The role of proteolysis in the processing and assembly of 11S seed globulins.

11S seed storage proteins are synthesized as precursors that are cleaved post-translationally in storage vacuoles by an asparaginyl endopeptidase. To study the specificity of the reaction catalyzed by this asparaginyl endopeptidase, we prepared a series of octapeptides and mutant legumin B and G4 glycinin subunits. These contained amino acid mutations in the region surrounding the cleavage site. The endopeptidase had an absolute specificity for Asn on the N-terminal side of the severed peptide bond but exhibited little specificity for amino acids on the C-terminal side. The ability of unmodified and modified subunits to assemble into hexamers after post-translational modification was evaluated. Cleavage of subunits in trimers is required for hexamer assembly in vitro. Products from a mutant gene encoding a noncleavable prolegumin subunit (LeBDeltaN281) accumulated as trimers in seed of transgenic tobacco, but products from the unmodified prolegumin B gene accumulated as hexamers. Therefore, the asparaginyl endopeptidase is required for hexamer assembly.     

Role of the sulfhydryl redox state and disulfide bonds in processing and assembly of 11S seed globulins.

Seed legumins contain two conserved disulfide bonds: an interchain bond (IE) connecting the acidic and basic chains and an intrachain bond (IA) internal to the acidic chain. Mutant subunits were constructed in which these disulfide bonds were disrupted. Oxidized glutathione stimulated the rate of assembly of trimers with unmodified prolegumin subunits. Stimulation was not detected during assembly of IE mutant subunits and was diminished for the IA mutant. Hexamer assembly with trimers of mature unmodified subunits required oxidizing conditions. Trimers composed of mature IE mutants did not form hexamers. Both mutant and non-mutant subunits accumulated in hexamers when the cDNAs were expressed in tobacco. Hexamer assembly in seeds probably involved trimers with a mixture of mutant and non-mutant subunits. Similarly, mixed trimers that were a mixture of mutant and non-mutant subunits assembled into hexamers in vitro. The results demonstrate the importance of disulfide bonds during the assembly of 11S globulins.     

Role of posttranslational cleavage in glycinin assembly.

Glycinin, like other 11S seed storage proteins, undergoes a complex series of posttranslational events between the time proglycinin precursors are synthesized in endoplasmic reticulum and the mature glycinin subunits are deposited in vacuolar protein bodies. According to the current understanding of this process, proglycinin subunits aggregate into trimers in endoplasmic reticulum, and then the trimers move to the vacuolar protein bodies where a protease cleaves them into acidic and basic polypeptide chains. Stable glycinin hexamers, rather than trimers, are isolated from mature seeds. We used a re-assembly assay in this study to demonstrate that proteolytic cleavage of the proglycinin subunits is required for in vitro assembly of glycinin oligomers beyond the trimer stage. The possibility that the cleavage is a regulatory step and that it triggers the deposition of 11S seed storage proteins as insoluble aggregates in vivo is considered.     

Accumulation of high levels of free amino acids in soybean seeds through integration of mutations conferring seed protein deficiency

Soybean ( Glycine max [L.] Merr.) seeds are rich in protein, most of which is contributed by the major storage proteins glycinin (11S globulin) and beta-conglycinin (7S globulin). Null mutations for each of the subunits of these storage proteins were integrated by crossbreeding to yield a soybean line that lacks both glycinin and beta-conglycinin components. In spite of the absence of these two major storage proteins, the mutant line grew and reproduced normally, and the nitrogen content of its dry seed was similar to that for wild-type cultivars. However, protein bodies appeared underdeveloped in the cotyledons of the integrated mutant line. Furthermore, whereas free amino acids contribute only 0.3-0.8% of the seed nitrogen content of wild-type varieties, they constituted 4.5-8.2% of the seed nitrogen content in the integrated mutant line, with arginine (Arg) being especially enriched in the mutant seeds. Seeds of the integrated mutant line thus appeared to compensate for the reduced nitrogen content in the form of glycinin and beta-conglycinin by accumulating free amino acids as well as by increasing the expression of certain other seed proteins. These results indicate that soybean seeds are able to store nitrogen mostly in the form of either proteins or free amino acids.     

