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Neprilysin (NEP) and NEPLs (NEPL-alpha, -beta, and Gamma), damage-induced neuronal endopeptidase (DINE)/ECEL1 and Phosphate regulating gene with homologies to endopeptidases on the X chromosome (PHEX)/PEX) Antibodies
    

M= Mouse; R=Rat; H=Human; Rb=Rabbit; G=goat; B=Bovine, MO=Monkey; P=pig; CT= near C-terminus; NT=near N-terminus; Internal=Middle of protein. EC=extracellular; CP=cytoplasmic domains *


Neprilysin (NEP) and NEPLs (NEPL-alpha, -beta, and Gamma), damage-induced neuronal endopeptidase (DINE)/ECEL1
and Phosphate regulating gene with homologies to endopeptidases on the X chromosome (PHEX)/PEX) Antibodies

The amyloid beat-peptide (Ab) of 39 to 43aa (Ab40, Ab42, Ab43) is constitutively produced in brain upon proteolysis of the b-amyloid precursor protein (APP). In the young and healthy humans, Ab is rapidly catabolized before it can be deposited in the brain. However, upon aging or the onset of familial Alzheimer's Disease, alterations in either synthesis or degradation/clearance of Ab may contribute to amyloid depositions in the brain. Ab is susceptible to a number of in vivo and in vitro proteases like cathepsin-D and M-13 metalloproteases. The M-13 family includes several members of zinc-dependent proteases like damage-induced neuronal endopeptidase (DINE), product of phosphate regulating gene with homologies to endopeptidases on the X chromosome (PHEX), neutral endopeptidase 24.11 or neprilysin (NEP) and neprilysin-like proteases (NEPLs). NEPLs (alpha, -beta, gamma) arise from the alternative splicing of a single NELPS gene and show ~54% sequence homology. M13 members are generally type II transmembrane proteins consisting of a single polypeptide chain with Zinc binding HEXXH motif, a short cytoplasmic tail, a transmembrane segment and an extra-cytoplasmic domain containing the enzyme active site.

NEP [variously termed as neutral endopeptidase-24.11 (NEP), neprilysin, enkephalinase, EC3.4.24.11, common acute lymphoblastic leukemia antigen (CALLA), CD10] is the key in vivo enzyme degrading biopeptides Ab, substance-P and enkephalin in brain; atrial natriuretic peptide, bradykinin and endothelin-1 in kidney. It is a 97 KDa ectoenzyme (mouse, rat, human 750-aa) with a large extracellular domain containing its catalytic site, which can degrade Ab on cell surface. NEP is ubiquitous but its high expression in brain is restricted to striatum, olfactory tubercle, substantia nigra, choroids plexes, endopeduncular nucleus, pontine nucleus, and cerebellum and in many peripheral tissues, particularly in brush-border membranes. The poor expression of NEP in the hippocampus and cerebral cortex is reflected in the selective deposition of Ab42 in these regions. NEP is also expressed in soluble form in human plasma and cerebrospinal fluid. NEP can degrade both synthetic and cell secreted Ab40 and Ab42 and may be a good therapeutic target for the treatment of Alzheimer's disease.

NEPL-alpha (variously called SEPD/splice 1) is a type II trans-membrane metallopeptidase (~100 kDa non-glycosylated and 120 kDa glycosylated) containing a highly glycosylated polypeptide chain of 742aa with a HEXXH zinc-binding consensus, cytoplasmic tail and a large extracellular catalytic domain. NPL-a lacks a 23aa sequence present in N-terminal of NEPL-b. In contrast to NEP and NEPL-b which exist in membrane-bound as well as soluble forms, NEPL-a is expressed as membrane-bound form only. In comparison to NEP, the NEPL-a has lower affinity and/or smaller Vmax for Ab and cannot proteolyze cell secreted Ab40 or Ab42 in vivo. Unlike NEP, NEPL-a is not a major in vivo peptidase for degrading endogenous Ab however, it might degrade Ab in Alzheimer patients where Ab is accumulated in excess and an Ab-degradative pathway, alternative to NEP, exists.

