In vitro endoproteolytic cleavage of castor bean lectin presursors

Harley, Suzanne M.; Lord, J. Michael

doi:10.1016/0168-9452(85)90111-6

Cited by 52 publications

(21 citation statements)

References 30 publications

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“…We found that the enzymic activities ofthe proteolytic cleavage of proglobulin exhibit the slightly acidic pH optimum around at 5.0, coinciding with the pH of the vacuolar sap. It has been reported that the endoproteolytic enzyme involved in the cleavage of the precursor molecules of ricin and R. communis agglutinin of castor bean endosperm had optimum pH 4.0 (9). However, in our present study the processing enzyme of pumpkin showed the lower activity at pH 4.0.…”

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confidence: 71%

“…However, the mechanism(s) involved in the posttranslational proteolytic processing of the single polypeptide precursor producing the mature form of protein consisting of disulfide-linked doublet polypeptide chains is not fully understood. There has been only one investigation concerning the endoproteolytic enzyme responsible for the posttranslational cleavage of the precursor proteins by Harley and Lord (9). They demonstrated that whole homogenates of the developing castor bean (Ricinus communis) endosperm contained the enzymic activities capable of converting the precursor peptides of ricin and R. communis agglutinin to their respective mature forms.…”

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Proglobulin Processing Enzyme in Vacuoles Isolated from Developing Pumpkin Cotyledons

Hara‐Nishimura

Nishimura

1987

Plant Physiol.

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The enzymic conversion of proglobulin to globulin catalyzed by the extracts of vacuoles isolated from developing pumpkin (Cucurbita sp. cv Kurokawa Amakuri Nankin) cotyledons was investigated. The endoplasmic reticulum fraction isolated from the developing cotyledons pulselabeled with I35Slmethionine was shown to contain mainly the radiolabeled proglobulin, which was used as a substrate for assaying the proteolytic processing in vitro. The vacuolar extracts catalyzed the proteolytic processing of the proglobulin molecule to produce globulin containing two kinds of polypeptide chains, 'y and 5. The pH optimum for the vacuole-mediated conversion was at pH 5.0. The proteolytic processing of proglobulin by the vacuolar extracts was inhibited in the presence of various thiol reagents, e.g. p-chloromercuribenzoate, N-ethylmaleimide, iodoacetic acid, Hg2", and Cu2", but not phenylmethylsulfonyl fluoride, EDTA, o-phenanthroline, leupeptin, antipain, pepstatin, chymostatin, or pumpkin trypsin inhibitor, and was activated in the presence of dithiothreitol and cysteine, indicating that the processing enzyme is a thiol protease. The suborganellar fractionation of the vacuoles showed that the processing activity was localized in the matrix fraction, but not in the membrane or crystalloid fractions. During the seed development, the enzyme was shown to increase, exhibiting the maximal activity at the late developmental stage. The matrix fraction of the protein bodies isolated from the dry castor bean (Ricinus communis) exhibited the processing activity toward the pumpkin proglobulin molecules in the same manner as that by the matrix fraction of pumpkin vacuoles.ing of two distinct polypeptides, y and 6, linked by a disulfide bond is synthesized as precursor of a single polypeptide chain (7) and that the disulfide bond is introduced in precursor molecule synthesized in rER (5). Several other seed proteins such as lectins which are composed of two distinct polypeptide chains linked together by disulfide bond(s) are also known to be synthesized as a single polypeptide precursor, and both the in vivo labeling and cellular fractionation studies have clearly demonstrated that the protein bodies are the site of the endoproteolytic cleavage of the precursor molecules (15). However, the mechanism(s) involved in the posttranslational proteolytic processing of the single polypeptide precursor producing the mature form of protein consisting of disulfide-linked doublet polypeptide chains is not fully understood. There has been only one investigation concerning the endoproteolytic enzyme responsible for the posttranslational cleavage of the precursor proteins by Harley and Lord (9). They demonstrated that whole homogenates of the developing castor bean (Ricinus communis) endosperm contained the enzymic activities capable of converting the precursor peptides of ricin and R. communis agglutinin to their respective mature forms.In the work reported in this communication, we have examined the nature of the endoproteolytic enzyme activit...

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confidence: 71%

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confidence: 99%

Proglobulin Processing Enzyme in Vacuoles Isolated from Developing Pumpkin Cotyledons

Hara‐Nishimura

Nishimura

1987

Plant Physiol.

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“…Although most proproteins of various seed proteins are well known to be post-translationally processed into the mature proteins (Higgins, 1984), the processing mechanism in plant vacuoles is very obscure. Three groups have investigated vacuolar processing activities that convert proglobulin into 11s globulin in maturing pumpkin cotyledons (Hara- Nishimura and Nishimura, 1987), proglycinin into glycinin in maturing soybean seeds (Scott et al, 1992), or proricin into ricin in maturing castor bean seeds (Harley and Lord, 1985), respectively.…”

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confidence: 99%

Molecular characterization of a vacuolar processing enzyme related to a putative cysteine proteinase of Schistosoma mansoni.

