Peptidases: structure, function and modulation of peptide‐mediated effects in the human lung (1999)

Peptidases are enzymes capable of cleaving, and thereby often inactivating, small peptides. They are widely distributed on the surface of many different cell types, with the catalytic site exposed only at the external surface. Peptidases are involved in a variety of processes, including peptide-mediated inflammatory responses, stromal cell-dependent B lymphopoiesis, and T-cell activation. In addition, some peptidases may have functions that are not based on their enzymatic activity. Peptidases are classified according to the location of the cleavage site in the putative substrate ( Table 1). Endopeptidases recognize specific amino acids in the middle of the peptide, whereas exopeptidases recognize one or two terminal amino acids. Exopeptidases that attack peptides from the N-terminus (removing either single amino acids or a dipeptide) are termed (dipeptidyl) aminopeptidases, whereas peptidases attacking the C-terminus are termed carboxypeptidases. Neutral endopeptidase (NEP, neprilysin, EC 3.4.24.11) was first characterized from rabbit kidney brush border. It soon became apparent that NEP was similar to enkephalinase, originally discovered in the brain. Furthermore, cloning of the NEP gene and subsequent cloning of the common acute lymphoblastic leukaemia antigen (CALLA, CD10) showed that both sequences were similar [ 1]. NEP is a glycoprotein of 750 amino acids, with a single 24 amino acid hydrophobic segment that functions as both a transmembrane region and a signal peptide [ 1]. The C-terminal 700 amino acids compose the extracellular domain, whereas the 25 N-terminal amino acids form the cytoplasmic tail. The extracellular domain contains six potential N-glycosylation sites. Tissue-specific glycosylation may result in different molecular masses, ranging from approximately 90–110 kDa. The extracellular domain contains the pentapeptide consensus sequence (His-Glu-[Ile, Leu, Met]-X-His) of zinc-binding metalloproteases, in which the two histidines co-ordinating zinc and the glutamic acid residue, together with an aspartic acid residue, are critically involved in the catalytic process. Characterization of the human NEP gene, which is located at chromosome 3 (q21-q27), showed that it spans more than 80 kb and is composed of 25 exons [ 2]. Exons 1, 1bis, and 2 encode 5′ untranslated sequences; exon 3 encodes the initiation codon and the transmembrane and cytoplasmic domain; 20 short exons (exons 4–23) encode most of the extracellular region; and exon 24 encodes the C-terminal 32 amino acids of the protein and contains the entire 3′ untranslated region (UTR). Within exon 24 are five poly (A) addition signals. Alternative splicing of exon 1, exon 1bis, exon 2 (2a), or part of exon 2 (2b) to the common exon 3, resulting in four different transcripts, may be the origin of the tissue-specific or stage of development-specific expression of NEP [ 3]. Indeed, two separate regulatory elements have been found in the NEP promoter region and these elements may be regulated by the transcription factor CBF/NF-Y in a tissue-specific manner. A cDNA clone lacking the complete exon 16 has been isolated from human lung tissue [ 4]. Deletion of this 27 amino acid segment was shown to reduce enzyme activity to barely detectable levels. However, the physiological relevance of this truncated enzyme remains to be determined. In the rat, an exon 5–18 deletion has been described, but no evidence was found to support the expression of this truncated variant in the human lung. NEP is expressed by a variety of haematopoietic and non-hematopoietic cells [ 5]. NEP is abundantly present in renal proximal tubular epithelial cells, small intestinal epithelium, and biliary canaliculae. In addition, NEP can be found in synaptic membranes of the central nervous system, bone marrow stromal cells, fibroblasts, placenta, lymphoid progenitors, and neutrophils. Given the expression of NEP on lymphoid progenitors, expression of NEP is used as a diagnostic marker for several lymphoid malignancies, including Burkitt's lymphomas and certain myelomas. In the human lung, NEP is expressed by bronchial epithelial cells, submucosal glands, bronchial smooth muscle, and endothelium [ 6, 7]. In addition, NEP can be found on alveolar epithelial cells. NEP is able to hydrolyse peptide bonds on the N-terminal site of hydrophobic amino acids, including Phe, Leu, Ile, Val, Tyr, Ala, and Trp ( Table 1). However, sub-site interactions and conformational factors greatly influence the efficiency of hydrolysis. Among the possible substrates of NEP are substance P (SP), neurokinin A (NKA), formyl-metheonyl-leucyl-phenylalanine (fMLP), atrial natriuretic factor (ANF), endothelin-1 (ET-1), bombesin-like peptides (BLP), angiotensins, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), bradykinin (BK), enkephalins, cholecystokinin, and neurotensin. Although NEP predominantly cleaves simple peptides, it has been reported that NEP may also be able to hydrolyse certain larger substrates, including cytokines such as interleukin (IL)-1β and IL-6. The general biological function of NEP is to reduce cellular responses to peptide hormones. Target cells express both NEP and the peptide-receptor; by degrading the peptide substrate, NEP reduces the local concentration of the peptide available for binding to the receptor. For example, NEP reduces ANF-mediated hypotension, fMLP-mediated chemotaxis of neutrophils, and enkephalin-mediated analgesia. Targeted disruption of the NEP locus in mice results in enhanced lethality to endotoxin, indicating an important protective role for NEP in septic shock. A role for NEP in lymphoid development has been suggested by studies showing that inhibition of NEP results in increased proliferation and maturation of B cells, both in vitro and in vivo [ 8]. Therefore, it has been suggested that NEP functions to regulate B-cell development by inactivating a peptide that stimulates B-cell proliferation and differentiation. Alternatively, NEP may activate a pro-peptide that inhibits proliferation and differentiation of B cells. The role of NEP in the regulation of cellular proliferation and differentiation in the lung is discussed next in more detail. The role of NEP in the modulation of neurogenic inflammation will be discussed further on. NEP plays an important role in the cellular differentiation and proliferation of bronchial epithelial cells by inactivating BLP [ 9]. BLP are potent growth factors for bronchial epithelial cells and are involved in lung development. The temporal and cellular patterns of NEP expression implicate the enzyme in the regulation of BLP-mediated fetal lung development. Indeed, both in vitro and in vivo, it was shown that inhibition of NEP resulted in increased maturation of the developing fetal lung [ 10]. Reduced NEP activity may also promote BLP-mediated proliferation of bronchial epithelial cells. Indeed, the growth and proliferation of BLP-dependent carcinomas is inhibited by NEP and potentiated by NEP inhibition [ 9]. NEP expression by epithelial cells is inversely correlated with cellular proliferation. Therefore, reduced NEP activity may promote BLP-mediated proliferation and facilitate the development of small-cell carcinomas of the lung [ 9]. A role for NEP in the regulation of tumour cell proliferation is also supported by studies using a human T-cell line (Jurkat). In these cells, NEP is required for phorbol ester-induced growth arrest. Aminopeptidase N (APN, EC 3.4.11.2) is a widely studied peptidase, which is known under a variety of names, including aminopeptidase M, alanine aminopeptidase, arylamidase, and microsomal α-aminoacyl-peptide hydrolase. APN is a glycoprotein of 967 amino acids with 11 potential sites of asparagine-linked oligosaccharide addition [ 11]. The unglycosylated protein has a molecular size of 110 kDa; post-translational modification results in the 130-kDa precursor (gp130) and the 150-kDa mature protein (gp150) [ 12]. The 23-amino acid retained signal also functions as the membrane-spanning segment, orientating the APN N-terminus inside and the C-terminus outside the cell (thereby defining APN as a type II integral membrane protein) [ 11]. The intracellular domain of APN is only nine amino acids long, whereas the extracellular domain contains 935 amino acids. Similar to NEP, the extracellular domain contains a pentapeptide consensus sequence characteristic of members of the zinc-binding metalloprotease family. On the surface of cells, APN is expressed as a non-covalently bound homodimer. Cloning of the APN cDNA revealed that its sequence was identical to the myeloid marker CD13 [ 11, 12]. The APN gene is located on the long arm of chromosome 15 (q25–26) and exists of 20 exons [ 13]. Northern blot analysis of RNA extracted from several tissues revealed two distinct APN transcripts: a 3.7-kb transcript expressed by monocytes, myeloid leukaemia cells, and fibroblasts, and a 3.4-kb transcript expressed by intestinal and kidney cells [ In epithelial cells, from the initiation codon and from a In the found in myeloid cells and from several sites in an exon located kb from the exon the initiation both encode the indicating that separate the tissue-specific expression of the APN gene [ In addition, a region with located kb of the site which is used in epithelial cells, may also be important for the tissue-specific The non-hematopoietic of APN a with NEP [ 7]. APN is expressed on renal proximal tubular epithelial cells, small intestinal epithelium, biliary synaptic membranes of the central nervous