NEPL-beta (variously called SEP/NL1/NEPII) is type II transmembrane enzyme containing a single polypeptide chain of 765aa (~110 KDa) with cytoplasmic and transmembrane domains and a large extracellular C-terminal core containing the peptidase active site. The aa sequence is 65.1 % identical to mouse NEP. In comparison to NEPL-a, NEPL-b has an additional and unique sequence of 23-aa (41-63 aa) after the TM domain. There are two splice variants of NEP-b; a secreted isoform of 126 KDa containing a 23 aa secretion signal sequence and a membrane associated isoform of 110 kDa. The secreted isoform is much more glycosylated than the membrane isoform. Testis is the only tissue where the soluble/secreted isoform of NEP-b is predominant. Unlike NEP, NEPL-b has no proteolytic activity to Ab however both enzymes can cleave Leu5-enkephalin.

NEPL-gamma is a 115 kDa (779 aa) with a Zinc binding HEXXH motif in its extracellular domain. NEPL-g appears to have a different conformation and substrate specificity than those of NEPL-a. In comparison to NEPL-a, NEPL-g has an additional sequence of 37aa (311-347) near the center of the polypeptide which possibly inhibits its activity by interfering with proper folding and interaction with the substrate. Unlike NEP, NEPL-g has no proteolytic activity to either synthetic or Ab peptides and does not appear to be a possible in vivo peptidase for endogenous Ab. Proteolytic activation of endopeptidase activity of NEPL-g by a partial/complete removal of the unique region (311-347aa) in its sequence has been proposed.

DINE (damage-induced neuronal endopeptidase) or Endothelin converting enzyme-like 1 (ECEL1) or X-converting enzyme (Xce) is a 95 Kda (mouse/rat/human 775 aa, chromosome 2q36-q37), type II integral membrane metalloprotease containing a conserved zinc-binding motif and an ENXADX consensus sequence, consistent with gluzinitin. The aa sequence of DINE is 36 and 32 % identical to ECE-1 and NEP, respectively. But, DINE is devoid of enzyme activity like ECE. Unlike NEP, DINE has no proteolytic activity to Ab. However, the enzyme can hydrolyze synthetic NEP substrates and thiorphan, EDTA and phosphoramidon inhibit its activity. DINE also inhibits C2-ceramide induced apoptosis in COS-7 cells. Although, the endogenous substrate for DINE is yet to be identified, its proteolytic activity activates, at least in part, free radical scavenging in damaged neurons. The DINE expression is restricted to brain tissue with predominant expression in hypothalamus, large cholinergic cells in striatum and low expression in virtually all regions of brain except in cerebral cortex, hippocampus and cerebellum. DINE, expression is markedly increased in response to optic, spinal sensory, cortical and thalamic nerve injury.

Phosphate regulating gene with homologies to endopeptidases on the X chromosome (PHEX) (formerly PEX) is a zinc-containing, type II integral membrane glycoprotein (~110 kDa; mouse/rat/human 749-aa; chromosome xp22.2-p22.1) with structural resemblance to M13 NEP proteases. In contrast to NEP, PHEX has very narrow substrate specificity and hydrolyzes parathroid hormone related peptide (PTHrP107-139). Compared to NEP, PHEX can proteolyze Ab40 (but not Ab42), at a very low rate and to a very poor extent and therefore is not a major in vivo peptidase for Ab; however, it might degrade Ab in Alzheimer patients where Ab is accumulated in excess and an Ab-degradative pathway, alternative to NEP, exists. PHEX is expressed lymphocytes and fetal brain but not in adult brain, placenta, skeletal muscle, pancreas, heart, liver and lung. Defects in PHEX are a cause of X-linked hypophosphatemic rickets (HYP). PHEX is implicated in bone and dentin mineralization and renal phosphate reabsorption.

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