Mucin-like Domain of Enteropeptidase Directs Apical Targeting in Madin-Darby Canine Kidney Cells

Zheng, X. Long; Sadler, J. Evan

doi:10.1074/jbc.m109857200

Cited by 29 publications

(32 citation statements)

References 33 publications

(51 reference statements)

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“…1). In this regard, corin differs from enteropeptidase that contains a mucin-like domain where O-linked oligosaccharides are abundant (24,25). Our results indicate that the N-glycosylation is required for corin zymogen activation.…”

Section: Discussionmentioning

confidence: 78%

“…Oberst et al (23) have shown that an N-glycosylation site in the protease domain is critical for the activation of matriptase, a type II transmembrane serine protease essential for the development of multiple epithelial tissues (37)(38)(39). Similarly, Zheng et al (24,25) have shown that N-glycans in the enteropeptidase protease domain are important for the apical targeting of this digestive enzyme. Therefore, it appears that N-glycans may have a general role in regulating the biosynthesis and activation of type II transmembrane serine proteases.…”

Section: Discussionmentioning

confidence: 99%

“…To date, the extent of N-linked oligosaccharides on corin protein and their functional importance have not been examined. Studies of other type II transmembrane serine proteases such as matriptase (23) and enteropeptidase (24,25) have shown that N-glycosylation is critical for zymogen activation and subcellular targeting. It is possible that carbohydrate moieties may play a similar role in corin.…”

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Role of Glycosylation in Corin Zymogen Activation

Liao

Wang

Chen

et al. 2007

Journal of Biological Chemistry

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The cardiac serine protease corin is the pro-atrial natriuretic peptide convertase. Corin is made as a zymogen, which is activated by proteolytic cleavage. Previous studies showed that recombinant human corin expressed in HEK 293 cells was biologically active, but activated corin fragments were not detectable, making it difficult to study corin activation. In this study, we showed that recombinant rat corin was activated in HEK 293 cells, murine HL-1 cardiomyocytes, and rat neonatal cardiomyocytes. In these cells, activated corin represented a small fraction of the total corin molecules. The activation of recombinant rat corin was inhibited by small molecule trypsin inhibitors but not inhibitors for matrix metalloproteinases or cysteine proteases, suggesting that a trypsin-like protease activated corin in these cells. Glycosidase digestion showed that rat and human corin proteins contained substantial N-glycans but little O-glycans. Treatment of HEK 293 cells expressing rat corin with tunicamycin prevented corin activation and inhibited its pro-atrial natriuretic peptide processing activity. Similar effects of tunicamycin on endogenous corin activity were found in HL-1 cells. Mutations altering the two N-glycosylation sites in the protease domain of rat corin prevented its activation in HEK 293 and HL-1 cells. Our results indicate that N-linked oligosaccharides play an important role in corin activation.

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Section: Discussionmentioning

confidence: 78%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Role of Glycosylation in Corin Zymogen Activation

Liao

Wang

Chen

et al. 2007

Journal of Biological Chemistry

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“…Recombinant proteins were visualized by SDS-PAGE and Western blotting with monoclonal anti-V5 antibody (Invitrogen), peroxidaseconjugated goat anti-mouse IgG (DAKO Corp., Carpinteria, CA), and the chemiluminescent ECL detection system (Amersham Biosciences) as described previously (20). The luminograms were scanned, and the relative amount of proteins detected was estimated by densitometry using NIH Image 1.62 (developed at the National Institutes of Health and available on the Internet at rsb.info.nih.gov/nih-image/).…”

Section: Methodsmentioning

confidence: 99%

Cleavage of von Willebrand Factor Requires the Spacer Domain of the Metalloprotease ADAMTS13

