDNA into COS-7 cells resulted in the secretion of the enzyme. In this study, recombinant A-LAP was expressed in Chinese hamster ovary cells, purified to homogeneity and its enzymatic properties were characterized. The purified enzyme was active towards a synthetic substrate, L-leucyl-p-nitroanilide, yielding a V(max) of 3.55 micromol/min/mg and a K(m) of 1.28 mM, and was shown to be a monomeric protein with molecular mass of 120 kDa in solution. By monitoring the sequential N-terminal amino acid liberation, it was found that the enzyme hydrolyzes a variety of bioactive peptides, including angiotensin II and kallidin. Immunohistochemical analysis indicated that the enzyme is expressed in the cortex of the human kidney, where tissue kallikrein is localized. Taken together, these results indicate that A-LAP possesses a broad substrate specificity towards naturally occurring peptide hormones and suggest that it plays a role in the regulation of blood pressure through the inactivation of angiotensin II and/or the generation of bradykinin in the kidney.

Human Leukocyte-derived Arginine Aminopeptidase

“…Considering that A-LAP, which cleaves angiotensin II and III and kallidin, regulates blood pressure (24,46), it is tempting to speculate that L-RAP also plays a role in the regulation of blood pressure by inactivating angiotensin III and generating bradykinin. Putative ER retention machinery may play a role in the biological function of substrate peptides by regulating the secretion of the enzyme.…”

“…Although substrate specificity of L-RAP toward synthetic substrates is rather restricted, L-RAP could process several antigenic precursors of MHC class I-presented antigen having various N-terminal residues like A-LAP (9,46). Several enzymes were purified either from cytoplasm or ER lumen as trimming enzymes.…”

Secretion of Endoplasmic Reticulum Aminopeptidase 1 Is Involved in the Activation of Macrophages Induced by Lipopolysaccharide and Interferon-γ

“…On the other hand, it shows relatively broad substrate specificity toward natural peptide hormones, such as angiotensin II, kallidin, and neurokinin A, which may reflect its role in the processing of various precursors of antigenic peptides presented to MHC class I molecules (11). On the basis of a preference for substrates of a specific length and C-terminal hydrophobic amino acid, the "molecular ruler" mechanism was proposed for the processing of antigenic peptides by the enzyme (12).…”

“…Because ERAP1 inactivates angiotensin II and converts kallidin to bradykinin, it was initially speculated that it might regulate blood pressure (11). Subsequently, by screening for polymorphisms in the human ERAP1 gene, Yamamoto et al (13) identified an association of K528R variant enzyme with essential hypertension.…”

MHC Class I Quality Control

“…Although both are members of the M1 family of zinc metolloproteases (Rawlings, Barrett et al 2010) ERAP1 and ERAP2 show striking differences in their substrate preferences. ERAP1 has a preference for large hydrophobic residues while ERAP2 prefers basic residues (Hattori, Kitatani et al 2000;Tanioka, Hattori et al 2003;Saveanu, Carroll et al 2005). In contrast to most other aminopeptidases, that are more or less restricted to cleaving peptides shorter than four residues, ERAP1 shows a strong increase in cleaving activity towards peptides that are between 10-16 residues long (York, Chang et al 2002).…”