The moonlighting enzyme CD13: old and new functions to target

Abstract

Aminopeptidase N (CD13) is a widely expressed ectoenzyme with functions that do not always depend on its enzymatic activity: an aspect that has been overlooked. Numerous CD13-targeting tools have been developed in the last few years. Several of them are already undergoing clinical trials, and there are promising reports on the effectiveness of others in animal models of disease. However, their efficacy might be obscured by their effects on unrecognized functions of CD13, resulting in unexpected complications. The purpose of this review is (i) to discuss the various functions ascribed to CD13 and the possible mechanisms behind them and (ii) to consider some of the questions that need to be answered to achieve a better understanding of the biological relevance of these functions, a more precise interpretation of the results obtained after their manipulation and a more rational design of CD13-targeting agents.

Figure 1

The functions and three mechanisms of action of human CD13. As shown in (a), upon ligand binding, CD13 functions: (i) as an enzyme, (ii) as a receptor and/or (iii) as a signaling molecule. Each of these functions depends on at least one of the mechanisms of action listed in (b), namely: (i) peptide cleavage, (ii) endocytosis and (iii) signal transduction. Each of these mechanisms result in the biological phenomena listed on the right side of part (b). Some complex phenomena, such as angiogenesis, invasion and chemotaxis, must occur as a result of the interplay between enzymatic activity and signaling functions. Similarly, upon virus or maybe even cholesterol and NGR-peptide binding (especially NGR-targeted liposomal drugs), the receptor functions could mediate signal transduction required for endocytosis. The functional interplay between mechanisms of action is represented by the arrows on the right of part (b).

Figure 2

Hypothetical structure of human CD13. (a) Schematic seven domain organization of CD13. The active site is located between domains V and VI. Dimerization occurs between domains VII from each monomer, as indicated by ± signs. N-glycosylation sites are indicated with black arrows and O-glycosylation sites with pink arrows. Amino acids (aa) forming each domain are indicated on the left. Modified from Ref. , with kind permission of Springer Science and Business Media. According to the authors, the original model was based on electron microscopic studies of the purified enzyme, knowledge of the exon–intron organization of the gene and computer-aided structure predictions. (b) Schematic hypothetical four-domain model of human CD13, as proposed in Box 1. The possible conformational changes occurring upon substrate binding and the available data on the structure of other human enzymes were taken into account to modify the previous model. The active site (domain III) would be covered by the C-terminal domain (IV), and a conformational change would expose the catalytic site to allow the entrance of substrates. This process could be explained by the utilization, by substrates, of an initial recognition site such as the exopeptidase GXMEN motif, as has already been proposed for CD13 and other M₁ family members. Monomers are closely apposed to allow the predicted rollover mechanism occurring during the dynamic opening and closing of the active site, as proposed by Lendeckel et al. .

Figure 3

Possible structural modifications occurring upon ligation of CD13. (a) In the resting state, the CD13 enzymatic active site is cryptic (hidden). (b) Substrate or inhibitor binding to the initial contact site induces a conformational change that exposes the cryptic active site as well as other cryptic epitopes that lie close to it, resulting in activation or inhibition of the enzymatic activity . This conformational change might also involve the release of galectins and, consequently, the concomitant unmasking of the carbohydrate moieties involved in ligand binding, which could explain the potentiation of signaling functions by substrates . Upon the exposure of cryptic epitopes, anti-CD13 autoantibodies or ligands (c,d) bind to and crosslink CD13, resulting in oligomerization and signaling. In the absence of a pre-bound substrate, oligomerization by multivalent ligands, such as antibodies and viruses, might be sufficient to trigger signaling or internalization , . (e) Additionally, substrate, antibody or virus-associated proteins regulate the endocytosis of CD13 , , , which might constitute a mechanism of signaling and inhibition of its enzymatic activity and could be responsible for its dissociation from galectins and adaptor molecules. This would explain some of the functions modulated by mAbs that do not block the enzymatic activity.