Expression and characterization of a His-tagged 11S seed globulin from Amaranthus hypochondriacus in Escherichia coli

DNA encoding a His-tagged 11S globulin from Amaranthus hypochondriacus (amarantin) was successfully expressed in Escherichia coli strains BL21 (DE3) and Origami (DE3). The two strains produced different accumulation patterns. Whereas most of the proamarantin expressed in BL21 (DE3) was localized in inclusion bodies, that produced in Origami (DE3) was soluble (76 mg/L). Sucrose density gradient ultracentrifugation analysis of the expressed soluble proamarantin revealed that the protein was assembled into trimers. Treatment of proamarantin trimers in vitro using purified asparaginyl endopeptidase resulted in the appearance of peptides of the sizes expected for acidic and basic chains. Because the proamarantin assembles into trimers with the expected sedimentation characteristics and is cleaved into acidic and basic chains rather than being degraded, the results suggest that the protein folding occurring in E. coli is similar to that taking place in seeds. The His-tagged proamarantin was purified in a single step by immobilized metal affinity chromatography with a final yield of 48 mg/L. The overexpression of proamarantin in E. coli, together with the one-step purification will facilitate further investigation of this storage protein through site-directed mutagenesis.     

Characterization of Blue-Green Fluorescence in the Mesophyll of Sugar Beet (Beta vulgaris L.) Leaves Affected by Iron Deficiency

Protease C1, an enzyme from soybean (Glycine max [L.] Merrill cv Amsoy 71) seedling cotyledons, was previously determined to be the enzyme responsible for the initial degradation of the alpha' and alpha subunits, but not the beta subunit, of beta-conglycinin storage protein. The sizes of the proteolytic products generated by the action of protease C1 suggest that the cleavage sites on the alpha' and alpha subunits of beta-conglycinin may be located in their N-terminal domain, which is not found in the beta subunit of beta-conglycinin. To check this hypothesis, storage proteins from other plant species that are homologous to either the alpha'/alpha or the beta subunit of beta-conglycinin were tested as substrates. As expected, the convicilin from pea (Pisum sativum), a protein homologous to the alpha' and alpha subunits of beta-conglycinin, was digested by protease C1. The vicilins from pea as well as vicilins from adzuki bean (Vigna angularis), garden bean (Phaseolus vulgaris), black-eyed pea (Vigna unguiculata), and mung bean (Vigna radiata), storage proteins that are homologous to the beta subunit of soybean beta-conglycinin, were not degraded by protease C1. Degradation of soybean beta-conglycinin involves a sequential attack of the alpha subunit at multiple sites, culminating in the formation of a stable intermediate of 53.5 kD and a final product of 48.0 kD. The cleavage sites resulting in this formation of the intermediates and final product were determined by N-terminal analysis. These were compared to the known amino acid sequences of the three beta-conglycinin subunits. Results showed these two polypeptides to be generated by proteolysis of the alpha subunit at regions bearing long strings of acidic amino acid residues.     

