Biomedical subjects

J S Bond

Publications and source records attributed to J S Bond.

At least 37 records · Page 2Linked to original sources

PubMed1996

Expression of subunits of the metalloendopeptidase meprin in renal cortex in experimental hydronephrosis.

Meprin A is a metalloendopeptidase in the proximal tubular epithelium of rodents that is capable of hydrolyzing a great variety of peptides and proteins. The aim of the present investigation was to investigate effects of ureteral ligation on the expression of meprin subunits. Ureteral ligation resulted in marked decreases in the expression of both alpha- and beta-meprin subunits within 12 h of ureteral obstruction. Even greater downregulation of expression of meprin alpha- and beta-mRNA was noted at 24, 48, and 96 h after ureteral ligation. The greatest decrease in meprin mRNA expression in obstructed kidneys over contralateral unobstructed control kidneys (CUK) occurred at 24 h postunilateral ureteral obstruction (post-UUO) for the meprin alpha-subunit (20-fold decrease compared with controls) and at 48 h for the meprin beta-subunit (90-fold decrease). On immunolabeling, the intensity for the two meprin subunits at the corticomedullary junction was dramatically decreased at 24 to 96 h after ureteral ligation in contrast to the CUK specimens. Results of in situ hybridization indicated that the CUK specimens expressed meprin beta-mRNA at the corticomedullary junction, whereas the obstructed kidneys exhibited a decrease in mRNA signal for meprin beta-subunit. There was a steady increase in the interstitial macrophage number in UUO rat kidneys over the 96 h of evaluation post-UUO. ED-1-positive macrophages were observed almost exclusively in the peritubular cortical interstitial space in a ringlike pattern with a preponderance of macrophage clusters around glomeruli. Unexpectedly, after reversal of UUO, the interstitial macrophage number remained higher than controls, despite the demonstrable decompression of the renal pelvis and caliceal system. In summary, this investigation demonstrates downregulation of meprin alpha and beta within hours of UUO and indicates a novel tubular response to ureteral obstruction.

Animals↗

PubMed1995

Fine mapping of MEP1A, the gene encoding the alpha subunit of the metalloendopeptidase meprin, to human chromosome 6P21.

Meprins are kidney and intestinal proteases encoded by two distinct genes, MEP1A and MEP1B. MEP1A was previously mapped to human chromosome 6p, by the use of radiation and somatic cell hybrids, in the region containing the gene for autosomal recessive polycystic kidney disease (ARPKD). We now report the fine mapping of MEP1A using yeast artificial chromosome clones, and linkage analysis of ARPKD families. The results from both physical and genetic mapping exclude MEP1A as a candidate for ARPKD. These studies place MEP1A in a region more telomeric to 6p12 and closer to the HLA loci than previously reported. More specifically, MEP1A is localized between loci D6S272 and D6S282, close to D6S452, on human chromosome 6p21.2-p21.1. The more precise location of MEP1A will facilitate genetic studies of this locus and clarify the relation of this gene to others.

Alleles↗

PubMed1995

The role of meprin A in the pathogenesis of acute renal failure.

Meprin A is a zinc-dependent metallo-endopeptidase that is present in the brush border membrane of renal proximal tubular epithelial cells. Although this enzyme has been detected in the kidney of mammalian species and its biochemical structure and action have been characterized, the role of this endopeptidase in normal renal physiology and in disease states is unknown. Therefore, we assessed the susceptibility of inbred strains of mice with normal and low meprin A activity to ischemic and nephrotoxic acute renal failure. We report that two normal meprin A strains of mice, C57BL/6 and DBA/2, developed more severe renal functional and structural injury following either bilateral renal artery clamping or the injection of hypertonic glycerol, compared to two low meprin A strains, C3H/He and CBA. These findings suggest that meprin A plays a role in the pathophysiology of acute renal failure following a variety of insults to the kidney. We propose that redistribution of this metalloendopeptidase to the basolateral membrane domain during acute renal failure results in degradation of the extracellular matrix and damage to adjacent peritubular structures.

Acute Kidney Injury↗

PubMed1995

COOH-terminal proteolytic processing of secreted and membrane forms of the alpha subunit of the metalloprotease meprin A. Requirement of the I domain for processing in the endoplasmic reticulum.

