Original articleNatriuretic peptide pharmacogenetics: Membrane metallo-endopeptidase (MME): Common gene sequence variation, functional characterization and degradation
Research highlights
►Membrane metallo-endopeptidase (MME), also known as neutral endopeptidase 24.11 is involved in the metabolism of natriuretic peptides that play a key role in modulating cardiac structure and function. ►MME was resequenced in three ethnic groups resulting in identification of 90 polymorphisms of which 65 were novel, including 8 nonsynonymous single nucleotide polymorphisms (nsSNPs) of which 7 were not described before. ►The functional effects of the nsSNPs on expressed protein levels and enzyme activity was studied in an in-vitro cell based system leading to the identification of a significant reduction in enzyme activity (21% of wild-type) and immunoreactive protein (29% of wild-type) for the Met73Val variant allozyme ►Proteasome-mediated degradation and autophagy participated in the degradation of the Met73Val allozyme and was associated with increased expression of the chaperone proteins, BiP and GRP94 suggesting protein misfolding, compatible with conclusions based on the MME X-ray crystal structure. ►The Met73Val variant allozyme could have a significant effect on the metabolism of natriuretic peptides.
Introduction
The heart synthesizes two important peptides, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). ANP and BNP cause vasodilation and natriuresis by direct actions that are primarily cGMP-mediated. These two peptides play an important regulatory role in cardiovascular disease as well as a key role in modulating cardiac structure and function [1]. BNP is commercially available as nesiritide and is approved for intravenous use in the treatment of decompensated heart failure (HF). These peptides are degraded approximately equally by two mechanisms: enzymatic degradation catalyzed by membrane metallo-endopeptidase (MME), or neutral endopeptidase (NEP 24.11; EC 3.4.24.11), and clearance by the natriuretic peptide receptor C (NPR-C) [2]. Sequence variation in the gene encoding the precursor of BNP (NPPB) has been associated with altered serum BNP levels [3], but the impact on BNP levels of MME gene sequence variation remains unknown. Measurement of serum BNP is useful for diagnostic and prognostic purposes, especially in heart failure [4]. Nesiritide (BNP) is used to treat acute decompensated heart failure, but response to its use is variable [5]. MME plays an important role in the metabolism of nesiritide [6], and the potential importance of MME genetic polymorphisms on nesiritide degradation is also unknown. Furthermore, compounds that inhibit MME by increasing endogenous peptides like natriuretic peptides and bradykinin have been developed, and have been used to treat hypertension and heart failure [7]. MME polymorphisms and their possible effect on the pharmacological actions of these drugs have not been studied. Important steps toward these translational studies would be the identification and characterization of common MME polymorphisms in “normal”, randomly selected populations. DNA samples from such subjects are studied because common genetic variation needs to be defined initially in “normal” individuals prior to studying drug effect and response [8].
MME is a 100 kDa, type II integral membrane protein containing a highly conserved zinc binding motif in its extracellular C-terminal domain [9]. The enzyme is present in polymorphonuclear leucocytes, brush border cells of the proximal tubule and podocytes of the kidney, and epithelial cells of the liver, breast, lung and brain. MME cleaves substrates on the amino side of hydrophobic amino acids by hydrolyzing peptide bonds, and, as a result, it inactivates several peptide hormones including glucagon, enkephalins, substance P, neurotensin, oxytocin, bradykinin, and natriuretic peptides. The MME gene encodes a 750 amino acid protein, is 45 kb in length, consisting of 23 exons and maps to chromosome 3q25.1–q25.2. The 5′-untranslated region of the gene is alternatively spliced, resulting in four separate 5′-UTR sequences [10]. However, the coding region is not affected by this alternative splicing. Because of the importance of MME as the final common pathway for natriuretic peptide metabolism and because of the importance of natriuretic peptides in cardiovascular disease, we resequenced MME in DNA from 288 subjects, identified novel coding and non-coding genetic polymorphisms in the MME gene, and subsequently characterized the functional implications of the coding polymorphisms.
Section snippets
DNA samples
DNA samples from 96 European-American (EA), 96 African-American (AA), and 96 Han Chinese-American (HCA) unrelated, healthy subjects (sample sets HD100CAU, HD100AA, HD100CHI) were obtained from the Coriell Cell Repository (Camden, NJ). All of these DNA samples were collected, anonymized and deposited by the National Institute of General Medical Sciences. All subjects had provided written consent for the use of their DNA for research purposes. The present study was reviewed and approved by the
Human MME resequencing
MME was resequenced using 288 anonymized DNA samples from subjects of three different ethnic groups. Ninety polymorphisms, including 86 SNPs and 4 indels, were observed; 62 in AA, 40 in EA, and 36 in HCA subjects (Table 1 and Fig. 1). Thirty-eight of these polymorphisms were “common”, with a minor allele frequency (MAF) of > 1% in at least one ethnic group. There were 17 polymorphisms within exons, 8 of which were nonsynonymous SNPs (nsSNPs) resulting in the following changes in the encoded
Discussion
MME plays an important role in the degradation of glucagon, enkephalins, substance P, neurotensin, oxytocin, bradykinin, and natriuretic peptides [26]. MME is expressed in cardiac and kidney tissue and is upregulated, with an increase in enzymatic activity, in patients with aortic stenosis and heart failure [27], [28]. The upregulation in MME expression is thought to promote fibrosis in aortic stenosis [29]. MME also plays an important role in the local control of bradykinin in cardiac tissue
Disclosures
None.
Acknowledgements
This study was supported, in part by HL 84904 (Heart Failure Clinical Research Network), a Marie Ingalls Cardiovascular Career Development Award, a PhRMA Foundation “Center for Excellence in Clinical Pharmacology” Award (R.M. Weinshilboum), and NIH grants UL1RR24150 (N. L. Pereira), RO1 GM28157 (R.M. Weinshilboum), RO1 CA132780 (R.M. Weinshilboum) and UO1 GM61388 (The Pharmacogenetics Research Network). We thank Luanne Wussow for her assistance with the preparation of this manuscript.
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These authors contributed equally to this work.