1993

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Proproteins of various vacuolar proteins are post-translationally processed into mature forms by the action of a unique vacuolar processing enzyme. If such a processing enzyme is transported to vacuoles together with proprotein substrates, the enzyme must be a latent form. lmmunocytochemical localization of a vacuolar processing enzyme, a 37-kD cysteine proteinase, in the endosperm of maturing castor bean seeds places the enzyme in the vacuolar matrix, where a variety of proproteins is also present. To characterize a molecular structure of vacuolar processing enzyme, we isolated a cDNA for the enzyme. Deduced primary structure of a 55-kD precursor is 33% identical to a putative cysteine proteinase of the human parasite Schistosoma mansoni. The precursor is composed of a signal peptide, a 37-kD active processing enzyme domain, and a propeptide fragment. Although the precursor expressed in Escherichia coli has no vacuolar processing activity, a 36-kD immunopositive protein expressed in E. coli is active. These results suggest that the activation of the vacuolar processing enzyme requires proteolytic cleavage of a 1CkD C-terminal propeptide fragment of the precursor.

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“…The furin cleavage site lies between two cysteines that are joined by a disulfide bond so that after proteolytic cleavage the resulting fragments remain covalently linked. The catalytically active (or A) chain (RTA) and the cell binding (or B) chain (RTB) of the plant toxin ricin are also synthesized as part of a single precursor protein (6), but the proteolytic cleavage necessary to separate them occurs during their biosynthesis in the producing plant (7). When the appropriate intracellular compartment is reached by the endocytosed toxin, the enzymic peptide fragment translocates across the membrane to enter the cytosol.…”

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Ribosome-mediated Folding of Partially Unfolded Ricin A-chain

Argent

Parrott

Day³

et al. 2000

Journal of Biological Chemistry

110

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After endocytic uptake by mammalian cells, the cytotoxic protein ricin is transported to the endoplasmic reticulum, whereupon the A-chain must cross the lumenal membrane to reach its ribosomal substrates. It is assumed that membrane traversal is preceded by unfolding of ricin A-chain, followed by refolding in the cytosol to generate the native, biologically active toxin. Here we describe biochemical and biophysical analyses of the unfolding of ricin A-chain and its refolding in vitro. We show that native ricin A-chain is surprisingly unstable at pH 7.0, unfolding non-cooperatively above 37°C to generate a partially unfolded state. This species has conformational properties typical of a molten globule, and cannot be refolded to the native state by manipulation of the buffer conditions or by the addition of a stem-loop dodecaribonucleotide or deproteinized Escherichia coli ribosomal RNA, both of which are substrates for ricin A-chain. By contrast, in the presence of saltwashed ribosomes, partially unfolded ricin A-chain regains full catalytic activity. The data suggest that the conformational stability of ricin A-chain is ideally poised for translocation from the endoplasmic reticulum. Within the cytosol, ricin A-chain molecules may then refold in the presence of ribosomes, resulting in ribosome depurination and cell death.Bacterial proteins including diphtheria toxin (DT), 1 Pseudomonas exotoxin A (PE), Shiga toxin (ST), Shiga-like toxins (SLTs), and plant proteins such as ricin kill mammalian cells by catalytically inactivating key components of the translational machinery (1). DT and PE achieve this by the ADPribosylation of elongation factor-2 (2), whereas ST, SLTs, and ricin inhibit protein synthesis by removing a specific adenine residue from 28 S ribosomal RNA (3), leaving toxin-modified ribosomes unable to carry out protein synthesis. Since the target substrates for all these toxins are present in the cytosol, a key feature of toxicity is the delivery of a catalytically active polypeptide or fragment into this cellular location.Cellular entry by the protein toxins listed above involves the same generalized mechanism. The toxin binds to a normal cell surface component, which is thus utilized as a toxin receptor. Surface-bound toxin enters the cell by endocytosis in both clathrin-coated and uncoated pits/vesicles (2, 4). During subsequent intracellular transport, DT, PE, ST, and SLT are cleaved by the membrane-associated protease, furin, to separate the cell-binding domain from the catalytic domain (5). The furin cleavage site lies between two cysteines that are joined by a disulfide bond so that after proteolytic cleavage the resulting fragments remain covalently linked. The catalytically active (or A) chain (RTA) and the cell binding (or B) chain (RTB) of the plant toxin ricin are also synthesized as part of a single precursor protein (6), but the proteolytic cleavage necessary to separate them occurs during their biosynthesis in the producing plant (7). When the appropriate intracellular compartment is re...

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In vitro endoproteolytic cleavage of castor bean lectin presursors

Cited by 52 publications

References 30 publications

Proglobulin Processing Enzyme in Vacuoles Isolated from Developing Pumpkin Cotyledons

Proglobulin Processing Enzyme in Vacuoles Isolated from Developing Pumpkin Cotyledons

Molecular characterization of a vacuolar processing enzyme related to a putative cysteine proteinase of Schistosoma mansoni.

Ribosome-mediated Folding of Partially Unfolded Ricin A-chain

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