Cloning of the PABA peptide hydrolase alpha subunit (PPHα) from human small intestine and its expression in COS‐1 cells

Dumermuth, Eric; Eldering, Joyce A.; Grünberg, Jürgen; Jiang, Weiping; Sterchi, Erwin E.

doi:10.1016/0014-5793(93)80421-p

Cited by 55 publications

(48 citation statements)

References 37 publications

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“…Lepageet al (1992a); SpAN, Reynoldset al (1992);SuBMP, Hwangetal. (1994);HuBMP-I, Wozneyet al (1988); XeBMP-I, Maeno et al (1993); DrTld (Drosophila tolloid), Shimell et al (1991); HuTld, Takahara et al (1994); MuBMP-I, Fukagawa et al (1994); DrTlr-l (Drosophila rolloid-relared-l), Nguyen et al (1994); Mu meprin-a, ; Rt meprin-a, Corbeil et al (1992); Hu meprin-a, Dumermuth et al (1993);Mu meprin-0, Gorbeaet al (1993); Rt meprin-0, Johnson and Hersh (1992); Hu meprin-0, Sterchi (pers. comm bonds.…”

Section: Js Bond and R J Beynonmentioning

confidence: 99%

“…Studies with transfected meprin cDNAs have led to a similar picture of meprin organization (Milhiet et al, 1994). When fulllength meprin CY transcripts were transfected into COS-I, 293, or MDCK (Madin Darby canine kidney) cells, meprin CY subunits were secreted, and very little remained associated with cells (Corbeil et al, 1993;Dumermuth et al, 1993). In contrast, transfection of full-length (3 subunit transcripts led to plasma membrane-bound (3 subunits (Johnson & Hersh, 1994).…”

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: Js Bond and R J Beynonmentioning

confidence: 99%

Section: Oligomeric Structure and Membrane Associationmentioning

confidence: 99%

The astacin family of metalloendopeptidases

1995

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“…Meprin, also termed endopeptidase 24.1 8 is expressed in microvillus membranes of epithelial cells in kidney proximal tubules and small intestine of mouse and rat [ 13 -151, and has also been found to be excreted in a soluble form in mouse urine [16]. In contrast, PPH has so far only been identified in the human intestine [17], although a similar enzyme activity has recently been found in human kidney also [18].…”

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confidence: 99%

“…Expression studies in COS-1 (Simian virus 40 transformed African green monkey kidney) and MDCK (Madin-Darby canine kidney) cells have shown that rat meprin a and a mouse meprin-PPHn chimera form disulphide linked homodimers [ 17,19,201 that are secreted in a complex glycosylated form. However, the cell-associated a subunit was an immature and transport-incompetent protein which did not reach the cell surface.…”

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confidence: 99%

Polarised Expression of Human Intestinal N‐benzoyl‐l‐tyroxyl‐p‐aminobenzoic Acid Hydrolase (Human Meprin) α and β Subunits in Madin‐Darby Canine Kidney Cells

Eldering¹,

Grünberg²,

Hahn³

et al. 1997

European Journal of Biochemistry

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N-Benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (PPH, human meprin), is a peptidase found in the microvillus membrane of human small intestinal epithelial cells. PPH belongs to the astacin family of zinc-metalloendopeptidases and is a protein complex composed of two glycosylated subunits, a and P.The present report describes the cloning of the complete /? subunit and the remaining N,-terminal end of the a subunit for analysis of their primary structures in addition to the examination of their biogenesis in transfected cell cultures. The complete open reading frame of the PPHP cDNA translates into 700 amino acid residues compared with 746 residues for the PPHa cDNA. The primary structure of P and a subunits are 44 % identical and 61 % similar. As predicted from their primary structure, the two subunits of PPH have identical modular structures; starting at the N,-terminus both contain a signal peptide, a propeptide, a protease domain containing the astacin signature, a meprin A5 protein tyrosine phospatase p (MAM) and a meprin and TRAF homology domain (MATH) domain, an epidermal growth factor(EGF)-like domain, a putative transmembrane anchor domain and a short cytosolic tail. Pulsekhase labelling and immuno-Gold electronmicroscopy of recombinant PPH , l ?and a subunits expressed in transfected MadinDarby canine kidney (MDCK) cells show that post-translational processing and transport of the two subunits are very different. When expressed alone, the subunit acquired complex glycan residues, readily formed homodimers and was transported to the plasma membrane. Small amounts of PPHP were found in the culture medium. In contrast, the cell-bound a subunit, when expressed alone, remained primarily in the high-mannose form, was aggregated and not expressed at the cell surface. However, the bulk of mostly endo-P-N-acetylglucosaminidase H-resistant a subunit was found in the filtered culture medium. The proteolytic event that leads to the formation of this soluble transport-competent form occurs in the endoplasmic reticulum (ER). Coexpression of the a subunit with the P subunit allowed the localisation of the a subunit to the plasma membrane. These studies indicate that assembly of the two subunits of PPH is required for the localisation of the a subunit to the plasma membrane. In contrast to rodent meprin, both PPH subunits are apically secreted from MDCK cells.Keywords: human meprin; N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase; astacin family; processing ; intracellular transport. N-Benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (PPH)is a metalloendopeptidase isolated from the microvillus membrane of human enterocytes [I]. The enzyme was first identified using N-benzoyl-L-tyrosyl-p-aminobenzoic acid (Bz-TyrNBzOH), a substrate used to assess exocrine pancreatic function Abbreviations. Bz-Tyr-NBzOH, N-benzoyl-L-tyrosyl-p-aminobenzoic acid; PPH, N-benzoyl-L-tyrosyl-I,-aminobenzoic acid hydrolase; EGF, epidermal growth factor; ER, endoplasmic reticulum; MAM, meprin A5 protein tyrosine phosphatase p ; MATH, meprin...

