Extracellular cysteines define ectopeptidase (APN, CD13) expression and function

Abstract

Alanyl aminopeptidase (APN) is a surface-bound metallopeptidase that processes the N-terminals of biologically active peptides such as enkephalins, angiotensins, neurokinins, and cytokines. It exerts profound activity on vital processes such as immune response, cellular growth, and blood pressure control. Inhibition of either APN gene expression or its enzymatic activity severely affects leukocyte growth and function. We show here that oxidoreductase-mediated modulations of the cell surface thiol status affect the enzymatic activity of APN. Additional evidence for the pivotal role of extracellular cysteines in the APN molecule was obtained when substitution of any of these six cysteines caused complete loss of surface expression and enzymatic activity. In contrast, the transmembrane Cys24 appears to have no similar function. Enzymatically inactive cysteine mutants were retained in the endoplasmic reticulum as shown by high-resolution imaging and Endoglycosidase H digestion. In the absence of any crystal-structure data, the demonstration that individual extracellular cysteines contribute to APN expression and function appears to be of particular importance. The data are the first to show thiol-dependent modulation of the activity of a typical surface-bound peptidase at the cell surface, probably reflecting a general regulating mechanism. This may relate to various disease processes such as inflammation or malignant transformation.

Regulation of the protein disulfide proteome by mitochondria in mammalian cells

“…2d), suggesting that disulfide formation in endoglin depends on mitochondrial ROS. Such disulfide-dependent antibody recognition also was reported previously for the cell-surface protein CD13 (13).…”

“…2e), but not platelet/endothelial cell adhesion molecule (PECAM), was eliminated, whereas staining within the Golgi increased. The alteration of surface expression also has been reported for CD13 with a disulfide-forming cysteine site mutant (13). Similarly, previous studies showed that disulfidebound proteins are reduced and reversibly retained in the ER in DTT-treated cells, whereas proteins entering the secretory pathway are not (14).…”

CIP-13F, a novel aminopeptidase N (APN/CD13) inhibitor, inhibits Lewis lung carcinoma growth and metastasis in mice

“…Aminopeptidase N (APN/CD13), a Zn 2+ -dependent ectopeptidase localized on cell surface, is a transmembrane protein that cleaves N-terminal neutral amino acids of various peptides and proteins [1,2]. In a classic report, a signiWcant correlation between APN/CD13 expression and metastasis of lung carcinoma was observed in patients with squamous cell carcinoma and ovarian cancer.…”

“…Aminopeptidase N (APN/CD13) is a type II membranebound metalloproteinase, which is expressed on several types of cells including intestinal epithelium, liver, placenta and lung [1,2]. APN/CD13 has a variety of functions, such as inXammatory and immunological responses, signal transduction, antigen processing and cytokine degradation [3,4].…”

Design, Synthesis, and Activity Evaluation of a New 5-fluorouracil Prodrug Containing an Asn-Gly-Arg(NO2)COOCH3 Tripeptide

“…It is a ubiquitous enzyme expressed in many kinds of tissues, organs, and cells [2,3]. APN can remove the amino acid from the unsubstituted terminus of biologically active peptides, such as enkephalins, angiotensins, neurokinins, cytokines [4].…”