Enzyme replacement therapy prevents dental defects in a model of hypophosphatasia.

Hypophosphatasia (HPP) occurs from loss-of-function mutation in the tissue-non-specific alkaline phosphatase (TNALP) gene, resulting in extracellular pyrophosphate accumulation that inhibits skeletal and dental mineralization. TNALP-null mice (Akp2(-/-)) phenocopy human infantile hypophosphatasia; they develop rickets at 1 week of age, and die before being weaned, having severe skeletal and dental hypomineralization and episodes of apnea and vitamin B(6)-responsive seizures. Delay and defects in dentin mineralization, together with a deficiency in acellular cementum, are characteristic. We report the prevention of these dental abnormalities in Akp2(-/-) mice receiving treatment from birth with daily injections of a mineral-targeting, human TNALP (sALP-FcD(10)). sALP-FcD(10) prevented hypomineralization of alveolar bone, dentin, and cementum as assessed by micro-computed tomography and histology. Osteopontin--a marker of acellular cementum--was immuno-localized along root surfaces, confirming that acellular cementum, typically missing or reduced in Akp2(-/-) mice, formed normally. Our findings provide insight concerning how acellular cementum is formed on tooth surfaces to effect periodontal ligament attachment to retain teeth in their osseous alveolar sockets. Furthermore, they provide evidence that this enzyme-replacement therapy, applied early in post-natal life--where the majority of tooth root development occurs, including acellular cementum formation--could prevent the accelerated tooth loss seen in individuals with HPP.

Soluble recombinant neprilysin induces aggrecanase-mediated cleavage of aggrecan in cartilage explant cultures.

Neprilysin (neutral endopeptidase, enkephalinase, CALLA, CD10, NEP) is a regulatory Zn metallopeptidase expressed in the brush border membranes of the kidney and has been found in porcine chondrocytes and rat articular cartilage as well as other cell types and tissues. Although its function in cartilage is not currently known, previous observations of high levels of NEP enzymatic activity in the synovial fluid of arthritic patients and on the chondrocyte membranes of human osteoarthritic cartilage have led to the hypothesis that NEP is involved in the inflammation or degradation pathways in articular cartilage. Our study localized endogenous NEP to the membranes of mature bovine articular chondrocytes in a tissue explant model and demonstrated that the addition of soluble recombinant NEP (sNEP) to the culture medium of bovine cartilage explants leads to the degradation of aggrecan through the action of aggrecanase. A 6-day exposure to sNEP was necessary to initiate the degradation, suggesting that the chondrocytes were responding in a delayed manner to an altered composition of regulatory peptides. This NEP-induced degradation was completely inhibited by the NEP inhibitors thiorphan and phosphoramidon. These results suggest that NEP is present as a transmembrane enzyme on articular chondrocytes where it can cleave regulatory peptides and lead to the induction of aggrecanase.

Molecular cloning, tissue distribution, and chromosomal localization of MMEL2, a gene coding for a novel human member of the neutral endopeptidase-24.11 family.

Members of the neutral endopeptidase (NEP, also known as MME for membrane metallo-endopeptidase in the Human Gene Nomenclature database) family play significant roles in pain perception, arterial pressure regulation, phosphate metabolism, and homeostasis. In this paper, we report the cloning of a new human member of the NEP family that we named MMEL2 for membrane metallo-endopeptidase-like 2. The MMEL2 protein has the structural characteristics of type II transmembrane proteins, although the presence of a furin-like cleavage site in the ectodomain suggests that it may be released into the medium following proteolytic cleavage. The MMEL2 protein contains the zinc-binding consensus sequence HEXXH and all the residues known to be essential for the enzymatic activity of other members of the family. The MMEL2 mRNA was detected predominantly in testis, but weak expression also was observed in brain, kidney, and heart. The human MMEL2 gene was mapped to 1p36 by fluorescence in situ hybridization. It will be important to test whether MMEL2 defects are associated with diseases such as hereditary motor sensory neuropathy 2A, Schwartz-Jampel-Aberfeld syndrome, or neuroblastoma, which all map to this locus.