Cell surface isoforms of meprin A (EC 3.4.24.18) from mice and rats contain beta subunits that are type I integral membrane proteins and alpha subunits that are disulfide-linked to or noncovalently associated with membrane-anchored meprin subunits. Both alpha and beta subunits are synthesized with COOH-terminal domains predicted to be cytoplasmic, transmembrane, and epidermal growth factor-like; these domains are retained in beta subunits but are removed from alpha during maturation. The present studies establish that an inserted 56-amino acid domain (the "I" domain), present in alpha but not in beta, is necessary and sufficient for COOH-terminal proteolytic processing of the alpha subunit. This was demonstrated by expression of mutant meprin subunits (deletion mutants, chimeric alpha beta subunits, and beta mutants containing the I domain) in COS-1 cells. Mutations of two common processing sites present in the I domain (a dibasic site and a furin site) did not prevent COOH-terminal proteolytic processing, indicating that the proteases responsible for cleavage are distinct from those having these specificities. Deletion of the I domain from the alpha subunit resulted in accumulation of unprocessed subunits in a preGolgi compartment. Furthermore, COOH-terminal proteolytic processing of wild-type alpha subunits occurred before acquisition of endoglycosidase H resistance. Pulse-chase experiments and expression of an alpha subunit transcript containing a c-myc epitope tag, confirmed that proteolytic processing at the COOH terminus occurs in the endoplasmic reticulum. This work identifies the region of the alpha subunit that is essential for COOH-terminal processing and demonstrates that the differential processing of the evolutionarily-related subunits of meprin A that results in a structurally unique tetrameric protease begins in the endoplasmic reticulum.

Amino Acid Sequence↗

PubMed1995

The structural genes, MEP1A and MEP1B, for the alpha and beta subunits of the metalloendopeptidase meprin map to human chromosomes 6p and 18q, respectively.

Meprins are cell membrane, oligomeric metalloendopeptidases composed of two distinct but evolutionarily related subunits, alpha and beta. The structural genes for the meprin subunits, Mep-1 alpha and Mep-1 beta, have been previously mapped to chromosomes 17 and 18, respectively, of the mouse genome. We now report the localization of MEP1A and MEP1B in the human genome. MEP1A mapped to the short arm of chromosome 6 by the use of radiation and somatic cell hybrids. More specifically, it is localized between the centromere and GSTA2 in 6p11-p12. MEP1B mapped to chromosome 18, by the use of somatic cell hybrids, in 18q12.2-q12.3, proximal to the TTR/PALB gene. As in the mouse genome, the two homologous human structural genes for alpha and beta (50% identical on the cDNA level) are unlinked. These new markers on human chromosomes 6 and 18 extend the region of known linkage homology with mouse chromosomes 17 and 18, respectively, and provide new molecular access to regions of the human genome.

Animals↗

PubMed1995

The astacin family of metalloendopeptidases.

The astacin family of metalloendopeptidases was recognized as a novel family of proteases in the 1990s. The crayfish enzyme astacin was the first characterized and is one of the smallest members of the family. More than 20 members of the family have now been identified. They have been detected in species ranging from hydra to humans, in mature and in developmental systems. Proposed functions of these proteases include activation of growth factors, degradation of polypeptides, and processing of extracellular proteins. Astacin family proteases are synthesized with NH2-terminal signal and proenzyme sequences, and many (such as meprins, BMP-1, tolloid) contain multiple domains COOH-terminal to the protease domain. They are either secreted from cells or are plasma membrane-associated enzymes. They have some distinguishing features in addition to the signature sequence in the protease domain: HEXXHXXGFXHEXXRXDR. They have a unique type of zinc binding, with pentacoordination, and a protease domain tertiary structure that contains common attributes with serralysins, matrix metalloendopeptidases, and snake venom proteases; they cleave peptide bonds in polypeptides such as insulin B chain and bradykinin and in proteins such as casein and gelatin; and they have arylamidase activity. Meprins are unique proteases in the astacin family, and indeed in the animal kingdom, in their oligomeric structure; they are dimers of disulfide-linked dimers and are highly glycosylated, type I integral membrane proteins that have many attributes of receptors or integrins with adhesion, epidermal growth factor-like, and transmembrane domains. The alpha and beta subunits are differentially expressed and processed to yield latent and active proteases as well as membrane-associated and secreted forms. Meprins represent excellent models of hetero- and homo-oligomeric enzymes that are regulated at the transcriptional and posttranslational levels.

