Purification and immunological analysis of phospholipase D from castor bean endosperm

Phospholipase D (EC 3.1.4.4) has been implicated in diverse cellular processes, but its physiological role is not well established in plants. In order to develop immunological and molecular biology approaches to address the problem, we report here the immunological analysis and N-terminal amino acid sequence of a cytosolic phospholipase D from castor bean (Ricinus communis L.). The enzyme was purified to apparent homogeneity from germinating castor bean endosperm. The specific activity of the purified enzyme was enhanced by approximately 670-fold with an overall yield of 4%. Its molecular mass was estimated at 92 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of this enzyme was KLVENIEETVGFGKG. Polyclonal antibodies were raised against the purified enzyme. The antibodies inhibited the activity of transphosphatidylation more than that of hydrolysis of phospholipase D. The differential effect on the two activities of this enzyme implies that different active sites on this enzyme may be involved in the two reactions. Immunoblot analyses showed that the amounts of phospholipase D protein relative to the total endosperm proteins increased during the first 5 days of germination. The antibodies cross-reacted to proteins from several tested plant species, and those proteins had molecular masses similar to that of castor bean phospholipase D. These results indicate that the expression of phospholipase D in castor bean changes according to growth stages and that phospholipase D enzymes of different plant species are structurally related.     

Structure and analysis of phospholipase D gene from Ricinus communis L

Phospholipase D (PLD; EC 3.1.4.4) has been proposed to play a pivotal role in various cellular processes, but molecular understanding of this enzyme is rather limited. This report describes the nucleotide sequence, structure, and genomic organization of a PLD gene from castor bean (Ricinus communis L. cv. Hale). The PLD gene was isolated from a castor bean genomic library using the PLD cDNA as a hybridization probe. Sequence comparison with the PLD cDNA revealed that the PLD gene consisted of four exons and three introns, one of which interrupts the 5'-untranslated region. Southern blot analysis indicated that the cloned PLD gene was present as a single-copy gene, and yet there were other PLD or PLD-related sequences in the castor bean genome.     

Targeting of castor bean glyoxysomal isocitrate lyase to tobacco leaf peroxisomes

The cDNA encoding castor bean endosperm isocitrate lyase (ICL) was expressed under the control of the promoter of the small subunit of pea ribulose bisphosphate carboxylase in transformed tobacco. ICL protein was detected using anti-ICL antibodies on immunoblots of total leaf protein extracts. Nycodenz density gradient separation of the extracts from the transgenic tobacco leaves showed ICL co-fractionated with hydroxypyruvate reductase, a peroxisomal matrix marker protein, and away from lactate dehydrogenase, a cytosolic marker protein. Immunoelectron microscopy of ultrathin leaf sections demonstrated the location of ICL within the matrix of the leaf peroxisomes of the transgenic plants. In vitro transcribed and translated ICL was also imported into leaf peroxisomes isolated from germinating sunflower seeds. The in vivo and in vitro import of this protein into leaf peroxisomes provides strong support for the notion that the import machinery of glyoxysomes and peroxisomes is very similar.     

Binding of the peroxisomal targeting sequence SKL is specified by a low-affinity site in castor bean glyoxysomal membranes. A domain next to the SKL binds to a high-affinity site

The carboxyl-terminal amino acid sequence serine-lysine-leucine (SKL) is the consensus peroxisomal targeting sequence 1 (PTS1) and is sufficient to direct a polypeptide to peroxisomes in vivo in plants, animals, and yeasts. However, there are also two sites on alkali-stripped glyoxysomal membranes from castor bean (Ricinus communis) endosperm that bind the peptide YHKHLKPLQSKL (SKLp), the sequence of the last 12 amino acids of acyl-coenzyme A oxidase (N.E. Wollins, R.P. Donaldson [1994] J Biol Chem 289: 1149-1153). It was hypothesized that one of these sites interacts with information other than the PTS1. To explore the sequence requirements for each SKLp binding site, we tested the peptides YHKHLKPQSKG and YHKHLKPLQS and found that they bound to the high-affinity site, but not to the low-affinity site. When the high-affinity site was blocked with YHKHLKPQSKG, SKLp bound to the low-affinity site with a dissociation constant (Kd) of 8.5 microM. In an attempt to disrupt high-affinity binding, two the upstream, positively charged residues were replaced with negatively charged residues to make the peptide YHKETEPLQSKL. YHKETEPLQSKL did not bind to either site on the glyoxysomal membranes. These results indicate that the PTS1 binds to the low-affinity site and that the adjacent, positively charged domain binds to the high-affinity site.     

