Characterization and molecular cloning of a proenzyme form of a ribosome-inactivating protein from maize. Novel mechanism of proenzyme activation by proteolytic removal of a 2.8-kilodalton internal peptide segment.

Ribosome-inactivating proteins (RIPs) are a widely distributed family of plant enzymes that are remarkably potent catalytic inactivators of eukaryotic protein synthesis. All RIPs described to date, including the A-chain of the plant cytotoxin ricin, are polypeptides of 25-32 kDa and share significant amino acid sequence homologies. We have characterized and cloned an RIP from maize (Zea mays). In contrast to previously described RIPs, we have found that maize RIP is synthesized and stored in the kernel as a 34-kDa inactive precursor (isoelectric point = 6.5). During germination, this neutral precursor is converted into a basic, active form (isoelectric point greater than 9) by limited proteolysis, which removes 25 amino acids (2.8 kDa) of net charge -6 from the center of the polypeptide chain. Additional processing also occurs at the amino and carboxyl termini of the polypeptide. The sequence of the internal processed region is unique and it is equivalent to an insertion centered around Thr-156 in the amino acid sequence of ricin toxin A-chain, i.e. in the center of the enzymatically active domain. The generation of an active enzyme by removal of a large amino acid segment from the middle of a precursor polypeptide chain represents a novel mechanism of proenzyme activation that is distinct from more conventional activation mechanisms involving NH2-terminal proteolytic processing. A two-chain active RIP (comprised of 16.5- and 8.5-kDa fragments that remain tightly associated) is produced from this processing event.     

Substrate specificity of a maize ribosome-inactivating protein differs across diverse taxa.

The superfamily of ribosome-inactivating proteins (RIPs) consists of toxins that catalytically inactivate ribosomes at a universally conserved region of the large ribosomal RNA. RIPs carry out a single N-glycosidation event that alters the binding site of the translational elongational factor eEF1A and causes a cessation of protein synthesis that leads to subsequent cell death. Maize RIP1 is a kernel-specific RIP with the unusual property of being produced as a zymogen, proRIP1. ProRIP1 accumulates during seed development and becomes active during germination when cellular proteases remove acidic residues from a central domain and both termini. These deletions also result in RIP activation in vitro. However, the effectiveness of RIP1 activity against target ribosomes remains species-dependent. To determine the potential efficiency of maize RIP1 as a plant defense protein, we used quantitative RNA gel blots to detect products of RIP activity against intact ribosomal substrates from various species. We determined the enzyme specificity of recombinant maize proRIP1 (rproRIP1), papain-activated rproRIP1 and MOD1 (an active deletion mutant of rproRIP1) against ribosomal substrates with differing levels of RIP sensitivity. The rproRIP1 had no detectable enzymatic activity against ribosomes from any of the species assayed. The papain-activated rproRIP1 was more active than MOD1 against ribosomes from either rabbit or the corn pathogen, Aspergillus flavus, but the difference was much more marked when rabbit ribosomes were used as a substrate. The papain-activated rproRIP1 was much more active against rabbit ribosomes than homologous Zea mays ribosomes and had no detectable effect on Escherichia coli ribosomes.     

Solution Structure of an Active Mutant of Maize Ribosome-Inactivating Protein (MOD) and Its Interaction with the Ribosomal Stalk Protein P2

Ribosome-inactivating proteins (RIPs) are N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of ribosomal RNA. This modification renders the ribosome unable to bind the elongation factors, thereby inhibiting the protein synthesis. Maize RIP, a type III RIP, is unique compared to the other type I and type II RIPs because it is synthesized as a precursor with a 25-residue internal inactivation region, which is removed in order to activate the protein. In this study, we describe the first solution structure of this type of RIP, a  28-kDa active mutant of maize RIP (MOD). The overall protein structure of MOD is comparable to those of the other type I RIPs and the A-chain of type II RIPs but shows significant differences in specific regions, including (1) shorter β6 and αB segments, probably for accommodating easier substrate binding, and (2) an α-helix instead of an antiparallel β-sheet in the C-terminal domain, which has been reported to be involved in binding ribosomal protein P2 in some RIPs. Furthermore, NMR chemical shift perturbation experiments revealed that the P2 binding site on MOD is located at the N-terminal domain near the internal inactivation region. This relocation of the P2 binding site can be rationalized by concerted changes in the electrostatic surface potential and 3D structures on the MOD protein and provides vital clues about the underlying molecular mechanism of this unique type of RIP.     