system, bone marrow stromal cells, fibroblasts, placenta, and [ 5]. In with NEP, APN is also expressed on and myeloid of APN may be used as a marker for myeloid cells may also express whereas not express this However, expression of APN on can be or to cells, epithelial cells and In the human APN is present on and cells, and [ 7]. APN is a which or substrates with an N-terminal alanine ( Table 1). amino acids, may also be with the of APN substrates to be small peptides than larger the enzyme is more in from than Among the possible substrates for APN are enkephalins, and cytokines such as and However, in certain cleavage by may be functions of APN have been [ 5]. APN expressed on the brush of the may be involved in the of of small peptides. with NEP and often in with NEP, APN may function to reduce cellular responses to peptide hormones. a APN in the of peptides by the II [ APN may be involved in tumour and by of type APN as a for which are RNA that in EC is an of amino acids with type II membrane [ It contains a intracellular domain of six amino acids, a acid hydrophobic transmembrane region also functions as signal and a acid extracellular The extracellular domain, which contains nine potential glycosylation can be an N-terminal region glycosylation sites and with a acid a region; and a acid C-terminal domain the putative catalytic On the surface of cells, is present as a two identical of approximately molecular studies that several of can be found [ In with NEP and not zinc in its catalytic its with is to the family. of this a catalytic site in which the are in the sequence Cloning of the cDNA revealed that its sequence was identical to the T-cell antigen [ The human gene, located on chromosome 2 spans approximately kb and contains exons [ The site is two the first Trp is in exon and the is in exon The the catalytic site are present in a distinct in exon in exon and in exon exon also contains the codon and the 3′ untranslated region of the The 5′ domain of the gene contains a a but a region in and contains potential binding sites for several transcription including and protein The human gene encodes two RNA of approximately and which in sequence only at the 3′ untranslated region [ the two from the of different sites in the exon of the In many the non-hematopoietic tissue of that of NEP and is expressed on renal proximal tubular epithelial cells, epithelial cells in the small and biliary but can also be found on alveolar and [ 5]. In the human is present in submucosal [ 7]. The expression of on haematopoietic cells is regulated is from the of human but some of cells express the expression on is increased T-cell activation. is a marker for cells in that expression on cells may with [ expression was found on and cells, whereas cells expression of The of was for this [ cells have been reported to in the T-cell this was not found in is also expressed by in and can be on cells. is a with a it cleaves from the N-terminus of is at the with alanine in the may also be with a are not by the of is a in the of such peptides. peptides have the sequence at their N-terminus and may an important role in their peptides and of by two and which both are more potent than can be by A is also present at the of several cytokines and tumour factor and factor may have several the tissue in which it is plays an role in the renal and intestinal of However, most has been to the function of on Although the role of on cells is not studies that it may as a that can the signal of the T-cell [ of to the of of the cells, including and of the expression of the and enzymatic activity. Furthermore, of of several intracellular with a similar to that [ of and and increased in with indicating that is a [ In addition to T-cell cells enhanced responses, increased and increased activity. possible for the enhanced of cells to of was suggested by the that a that with [ may T-cell proliferation in part by of of activity results in reduced as as reduced of and of cells [ inhibition increased and of growth factor and a the in [ In a of reduced to than of the [ or responses to were in these showed reduced In with was by a in activity and by enhanced enzyme activity several results that plays an important role in responses in cells have been shown to their antigen with an that is by increased expression of it has been shown that to this increased antigen of cells. this may be by the of [ the may be for T-cell in of antigen and may of cells antigen is also found to be with and this is to as an important may to cell surface and the thereby its local may also function as an was found to to of the extracellular such as and of human cells to a signal in T-cell resulting in increased proliferation. inhibited this may be involved in the of the may as one of the for human Furthermore, the antigen has been