Characterization of PHEX endopeptidase catalytic activity: identification of parathyroid-hormone-related peptide107-139 as a substrate and osteocalcin, PPi and phosphate as inhibitors.

Mutations in the PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) are responsible for X-linked hypophosphataemia, and studies in the Hyp mouse model of the human disease implicate the gene product in the regulation of renal phosphate (P(i)) reabsorption and bone mineralization. Although the mechanism for PHEX action is unknown, structural homologies with members of the M13 family of endopeptidases suggest a function for PHEX protein in the activation or degradation of peptide factors involved in the control of renal P(i) transport and matrix mineralization. To determine whether PHEX has endopeptidase activity, we generated a recombinant soluble, secreted form of human PHEX (secPHEX) and tested the activity of the purified protein with several peptide substrates, including a variety of bone-related peptides. We found that parathyroid-hormone-related peptide(107-139) is a substrate for secPHEX and that the enzyme cleaves at three positions within the peptide, all located at the N-terminus of aspartate residues. Furthermore, we show that osteocalcin, PP(i) and P(i), all of which are abundant in bone, are inhibitors of secPHEX activity. Inhibition of secPHEX activity by osteocalcin was abolished in the presence of Ca(2+). We suggest that PHEX activity and mineralization may be controlled in vivo by PP(i)/P(i) and Ca(2+) and, in the latter case, the regulation requires the participation of osteocalcin.

Developmental expression and tissue distribution of Phex protein: effect of the Hyp mutation and relationship to bone markers.

Mutations in PHEX, a phosphate-regulating gene with homology to endopeptidases on the X chromosome, are responsible for X-linked hypophosphatemia (XLH). The murine Hyp homologue has the phenotypic features of XLH and harbors a large deletion in the 3' region of the Phex gene. We characterized the developmental expression and tissue distribution of Phex protein, using a monoclonal antibody against human PHEX, examined the effect of the Hyp mutation on Phex expression, and compared neprilysin (NEP), osteocalcin, and parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor gene expression in bone of normal and Hyp mice. Phex encodes a 100- to 105-kDa glycoprotein, which is present in bones and teeth of normal mice but not Hyp animals. These results were confirmed by in situ hybridization (ISH) and ribonuclease protection assay. Phex protein expression in femur and calvaria decreases with age, suggesting a correlation between Phex expression and bone formation. Immunohistochemical studies detected Phex protein in osteoblasts, osteocytes, and odontoblasts, but not in osteoblast precursors. In contrast to Phex, the abundance of NEP messenger RNA (mRNA) and protein is not significantly altered in Hyp bone. Similarly, osteocalcin and PTH/PTHrP receptor gene expression are not compromised in bone of Hyp mice. Our results are consistent with the hypothesis that loss of Phex function affects the mineralizing activity of osteoblasts rather than their differentiation.

Cellular localization of neprilysin in mouse bone tissue and putative role in hydrolysis of osteogenic peptides.

The regulation of osteoblast and osteoclast metabolism is mediated by both hormones and local bone peptide factors. Peptides and hormones are under control of membrane peptidases such as Neprilysin (NEP). NEP is a widely distributed cell-surface zinc-metallopeptidase that is involved in the regulation of several important physiological processes by controlling the half-life of bioactive peptides. Although NEP is known to be present in skeletal tissues, neither its cellular localization nor its function have been established. To address this question, we examined NEP distribution in bones of postnatal mouse. In situ hybridization (ISH) and immunohistochemistry showed that NEP messenger RNA (mRNA) and protein are associated with bone-forming cells including presumptive osteoblast precursors, preosteoblasts, osteoblasts, and osteocytes. NEP levels in newborn and adult mice bones also were compared by immunoblotting. Higher amounts of NEP immunoreactivity were observed in newborn as compared with adult bones, suggesting a relationship between NEP expression and bone growth. To further explore this hypothesis, we monitored in vitro NEP proteolytic activity using a series of synthetic osteogenic peptides such as parathyroid hormone-related peptide 1-43 (PTHrP1-34), osteostatin (PTHrP107-139), osteogenic growth peptide (OGP), calcitonin, alpha-calcitonin gene-related peptide (alpha-CGRP), and PTH1-34. Except for PTH1-34, all peptides were found to be NEP substrates.