Sequence comparison and phylogenetic analysis by the Maximum Likelihood method of ribosome-inactivating proteins from angiosperms

Ribosome-inactivating proteins (RIPs) from angiosperms are rRNA N-glycosidases that have been proposed as defence proteins against virus and fungi. They have been classified as type 1 RIPs, consisting of single-chain proteins, and type 2 RIPs, consisting of an A chain with RIP properties covalently linked to a B chain with lectin properties. In this work we have carried out a broad search of RIP sequence data banks from angiosperms in order to study their main structural characteristics and phylogenetic evolution. The comparison of the sequences revealed the presence, outside of the active site, of a novel structure that might be involved in the internal protein dynamics linked to enzyme catalysis. Also the B-chains presented another conserved structure that might function either supporting the beta-trefoil structure or in the communication between both sugar-binding sites. A systematic phylogenetic analysis of RIP sequences revealed that the most primitive type 1 RIPs were similar to that of the actual monocots (Poaceae and Asparagaceae). The primitive RIPs evolved to the dicot type 1 related RIPs (like those from Caryophyllales, Lamiales and Euphorbiales). The gene of a type 1 RIP related with the actual Euphorbiaceae type 1 RIPs fused with a double beta trefoil lectin gene similar to the actual Cucurbitaceae lectins to generate the type 2 RIPs and finally this gene underwent deletions rendering either type 1 RIPs (like those from Cucurbitaceae, Rosaceae and Iridaceae) or lectins without A chain (like those from Adoxaceae).     

Maize Ribosome-Inactivating Protein Uses Lys158–Lys161 to Interact with Ribosomal Protein P2 and the Strength of Interaction Is Correlated to the Biological Activities

Ribosome-inactivating proteins (RIPs) inactivate prokaryotic or eukaryotic ribosomes by removing a single adenine in the large ribosomal RNA. Here we show maize RIP (MOD), an atypical RIP with an internal inactivation loop, interacts with the ribosomal stalk protein P2 via Lys158–Lys161, which is located in the N-terminal domain and at the base of its internal loop. Due to subtle differences in the structure of maize RIP, hydrophobic interaction with the ‘FGLFD’ motif of P2 is not as evidenced in MOD-P2 interaction. As a result, interaction of P2 with MOD was weaker than those with trichosanthin and shiga toxin A as reflected by the dissociation constants (K_D) of their interaction, which are 1037.50±65.75 µM, 611.70±28.13 µM and 194.84±9.47 µM respectively.Despite MOD and TCS target at the same ribosomal protein P2, MOD was found 48 and 10 folds less potent than trichosanthin in ribosome depurination and cytotoxicity to 293T cells respectively, implicating the strength of interaction between RIPs and ribosomal proteins is important for the biological activity of RIPs. Our work illustrates the flexibility on the docking of RIPs on ribosomal proteins for targeting the sarcin-ricin loop and the importance of protein-protein interaction for ribosome-inactivating activity.     

Molecular cloning, sequencing, deletion, and overexpression of a methionine aminopeptidase gene from Saccharomyces cerevisiae.