Zheng

Nishio

Majerus

et al. 2003

Journal of Biological Chemistry

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ADAMTS13 consists of a reprolysin-type metalloprotease domain followed by a disintegrin domain, a thrombospondin type 1 motif (TSP1), Cys-rich and spacer domains, seven more TSP1 motifs, and two CUB domains. ADAMTS13 limits platelet accumulation in microvascular thrombi by cleaving the Tyr 1605 -Met 1606 bond in von Willebrand factor, and ADAMTS13 deficiency causes a lethal syndrome, thrombotic thrombocytopenic purpura. ADAMTS13 domains required for substrate recognition were localized by the characterization of recombinant deletion mutants. Constructs with C-terminal His 6 and V5 epitopes were expressed by transient transfection of COS-7 cells or in a baculovirus system. No association with extracellular matrix or cell surface was detected for any ADAMTS13 variant by immunofluorescence microscopy or chemical modification. Both plasma and recombinant full-length ADAMTS13 cleaved von Willebrand factor subunits into two fragments of 176 kDa and 140 kDa. Recombinant ADAMTS13 was divalent metal ion-dependent and was inhibited by IgG from a patient with idiopathic thrombotic thrombocytopenic purpura. ADAMTS13 that was truncated after the metalloprotease domain, the disintegrin domain, the first TSP1 repeat, or the Cys-rich domain was not able to cleave von Willebrand factor, whereas addition of the spacer region restored protease activity. Therefore, the spacer region is necessary for normal ADAMTS13 activity toward von Willebrand factor, and the more Cterminal TSP1 and CUB domains are dispensable in vitro.Thrombotic thrombocytopenic purpura (TTP) 1 is a syndrome characterized by microangiopathic hemolytic anemia and thrombocytopenia, and it may be accompanied by neurological dysfunction, renal failure, and fever (1-3). If untreated, the mortality can exceed 90%, but plasma-exchange therapy has reduced the mortality to less than 20% (4). Although the pathophysiology of TTP is not fully understood, a plausible model has been proposed in which the proteolysis of von Willebrand factor (VWF) plays a central role (5). VWF is a multimeric protein that binds receptors on the surface of platelets and in connective tissue, thereby mediating the adhesion of platelets to sites of vascular injury. If unchecked, the process can lead to microvascular thrombosis. A plasma VWF-cleaving protease has been described that acts on the Tyr 1605 -Met 1606 bond in the central A2 domain of the VWF subunit, and cleavage is stimulated by shear forces like those occurring at sites of thrombosis or by low concentrations of urea or guanidine (6, 7). This proteolytic reaction limits VWF-dependent platelet adhesion, and most adults with idiopathic TTP have an acquired autoantibody that inhibits the VWF-cleaving protease (8, 9). Therefore, therapeutic plasma exchange may ameliorate TTP by replacing the missing protease and removing the inhibitory antibody.The VWF-cleaving protease was recently purified and identified as a new member of the ADAMTS family of metalloproteases (10, 11), so named for the combination of a disintegrin-like and metalloprote...

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“…The study demonstrated the involvement of Lys-99, which is situated in a unique exosite on the enzyme surface, in the specific cleavage of trypsinogen and similar peptidyl substrates. More recently, a mucin-like domain found in the heavy chain of EP has been shown to be a possible targeting signal for apical sorting of the protein (12).…”

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confidence: 99%

Specificity of the medaka enteropeptidase serine protease and its usefulness as a biotechnological tool for fusion-protein cleavage

Ogiwara

Takahashi²

2007

Proc. Natl. Acad. Sci. U.S.A.

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We cloned two distinct cDNAs for enteropeptidase (EP) from the intestine of the medaka, Oryzias latipes, which is a small freshwater teleost. The mRNAs code for EP-1 (1,036 residues) and EP-2 (1,043 residues), both of which have a unique, conserved domain structure of the N-terminal heavy chain and C-terminal catalytic serine protease light chain. When compared with mammalian EP serine proteases, the medaka enzyme exhibited extremely low amidolytic activity for small synthetic peptide substrates. Twelve mutated forms of the medaka EP protease were produced by site-directed mutagenesis. Among them, one mutant protease, E173A, was found to have considerably reduced nonspecific hydrolytic activities both for synthetic and protein substrates without serious reduction of its Asp-Asp-Asp-Asp-Lys (D4K)-cleavage activity. For the cleavage of fusion proteins containing a D4K-cleavage site, the medaka EP proteases were shown to have advantages over their mammalian counterparts. Based on our present data, we propose that the E173A mutant is the most appropriate protease to specifically cleave proteins containing the D4K cleavage sequence.cleavage specificity ͉ medaka fish E nteropeptidase (EP) (enterokinase, EC 3.4.21.9) is a twochain, membrane-bound serine protease of the duodenal mucosa that converts trypsinogen to active trypsin (1). Trypsin thus produced then cleaves and activates other zymogens in pancreatic secretions, including chymotrypsinogen, proelastase, procarboxypeptidases, and some prolipases. Therefore, EP has been recognized to play a critical role in regulating protein digestion within the lumen of the gut. The biological importance of EP is demonstrated by the malabsorption and malnutrition of patients with primary EP deficiency, a genetic disorder resulting in no or little EP activity in the duodenum (2).EP has been intensively studied in the last decade. To date, EP has been molecularly cloned from several sources, including cattle (3, 4), humans (5), pigs (6), rats (7), and mice (8). These studies provided much information on the structural details and organization of EP, and opened a path to further investigation of the molecular properties of this protease. For example, it was demonstrated that the N-terminal heavy chain is required for efficient activation of trypsinogen by the serine protease domain of the C-terminal light chain (9, 10). In addition, a recent study by Lu et al. (11) established the tertiary structure of the bovine EP catalytic domain. The study demonstrated the involvement of Lys-99, which is situated in a unique exosite on the enzyme surface, in the specific cleavage of trypsinogen and similar peptidyl substrates. More recently, a mucin-like domain found in the heavy chain of EP has been shown to be a possible targeting signal for apical sorting of the protein (12).EP is also of biotechnological interest because of the unique substrate specificity of the serine protease domain. The high degree of specificity exhibited by EP makes it a suitable reagent for cleaving bacterially pr...

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Mucin-like Domain of Enteropeptidase Directs Apical Targeting in Madin-Darby Canine Kidney Cells

Cited by 29 publications

References 33 publications

Role of Glycosylation in Corin Zymogen Activation

Role of Glycosylation in Corin Zymogen Activation

Cleavage of von Willebrand Factor Requires the Spacer Domain of the Metalloprotease ADAMTS13

Specificity of the medaka enteropeptidase serine protease and its usefulness as a biotechnological tool for fusion-protein cleavage

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