Cleavage specificity of the subtilisin-like protease C1 from soybean

The cleavage specificity of protease C1, isolated from soybean (Glycine max (L.) Merrill) seedling cotyledons, was examined using oligopeptide substrates in an HPLC based assay. A series of peptides based on the sequence Ac-KVEKEESEEGE-NH2 was used, mimicking a natural cleavage site of protease C1 in the alpha subunit of the storage protein beta-conglycinin. A study of substrate peptides truncated from either the N- or C-terminus indicates that the minimal requirements for cleavage by protease C2 are three residues N-terminal to the cleaved bond, and two residues C-terminal (i.e. P3-P2'). The maximal rate of cleavage is reached with substrates containing four to five residues N-terminal to the cleaved bond and four residues C-terminal (i.e. P4 or P5 to P4'). The importance of Glu residues at the P1, P1', and P4 positions was examined using a series of substituted nonapeptides (P5-P4') with a base sequence of Ac-KVEKEESEE-NH2. At the P1 position, the relative ranking, based on kcat/Km, was E>Q>K>A>D>F>S. Substitutions at the P1' position yield the ranking E congruent withQ>A>S>D>K>F, while those at P4' had less effect on kcat/Km, yielding the ranking F congruent with S congruent with E congruent withD>K>A congruent withQ. These data show that protease C1 prefers to cleave at Glu-Glu and Glu-Gln bonds, and that the nature of the P4' position is less important. The fact that there is specificity in the cleavage of the oligopeptides suggests that the more limited specific cleavage of the alpha and alpha' subunits of beta-conglycinin by protease C1 is due to a combination of the sequence cleavage specificity of the protease and the accessibility of appropriate scissile peptide bonds on the surface of the substrate protein.     

Proteolytic Cleavage at Twin Arginine Residues Affects Structural and Functional Transitions of Lupin Seed 11S Storage Globulin

The 11S storage globulin of white lupin seeds binds to a metal affinity chromatography matrix. Two unusual stretches of contiguous histidine residues, reminiscent of the multiple histidines forming metal binding motifs, at the C-terminal end of 11S globulin acidic chains were hypothesized as candidate elements responsible for the binding capacity. To prove this, the protein was incubated with a lupin seed endopeptidase previously shown to cleave at twin arginine motifs, recurrent in the sequence region of interest. Upon incubation with this enzyme, the loss of metal binding capacity paralleled that of the anti-his-tag reactive polypeptides. The recovered small proteolytic fragment was analyzed by mass spectrometry and N-terminal sequencing and found to correspond to the 24-mer region cleaved off at twin arginine residues and containing the natural his-tag-like region. Similarly, when lupin seeds were germinated for a few days, the his-tag containing 11S globulin chain was converted to a form devoid of such region, suggesting that this mechanism is a part of the natural degradatory process of the protein. The hypothesis that the ordered and controlled dismantling of storage proteins may generate peptide fragments with potential functional roles in plant ontogenesis is presented and discussed.     

In vitro proteolytic processing of pea and jack bean storage proteins by an endopeptidase from lupin seeds

The highly specific proteolytic breakdown observed upon prolonged treatment of pea legumin and pea and jack bean vicilin with a thiol endopeptidase purified from mature lupin seeds has been studied in detail. Proteolytic cleavage occurred in the acidic subunits of pea legumin, whereas the basic subunits were unaffected. Jack bean vicilin (M, 47 K) was cleaved near the middle of the polypeptide chain, whereas pea vicilin (M, 50 K) was cleaved into two fragments of M, 30 K and 20 K, respectively. The 30 K M, polypeptide chain contained covalently linked carbohydrate and had an N-terminal sequence suggesting that cleavage had taken place between the α and β region of the vicilin 50 K M, polypeptide as previously described in vivo. These results suggested that the cleavage specificity of lupin endopeptidase was in the proximity of paired arginine amino acid residues.The changes in the vicilin polypeptides due to proteolytic cleavage by lupin enzyme and those occurring during germination of pea seeds are also reported and discussed.     

