Homology within the <i>N</i>-terminal extension of cysteine proteinases

North, Michael

doi:10.1042/bj2380623

Cited by 6 publications

(6 citation statements)

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“…Most vacuolar, lysosomal and extracellular proteinases are initially synthesized as higher molecular mass precursors, zymogens. Papain superfamily cysteine proteinases are also synthesized as proforms with large N‐terminal propeptides [1] and in some cases with C‐terminal extensions [2–4]. It is known that the proregion of the proteinases acts as an intramolecular chaperone to ensure correct folding of the enzymes [5], contains signals for intracellular transport [6] and plays a role in inactivation of the proteinases [7–9].…”

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confidence: 99%

Asparaginyl endopeptidase (VmPE‐1) and autocatalytic processing synergistically activate the vacuolar cysteine proteinase (SH‐EP)

Okamoto

Yûki

Mitsuhashi

et al. 1999

European Journal of Biochemistry

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A vacuolar cysteine proteinase, designated SH-EP, is synthesized in cotyledons of germinated Vigna mungo seeds and is responsible for degradation of the seed proteins accumulated in protein bodies (protein storage vacuoles). SH-EP belongs to the papain proteinase family and has a large N-terminal prosegment consisting of 104 amino acid residues and a C-terminal prosegment of 10 amino acid residues. It has been suggested that an asparaginyl endopeptidase, V. mungo processing enzyme 1 (VmPE-1), is involved in the N-terminal post-translational processing of SH-EP. The recombinant proform of SH-EP (rSH-EP) was produced in Escherichia coli cells, purified to homogeneity and refolded by stepwise dialysis. 31 P-NMR analysis of intact germinated cotyledons revealed that the vacuolar pH of cotyledonary cells changes from 6.04 to 5.47 during seed germination and early seedling growth. rSH-EP was converted in vitro to the mature form through autocatalytic processing at a pH mimicking the vacuolar pH at the mid and late stages of seed germination, but not at the pH of the early stage. VmPE-1 accelerated the rate of processing of rSH-EP in vitro at the pH equivalent to the vacuolar pH at the early and mid stages of germination. In addition, the cleavage sites of the in vitro processed intermediates and the mature form of SH-EP were identical to those of SH-EP purified from germinated cotyledons of V. mungo. We propose that the asparaginyl endopeptidase (VmPE-1)-mediated processing mainly functions in the activation of proSH-EP at the early stage of seed germination, and both VmPE-1-mediated and autocatalytic processings function synergistically in the activation of proSH-EP in cotyledons at the mid and late stages. The activation of propapain [5,8] and human procathepsin B [13] has been intensively studied, and both were found to be activated by both intramolecular and intermolecular autocatalytic processings. Rat procathepsin B is completely processed to the mature form intermolecularly by mature cathepsin B and subsequent trimming by a dipeptidylpeptidase [14]. Procathepsin L [15,16] and procathepsin K [17] are also activated to the mature enzymes in a self-catalytic manner. In contrast, precursors of plant vacuolar cysteine proteinases, SH-EP [18] and aleurain [19], and an extracellular proteinase, EPB [20], are thought to be processed by other proteinases.SH-EP is a cysteine proteinase that plays a major role in the degradation of seed storage proteins accumulated in cotyledonary vacuoles of Vigna mungo seedlings [21]. It was previously reported that the enzyme is synthesized on membrane-bound ribosomes as a 45-kDa polypeptide, which is cotranslationally processed to a 43-kDa intermediate through cleavage of a 2-kDa signal peptide. The intermediate is further processed to the 33-kDa mature enzyme via 39-and 36-kDa intermediates, and the conversion from the 36-kDa intermediate to the 33-kDa mature enzyme results in full activation [22]. Okamoto and Minamikawa [18] isolated a processing enzyme, designated VmPE-1, that is...

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confidence: 99%

Asparaginyl endopeptidase (VmPE‐1) and autocatalytic processing synergistically activate the vacuolar cysteine proteinase (SH‐EP)

Okamoto

Yûki

Mitsuhashi

et al. 1999

European Journal of Biochemistry

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“…All cysteine proteases are initially synthesized as larger precursor proteins with NH 2 -terminal prodomains of approximately 120 amino acids (11). The prodomain is an inhibitor of the protease (12) and is essential in the correct folding of the protein (13).…”

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confidence: 99%

Posttranslational Removal of the Carboxyl-terminal KDEL of the Cysteine Protease SH-EP Occurs Prior to Maturation of the Enzyme

