N -lactoyl-amino acids are ubiquitous metabolites that originate from CNDP2-mediated reverse proteolysis of lactate and amino acids

Abstract

Despite technological advances in metabolomics, large parts of the human metabolome are still unexplored. In an untargeted metabolomics screen aiming to identify substrates of the orphan transporter ATP-binding cassette subfamily C member 5 (ABCC5), we identified a class of mammalian metabolites, N-lactoyl-amino acids. Using parallel protein fractionation in conjunction with shotgun proteomics on fractions containing N-lactoyl-Phe-forming activity, we unexpectedly found that a protease, cytosolic nonspecific dipeptidase 2 (CNDP2), catalyzes their formation. N-lactoyl-amino acids are ubiquitous pseudodipeptides of lactic acid and amino acids that are rapidly formed by reverse proteolysis, a process previously considered to be negligible in vivo. The plasma levels of these metabolites strongly correlate with plasma levels of lactate and amino acid, as shown by increased levels after physical exercise and in patients with phenylketonuria who suffer from elevated Phe levels. Our approach to identify unknown metabolites and their biosynthesis has general applicability in the further exploration of the human metabolome.unknown metabolites | untargeted metabolomics | ABCC5 | MRP5 | physical exercise U ntargeted metabolomics aims to provide a comprehensive snapshot of the metabolome and is becoming a mainstream technique to discover biomarkers, to study the effects of interventions, and to discover the function of enzymes (1).Recent technical improvements now make it possible to detect several thousand metabolites in a single untargeted metabolomics analysis, but the identity of these metabolites is initially not known beyond their molecular mass (1). Knowing the chemical identity of metabolites is crucial for the proper interpretation of metabolomic studies, however. Online metabolite databases like METLIN (∼240,000 entries) and the Human Metabolome Database (HMDB; ∼42,000 entries) contain vast numbers of metabolites and are extremely useful to annotate the detected molecular masses (2, 3). These databases cover large parts of the metabolome, but significant gaps remain. Due to poorly characterized and promiscuous enzymes, the human metabolome is much larger than initially anticipated (4). State-of-the-art untargeted metabolomics studies still report up to 40% unidentified, but potentially important, metabolites that can be detected reproducibly (5-8). Nevertheless, unknown metabolites are only rarely characterized because of the extensive work required for de novo structure elucidation (9-11). Here, we describe the characterization of a class of mammalian metabolites that we discovered in an untargeted metabolomic screen aimed to identify substrates of the orphan transporter ATP-binding cassette subfamily C member 5 (ABCC5).ABCC5 is a member of the C-branch of the superfamily of ABC transporters, which use the energy provided by the hydrolysis of ATP to transport substrates across the plasma membrane (12). ABCC5, also known as multidrug resistance-associated protein 5 (MRP5), is ubiquitously expressed, but levels in b...

ATP-binding Cassette Subfamily C Member 5 (ABCC5) Functions as an Efflux Transporter of Glutamate Conjugates and Analogs

“…Regardless of their diverse functions, all metabolites were transported into ABCC5-containing vesicles. Vesicular transport of NAAG and ZJ43 shows that ABCC5 has a relatively low affinity for glutamate conjugates, which is comparable to the K m reported for folic acid (1.3 mM) (14) and N-lactoyl-phenylalanine (ϳ1 mM) (16). As the physiological levels of NAAG and BCG can reach millimolar concentrations in the brain, these substrates can still be transported by ABCC5 at considerable rates.…”

“…Folate accumulated in Abcc5 Ϫ/Ϫ tissues, but this accumulation was nonsignificant in all tissues except brain. The lack of difference in levels of N-lactoyl-amino acids can be explained by the fact that it is in intracellular equilibrium with the levels of lactate and amino acids (16).…”

“…In terms of drug resistance, ABCC5 is best known for its ability to transport nucleotide analogs (12,13) and antifolates like methotrexate (14) but elevated ABCC5 levels have not been linked to clinical drug resistance (1). Although ABCC5 transports endogenous metabolites like cyclic nucleotides (15), folic acid (14), and the recently identified N-lactoyl-amino acids (16) in vitro, the physiological relevance of this transport is unclear. Abcc5 Ϫ/Ϫ mice do not have an overt phenotype and do not provide clues about the in vivo substrates of ABCC5 either (12).…”

Exploring conformational equilibria of a heterodimeric ABC transporter

“…The same transporters have received increasing attention in past years, playing a central role in the absorption, distribution, metabolism and elimination of pharmaceuticals in the human body (Nigam, 2015). In addition, ABC exporters play a major physiological role in the transport of metabolites such as urate, glucuronides and N-lactoyl-amino acids (Jansen et al, 2015; Krumpochova et al, 2012; Woodward et al, 2009). …”

ABCC5 is required for cAMP-mediated hindgut invagination in sea urchin embryos

“…Among these ABC transporters, ABCC5 (also known as MRP5, MOAT-C, pABC11, sMRP) is unique in that, although being related to proteins involved in protective efflux and drug resistance (Belinsky et al, 1998;Chen and Tiwari, 2011;Kool et al, 1997;McAleer et al, 1999), it has not been demonstrated to be toxicologically important (Chen and Tiwari, 2011;Leslie et al, 2001). That ABCC5 effluxes cGMP and cAMP has long been known (Jedlitschky et al, 2000;Sager and Ravna, 2009;Wielinga et al, 2003); however, more recently, heme (Korolnek et al, 2014) and N-lactoyl-amino acids (Jansen et al, 2015) were reported to be possible substrates. Hyaluronan may also be transported by ABCC5 (Schulz et al, 2007), although it is more likely that it is translocated by hyaluronan synthase itself (Medina et al, 2012).…”