A yeast gene for a methionine aminopeptidase, one of the central enzymes in protein synthesis, was cloned and sequenced. The DNA sequence encodes a precursor protein containing 387 amino acid residues. The mature protein, whose NH2-terminal sequence was confirmed by Edman degradation, consists of 377 amino acids. The function of the 10-residue sequence at the NH2 terminus, containing 1 serine and 6 threonine residues, remains to be established. In contrast to the structure of the prokaryotic enzyme, the yeast methionine aminopeptidase consists of two functional domains: a unique NH2-terminal domain containing two motifs resembling zinc fingers, which may allow the protein to interact with ribosomes, and a catalytic COOH-terminal domain resembling other prokaryotic methionine aminopeptidases. Furthermore, unlike the case for the prokaryotic gene, the deletion of the yeast MAP1 gene is not lethal, suggesting for the first time that alternative NH2-terminal processing pathway(s) exist for cleaving methionine from nascent polypeptide chains in eukaryotic cells.     

Maize ribosome-inactivating protein (b-32). Homologs in related species, effects on maize ribosomes, and modulation of activity by pro-peptide deletions.

The ribosome-inactivating protein (RIP) from maize (Zea mays L.) is unusual in that it is produced in the endosperm as an inactive pro-form, also known as b-32, which can be converted by limited proteolysis to a two-chain active form, alpha beta RIP. Immunological analysis of seed extracts from a variety of species related to maize showed that pro/alpha beta forms of RIP are not unique to maize but are also found in other members of the Panicoideae, including Tripsacum and sorghum. Ribosomes isolated from maize were quite resistant to both purified pro- and alpha beta maize RIPs, whereas they were highly susceptible to the RIP from pokeweed. This suggests that the production of an inactive pro-RIP is not a mechanism to protect the plant's own ribosomes from deleterious action of the alpha beta RIP. RIP derivatives with various pro-segments removed were expressed at high levels in Escherichia coli. Measurement of their activity before and after treatment with subtilisin Carlsberg clearly identified the 25-amino acid intradomain insertion, rather than the N- or C-terminal extensions, as the major element responsible for suppression of enzymatic activity. A RIP with all three processed regions deleted had activity close to that of the native alpha beta form.     

Purification and complete sequence of a small proteolipid associated with the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae.

The purified plasma membrane H(+)-ATPase of Schizosaccharomyces pombe and Saccharomyces cerevisiae display, in addition to the catalytic subunit of 100 kDa, a highly mobile component, soluble in chloroform/methanol. Chloroform/methanol extraction of S. cerevisiae plasma membranes led to isolation of a low molecular weight proteolipid identical to that present in purified H(+)-ATPase. NH2-terminal amino acid sequencing revealed a 38-residue polypeptide with a calculated molecular mass of 4250 Da. The polypeptide lacks the first two NH2-terminal amino acids as compared with the deduced sequence of the PMP1 gene (for plasma membrane proteolipid) isolated by hybridization with an oligonucleotide probe corresponding to an internal amino acid sequence of the proteolipid. The polypeptide is predicted to contain an NH2-terminal transmembrane segment followed by a very basic hydrophilic domain.     

A switch-on mechanism to activate maize ribosome-inactivating protein for targeting HIV-infected cells

Maize ribosome-inactivating protein (RIP) is a plant toxin that inactivates eukaryotic ribosomes by depurinating a specific adenine residue at the α-sarcin/ricin loop of 28S rRNA. Maize RIP is first produced as a proenzyme with a 25-amino acid internal inactivation region on the protein surface. During germination, proteolytic removal of this internal inactivation region generates the active heterodimeric maize RIP with full N-glycosidase activity. This naturally occurring switch-on mechanism provides an opportunity for targeting the cytotoxin to pathogen-infected cells. Here, we report the addition of HIV-1 protease recognition sequences to the internal inactivation region and the activation of the maize RIP variants by HIV-1 protease in vitro and in HIV-infected cells. Among the variants generated, two were cleaved efficiently by HIV-1 protease. The HIV-1 protease-activated variants showed enhanced N-glycosidase activity in vivo as compared to their un-activated counterparts. They also possessed potent inhibitory effect on p24 antigen production in human T cells infected by two HIV-1 strains. This switch-on strategy for activating the enzymatic activity of maize RIP in target cells provides a platform for combating pathogens with a specific protease.     