Amino Acid Sequence↗

PubMed1995

Meprins A and B.

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Amino Acid Sequence↗

PubMed1994

Membrane association and oligomeric organization of the alpha and beta subunits of mouse meprin A.

Meprins are oligomeric cell surface metalloproteinases of the "astacin family." They consist of two types of subunits (alpha and beta), which are evolutionarily related and whose cDNA sequences predict a similar domain structure. The present work shows that reducing agents solubilized meprin alpha subunits (approximately 90%), but not beta subunits, from mouse kidney brush border membranes. In addition, immunoblotting of membranes or purified meprins with an antibody raised to the alpha subunit epidermal growth factor-like domain, predicted to be near the COOH terminus from the cDNA-deduced amino acid sequence, indicated that this domain is not present in the mature alpha subunit. By contrast, an epitope predicted to be near the COOH terminus of the beta subunit was present in the mature form of beta. When meprins were solubilized from brush border membranes by papain, the size of the alpha subunit (approximately 90 kDa) did not change, while the beta subunit decreased from 110 to 90 kDa with concomitant loss of the COOH-terminal epitope. These data indicate that beta is a type I transmembrane protein, while alpha does not transverse the membrane and its association is dependent on disulfide bonds. The oligomeric organization of purified meprin A (EC 3.4.24.18), examined by sedimentation equilibrium analysis and native gradient gel electrophoresis, is that of disulfide-bridged dimers which aggregate noncovalently to form higher molecular weight complexes, predominantly tetramers. Western blotting of ICR kidney brush border membrane proteins identified alpha 2 homodimers and alpha beta heterodimers. Treatment of mouse or rat kidney brush border membranes with 7 M urea solubilized alpha 2, but not alpha beta dimers. Thus, the mature alpha subunit exists in alpha 2 and alpha beta disulfide-linked dimers which form tetramers, and the alpha 2 homodimers associate with the membrane through noncovalent interactions with alpha beta.

Amino Acid Sequence↗

PubMed1993

Biosynthesis and degradation of meprins, kidney brush border proteinases.

Meprins, which are cell surface metalloendopeptidases, consist of two types of subunits, alpha and beta. Genetic factors determine which subunits and oligomeric forms of the enzyme exist in kidney in different strains of mice. In order to further explore factors that determine the concentration and activity of meprins, the rates of biosynthesis and degradation of the meprin subunits were determined in an organ culture system using ICR and C3H/He mouse kidneys. In biosynthesis experiments, the rate of incorporation of radiolabeled amino acids into immunoprecipitable forms of the alpha and beta subunits was determined. The rate of loss of radiolabel from the subunits was measured in pulse-chase experiments to determine the half-lives of the subunits. The rate of synthesis of the alpha subunit was twofold greater than that of the beta subunit: 861 +/- 32 vs 361 +/- 23 dpm/micrograms subunit protein/min for alpha vs beta. The rate of synthesis for total kidney protein in both strains was approximately 700 dpm/micrograms/min. The half-life for alpha was 8.9 +/- 0.24 h compared to 12.1 +/- 0.7 h for beta; the half-life for total protein in kidney was approximately 35 h. Thus, the half-lives of alpha and beta were similar and shorter than the half-lives of the average protein in kidney cells. The higher rate of synthesis of alpha is probably responsible for the greater abundance of this protein compared to beta in microvillus membranes.

Animals↗

PubMed1993

Cloning, expression, and chromosomal localization of the mouse meprin beta subunit.