Protease C2, a cysteine endopeptidase involved in the continuing mobilization of soybean beta-conglycinin seed proteins

The protease that degrades the beta subunit of the soybean (Glycine max (L.) Merrill) storage protein beta-conglycinin was purified from the cotyledons of seedlings grown for 12 days. The enzyme was named protease C2 because it is the second enzyme to cleave the beta-conglycinin storage protein, the first (protease C1) being one that degrades only the alpha' and alpha subunits of the storage protein to products similar in size and sequence to the remaining intact beta subunit. Protease C2 activity is not evident in vivo until 4 days after imbibition of the seed. The 31 kDa enzyme is a cysteine protease with a pH optimum with beta-conglycinin as substrate of 5.5. The action of protease C2 on native beta-conglycinin produces a set of large fragments (52-46 kDa in size) and small fragments (29-25 kDa). This is consistent with cleavage of all beta-conglycinin subunits at the region linking their N- and C-domains. Protease C2 also cleaves phaseolin, the Phaseolus vulgaris vicilin homologous to beta-conglycinin, to fragments in the 25-28 kDa range. N-Terminal sequences of isolated beta-conglycinin and phaseolin products show that protease C2 cleaves at a bond within a very mobile surface loop connecting the two compact structural domains of each subunit. The protease C2 cleavage specificity appears to be dictated by the substrate's three-dimensional structure rather than a specificity for a particular amino acid or sequence.     

Electron microscopy of seed-storage globulins

The quaternary structures of a range of seed globulins, including examples of both the so-called 7 S and 11 S types, have been examined by electron microscopy. The legume 7 S proteins, phaseolin (bean), beta-conglycinin (soybean), and vicilin (pea), appear as flat discs of diameter ca. 8.5 nm and thickness ca. 3.5 nm formed by association of three subunit domains. Phaseolin converts to an 18 S tetramer at acid pH, and images recorded under these conditions suggest that four of the 7 S protomer discs associate to form the faces of a regular tetrahedron. The classical 11 S seed globulins, cucurbitin (pumpkin) and legumin (pea), are approximately spherical molecules of diameter ca. 8.8 nm composed of six subunits. In contrast, the hexameric 10 S storage protein from lupin seed, conglutin gamma, appears toroidal in shape with outer diameter ca. 10.3 nm and thickness ca. 2.2 nm. These results indicate that constraints imposed on seed proteins by their role in sustaining the germinating plant may have allowed a variety of different globulin structures to accumulate in the protein-storage bodies of seeds.     

Effect of structural modifications on the assembly of a glycinin subunit.

A Gy4 glycinin cDNA was modified and used to produce structurally altered 11S storage protein subunits. We evaluated these modified subunits for their ability to assemble into oligomers. Alterations made in the acidic polypeptide changed the subunit solubility characteristics but did not eliminate assembly. Modifications in the basic polypeptide usually eliminated assembly of subunits into trimers. A region exhibiting high natural variability located at the COOH terminus of the acidic polypeptide that we have designated the hypervariable region was also studied. Extensive deletions and insertions were tolerated in the hypervariable region without perturbing subunit assembly. Some of the insertions significantly increased the methionine content in the Gy4 glycinin subunit. Together, our results indicated that the structure of the basic polypeptide was more critical for assembly of trimers than that of the acidic polypeptide, an observation that implies that the basic polypeptides direct trimer formation. The assembly assays described here will be useful in efforts to improve seed quality. Using them, the effects of modifications to the storage protein subunits can be rapidly evaluated before introducing the mutated genes into plants.     

Activation of Asparaginyl Endopeptidase Leads to Tau Hyperphosphorylation in Alzheimer Disease