Ebulitins: A new family of type 1 ribosome-inactivating proteins (rRNA N-glycosidases) from leaves of Sambucus ebulus L. that coexist with the type 2 ribosome-inactivating protein ebulin 1

A new family of single chain (type 1) ribosome-inactivating proteins (RIPs), that we have named ebulitins, have been found in mature leaves of Sambucus ebulus L., a caprifoliaceae plant also known to contain a non-toxic two chain (type 2) RIP named ebulin 1 in its leaves. Ebulitins are basic proteins of M_r 32,000, 29,000 and 29,000 for ebulitins , β and γ, respectively. The simultaneous presence of different basic type 1 and acidic type 2 RIPs in the same plant and in the same tissue is described here for the first time and opens a new door in research into RIPs.     

Biosynthesis of human preapolipoprotein A-IV

The primary translation product of human intestinal apolipoprotein A-IV mRNA was purified from ascites and wheat germ cell-free systems. Comparison of its NH2-terminal sequence with mature, chylomicron-associated apo-A-IV revealed that apo-A-IV was initially synthesized with a 20-amino acid long NH2-terminal extension: Met-X-Leu-X-Ala-Val-Val-Leu-X-Leu-Ala-Leu-Val-Ala-Val-Ala-Leu-X-X-Ala. Co-translational cleavage of the cell-free product as well as Edman degradation of the stable intracellular form of the protein recovered from Hep G2 cells indicated that this entire 20-amino acid sequence behaved as a signal peptide. There is at least 55% sequence homology between the rat and human apo-A-IV signal peptides and 33% homology between the human A-I and A-IV presegments. Agarose gel chromatography of Hep G2 culture media indicated that neither apo-A-IV nor -A-I is associated with particles that have physical properties resembling any of the plasma lipoprotein density classes. Incubation of plasma with Hep G2 media resulted in transfer of A-I but not A-IV to lipoproteins. Since the NH2 termini of co-translationally cleaved and chylomicron-associated apo-A-IV are identical, it is apparent that 1) this polypeptide does not undergo NH2-terminal post-translational proteolysis like proapo-A-II or proapo-A-I, and 2) regulation of A-IV-lipoprotein interaction is not dependent on any NH2-terminal proteolytic processing event.     

Interchain disulfide bonding in the regulatory subunit of cAMP-dependent protein kinase I

The two protomers of the purified regulatory subunit from porcine cAMP-dependent protein kinase I have been shown to be covalently cross-linked by interchain disulfide bonding. Limited proteolysis which cleaves the polypeptide chain into two fragments demonstrated that the disulfide bonding was associated exclusively with the fragment that corresponded to the NH2-terminal region of the polypeptide chain. This NH2-terminal fragment accounted for approximately 15 to 20% of the molecule. The disulfide bonding was further characterized by alkylating the cysteines in the native regulatory subunit. Following oxidation with performic acid, each regulatory subunit contained 7 cysteic acid residues; however, under denaturing conditions, but without prior reduction, only 5 cysteine residues could be alkylated with iodoacetic acid. Following limited proteolysis, all five of these cysteines were associated with the larger COOH-terminal, cAMP binding domain. In contrast, if the denatured subunit was first reduced prior to alkylation, all 7 cysteine residues were alkylated. The 2 cysteines that were only accessible to alkylation after prior reduction were both associated with the NH2-terminal end of the polypeptide chain ultimately with a 5,400 peptide. Alkylation of the isolated, denatured NH2-terminal domain with iodoacetic acid resulted in no covalent modification unless the fragment was first reduced with dithiothreitol. The NH2-terminal and COOH-terminal domains were shown to be linked by a region of the polypeptide chain that is rich in both proline and arginine. It is the arginine-rich site that is readily prone to proteolytic cleavage.     

MAK3 encodes an N-acetyltransferase whose modification of the L-A gag NH2 terminus is necessary for virus particle assembly.