Meprins are plasma membrane homo- or hetero-oligomeric metalloendopeptidases that contain glycosylated alpha and/or beta subunits. This paper reports the cloning and sequencing of the mouse kidney beta subunit. The primary translation product is composed of 704 amino acids which includes a transient signal sequence of 20 amino acids at the NH2 terminus. The protease domain (Asn-63 to Leu-260) contains the putative zinc-binding motif characteristic of metalloendopeptidases of the "astacin family." The COOH terminus contains an epidermal growth factor-like domain, a potential membrane-spanning domain, and an additional 26 amino acids. The beta subunit has an overall 42% identity to the alpha subunit, however, a 56-amino acid segment near the COOH terminus of alpha is missing in beta, and the putative transmembrane and cytoplasmic domains of the subunits share no significant sequence similarity. NH2-terminal analyses of detergent-solubilized mature forms revealed that, unlike alpha, the prosequence (Leu-21 to Lys-62) is not removed from the beta subunit. Northern blot analysis revealed a 2.5-kilobase message for the beta subunit in the kidney and intestine of C57BL/6 and C3H/He mice. The gene for the beta subunit was localized to mouse chromosome 18. These studies indicate that alpha and beta probably derived from a common ancestral gene, but have evolved so that their genes are on two different chromosomes, and their tissue-specific expression and post-translational processing differ.

Amino Acid Sequence↗

PubMed1993

Tissue-specific expression and chromosomal localization of the alpha subunit of mouse meprin A.

Meprins, membrane-bound oligomeric metalloendopeptidases, contain alpha and/or beta subunits. Their activities have been found in the mouse and rat kidney. The cloned cDNA for the mouse alpha subunit of meprin A (EC cloned cDNA for the mouse alpha subunit of meprin A (EC 3.4.24.18) was used here to survey mRNA expression in kidney of different mouse strains and in various tissues of mice and rats. A single message of 3.6 kilobases was found in kidney of random bred (ICR) and inbred mice (C57BL/6, DBA/2) that contain high meprin A activity and in Sprague-Dawley rat kidney. The alpha subunit message was undetectable in the kidney of C3H/He and CBA mice, inbred strains that do not express meprin A activity. Therefore, meprin A activity in the kidney of mouse strains correlates with the amount of alpha subunit mRNA present. The 3.6-kilobase mRNA meprin alpha subunit message was also detected in the small intestine of the rat but not in mice. No message was detected in brain, heart, skeletal muscle, liver, lung, or spleen of mice or rats. Polymerase chain reaction amplification or Southern blot analysis of genomic DNA revealed that the gene for the alpha subunit is present in all mouse strains as well as in human, monkey, rat, mouse, dog, cow, rabbit, and chicken, but it was not detected in yeast. There is one gene copy present in the mouse genome. The gene was localized to mouse chromosome 17 centromeric to the major histocompatibility complex (H-2) by the interspecific backcrossing method. The localization of this allele to Mep-1, the gene previously found to regulate the expression of meprin A activity in mice, supports the proposal that Mep-1 is the structural gene for the alpha subunit.

Animals↗

PubMed1993

Meprin activity in rats with experimental renal disease.

Activity of renal meprin, a membrane-bound proteinase in the proximal tubule brush border, was measured in normal rats and in two disease groups: chronic puromycin aminonucleoside nephropathy for 12 weeks and streptozocin-induced diabetes for 6 months. Enzyme activity in kidney homogenates was assayed using azocasein as substrate. The mean activity of mephrin was 3.22 +/- 0.34 U/g kidney weight in normal rats. In diabetic animals, enzyme activity was 8.58 +/- 2.11 U/g kidney weight, P < 0.01. In contrast, meprin activity was decreased in rats with puromycin-induced glomerulopathy, 2.13 +/- 0.17 U/g kidney weight, P < 0.01. These findings indicate that meprin activity is elevated in experimental diabetes. Diminished activity of this luminal membrane enzyme in puromycin aminonucleoside nephropathy may contribute to renal injury in this disease model associated with massive urinary protein excretion.

Animals↗

PubMed1992

Families of metalloendopeptidases and their relationships.

Crystal structures available for four metalloendopeptidases have revealed zinc ligands for these enzymes. New sequence information has made it possible to compare the primary structures of the zinc-binding site in metalloendopeptidases. A scheme based on the zinc-binding site is proposed to classify metalloendopeptidases into five distinct families: thermolysin, astacin, serratia, matrixin, and snake venom metalloproteinases. Two histidines and one glutamate are zinc-ligands in the thermolysin family. Three histidines and one tyrosine are zinc ligands in the other four families, which are further distinguished by the identity of the residue following the third histidine and by the environment surrounding the tyrosine.

