COOH-terminal Proteolytic Processing of Secreted and Membrane Forms of the α Subunit of the Metalloprotease Meprin A

Marchand, Petra; Tang, Jie; Johnson, Gary D.; Bond, Judith S.

doi:10.1074/jbc.270.10.5449

Cited by 74 publications

(106 citation statements)

References 42 publications

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“…1,7). Removal of this domain by genetic engineering results in no COOH-terminal processing of meprin a, and no transport of the subunit to the cell surface; the subunit remains in the endoplasmic reticulum (Marchand et al, 1995). This is in contrast to the meprin p subunit, which is not COOH-terminally processed in vivo, and is found as a type I integral membrane protein localized to the cell surface.…”

Section: Oligomeric Structure and Membrane Associationmentioning

confidence: 97%

“…The cDNA-deduced amino acid sequences COOH-terminal to the X domain differ in the a and P subunit of meprin, except that both contain EGF-like domains that are 34% identical. The a subunit contains a 56-amino acid inserted domain (I) that has no counterpart in the P subunit, and there is no homology between the putative transmembrane or cytoplasmic domains of a and (Marchand et al, 1995). The cytoplasmic domains are deduced to be 6 residues for a and 26 residues for 0.…”

Section: Comm)mentioning

confidence: 99%

“…The subcellular site of the COOH-terminal processing for meprins is the endoplasmic reticulum, but the proteases responsible for processing are unknown. Proteolytic processing of the meprin 01 subunit is not autolytic and is not due to furin-type enzymes (Marchand et al, 1995). There is evidence for an active proteolytic system in the ER because misfolded or incompletely assembled proteins are rapidly degraded, and antigen processing is proposed to occur in this subcellular compartment.…”

Section: Oligomeric Structure and Membrane Associationmentioning

confidence: 99%

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The astacin family of metalloendopeptidases

1995

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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 NH,-terminal signal and proenzyme sequences, and many (such as meprins, BMP-1, folloid) 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 01 and /3 subunits are differentially expressed and processed to yield latent and active proteases as well as membraneassociated and secreted forms. Meprins represent excellent models of hetero-and homo-oligomeric enzymes that are regulated at the transcriptional and posttranslational levels.

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Section: Oligomeric Structure and Membrane Associationmentioning

confidence: 97%

Section: Comm)mentioning

confidence: 99%

Section: Oligomeric Structure and Membrane Associationmentioning

confidence: 99%

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The astacin family of metalloendopeptidases

1995

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“…For transient expression of recombinant wild-type and mutant meprin ␣, the expression plasmids were transfected into human embryonic kidney 293 cells by the calcium phosphate precipitation method using 10 g of expression plasmid and 1 g of helper plasmid pVA1 per 100-mm tissue culture plate (24). Cells were grown to ϳ90% confluency by overnight incubation in complete DMEM.…”

Section: Methodsmentioning

confidence: 99%

“…The meprins are oligomers composed of ␣ and/or ␤ subunits that are evolutionarily related, but differ in function (1,3). Meprin ␤ subunits are integral membrane proteins; mature ␣ subunits are secreted from cells unless associated with ␤ subunits (4,5). Meprin A (EC 3.4.24.18) is defined as those isoforms that contain the ␣ subunit; this includes membrane-bound ␣/␤ hetero-oligomeric forms and a secreted ␣ homo-oligomeric form (1,6).…”

mentioning

confidence: 99%

N-Linked Oligosaccharides on the Meprin A Metalloprotease Are Important for Secretion and Enzymatic Activity, but Not for Apical Targeting

Kadowaki

Tsukuba

Bertenshaw

et al. 2000

Journal of Biological Chemistry

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The ␣ and ␤ subunits of meprins, mammalian zinc metalloendopeptidases, are extensively glycosylated; ϳ25% of the total molecular mass of the subunits is carbohydrate. The aim of this study was to investigate the roles of the N-linked oligosaccharides on the secreted form of mouse meprin A. Recombinant meprin ␣ and mutants in which one of the 10 potential Asn glycosylation sites was mutated to Gln were all secreted and sorted exclusively into the apical medium of polarized Madin-Darby canine kidney cells, indicating that no specific N-linked oligosaccharide acts as a determinant for apical targeting of meprin ␣. Several of the mutant proteins had decreased enzymatic activity using a bradykinin analog as substrate, and deglycosylation of the wildtype protein resulted in loss of 75-100% activity. Some of the mutants were also more sensitive to heat inactivation. In studies with agents that inhibit glycosylation processes in vivo, tunicamycin markedly decreased secretion of meprin, whereas castanospermine and swainsonine had little effect on secretion, sorting, or enzymatic properties of meprin. When all the potential glycosylation sites on a truncated form of meprin ␣ (␣-(1-445)) were mutated, the protein was not secreted into the medium, but was retained within the cells even after 10 h. These results indicate that there is no one specific glycosylation site or type of oligosaccharide (high mannose-or complex-type) that determines apical sorting, but that core N-linked carbohydrates are required for optimal enzymatic activity and for secretion of meprin ␣.

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Development of high throughput screening assays and pilot screen for inhibitors of metalloproteases meprin α and β

Madoux

Tredup²,

Spicer

et al. 2014

Biopolymers

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Zinc metalloproteinases meprin α and meprin β are implicated in a variety of diseases, such as fibrosis, inflammation and neurodegeneration, however, there are no selective small molecule inhibitors that would allow to study their role in these processes. To address this lack of molecular tools we have developed high throughput screening (HTS) assays to enable discovery of inhibitors of both meprin α and meprin β and screened a collection of well characterized pharmaceutical agents (LOPAC, n = 1,280 compounds). Two compounds (PPNDS, NF449) confirmed their activity and selectivity for meprin β. Kinetic studies revealed competitive (PPNDS) and mixed competitive/non-competitive (NF449) inhibition mechanisms suggesting that binding occurs in meprin β active site. Both PPNDS and NF449 exhibited low nanomolar IC50 and Ki values making them the most potent and selective inhibitors of meprin β reported to the date. These results demonstrate the ability of meprin α and β assays to identify selective compounds and discard artifacts of primary screening.

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COOH-terminal Proteolytic Processing of Secreted and Membrane Forms of the α Subunit of the Metalloprotease Meprin A

Cited by 74 publications

References 42 publications

The astacin family of metalloendopeptidases

The astacin family of metalloendopeptidases

N-Linked Oligosaccharides on the Meprin A Metalloprotease Are Important for Secretion and Enzymatic Activity, but Not for Apical Targeting

Development of high throughput screening assays and pilot screen for inhibitors of metalloproteases meprin α and β

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