Neurofibrillary pathology of abnormally hyperphosphorylated Tau is a key lesion of Alzheimer disease and other tauopathies, and its density in the brain directly correlates with dementia. The phosphorylation of Tau is regulated by protein phosphatase 2A, which in turn is regulated by inhibitor 2, I₂^PP2A. In acidic conditions such as generated by brain ischemia and hypoxia, especially in association with hyperglycemia as in diabetes, I₂^PP2A is cleaved by asparaginyl endopeptidase at Asn-175 into the N-terminal fragment (I_2NTF) and the C-terminal fragment (I_2CTF). Both I_2NTF and I_2CTF are known to bind to the catalytic subunit of protein phosphatase 2A and inhibit its activity. Here we show that the level of activated asparaginyl endopeptidase is significantly increased, and this enzyme and I₂^PP2A translocate, respectively, from neuronal lysosomes and nucleus to the cytoplasm where they interact and are associated with hyperphosphorylated Tau in Alzheimer disease brain. Asparaginyl endopeptidase from Alzheimer disease brain could cleave GST-I₂^PP2A, except when I₂^PP2A was mutated at the cleavage site Asn-175 to Gln. Finally, an induction of acidosis by treatment with kainic acid or pH 6.0 medium activated asparaginyl endopeptidase and consequently produced the cleavage of I₂^PP2A, inhibition of protein phosphatase 2A, and hyperphosphorylation of Tau, and the knockdown of asparaginyl endopeptidase with siRNA abolished this pathway in SH-SY5Y cells. These findings suggest the involvement of brain acidosis in the etiopathogenesis of Alzheimer disease, and asparaginyl endopeptidase-I₂^PP2A-protein phosphatase 2A-Tau hyperphosphorylation pathway as a therapeutic target.     

Purification and characterization of mRNA from soybean seeds. Identification of glycinin and beta-conglycinin precursors

Poly(A)-rich RNA was isolated from developing soybean seeds (Glycine max (L.) Merr.) and fractionated on linear log sucrose gradients. Two major fractions sedimenting at 18 S and 20 S were separated and then purified by further sucrose gradient fractionation. Both fractions were active as messengers when added to a rabbit reticulocyte lysate protein synthesis system. The 18 S fraction caused proteins migrating primarily to the 60,000-dalton region of a sodium dodecyl sulfate gel to be produced, while translation of the 20 S fraction preferentially directed the synthesis of polypeptides similar in size to the alpha and alpha' subunits of beta-conglycinin. Evidence that many of the 60,000-dalton polypeptides were related to glycinin and the high molecular weight 20 S translation products were related to beta-conglycinin was obtained by immunoprecipitation using monospecific antibodies against glycinin and beta-conglycinin, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitated products revealed that the glycinin precursor region contained at least three different size components and that the family of glycinin precursors had larger apparent molecular weight (58,000-63,000) than the disulfide-linked complexes between acidic and basic glycinin subunits (57,000). Unlike the disulfide-linked glycinin complexes which were cleaved by disulfide reduction, glycinin precursors were insensitive to reducing agents. The alpha and alpha' subunits synthesized in vitro also had slightly larger apparent molecular weights than purified alpha and alpha' standards.     

Characterization of a novel intramolecular chaperone domain conserved in endosialidases and other bacteriophage tail spike and fiber proteins

Folding and assembly of endosialidases, the trimeric tail spike proteins of Escherichia coli K1-specific bacteriophages, crucially depend on their C-terminal domain (CTD). Homologous CTDs were identified in phage proteins belonging to three different protein families: neck appendage proteins of several Bacillus phages, L-shaped tail fibers of coliphage T5, and K5 lyases, the tail spike proteins of phages infecting E. coli K5. By analyzing a representative of each family, we show that in all cases, the CTD is cleaved off after a strictly conserved serine residue and alanine substitution prevented cleavage. Further structural and functional analyses revealed that (i) CTDs are autonomous domains with a high alpha-helical content; (ii) proteolytically released CTDs assemble into hexamers, which are most likely dimers of trimers; (iii) highly conserved amino acids within the CTD are indispensable for CTD-mediated folding and complex formation; (iv) CTDs can be exchanged between proteins of different families; and (v) proteolytic cleavage is essential to stabilize the native protein complex. Data obtained for full-length and proteolytically processed endosialidase variants suggest that release of the CTD increases the unfolding barrier, trapping the mature trimer in a kinetically stable conformation. In summary, we characterize the CTD as a novel C-terminal chaperone domain, which assists folding and assembly of unrelated phage proteins.     