Molecular cloning and biochemical characterization of a new mouse testis soluble-zinc-metallopeptidase of the neprilysin family.

Because of their roles in controlling the activity of several bio-active peptides, members of the neprilysin family of zinc metallopeptidases have been identified as putative targets for the design of therapeutic agents. Presently, six members have been reported, these are: neprilysin, endothelin-converting enzyme (ECE)-1 and ECE-2, the Kell blood group protein, PHEX (product of the phosphate-regulating gene with homologies to endopeptidase on the X chromosome) and X-converting enzyme (XCE). In order to identify new members of this important family of peptidases, we designed a reverse transcriptase-PCR strategy based on conserved amino acid sequences of neprilysin, ECE-1 and PHEX. We now report the cloning from mouse testis of a novel neprilysin-like peptidase that we called NL1. NL1 is a glycoprotein that, among the members of the family, shows the strongest sequence identity with neprilysin. However, in contrast with neprilysin and other members of the family which are type II integral membrane proteins, NL1 was secreted when expressed in cultured mammalian cells, likely due to cleavage by a subtilisin-like convertase at a furin-like site located 22 amino acid residues in the C-terminus of the transmembrane domain. The recombinant enzyme exhibited neprilysin-like peptidase activity and was efficiently inhibited by phosphoramidon and thiorphan, two inhibitors of neprilysin. Northern blot analysis and in situ hybridization showed that NL1 mRNA was found predominantly in testis, specifically in round and elongated spermatids. This distribution of NL1 mRNA suggests that it could be involved in sperm formation or other processes related to fertility.

A novel, 85 kDa endothelial antigen differentiates plasma membrane macrodomains in lung alveolar capillaries.

A monoclonal antibody raised against purified rat lung endothelial plasma membranes was found to recognize an apparently novel, 85 kD, integral endothelial plasma membrane glycoprotein. By immunofluorescence and electron microscope immunocytochemistry, this endothelial antigen was detected at the luminal and tissue fronts of all rat endothelia, except those of discontinuous type (liver and spleen sinusoids). In lung alveolar capillaries the antigen appeared to be uniquely associated with the very attenuated endothelial processes forming the blood-air barrier, and virtually absent on the surface of the rest of the cell, where the nucleus and the organelles are located. No other cells, except fibroblasts, appeared to be labeled by this monoclonal antibody.

The N-terminal segment of endothelin-converting enzyme (ECE)-1b contains a di-leucine motif that can redirect neprilysin to an intracellular compartment in Madin-Darby canine kidney (MDCK) cells.

Endothelin-converting enzyme (ECE)-1 is a membrane-bound metallopeptidase of the neprilysin (NEP) family. ECE-1 is responsible for the conversion of inactive big-endothelins into active endothelins. Three different isoforms of human ECE-1 (ECE-1a, ECE-1b and ECE-1c) have been identified. They differ in their N-terminal cytosolic regions, have distinct tissue distribution and intracellular localization. ECE-1a and ECE-1c are both located at the cell surface whereas ECE-1b is targeted to an intracellular compartment. To better understand the nature of the signal responsible for the targeting of ECE-1b to the intracellular compartment, we have constructed several ECE/NEP chimaeric proteins and expressed them by transfection into Madin-Darby canine kidney (MDCK) cells. This allowed us to identify a nine amino acid segment in the cytosolic tail of ECE-1b that is sufficient to relocate NEP from the cell surface to an intracellular compartment. Site-directed mutagenesis on these chimaeras led to the identification of two leucine residues as part of the intracellular retention signal.

Interaction of mammalian neprilysin with binding protein and calnexin in Schizosaccharomyces pombe.