shown to the enzymatic activity of resulting in the inhibition of T-cell responses to antigen and the of the protein may be by In addition to the peptidases peptidases are involved in the of enzyme enzyme aminopeptidases, and carboxypeptidases. also known as A or is a type II integral membrane endopeptidase to the of in [ of are present the human a form with a molecular which is found in epithelial and cells, and a found in cells. are from a single gene by the of two separate a and a The form is composed of two by gene in the of The only contains one of the two amino acids with potential glycosylation sites. domain has a catalytic which functions is widely distributed in human it is present on cells, in the brush of of the small and the renal proximal and in monocytes, and its location is to be the surface of the lung. The enzyme cleaves peptides an in the ( Table but the enzyme is than It is capable of inactivating bradykinin and enkephalins, and to the peptide to a role in and and In addition, substance but neurokinin A is not a is a type II integral membrane protein with NEP [ NEP, exists as a of molecular kDa. to its ( Table which is a potent and that may regulate and of can be from splicing of a single and In the human lung, has been found in epithelium, and smooth muscle, and bronchial [ Although may a role in peptides, it remains to be it is involved in the of in increased of have been found in and bronchial epithelial cells with tissues an of and The aminopeptidase is are aminopeptidase A specific for N-terminal and and aminopeptidase which will an N-terminal to a ( Table 1). The role of these peptidases in the of peptides has been but it may be that these enzymes are involved in the of a variety of substrates, with or cleavage by an A role for aminopeptidase A in the of peptides binding to the neurokinin has been may also be involved in the regulation of as deletion of the from an of to an N cleaves the C-terminal and of peptides such as bradykinin [ of the functions of is to the from potent vasoactive and inflammatory peptides or which are the In the human lung, has been in alveolar type cells, in the of the epithelium, in some and in the epithelial membrane [ activity in has been shown to be enhanced in that and the are the its in has also been found in increased activity was found in with lung or lung it was that activity in may be an of type cell Although the peptidases are integral membrane peptidases with enzymatic activity can be in may either be from of or may be by post-translational cleavage of the For most the physiological role of the remains to be endopeptidase activity from of the [ activity of NEP has been in exposed to [ and in with or Although the of the increased NEP remains to be it has been suggested that increased NEP may local tissue with subsequent of Furthermore, activity of NEP is increased in acute renal in with renal and in contains an of aminopeptidase including alanine aminopeptidase and alanine aminopeptidase activity predominantly a of CD13 [ activity of aminopeptidase has been in of with and it was shown that this be to lung tissue is present in several in human and may T-cell proliferation [ studies that is a of and that this which is a potent T-cell is not a of [ Furthermore, the form of found in is identical with a found to be expressed on the surface of cells [ cells to be the of activity [ activity in is in with and a was activity and cells in of but not of were no activity and or such as or Reduced activity has also been in with and in In the a activity and or cells was In addition to the two nervous and that the a is are the activity of and from are and the of and peptide from [ of either by or results in an and subsequent of from the of the these a variety of of specific neurokinin including increased and submucosal smooth and of sequence of is known as [ the neurogenic inflammatory many of the of a role for in the of has been In the the of peptides and may be whereas the of peptides may be it became apparent that were for the neurogenic inflammatory responses, it was that which may the of with the role of in the of studies have that peptidases a role in the modulation of peptide-mediated in the in [ has on the of the and and the enzyme The physiological relevance of by enzymatic has been from studies of the of enzyme inhibition on the physiological of or peptides. In the first it was shown that inhibition of NEP potentiated the of on submucosal in the in that inhibition of NEP potentiated the of on and smooth In it was shown that both NEP and in the of by was by NEP In addition, the not bronchial smooth in Therefore, is to an important role in the biological activity of peptides, whereas NEP is also involved in the of peptides present lung tissue or the bronchial The of NEP in is further supported by