Asparaginyl endopeptidase cleaves TDP-43 in brain

TAR DNA-binding protein 43 (TDP-43) is a nuclear protein involved in RNA splicing and a major protein component in ubiquitin-positive, tau-negative inclusions of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Under disease conditions, TDP-43 redistributes to the cytoplasm where it can be phosphorylated, ubiquitinated, and proteolytically cleaved. Enzymes responsible for TDP-43 proteolytic processing in brain remain largely unreported. Using a MS approach, we identified two truncated TDP-43 peptides, terminating C-terminal to asparagines 291 (N291) and 306 (N306). The only documented mammalian enzyme capable of cleaving C-terminal to asparagine is asparaginyl endopeptidase (AEP). TDP-43-immunoreactive fragments (～35 and 32 kDa) predicted to be generated by AEP cleavage at N291 and N306 were observed by Western blot analyses of postmortem frontotemporal lobar degeneration brain tissue and cultured human cells over-expressing TDP-43. Studies in vitro determined that AEP can directly cleave TDP-43 at seven sites, including N291 and N306. Western blots of brain homogenates isolated from AEP-null mice and wild-type littermate controls revealed that TDP-43 proteolytic fragments were substantially reduced in the absence of AEP in vivo. Taken together, we conclude that TDP-43 is cleaved by AEP in brain. Moreover, these data highlight the utility of combining proteomic strategies in vitro and in vivo to provide insight into TDP-43 biology that will fuel the design of more detailed models of disease pathogenesis.     

Biochemical dissection of the role of the one-kilodalton carboxyl-terminal moiety of tubulin in its assembly into microtubules

The 4-kDa C-terminal domain of both tubulin subunits plays a major role in the regulation of microtubule assembly [Serrano et al. (1984) Biochemistry 23, 4675]. Controlled proteolysis of tubulin with subtilisin produces the selective cleavage of this 4-kDa moiety from alpha- and beta-tubulin with a concomitant enhancement of the assembly. Here we show that gradual removal of the last six to eight amino acid residues of the C-terminal region of alpha and beta subunits by an exopeptidase, carboxypeptidase Y, produces a modified protein (C-tubulin) without relieving the modulatory effect of the C-terminal domain and the usual need of MAPs for microtubule assembly. Actually, treatment with this proteolytic enzyme did not change tubulin assembly as promoted by either MAP-2, taxol, MgCl2, dimethyl sulfoxide, or glycerol. The critical concentration for the assembly of C-tubulin remained the same as that for the unmodified tubulin control. Microtubule-associated proteins MAP-2 and tau incorporated into C-tubulin polymers. Clearly, pure C-tubulin did not assemble in the absence of MAPs or without addition of assembly-promoting compounds. However, proteolysis with the exopeptidase induced changes in tubulin conformation as assessed by biophysical methods and double-limited proteolysis. The cleavage with subtilisin after carboxypeptidase digestion did not result in enhancement of the assembly to the levels observed after the treatment of native tubulin with subtilisin. Interestingly, Ca2+ ions affected neither C-tubulin assembly nor depolymerized microtubules assembled from C-tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of post-translational processing on dystroglycan synthesis and trafficking

Dystroglycan is a component of the dystrophin glycoprotein complex that is cleaved into two polypeptides by an unidentified protease. To determine the role of post-translational processing on dystroglycan synthesis and trafficking we expressed the dystroglycan precursor and mutants thereof in a heterologous system. A point mutant in the processing site, S655A, prevented proteolytic cleavage but had no effect upon the surface localisation of dystroglycan. Mutation of two N-linked glycosylation sites that flank the cleavage site inhibited proteolytic processing of the precursor. Furthermore, chemical inhibition of N- and O-linked glycosylation interfered with the processing of the precursor and reduced the levels of dystroglycan at the cell surface. Dystroglycan processing was also inhibited by the proteasome inhibitor lactacystin. N-linked glycosylation is a prerequisite for efficient proteolytic processing and cleavage and glycosylation are dispensable for cell surface targeting of dystroglycan.