Neutral endopeptidase (neprilysin or NEP, EC 3.4.24.11) is a zinc metallo-endopeptidase expressed in many eukaryotic cell types and displaying several important physiological roles. In the brain (and central nervous system), this enzyme is involved in the molecular mechanism of pain by its action in the degradation of enkephalin molecules. In the kidney, NEP is implicated in the degradation of regulatory factors involved in the control of arterial pressure, including atrial natriuretic peptide and bradykinin. In this study we assessed the potential of the fission yeast Schizosaccharomyces pombe to overproduce rabbit NEP and secreted NEP (sNEP, a soluble derivative of this integral membrane protein). Both recombinant NEP and sNEP were produced at high levels (5 mg/l) in this system. Enzymic studies revealed that these recombinant proteins were fully active and exhibit kinetic parameters similar to those of the bona fide enzyme. Immunofluorescence microscopy and enzymic assays demonstrated that recombinant NEP is correctly targeted to the cell membrane. Furthermore, co-immunoprecipitation studies showed that folding intermediates of NEP and sNEP, produced in S. pombe, interact in the endoplasmic reticulum (ER) with binding protein (BiP) and calnexin (Cnx1p). The amount of sNEP coprecipitated with both BiP and Cnx1p augmented when cells were subjected to various stresses causing the accumulation of unfolded proteins in the ER. The interactions of NEP with BiP and Cnx1p were, however, more refractive to the same stresses.

Bradykinin metabolism in the postinfarcted rat heart: role of ACE and neutral endopeptidase 24.11.

The respective role of angiotensin-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) in the degradation of bradykinin (BK) has been studied in the infarcted and hypertrophied rat heart. Myocardial infarction (MI) was induced in rats by left descendant coronary artery ligature. Animals were killed, and hearts were sampled 1, 4, and 35 days post-MI. BK metabolism was assessed by incubating synthetic BK with heart membranes from sham hearts and infarcted (scar) and noninfarcted regions of infarcted hearts. The half-life (t1/2) of BK showed significant differences among the three types of tissue at 4 days [sham heart (114 +/- 7 s) > noninfarcted region (85 +/- 4 s) > infarcted region (28 +/- 2 s)] and 35 days post-MI [sham heart (143 +/- 6 s) = noninfarcted region (137 +/- 9 s) > infarcted region (55 +/- 4 s)]. No difference was observed at 1 day post-MI. The participation of ACE and NEP in the metabolism of BK was defined by preincubation of the membrane preparations with enalaprilat, an ACE inhibitor, and omapatrilat, a vasopeptidase inhibitor that acts by combined inhibition of NEP and ACE. Enalaprilat significantly prevented the rapid degradation of BK in every tissue type and at every sampling time. Moreover, omapatrilat significantly increased the t1/2 of BK compared with enalaprilat in every tissue type and at every sampling time. These results demonstrate that experimental MI followed by left ventricular dysfunction significantly modifies the metabolism of exogenous BK by heart membranes. ACE and NEP participate in the degradation of BK since both enalaprilat and omapatrilat have potentiating effects on the t1/2 of BK.

Role of the glycosyl-phosphatidylinositol anchor in the intracellular transport of a transmembrane protein in Madin-Darby canine kidney cells.

In order to compare the trafficking of proteins with different membrane anchors, we have constructed and expressed three different recombinant forms of neutral endopeptidase (NEP) in MDCK cells. The wild type form of NEP (WT-NEP) is attached to the plasma membrane by a single N-terminal membrane spanning domain, whereas the glycosylphosphatidylinositol-anchored form of the protein (GPI-NEP) contains a C-terminal GPI anchor. A double anchored form of NEP (DA-NEP) was also constructed, that contains both the original N-terminal membrane spanning domain and a C-terminal GPI anchor. We show here that WT-NEP, GPI-NEP and DA-NEP, which are all apically targeted in MDCK cells, behave differently when subjected to Triton X-100 solubilisation: despite the presence of the transmembrane anchor DA-NEP behaves as a GPI-anchored protein. This suggests that the GPI anchor of DA-NEP is dominant over the transmembrane anchor of the native protein to determine its pattern of solubility in Triton X-100.

Recombinant human endothelin-converting enzyme ECE-1b is located in an intracellular compartment when expressed in polarized Madin-Darby canine kidney cells.