the that of of aminopeptidases, and not in the The of NEP in the of has also been shown in in vivo studies in studies showed that both and be potentiated by NEP inhibition [ Furthermore, these studies that but not increased the to that inflammatory are to [ In with the studies in which the of were increased to the inhibition of some studies have shown that of NEP may neurogenic of NEP inhibited the and to in [ and studies have shown that NEP is present on epithelial cells [ of the was further shown to result in increased responses to or [ However, NEP is also present at sites the and also of the NEP [ NEP expressed by epithelial cells may more be by than NEP located at sites. may thereby responses to and and antigen In may their in part by NEP activity. may neurogenic inflammatory responses inhibition of NEP activity. In with or was shown to result in enhanced responses to an that was by epithelial NEP activity [ In and many to results in the of to the and increased to can be by which from to also results in increased for and this not be enhanced by inhibition of NEP [ that NEP, which is supported by the that the NEP activity in was than the NEP activity in [ is a widely used that may In it was shown that at increased to and endopeptidase [ of the to in [ of NEP by increased the by in but not in exposed to NEP activity in of was inhibited by However, in no of on NEP activity in vivo be A possible for this may be that the NEP inhibited by only a small of the of NEP in the is an important factor to the development of small-cell lung carcinomas of the lung. NEP activity is in lung [ 9]. Therefore, one may that to the development of lung in part by NEP, thereby the of peptides and on bronchial epithelial cells. inflammation may be to the of by an on activity. In antigen results in inflammation and to [ It was shown that with inflammation were more to the of and the of than from In addition, the of enzyme on physiological responses and peptide cleavage were with NEP and enhanced activity [ In a human in vivo no of NEP on responses in was [ that either not a role in responses, or that in in However, in it has been shown that to [ an that is the of and have potent and are widely used in the of The may be in by an of NEP thereby neurogenic inflammatory Indeed, NEP activity by a human cell line and a bronchial epithelial cell line was shown to be increased with However, no of was in using the bronchial epithelial cell In were shown to reduce which be to NEP was supported by the that of with NEP and the of The of in vivo on NEP expression in the human has been reported [ In that it was shown that NEP expression in the bronchial and of was than the expression in [ shown many of the that to of to reduce the activity of NEP at the to responses to and neurogenic inflammation ( 1). However, most of these studies have been in the and have not been in Furthermore, in many studies the NEP was not only inhibits NEP, but was also shown to it that can the of the NEP from using is inflammation in from are by the of these are In the several factors may result in a thereby the neuropeptide from [ Although and peptidases have been shown to be present in the human their role in remains to be However, several may support the that and peptidases are involved in the of and have been shown in several in vivo studies to and these be potentiated by inhibition of NEP in [ Furthermore, these studies that is in with However, the of the was similar in and that the activity of NEP not both [ used in the were and it be that reduced NEP activity may of of can be in of and in whereas not of with The that are in vivo has also been supported by the that in can be by a [ this not be in some and can be potentiated by NEP which is present in the and is in can to of of in the of may both to acute and inflammatory In addition to their acute such as the of smooth muscle, the of and [ several studies have shown that a of and may be involved in tissue responses [ several of including and are able to and further a role for these in of peptidases may result in responses and thereby to the development of the inflammatory process. of NEP, either in or has been shown to the of known to be as and However, inhibition of NEP at shown to the of not the and in [ may that not a role in Alternatively, antigen may result in a of NEP activity. further peptidases and to in vivo studies using neurokinin be both in the and of NEP or first be in to the of the the of these be in both in the and in the of Furthermore, it be to with neurokinin or by for a of and to this and bronchial inflammation by analysis of bronchial and will in the of to the inflammatory in the of the of and peptidases in may be by with NEP, and the on by or such as it that and peptidases are involved in the of or peptidases may be in the of for the