A. John Kenny

Role of endopeptidase-24.11 in the inactivation of atrial natriuretic peptide.

The circulating form of atrial natriuretic factor is a 28‐residue peptide containing a 17‐residue disulphide‐linked ring. It has important actions on the kidney, largely on its haemodynamics, and at other sites including the adrenal cortex and CNS. It has a short half‐life in vivo and is rapidly inactivated when incubated with kidney microvillar membranes. Of the battery of peptidases present in that membrane, only one, endopeptidase‐24.11, is responsible for initiating the attack, and this commences with hydrolysis of the Cys7‐Phe8 bond within the ring. Hydrolysis at this and other points has been shown to inactivate the peptide and this information has pointed the way to the synthesis of resistant analogues.

Endopeptidase-24.11 and aminopeptidase activity in brain synaptic membranes are jointly responsible for the hydrolysis of cholecystokinin octapeptide (CCK-8)

Endopeptidase‐24.11 (EC 3.4.24.11) from pig kidney hydrolysed CCK‐8 (sulphated) at two distinct sites: Asp‐Tyr(SO3H)‐Met‐Gly↓Trp‐Met‐Asp↓PheNH2. Under initial conditions, the splitting of the Asp7‐Phe8NH2 bond proceeded 4‐times more rapidly than the Gly4‐Trp5 bond. Pig brain striatal synaptic membranes attacked this substrate at the same sites and this activity was inhibited by phosphoramidon. However, other products were detected even in the presence of phosphoramidon. One of these products was identified as free tryptophan. Since their formation was inhibited by bestatin, one or more membrane aminopeptidases is also implicated in the degradation of CCK‐8.

Fate of injected interleukin 1 in rats: Sequestration and degradation in the kidney

The tissue distribution and route of clearance of human recombinant interleukin 1 alpha (IL 1 alpha) injected intravenously in rats was studied. The plasma half-life was approximately 2.5 min, and this was increased after nephrectomy, the kidney being the major organ through which the IL 1 alpha was excreted. Two iodinated fragments of IL 1 alpha, of approximately 5 and 9 kDa, were excreted by the kidneys whereas only intact, 17-kDa IL 1 alpha was detected in plasma, suggesting that the protein was being degraded after uptake by the kidney. The results of in vivo experiments in which surface endopeptidase-24.11 was inhibited with phosphoramidon and in vitro experiments in which rat kidney homogenates were incubated with radiolabeled IL 1 alpha suggest that the cytokine was endocytosed and then hydrolysed by lysosomal proteinases.

Membrane Localization of Endopeptidase-24.11 and Peptidyl Dipeptidase A (Angiotensin Converting Enzyme) in the Pig Brain: A Study Using Subcellular Fractionation and Electron Microscopic Immunocytochemistry

Abstract: Brains from piglets were dissected and a block of tissue including the substantia nigra, globus pallidus, and entopeduncular nucleus was homogenized and then fractionated on discontinuous Percoll gradients. Ligand‐binding assays using (–)‐[3H]nicotine and [3H]quinuclidinyl benzilate served to delineate fractions containing nicotinic and muscarinic acetylcholine receptors. In this system endopeptidase‐24. 11 exhibited a biphasic distribution, consistent with its presence on both pre‐ and postsynaptic membranes. Peptidyl dipeptidase A (angiotensin converting enzyme; ACE) was associated with membrane fractions containing muscarinic receptors. An immunoblot of these fractions with an affinity‐purified polyclonal antibody to ACE revealed only the neuronal form of ACE (Mr 170,000), the endothelial form (Mr 180,000) being undetectable. Electron microscopic immunoperoxidase staining of the substantia nigra, with an affinity‐purified antibody to endopeptidase‐24. 11 at the preembedding stage, showed this antigen to be confined to the plasma membranes of boutons, axons, and some dendrites. Both pre‐ and postsynaptic membranes were stained, and occasionally other regions of the dendritic membrane were positive. No staining of synaptic vesicles within the boutons was observed. Thus, two independent approaches indicate that endopeptidase‐24.11 is present on both pre‐ and postsynaptic membranes in the pig substantia nigra. The subcellular fractionation suggests that neuronal ACE is confined to dendritic membranes.

Electronmicroscopic immunocytochemistry of pig brain shows that endopeptidase-24.11 is localized in neuronal membranes.

An ultrastructural study of endopeptidase-24.11 in the globus pallidus from the brain of a newborn pig is reported. The antigen was localized by an immunoperoxidase method using an affinity-purified polyclonal antibody in which staining was performed on thick vibratome sections prior to osmication and flat embedding. When areas selected by light microscopy for re-embedding were examined in the electron microscope a minority of the axonal membranes in the fields examined were observed to be immunostained for endopeptidase-24.11. These were unmyelinated fibres and the membrane staining included not only the length of an axon but also some boutons synapsing with dendrites. Positively staining dendritic and glial membranes were not observed. These results support the view that endopeptidase-24.11 may play a role in inactivating some neuropeptides after their release at the synapse.