Endothelin-converting enzyme (ECE) is a phosphoramidon-sensitive membrane-bound metalloprotease responsible for the conversion of big-endothelins into endothelins [Yanagisawa, Kurihara, Kimura, Tomobe, Kobayashi, Mitsui, Yazaki, Goto and Masaki (1988) Nature (London) 332, 411-415]. Several distinct isoforms of ECE have been cloned and identified. ECE-1a, b and c have the same ectodomain and differ only by their cytosolic tails [Schweizer, Valdenaire, Nelböck, Deuschle, Edwards, Stumpf and Löffler (1997) Biochem. J. 328, 871-877]. The ectodomain common to ECE-1 a, b and c shares extensive sequence similarities with neprilysin, a major kidney brush border metallopeptidase. To study the sorting of ECE in polarized cells, ECE-1bcDNA was expressed by transfection in polarized Madin-Darby canine kidney (MDCK) cells. Cell-surface biotinylation and immunofluorescence studies showed that ECE-1b is not expressed on the cell-surface but was rather located in intracellular compartments that could also be labelled with anti-Rab-5 and Rab-7 antibodies and was thus tentatively identified as early and late endosomes. Similar results were also obtained when ECE-1b was expressed in non-polarized Chinese hamster ovary cells for comparison purposes. When MDCK or Chinese hamster ovary transfected cells were pre-treated with the ECE inhibitor phosphoramidon, a 3-fold increase in the level of ECE-1b was observed both by Western blotting and by enzymic activity. However, no change in the level of neprilysin or the beta-chain of meprin, two apical membrane metallopeptidases, was observed in MDCK cells transfected under similar conditions. Northern blotting showed that the increase in the level of ECE-1b was not owing to changes in the ECEmRNA transcription rate or stability. Rather, pulse-chase experiments followed by immunoprecipitation showed a decrease in the rate of degradation of ECE-1b in phosphoramidon-treated cells. Half-lives were determined to be 2.8 and 7.5 h for non-treated and phosphoramidon-treated cells, respectively. Confocal microscopy showed accumulation of ECE-1b immunoreactive material in the lysosomes of phosphoramidon-treated cells. Taken together, these results suggest that ECE-1b turns over very rapidly between endosomal and lysosomal compartments and that lysosomal degradation of the enzyme is slowed down by phosphoramidon.

Pex mRNA is localized in developing mouse osteoblasts and odontoblasts.

Mutations in PEX, a phosphate-regulating gene with homology to endopeptidase on the X chromosome, were recently identified in patients with X-linked hypophosphatemia (XLH), an inherited disorder of phosphate homeostasis characterized by growth retardation and rachitic and osteomalacic bone disease. To understand the mechanism by which loss of PEX function elicits the mutant phenotype, a study of its mRNA localization and ontogenesis was undertaken. Using the reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) with polyA+ RNA purified from mouse testis, a 337-bp Pex cDNA fragment was generated and cloned in the pCRII plasmid. The cDNA was used to generate sense and anti-sense Pex riboprobes for in situ hybridization (ISH) and Northern analysis. To survey a large number of different tissues, sagittal sections of embryos and newborn mice were examined. ISH showed the presence of Pex mRNA in osteoblasts and odontoblasts. Pex gene expression was detectable on Day 15 of embryonic development, which coincides with the beginning of intercellular matrix deposition in bones. Finally, Northern analysis of total RNA from calvariae and teeth of 3-day-old and adult mice showed that the abundance of the 7-kb Pex transcript is decreased in adult bones and in nongrowing teeth. The present study demonstrates that Pex mRNA is expressed in bones and teeth and suggests that this putative endopeptidase plays an important role in the development of these tissues.

Trophic and tropic effects of striatal astrocytes on cografted mesencephalic dopamine neurons and their axons.

