Developmental retardation, microcephaly, and peptiduria in mice without aminopeptidase P1

Abstract

Cytosolic aminopeptidase P1 (APP1) is one of the three known mammalian aminopeptidase Ps (APPs) that cleave the N-terminal amino acid residue of peptides in which the penultimate amino acid is proline. In mammals, many biologically active peptides have a highly conserved N-terminal penultimate proline. However, little is known about the physiological role of APP1. In addition, there is no direct evidence to associate a deficiency in APP1 with metabolic diseases. Although two human subjects with reduced APP activity exhibited peptiduria, it is unclear which of the three APP isoforms is responsible for this disorder. In this study, we generated APP1-deficient mice by knocking out Xpnpep1. Mouse APP1 deficiency causes severe growth retardation, microcephaly, and modest lethality. In addition, imino-oligopeptide excretion was observed in urine samples from APP1-deficient mice. These results suggest an essential role for APP1-mediated peptide metabolism in body and brain development, and indicate a strong causal link between APP1 deficiency and peptiduria.

Introduction

Many biologically active peptides in mammals have a highly conserved proline residue at the penultimate N-terminal position. This configuration forms an X-Pro motif in which X is any amino acid. More than 23 bioactive peptides with this structure, including cytokines, growth hormones, and neuropeptides, have been identified [1]. The unique cyclic structure of proline in the X-Pro motif confers both resistance to non-specific proteolytic degradation and biological activity [2].

Proteolytic cleavage of the first residue in the X-Pro motif is mediated by aminopeptidase Ps (APPs), which are also known as X-prolyl aminopeptidases (EC 3.4.11.9). APPs have been found in a wide range of organisms including vertebrates, insects, nematodes, bacteria, and plants [3], [4], [5], [6], [7]. There are three known APPs (APP1-3) in mammals. Each APP isoform is encoded by a distinct gene (Xpnpep1-3) and has a specific subcellular localization. Soluble cytosolic isoform APP1 has been identified in human leukocytes [8], human platelets [9], rat brain [10], and guinea pig serum [11]. This enzyme exhibits broad substrate specificity for peptides of diverse sizes [10]. In contrast, APP2 is a glycosylphosphatidylinositol (GPI)-anchored ectoenzyme that is ubiquitously expressed in human tissues [12], [13]. In comparison to APP1, APP2 has a much more restricted substrate specificity [14], [15]. The third isoform, APP3, is mainly localized to mitochondria and shows a ubiquitous tissue distribution pattern in humans [16], [17].

Deficiency in APP activity has been known to cause human peptiduria, which is the massive urinary excretion of undigested imino-oligopeptide. In addition, one human subject that was deficient in APP activity exhibited severe developmental retardation and microcephaly [18], suggesting that APP activity might be critical for the regulation of body and brain development. However, a direct causal relationship between APP deficiency and developmental retardation has not been established in vivo. Additionally, it is still not known which of the three APP isoforms is responsible for such dysfunction in these subjects.

In this study, we genetically disrupted mouse APP1 function and investigated the consequences. APP1-deficient mice displayed severe developmental retardation and microcephaly. In addition, APP1 deficiency caused peptiduria in mice. Our results indicate a strong causal link between APP1 deficiency and developmental dysfunction as well as peptiduria, and we identify APP1 as an important regulator of body and brain development.