Skeletal Effects of Bone-Targeted TGFbeta Inhibition in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease that is frequently associated with secondary osteoporosis. Previous studies have shown that TGFbeta inactivating antibody improves the muscle phenotype in mice, a model of DMD. In the present study, we assessed the skeletal effects of treatment with a bone-targeted TGFbeta antibody (PCT-011) in mice.

Enzyme-replacement therapy in life-threatening hypophosphatasia.

Background: Hypophosphatasia results from mutations in the gene for the tissue-nonspecific isozyme of alkaline phosphatase (TNSALP). Inorganic pyrophosphate accumulates extracellularly, leading to rickets or osteomalacia. Severely affected babies often die from respiratory insufficiency due to progressive chest deformity or have persistent bone disease.

Dose response of bone-targeted enzyme replacement for murine hypophosphatasia.

Hypophosphatasia (HPP) features rickets or osteomalacia from tissue-nonspecific alkaline phosphatase (TNSALP) deficiency due to deactivating mutations within the ALPL gene. Enzyme replacement therapy with a bone-targeted, recombinant TNSALP (sALP-FcD(10), renamed ENB-0040) prevents manifestations of HPP when initiated at birth in TNSALP knockout (Akp2(-/-)) mice. Here, we evaluated the dose-response relationship of ENB-0040 to various phenotypic traits of Akp2(-/-) mice receiving daily subcutaneous (SC) injections of ENB-0040 from birth at 0.

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.

MEPE-ASARM peptides control extracellular matrix mineralization by binding to hydroxyapatite: an inhibition regulated by PHEX cleavage of ASARM.

Hyp mice having an inactivating mutation of the phosphate-regulating gene with homologies to endopeptidases on the X-chromosome (Phex) gene have bones with increased matrix extracellular phosphoglycoprotein (MEPE). An acidic, serine- and aspartic acid-rich motif (ASARM) is located in the C terminus of MEPE and other mineralized tissue matrix proteins. We studied the effects of ASARM peptides on mineralization and how PHEX and MEPE interactions contribute to X-linked hypophosphatemia (XLH).

Enzyme replacement therapy for murine hypophosphatasia.

Introduction: Hypophosphatasia (HPP) is the inborn error of metabolism that features rickets or osteomalacia caused by loss-of-function mutation(s) within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNALP). Consequently, natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5'-phosphate (PLP), a co-factor form of vitamin B6. Babies with the infantile form of HPP often die with severe rickets and sometimes hypercalcemia and vitamin B6-dependent seizures.

Human recombinant endopeptidase PHEX has a strict S1' specificity for acidic residues and cleaves peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein.

The PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) encodes a protein (PHEX) with structural homologies to members of the M13 family of zinc metallo-endopeptidases. Mutations in the PHEX gene are responsible for X-linked hypophosphataemia in humans. However, the mechanism by which loss of PHEX function results in the disease phenotype, and the endogenous PHEX substrate(s) remain unknown.

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.

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.

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.

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.

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.

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.

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.

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.

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

Neutral endopeptidase (neprilysin or NEP, EC 3.4.24.

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.

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.

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.

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.

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.

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.

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.

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.