Astrocytes from the ventral mesencephalon and from the striatum respectively promote the dendritic and axonal arborization of dopamine (DA) neurons in vitro. To test this response in vivo, astrocytes in primary cultures from the neonatal cerebral cortex, ventral mesencephalon, or striatum were coimplanted with fetal ventral mesencephalic tissue into the intact or DA-denervated striatum of adult rats and these cografts examined after 3-6 months by tyrosine hydroxylase (TH) immunohistochemistry (intact recipients) or after 5-6 months by in vitro [3H]DA-uptake autoradiography (DA-denervated recipients). In contrast with single ventral mesencephalic grafts, all types of cograft displayed a rather uniform distribution of TH-immunoreactive perikarya. The average size of TH-immunoreactive cell bodies was not significantly different in cografts containing cortical or mesencephalic astrocytes and in single ventral mesencephalic grafts, but it was significantly larger in cografts containing striatal astrocytes. Nevertheless, the number of [3H]DA-labeled terminals in the DA-lesioned host striatum was clearly smaller with cografts of striatal astrocytes than with single mesencephalic grafts or with cografts containing cortical astrocytes. On the other hand, cografts of striatal astrocytes contained much higher numbers of [3H]DA-labeled terminals than the other types of graft or cograft. Thus, while cografted astrocytes in general influence the distribution of DA neurons within the graft, astrocytes from the neonatal striatum have a trophic effect on DA perikarya and a tropic effect on DA axons, keeping the latter within the graft.

Predominant basolateral proteolytic processing of prosomatostatin into somatostatin-28 in polarized LLC-PK1 cells.

Polarized epithelial cells secrete specific proteins through their apical or basolateral membrane. In the present study, we have expressed the human prosomatostatin cDNA in the pig kidney epithelial cell line (LLC-PK1) and monitored the processing and release of the somatostatin-related peptides. Analysis by high-performance liquid chromatography and radioimmunoassay of the somatostatin-related peptides synthesized by the transfected cells showed that the LLC-PK1 cells released prosomatostatin and somatostatin-28 (S-28) in the culture medium. Furthermore, when the cells were polarized, we observed release of prosomatostatin from both membrane domains (apical and basolateral), while liberation of S-28 was mostly from the basolateral side. This observation suggests that, in these cells, the proprotein convertase(s) responsible for prosomatostatin processing is(are) associated with the basolateral secretory pathway.

Characterisation of neprilysin (EC 3.4.24.11) S2' subsite.

Neprilysin is a neutral peptidase that cleaves small peptide substrates on the amino-side of hydrophobic amino acid residues. In the present study, we have used inhibition of non-mutated and mutated enzymes with dipeptide inhibitors and hydrolysis of the substrate [Leu5, Arg6]enkephalin in order to evaluate the contribution of the S2' subsite to substrate and inhibitor binding. Our results suggest that (1) Arg-102 and Asn-542 provide major contributions to the interaction of the enzyme with the P2' residue of the substrate, (2) the S2' subsite is vast and can accommodate bulky side chains, and (3) Arg-102 restricts access to the S2' subsite to some side chains such as arginine.

Antibodies specific to the plasma membrane of rat lung microvascular endothelium.

The highly purified, luminal domain of rat lung endothelial plasma membranes was used as an immunogen to obtain monoclonal antibodies to the endothelial cell surface. The procedure was highly efficient, yielding antibodies which recognize three seemingly novel endothelial integral membrane glycoproteins of molecular weights of 170, 114, and 95 kDa, respectively. By immunofluorescence, two of these antigens (170 and 95 kDa) appeared to be uniquely expressed by the lung microvascular endothelium. The third one, the 114-kDa polypeptide, was detected in the continuous endothelium of the lung, but also in the fenestrated endothelia of pancreas, intestinal villi, and kidney peritubular capillaries. Partition in Triton X-100-soluble and -insoluble plasmalemmal components suggests that two of these novel endothelial antigens (170 and 114 kDa) are specific for the plasma membrane proper only, while that of 95 kDa is present both in the caveolae and on the rest of the cell surface.

Secretion of a type II integral membrane protein induced by mutation of the transmembrane segment.