Localization of aminopeptidase N and dipeptidyl peptidase IV in pig striatum and in neuronal and glial cell cultures

The subcellular distribution of the plasma membrane ectoenzymes, aminopeptidase N (aminopeptidase M) and dipeptidyl peptidase IV, has been examined by fractionating homogenates of porcine striata by a discontinuous Percoll gradient centrifugation procedure which distinguishes fractions containing pre‐ and post‐synaptic elements. The two enzymes showed different distributions–dipeptidyl peptidase IV did not show a significant pre‐synaptic location, whereas aminopeptidase N was present on both pre‐ and post‐synaptic fractions. Immunofluorescent staining on mixed and neuron‐enriched primary cultures of pig striatal tissue using affinity purified antibodies to the aminopeptidase and to the dipeptidyl peptidase revealed the ectoenzymes on distinct populations of cells. The astrocytic identity of the aminopeptidase N‐staining cells was established by correlation with immunostaining for glial fibrillary acidic protein and for vimentin by confocal microscopy. Ultracryosections of striatum immunostained with gold‐labelled immunoglobulins of differing diameters demonstrated aminopeptidase N on pericytes and confirmed its location on endothelial and astrocytic glial cells. Thus, several independent approaches indicated that aminopeptidase N, in addition to being present on endothelial and synaptic membranes, is found on astrocytes and pericytes in the perivascular neuropil, whereas dipeptidyl peptidase IV is less widely distributed on microvessels and appears not to have a synaptosomal location.

Molecular cloning of the α-subunit of rat endopeptidase-24.18 (endopeptidase-2) and co-localization with endopeptidase-24.11 in rat kidney by in situ hybridization

Endopeptidase‐24.18 (endopeptidase‐2, EC 3.4.24.18, E‐24.18) is a Zn‐ectoenzyme of rat renal and intestinal microvillar membranes exhibiting an oligometric structure, α2‐β2. The primary structure of the α‐subunit of E‐24.18 has been defined by molecular cloning and its expression mapped in rat kidney by in situ hybridization. A 2.9‐kb cDNA coding for the α‐subunit was isolated and sequenced. It had an open reading frame of 2.244 base pairs coding for a type I membrane protein of 748 amino acids. The deduced amino acid sequence showed 87% identity with that of meprin A, a mouse metallo‐endopeptidase, sharing common properties with the rat enzyme, and 85% identity with the human intestinal enzyme, ‘PABA‐peptide hydrolase’. Northern blot analysis revealed the α‐subunit to be encoded by a single mRNA species of 3.2‐kb. In situ hybridization performed on rat kidney showed a co‐localization of E‐24.18 with endopeptidase‐24.11 in proximal tubules of juxtamedullary nephrons, suggesting that the two enzymes have similar or complementary physiological functions in kidney.

Mosaic expression of membrane peptidases by confluent cultures of Caco-2 cells

The cell‐surface expression of endopeptidase‐24.11 (EC 3.4.24.11) on Caco‐2 cells cultured to confluency is markedly heterogeneous unlike that of dipeptidylpeptidase IV (EC 3.4.14.5). Here we have investigated the cell‐surface expression of three other ectopeptidases: angiotensin converting enzyme (EC 3.4.15.1), aminopeptidase N (EC 3.4.11.2) and aminopeptidase W (EC 3.4.11.16). We show by indirect immunofluorescent staining that these three enzymes are present on the surface of some cells but not on others. However, these enzymes were detected in the majority of detergent‐permeabilised Caco‐2 cells indicating the presence of intracellular pools of these enzymes. This suggests that there may either be differential regulation of apical transport for these peptidases or that they recycle at different rates.

Rat endopeptidase-24.18 α subunit is secreted into the culture medium as a zymogen when expressed by COS-1 cells

Endopeptidase‐24.18 (EC 3.4.24.18, E‐24.18) is an oligomeric Zn‐ectoenzyme. The α and β submits have been cloned from both rat and mouse kidneys. The primary structure of these subunits revealed that they both contain the consensus Zn binding site and that they are members of the astacin family. Analysis of the hydropathy plot also suggested that they are anchored by a C‐terminal hydrophobic domain. In order to verify the mode of anchoring of the rat E‐24.18 α subunit and to test the functionality of the astacin‐like domain in the α subunit when expressed alone, COS‐1 cells were transfected with a cloned cDNA for rat α subunit. Despite the presence of its putative transmembrane domain, the α subunit was not anchored in the plasma membrane but rather secreted as a dimer into the culture medium. When the enzymatic activity of the secreted recombinant protein was tested in the azocasein degradation assay, the α subunit was found to be inactive. Activity could, however, be revealed after mild trypsin digestion. This activity was abolished by replacing the Glu‐157 in the active site by Val. Taken together our results suggest that the α subunit of Endopeptidase‐24.18 contains a latent astacin‐like Zn metallopeptidase activity which could be secreted as a soluble enzyme by kidney and intestine.

Endopeptidase-24.11 in the adenohypophysis of the pig is localized in the gonadotrophic cells

Endopeptidase-24.11, an ectoenzyme with a key role in metabolizing peptides at cell surfaces, is present in the adenohypophysis. A specific polyclonal antibody to the endopeptidase has been used to explore its localization in cryostat sections of pig pituitary glands by an immunoperoxidase method. Immunoreactivity was symmetrically but not uniformly distributed over the anterior lobe, with the highest intensity a zone just beneath the capsule along the anterior surface. In detail, the staining was observed to be in the cell membrane, but in some cells a small area of intense paranuclear staining was also observed. Serial 5 micron sections were immunostained alternately for endopeptidase-24.11 and for pituitary proteohormone. Luteinizing hormone (LH), follicular stimulating hormone (FSH), thyrotropin, adrenocorticotropin, prolactin and growth hormone were studied in this way. It was possible to identify groups of cells in adjacent sections and a good correlation was observed for endopeptidase-24.11-immunoreactivity with that for LH and FSH. The association of the endopeptidase with gonadotrophs was confirmed by double labelling. No evidence of colocalization was observed with the other proteohormone antibodies. We conclude that among the cells of the adenohypophysis only the gonadotrophs express endopeptidase-24.11 and discuss the possible significance of this observation in regard to the termination of peptide signals, such as that of luteinizing hormone-releasing hormone (LHRH) acting at this site.