The MAK3 gene is necessary for propagation of the L-A double-stranded RNA virus of Saccharomyces cerevisiae. MAK3 encodes a protein with substantial homology to the Escherichia coli rimI N-acetyltransferase that acetylates the NH2 terminus of ribosomal protein S18, and shares consensus sequences with a group of N-acetyltransferases. The NH2 terminus of the viral major coat protein encoded by L-A is normally blocked, but we find that it is unblocked in a mak3-1 mutant. L-A virus-encoded proteins produced from a cDNA clone of L-A can encapsidate the L-A (+)-strands in a wild-type host, but not in a mak3-1 mutant strain. The amount of major coat protein found in the particle fraction is reduced greater than 100-fold, and the amount in the total cell extract is reduced 5-10-fold. A modified beta-galactosidase, having as its NH2-terminal the NH2-terminal 13 residues of the L-A-encoded major coat protein, is blocked in a wild-type host, but not in a mak3-1 host. We propose that MAK3 encodes an N-acetyltransferase whose modification of the L-A major coat protein NH2 terminus is essential for viral assembly, and that unassembled coat protein is unstable.     

Novel role for pectin methylesterase in Arabidopsis: A new function showing ribosome-inactivating protein (RIP) activity

Ribosome-inactivating proteins (RIPs, EC 3.2.2.22) are plant enzymes that can inhibit the translation process by removing single adenine residues of the large rRNA. These enzymes are known to function in defense against pathogens, but their biological role is unknown, partly due to the absence of work on RIPs in a model plant. In this study, we purified a protein showing RIP activity from Arabidopsis thaliana by employing chromatography separations coupled with an enzymatic activity. Based on N-terminal and internal amino acid sequencing, the RIP purified was identified as a mature form of pectin methylesterase (PME, At1g11580). The purified native protein showed both PME and RIP activity. PME catalyzes pectin deesterification, releasing acid pectin and methanol, which cause cell wall changes. We expressed the full-length and mature form of cDNA clones into an expression vector and transformed it in Escherichia coli for protein expression. The recombinant PME proteins (full-length and mature) expressed in E. coli did not show either PME or RIP activity, suggesting that post-translational modifications are important for these enzymatic activities. This study demonstrates a new function for an old enzyme identified in a model plant and discusses the possible role of a protein's conformational changes corresponding to its dual enzymatic activity.     

Unique precursor structure of an extracellular protease, aqualysin I, with NH2- and COOH-terminal pro-sequences and its processing in Escherichia coli

Aqualysin I is a subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extremely thermophilic Gram-negative bacterium. The nucleotide sequence of the entire gene for aqualysin I was determined, and the deduced amino acid sequence suggests that aqualysin I is produced as a large precursor, consisting of at least three portions, an NH2-terminal pre-pro-sequence (127 amino acid residues), the protease (281 residues), and a COOH-terminal pro-sequence (105 residues). When the cloned gene was expressed in Escherichia coli cells, aqualysin I was not secreted. However, a precursor of aqualysin I lacking the NH2-terminal pre-pro-sequence (38-kDa protein) accumulated in the membrane fraction. On treatment of the membrane fraction at 65 degrees C, enzymatically active aqualysin I (28-kDa protein) was produced in the soluble fraction. When the active site Ser residue was replaced with Ala, cells expressing the mutant gene accumulated a 48-kDa protein in the outer membrane fraction. The 48-kDa protein lacked the NH2-terminal 14 amino acid residues of the precursor, and heat treatment did not cause any subsequent processing of this precursor. These results indicate that the NH2-terminal signal sequence is cleaved off by a signal peptidase of E. coli, and that the NH2- and COOH-terminal pro-sequences are removed through the proteolytic activity of aqualysin I itself, in that order. These findings indicate a unique four-domain structure for the aqualysin I precursor; the signal sequence, the NH2-terminal pro-sequence, mature aqualysin I, and the COOH-terminal pro-sequence, from the NH2 to the COOH terminus.