Signal peptide/membrane anchor (SA) domains of type II membrane proteins initiate the translocation of downstream polypeptides across the endoplasmic reticulum (ER) membrane. In contrast with signal peptides, however, SA domains are not cleaved by signal peptidase and thus anchor the protein in the membrane. In the present study we have introduced mutations in the SA domain of neprilysin (neutral endopeptidase-24.11; NEP) to identify structural elements that would favour the processing of SA domains by signal peptidase. Mutants of full-length and truncated (without cytoplasmic domain) protein were constructed by substitution of the sequences SQNS, QQTT or YPGY for VTMI starting at position 15 of the NEP SA domain. In addition, a Pro residue was substituted for Thr at position 16 of the SA domain. The rationale for the use of these sequences was decided from our previous observation that substitution in the NEP SA domain of the sequence SQNS, which is polar and has alpha-helix-breaking potential, could promote SA domain processing under certain conditions (Roy, Chatellard, Lemay, Crine and Boileau (1993) J. Biol. Chem. 268. 2699-2704; Yang. Chatellard, Lazure, Crine and Boileau (1994) Arch. Biochem. Biophys. 315, 382-386). The QQTT sequence is polar but, according to secondary structure predictions, is compatible with the alpha-helix structure of the NEP SA domain. The YPGY sequence and single Pro residue are less polar and have alpha-helix-breaking potential. The predicted effects of these mutations on the structure of the NEP SA domain were confirmed by CD analysis of 42-residue peptides encompassing the hydrophobic segment and flanking regions. Wild-type and mutated proteins were expressed in COS-I cells and their fate (membrane-bound or secreted) was determined by immunoblotting and by endoglycosidase digestions. Our biochemical and structural data indicate that: (I) the cytosolic domain of NEP restricts the conformation of the SA domain because mutants not secreted in their full-length form are secreted in their truncated form; (2) alpha-helix-breaking residues are not a prerequisite for cleavage; (3) the presence, in close proximity to a putative signal peptidase cleavage site, of a polar sequence that maintains the alpha-helical structure of the SA domain is sufficient to promote cleavage. Furthermore pulse chase studies suggest that cleavage is performed in the ER by signal peptidase and indicate that cleavage is not a limiting step in the biosynthesis of the soluble form of the protein.

Identification of the cysteine residues implicated in the formation of alpha 2 and alpha/beta dimers of rat meprin.

Meprin (endopeptidase-24.18; EC 3.4.24.18) is a multisubunit zinc-metallopeptidase found in the brush-border membranes of rodent kidney and human intestine. The alpha and beta subunits of meprin are disulphide-linked to form either soluble alpha 2 homodimers or membrane-associated alpha/beta heterodimers. The aim of the present study was to identify the cysteine residue(s) implicated in the formation of alpha 2 and alpha/beta dimers and to investigate the effects of dimerization on intracellular transport and processing of the alpha subunit. Three cysteine residue candidates for the formation of disulphide bonds in the alpha subunit were selected by hydrophobic cluster analysis. These residues, located at positions 309, 560 and 562, were mutated to serine residues. When the resulting alpha subunit mutants were expressed alone in COS-1 cells, the alpha C560S and alpha C562S mutants were found to be secreted as alpha 2 homodimers whereas the alpha C309S mutant was found as monomers in the culture medium. In double-transfection experiments with the wild-type beta subunit, the alpha C560S and alpha C562S mutants behaved exactly as the wild-type alpha subunit and formed membrane-bound alpha/beta heterodimers. In contrast, the alpha C309S mutant was not retained at the cell surface but rather secreted as monomers in the culture medium, as was found in the simple transfection experiment. These results show that, despite the normal expression level and folding of the protein in a transport-competent from, the alpha C309S mutant is unable to form alpha 2 homodimers or alpha/beta heterodimers. This suggests that Cys309 is the unique residue of the alpha subunit implicated in the alpha 2 and alpha/beta dimerizations. Hydrophobic cluster analysis of the alpha and beta subunit sequences predicts that Cys309 is similar to Cys306 of the beta subunit. We mutated the latter residue to a serine and expressed the beta C306S mutant and the wild-type alpha subunit in the same COS-1 cells. No beta 2 or alpha/beta dimers were observed on immunoblotting, showing that Cys306 of the beta subunit is required for the formation of intermolecular disulphide bonds both in beta 2 homodimers and in alpha/beta heterodimers. Taken together, these results suggest that the alpha/beta heterodimeric form of meprin is held together by a single disulphide bond linking Cys309 in the alpha subunit to Cys306 in the beta subunit.