Okomoto

Minamikawa

Edward

et al. 1999

Journal of Biological Chemistry

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SH-EP is a cysteine protease from germinating mung bean (Vigna mungo) that possesses a carboxyl-terminal endoplasmic reticulum (ER) retention sequence, KDEL. In order to examine the function of the ER retention sequence, we expressed a full-length cDNA of SH-EP and a minus-KDEL control in insect Sf-9 cells using the baculovirus system. Our observations on the synthesis, processing, and trafficking of SH-EP in Sf-9 cells suggest that the KDEL ER-retention sequence is posttranslationally removed either while the protein is still in the ER or immediately after its exit from the ER, resulting in the accumulation of proSH-EP minus its KDEL signal. It is this intermediate form that appears to progress through the endomembrane system and is subsequently processed to form mature active SH-EP. The removal of an ER retention may regulate protein delivery to a functional site and present an alternative role for ER retention sequences in addition to their well established role in maintaining the protein composition of the ER lumen.SH-EP is a thiol protease expressed in germinating mung bean (Vigna mungo) cotyledons (1-3) that is among a large number of similar cysteine proteases that are synthesized after germination in order to degrade seed storage proteins (2, 4 -10). All cysteine proteases are initially synthesized as larger precursor proteins with NH 2 -terminal prodomains of approximately 120 amino acids (11). The prodomain is an inhibitor of the protease (12) and is essential in the correct folding of the protein (13). Processing of the proenzyme occurs by self-catalysis (14) in post-Golgi compartments of the secretory system. With intracellular proteases, this processing occurs in the lytic compartment (lysosomes or vacuoles) (15); with extracellular proteases, the processing occurs during secretion (16). Targeting to the correct compartment for processing is mediated by a peptide (17) or phosphomannose (18) targeting signal on the NH 2 -terminal precursor domain that is recognized by a specific receptor that is presumed to be localized in the Golgi apparatus (19,20) to target the protein to the activating compartment.SH-EP is unusual because it and a few closely related thiol proteases (21-23) are the only cysteine proteases known to possess a carboxyl-terminal ER 1 retention sequence KDEL (24 -26). Although papain superfamily proteases are widely distributed among eukaryotes (27, 28), only these plant cysteine proteases have been identified as possessing a carboxylterminal ER retention sequence. Several other legume cysteine proteases have been cloned that do not possess a KDEL tail (28 -34), indicating that even among legumes and more broadly in plants these KDEL-proteases appear to constitute a special class. Plant cells utilize both HDEL and KDEL as ER retention sequences (35)(36)(37)(38). The presence of a carboxyl-terminal KDEL in SH-EP raises a question as to whether this sequence is functional in vivo. A papain superfamily cysteine protease would appear to be an unusual putative constituent of the ER lumen, ...

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“…The cysteine proteinases are highly active enzymes and, as expected, some have been shown to be localised in membrane-bound organelles, including mammalian cathepsin B [9] and the cysteine proteinases from Dictyostelium [27]. Consistent with their location, signal peptides were identified by analysis at the DNA level of the two Dictyostelium proteinases [27], and of aleurain, which is secreted [26]. In addition, cDNA cloning of these enzymes, but also of papain [7] and cathepsin B from rat [32], revealed the presence of an extra 100 to 120 amino acids N-terminal to the region of homology with actinidin and to the mature N-termini of papain and cathepsin B.…”

Section: Introductionmentioning

confidence: 55%

“…Carboxyterminal extensions of unrelated enzymes, such as cathepsin D and ~3-glucuronidase, have been implicated in intracellular transport to the lysosomes [11, 121. The considerable sequence homology of the actinidin prosegment with those of other cysteine proteinases indicates that these extensions may have structural and functional similarities [26]. There is strong evidence that papain is synthesised in the form of a zymogen which becomes activated by the removal of the amino-terminal extension [7], and the precursor of cathepsin B is thought to have very limited proteolytic activity [9].…”

Section: Northern Blot Analysis Of Rna At Different Developmental Stagesmentioning

confidence: 99%

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Molecular analysis of actinidin, the cysteine proteinase of Actinidia chinensis

1988

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We have isolated and sequenced two very similar cDNA clones of 1145 and 809 bp length, from a fruit-specific library of Actinidia chinensis, the larger encoding all 220 amino acids of actinidin, showing 91% homology to the published amino acid sequence. Both cDNAs code for an additional 25 amino acids following the mature carboxy terminus of actinidin. The larger clone has coding potential for 57 residues of an amino-terminal extension with considerable homology to amino-terminal sequences of other cysteine proteinases. From size determination of both mRNA (1.4 kb) and immunoprecipitated in vitro translation product (39 kDa) it was estimated that actinidin is synthesised as a precursor approximately 15 kDa larger than the mature protein. Both proteolytic cleavage sites are located on the surface of the molecule as illustrated by the hydropathy profile of the deduced amino acid sequence. Features of the prosegment primary sequence are considered with regard to a possible mechanism of inactivation of the proteinase, by analogy with other proteolytic zymogens. The presence of three potential glycosylation sites, one within the carboxy-terminal and two in the amino-terminal extension, are consistent with subcellular location of the enzyme within membrane-bound organelles. Results from a Southern blot suggest that actinidin is encoded by a multigene family of up to ten members. Actinidin gene expression, both at the level of mRNA and protein, is largely restricted to the fruit of the plant, where the level of actinidin mRNA accumulates early during development.

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Homology within the N-terminal extension of cysteine proteinases

Cited by 6 publications

References 10 publications

Asparaginyl endopeptidase (VmPE‐1) and autocatalytic processing synergistically activate the vacuolar cysteine proteinase (SH‐EP)

Asparaginyl endopeptidase (VmPE‐1) and autocatalytic processing synergistically activate the vacuolar cysteine proteinase (SH‐EP)

Posttranslational Removal of the Carboxyl-terminal KDEL of the Cysteine Protease SH-EP Occurs Prior to Maturation of the Enzyme

Molecular analysis of actinidin, the cysteine proteinase of Actinidia chinensis

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