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“…Meprin is a membranal metalloendoproteinase found in the intestinal and renal brush-border membranes of the mouse (14,15), rat (16 -18), and man (19,20). Meprins belong to the astacin family of endopeptidases (21)(22)(23)(24) and are usually composed of two types of subunits, ␣ and ␤, that exist as homo-and heterotetramers bound to each other through disulfide bridges (25,26).…”

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confidence: 99%

Unveiling the Substrate Specificity of Meprin β on the Basis of the Site in Protein Kinase A Cleaved by the Kinase Splitting Membranal Proteinase

Chestukhin¹,

Litovchick²,

Muradov

et al. 1997

Journal of Biological Chemistry

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The kinase splitting membranal proteinase (KSMP) is a metalloendopeptidase that inactivates the catalytic (C) subunit of protein kinase A (PKA) by clipping off its carboxyl terminal tail. Here we show that this cleavage occurs at Glu 332 -Glu 333 , within the cluster of acidic amino acids (Asp 328 -Glu 334 ) of the kinase. The K m values of KSMP and of meprin ␤ (which reproduces KSMP activity) for the C-subunit are below 1 M. The K m for peptides containing a stretch of four Glu residues are in the micromolar range, illustrating the significant contribution of this cluster to the substrate recognition of meprin ␤. This conclusion is supported by a systematic study using a series of the C-subunit mutants with deletions and mutations in the cluster of acidics. Hydrophobic amino acids vicinal to the cleavage site increase the K cat of the proteinase. These studies unveil a new specificity for meprin ␤, suggesting new substrates that are 1-2 orders of magnitude better in their K m and K cat than those commonly used for meprin assay. A search for substrates having such a cluster of acidics and hydrophobics, which are accessible to meprin under physiological conditions, point at gastrin as a potential target. Indeed, meprin ␤ is shown to cleave gastrin at its cluster of five glutamic acid residues and also at the M-D bond within its WMDF-NH 2 sequence, which is indispensable for all the known biological activities of gastrins. The latter meprin cleavage will lead to the inactivation of gastrin and thus to the control of its activity.The presence of a kinase splitting membranal proteinase (KSMP) 1 in the brush-border membranes of the rat small intestine was demonstrated as early as 1979 (1). This proteinase was shown to clip the catalytic (C) subunit of PKA, yielding a distinct cleavage product (CЈ) that was found to be devoid of the kinase activity. The biochemical characterization of KSMP as a proteinase revealed that it is an intriguing enzyme with a combination of the following unique features. (a) KSMP cleaves the C-subunit when it is free but not when inhibited by its regulatory (R) subunits, as in the R 2 C 2 complex (1, 2). (b) This cleavage could not be simulated by other proteinases (trypsin, chymotrypsin, clostripain, and papain (2)), suggesting that its specificity is not due merely to an interdomain exposure in C. (c) The proteinase was found to single out and selectively cleave the C-subunit in the presence of the large number of other proteins found in crude extracts of different tissues (brain, liver, or muscle) (3). (d) KSMP was found to cleave the Csubunit in its native conformation but not if the kinase is pre-denatured (2). (e) It distinguishes between the "open" and "closed" conformations of the C-subunit (4) that were recently identified by x-ray crystallography of this kinase (5).The cleavage of the C-subunit by KSMP leads to the removal of the carboxyl terminus tail of this kinase and seemed to occur at a distinct site (6 -8). Interestingly, two other kinases, the EGF-and the insulin-receptor ki...

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Cloning of the PABA peptide hydrolase alpha subunit (PPHα) from human small intestine and its expression in COS‐1 cells

Cited by 55 publications

References 37 publications

The astacin family of metalloendopeptidases

The astacin family of metalloendopeptidases

Polarised Expression of Human Intestinal N‐benzoyl‐l‐tyroxyl‐p‐aminobenzoic Acid Hydrolase (Human Meprin) α and β Subunits in Madin‐Darby Canine Kidney Cells

Unveiling the Substrate Specificity of Meprin β on the Basis of the Site in Protein Kinase A Cleaved by the Kinase Splitting Membranal Proteinase

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