Stabilisation of cathepsin E by ATP

Thomas, Dominique; Richards, Anthony D.; Jupp, R A; Ueno, Eiko; Yamamoto, Kazuo; Samloff, I. Michael; Dunn, Ben M.; Kay, John

doi:10.1016/0014-5793(89)80117-6

Cited by 33 publications

(15 citation statements)

References 15 publications

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“…The concentration of cathepsin E at the site of early endocytosis is particularly noticeable in DC adhered to fibronectin, which extend long dendrites, the tips of which demonstrate extensive membrane ruffling and are the site of avid pinocytosis. The localization of cathepsin E at a very early stage of endocytosis is consistent with enzymatic studies showing that, in contrast to cathepsin D, this protease retains activity at near neutral pH (24), albeit with a somewhat different peptide selectivity (25). Although cathepsin E could not be detected in the classical late endosomal class II-loading compartment (26), we cannot rule out the possibility that some enzyme is transported into this compartment along with Ag during active endocytosis.…”

Section: Discussionsupporting

confidence: 72%

The Expression and Function of Cathepsin E in Dendritic Cells

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Medd³

et al. 2005

The Journal of Immunology

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Cathepsin E is an aspartic proteinase that has been implicated in Ag processing within the class II MHC pathway. In this study, we document the presence of cathepsin E message and protein in human myeloid dendritic cells, the preeminent APCs of the immune system. Cathepsin E is found in a perinuclear compartment, which is likely to form part of the endoplasmic reticulum, and also a peripheral compartment just beneath the cell membrane, with a similar distribution to that of Texas Red-dextran within 2 min of endocytosis. To investigate the function of cathepsin E in processing, a new soluble targeted inhibitor was synthesized by linking the microbial aspartic proteinase inhibitor pepstatin to mannosylated BSA via a cleavable disulfide linker. This inhibitor was shown to block cathepsin D/E activity in cell-free assays and within dendritic cells. The inhibitor blocked the ability of dendritic cells from wild-type as well as cathepsin D-deficient mice to present intact OVA, but not an OVA-derived peptide, to cognate T cells. The data therefore support the hypothesis that cathepsin E has an important nonredundant role in the class II MHC Ag processing pathway within dendritic cells.

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Section: Discussionsupporting

confidence: 72%

The Expression and Function of Cathepsin E in Dendritic Cells

Chain¹,

Free

Medd³

et al. 2005

The Journal of Immunology

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“…We have previously shown that CE from human erythrocyte membranes is activated by ATP [11,28], the nonhydrolyzable ATP analogues [28] and RNA [29] and restored to virtual activity at weakly acidic pH values (pH 5.5–5.8), at which the enzyme is essentially inactive by itself. This was considered to be due to the stabilization of the enzyme by these compounds in such a way as to maintain its active conformation even at weakly acidic pH values.…”

Section: Resultsmentioning

confidence: 99%

Role of N‐glycosylation in cathepsin E

Yasuda

Ikeda

Sakai

et al. 1999

European Journal of Biochemistry

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Cathepsin E (CE), a nonlysosomal, intracellular aspartic proteinase, exists in several molecular forms that are N-glycosylated with high-mannose and/or complex-type oligosaccharides. To investigate the role of N-glycosylation on the catalytic properties and molecular stability of CE, both natural and recombinant enzymes with distinct oligosaccharides were purified from different sources. An N-glycosylation minus mutant, that was constructed by site-directed mutagenesis (by changing asparagine residues to glutamine and aspartic acid residues at positions 73 and 305 in potential N-glycosylation sites of rat CE) and expressed in normal rat kidney cells, was also purified to homogeneity from the cell extracts. The kinetic parameters of the nonglycosylated mutant were found to be essentially equivalent to those of natural enzymes N-glycosylated with either highmannose or complex-type oligosaccharides. In contrast, the nonglycosylated mutant showed lower pH and thermal stabilities than the glycosylated enzymes. The nonglycosylated mutant exhibited particular sensitivity to conversion to a monomeric form by 2-mercaptoethanol, as compared with those of the glycosylated enzymes. Further, the high-mannose-type enzymes were more sensitive to this agent than the complex-type proteins. A striking difference was found between the high-mannose and complex-type enzymes in terms of activation by ATP at a weakly acidic pH. At pH 5.5, the complex-type enzymes were stabilized by ATP to be restored to the virtual activity, whereas the high-mannose-type enzymes as well as the nonglycosylated mutant were not affected by ATP. These results suggest that N-glycosylation in CE is important for the maintenance of its proper folding upon changes in temperature, pH and redox state, and that the complex-type oligosaccharides contribute to the completion of the tertiary structure to maintain its active conformation in the weakly acidic pH environments.Keywords: aspartic proteinase; cathepsin E; role of N-glycosylation; intracellular stability.Cathepsin E (CE) is a major intracellular aspartic proteinase. It exists as a homodimer of two catalytically active monomers with a molecular mass of 42 kDa in the active form, but is easily converted to a monomeric form that retains catalytic activity under mild reducing conditions (reviewed in [1,2] [17] have been reported. The sequences of CE from these species show a high degree of similarity (more than 80% identity) and are apparently different from those of other aspartic proteinases, such as cathepsin D (47% identity), pepsinogen (48%), and renin (41%). The unique structural characteristics of pro-CE are conserved in all of the species; the amino (N)-terminal portion of CE contains a Cys residue at position 43 which is responsible for the formation of a disulfide bond between the two identical subunits. The highly conserved tripeptide sequence Asp-Thr-Gly at the active sites of aspartic proteinases is also found in all of these CE species, except that the sequence near the N-terminal region of rabb...

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“…Influence of reducing agent on the substrate dependence for hydrolysis by naturally-occurring cathepsin E. Measurements of initial velocity (pmol/s x 10 -2) were made at differing concentrations of substrate in the absence (1) and presence (©) of reducing agent. In (a) the substrate was Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu, pH was 3.1 and dithiothreitol (20 mM) was the reducing agent; in (b) the substrate was Pro-Pro-Thr-Ile-Phe*Nph-Arg-Leu, the pH was 5.8, 6.25 mM ATP [15] was included in all assays and mercaptoethanol (50 mM) was the reducing agent.…”

Section: Resultsmentioning

confidence: 99%

Monomeric human cathepsin E

et al. 1995

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Cathepsin E is a homodimer, consisting of two monomers linked by an inter-molecular disulphide bond. The cysteine residue involved is located near to the N-terminus of the mature proteinase. By mutating this residue to alanine, a monomeric form of human cathepsin E was engineered and purified. The activity of the resultant enzyme was not altered significantly (in terms of its ability to hydrolyse two chromogenic peptide substrates; and its susceptibility to inhibition by pepstatin). However, the stability of the mutant enzyme to alkaline pH and to temperature was markedly reduced.

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Stabilisation of cathepsin E by ATP

Cited by 33 publications

References 15 publications

The Expression and Function of Cathepsin E in Dendritic Cells

The Expression and Function of Cathepsin E in Dendritic Cells

Role of N‐glycosylation in cathepsin E

Monomeric human cathepsin E

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