Amino Acid Sequence↗

PubMed1992

The alpha subunit of meprin A. Molecular cloning and sequencing, differential expression in inbred mouse strains, and evidence for divergent evolution of the alpha and beta subunits.

Meprin A, a membrane-bound oligomeric metalloendopeptidase, contains two different subunits, alpha and beta. We report here the cloning and sequencing of the alpha subunit cDNA. The translated polypeptide consists of 760 amino acids, including a preprosequence (77 amino acids) that precedes the NH2 terminus of the purified enzyme. The next 198 amino acids constitute the "astacin family" protease domain, which includes the astacin family signature sequence, HE(L,I)XHXXGFXHE(Q,H)XRXDRDX(Y,H)(V,I)X(I,V). An immunoglobulin/major histocompatibility complex protein signature was found at the end of the protease domain. At the COOH terminus of the alpha subunit, there is an epidermal growth factor-like domain, followed by a transmembrane domain, and six additional amino acids. Ten potential glycosylation sites have been identified, and at least three of those sites are glycosylated. Northern blot analyses of kidney tissue from C57BL/6 and C3H/He mice indicate that variations in meprin A activity in these strains reflect differences in the levels of the alpha subunit mRNA. Several internal peptide sequences obtained from the beta subunit indicate that it is approximately 50% identical to the alpha subunit. Furthermore, NH2-terminal sequence analyses (39 residues) indicate that rat and mouse alpha are 79% identical, rat and mouse beta are 74% identical, and that alpha and beta subunits for both species are 47% identical. These data indicate that alpha and beta are closely related products of divergent evolution.

Amino Acid Sequence↗

PubMed1991

The astacin family of metalloendopeptidases.

Molecular cloning of a human intestinal brush border metalloendopeptidase (N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase, PPH) and a mouse kidney brush border metalloendopeptidase (meprin A) has revealed 82% identity in the NH2-terminal amino acid sequences (198 residues) of the mature enzymes. Furthermore, searching of protein sequence data bases with the inferred peptide sequences as probes revealed strong similarities to astacin, a crayfish digestive protease, and an NH2-terminal domain of a human bone morphogenetic protein (BMP-1). Meprin A and PPH both have approximately 30% identity with astacin and BMP-1. Multiple alignment analysis indicated that 37 residues, including 3 cysteine residues, are strictly conserved for the four proteins in a sequence frame equivalent to the complete 200-amino acid astacin sequence. The four proteins contain a zinc-binding motif (HEXXH), found at the active site of most metalloendopeptidases, within an extended sequence of HEXXHXXGFXHE which is unique to this subgroup of metalloendopeptidases. In addition, the four proteins have 54% identity in a 24-amino acid sequence that includes the putative active site. A fifth protein, Xenopus laevis developmentally regulated protein UVS.2, also shares sequence identity with the metalloendopeptidases. These data provide strong evidence for an evolutionary relationship of these proteins. It is suggested that this new family of metalloendopeptidases be called the "astacin family."

Amino Acid Sequence↗

PubMed1991

Homo- and heterotetrameric forms of the membrane-bound metalloendopeptidases meprin A and B.

Meprin A and B are disulfide-linked, tetrameric metalloendopeptidases in renal brush border membranes. Meprin A contains 90-kDa subunits (alpha subunits) and is expressed in random-bred and some inbred strains of mice. Meprin B contains subunits of 110 kDa (beta subunits) in situ, and the enzyme from C3H mice, a strain that does not express alpha subunits, has been characterized. Evidence from this and previous studies indicate that beta subunits are expressed in all mouse strains. The tetrameric organization of these meprins was examined in brush border membrane fractions from a random-bred strain (ICR) and two inbred strains of mice (C57BL/6 and C3H/He). Lectin blotting using biotinylated concanavalin A revealed that membranes from the random-bred strain contained three oligomeric complexes of approximately 390, 440, and 490 kDa as determined after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of reducing agents. The subunits in all three oligomers were linked by disulfide bridges. Western blotting using an anti-alpha monoclonal antibody indicated that alpha subunits (90 kDa) were present in the 390- and 440-kDa complexes. Western blotting with a polyclonal antibody specific for beta subunits (110 kDa) indicated the presence of these subunits in the 440- and 490-kDa complexes. Electroelution of the individual oligomers followed by SDS-PAGE under reducing conditions confirmed that the 390- and 490-kDa molecules are homotetramers of alpha and beta subunits, respectively, and that the 440-kDa molecule is a heterotetramer consisting of disulfide-bridged alpha and beta subunits. C57BL/6 mice expressed both alpha and beta subunits and contained tetramers composed of alpha 4 and alpha 2 beta 2. C3H/He mice expressed only the 110-kDa beta subunits and the beta 4 oligomer. This type of multimeric organization of disulfide-linked subunits is unique for the known endopeptidases.

Animals↗

PubMed1991

Meprin-A and -B. Cell surface endopeptidases of the mouse kidney.

The proteinase meprin-A is a disulfide-linked tetramer of 90-kDa glycoprotein subunits. It is expressed at high levels in kidney brush border membranes of random bred and certain inbred strains of mice. Some mouse strains (e.g. C3H/He) do not express meprin-A subunits, but do produce a similar but less well characterized metalloendopeptidase, meprin-B. In the present study, meprin-B was purified from C3H/He mouse kidneys to electrophoretic homogeneity, and the relationship between it and meprin-A was investigated. The papain-solubilized form of meprin-B was similar to meprin-A in amino acid composition, molecular mass, secondary, and quaternary structure. However, immunoblots indicated that the enzymes have some common and some distinct epitopes. Lectin blots indicated both enzymes have high mannose and/or complex biantennary oligosaccharides, but there are differences in the complex-type glycosylation. Peptide maps and sequencing of cyanogen-bromide fragments of the enzymes revealed some different amino acid sequences. Thermal inactivation studies indicated that meprin-B was much less stable than meprin-A; the half-life for inactivation at 58 degrees C for meprin-A was 50 min, whereas for meprin-B it was less than 3 min. Both enzymes hydrolyzed azocasein and insulin B chain, but limited proteolysis of the enzymes with trypsin activated meprin-B 5-20-fold, whereas meprin-A was activated 2-fold at most. Analysis of hydrolysis products of the oxidized insulin B chain revealed some common and some distinct sites of cleavage. Bradykinin was a good substrate for meprin-A, while it was not hydrolyzed by meprin-B. A synthetic peptide, YLVC(SO3-)GERG, derived from insulin B chain was hydrolyzed faster by meprin-B than meprin-A, and neither enzyme was activated by trypsin treatment against this substrate. Taken together, the data indicate that the two metalloendopeptidases have many similarities but are distinct enzymes.

Amino Acid Sequence↗

PubMed1991

Mapping the active site of meprin-A with peptide substrates and inhibitors.

The extended substrate-binding site of meprin-A, a tetrameric metalloendopeptidase from brush border membranes of mouse kidney proximal tubules, was mapped with a series of peptide substrates. Previous studies led to the development of the chromogenic substrate Phe5(4-nitro)bradykinin for meprin-A. With this substrate, several biologically active peptides were screened as alternate substrate inhibitors, and, of these, bradykinin (RPPGFSPFR) was found to be the best substrate with a single cleavage site (Phe5-Ser6). Three types of bradykinin analogues were used for a systematic investigation of substrate specificity: (1) nonchromogenic bradykinin analogues with substitutions in the P3 to P3' subsites were used as alternative substrate inhibitors of nitrobradykinin hydrolysis, (2) analogues of nitrobradykinin with variations in the P1' position were tested as substrates, and (3) intramolecularly quenched fluorogenic bradykinin analogues with substitutions in the P1 to P3 sites were tested as substrates. A wide variety of substitutions in P1' had little effect on KM (174-339 microM) but markedly affected kcat (51.5 s-1 = A greater than S greater than R greater than F greater than K greater than T greater than E = 0). Substitutions in P1 had a greater effect on KM (366 microM-2.46 mM) and also strongly affected kcat (98.5 s-1 = A greater than F much greater than L greater than E greater than K = 2.4 s-1). The variety of allowed cleavages indicates that meprin-A does not have strict requirements for residues adjacent to the cleavage site. Substitutions farther from the scissle bond also affected binding and hydrolysis, demonstrating that multiple subsite interactions are involved in meprin-A action.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