Structural characterization of argingipain, a novel arginine-specific cysteine proteinase as a major periodontal pathogenic factor from Porphyromonas gingivalis

Argingipain, so termed due to its peptide cleavage specificity at arginine residue, is a unique extracellular cysteine proteinase produced by the anaerobic rod Porphyromonas gingivalis, which is known as a major pathogenic factor of the progressive periodontal disease (T. Kadowaki, M. Yoneda, K. Okamoto, K. Maeda, and K. Yamamoto (1994) J. Biol. Chem. 269, 21371-21378). The catalytic specificity and functional importance of this enzyme prompted us to elucidate its structural features. A DNA fragment for argingipain was selectively amplified by polymerase chain reaction using mixed oligonucleotide primers designed from the NH2-terminal amino acid sequence of the purified enzyme. Although the extracellular mature enzyme was shown to have an apparent molecular mass of 44 kDa in gels, the nucleotide sequence of the isolated gene revealed a single gene coding for a 109-kDa precursor of argingipain. The deduced amino acid sequence exhibited no significant similarity to the sequences of representative members of the cysteine protease family. The precursor contained four functional domains: the NH2-terminal signal peptide required for the inner membrane transport; the NH2-terminal prosequence, which is assumed to stabilize the precursor structure; the proteinase domain; and the COOH-terminal hemagglutinin domain, which appears to be essential for extracellular secretion of the proteinase domain. Experiments involving the addition of the argingipain inhibitors to the culture medium of P. gingivalis suggested that the maturation of argingipain occurs intracellularly via an autocatalytic cleavage of the pro-argingipain propeptide.     

Cathepsin L2, a novel human cysteine proteinase produced by breast and colorectal carcinomas

We have identified and cloned a new member of the papain family of cysteine proteinases from a human brain cDNA library. The isolated cDNA codes for a polypeptide of 334 amino acids that exhibits all of the structural features characteristic of cysteine proteinases, including the active site cysteine residue essential for peptide hydrolysis. Pairwise comparisons of this amino acid sequence with the remaining human cysteine proteinases identified to date showed a high percentage of identity (78%) with cathepsin L; the percentage of identity with all other members of the family was much lower (<40%). On the basis of these structural characteristics, we have tentatively called this novel protein cathepsin L2. The cDNA encoding the mature cathepsin L2 was expressed in Escherichia coli, and after purification, the recombinant protein was able to degrade the synthetic peptide benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin, which is commonly used as a substrate for cysteine proteinases. Cathepsin L2 proteolytic activity on this substrate was abolished by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cysteine proteinases, thus providing additional evidence that the isolated cDNA encodes a functional cysteine proteinase. Northern blot analysis of polyadenylated RNAs isolated from a variety of human tissues demonstrated that cathepsin L2 is predominantly expressed in the thymus and testis. This finding is in marked contrast with the wide tissue distribution of most cysteine proteinases characterized to date, including cathepsin L, and suggests that cathepsin L2 may play a specialized role in the thymus and testis. Expression analysis of cathepsin L2 in human tumors revealed a widespread expression in colorectal and breast carcinomas but not in normal colon or mammary gland or in peritumoral tissues. Cathepsin L2 was also expressed by colorectal and breast cancer cell lines as well as by some tumors of diverse origin, including ovarian and renal carcinomas. These results open the possibility that this novel enzyme may be involved in tumor processes, as already reported for other cysteine proteinases, including cathepsin L.     

Factors related to the development of sensitization to green coffee and castor bean allergens among coffee workers

BACKGROUND: Occupational allergic respiratory symptoms in coffee workers have been frequently reported, but the ultimate cause of sensitization is still debated, castor bean being considered besides green coffee beans. Atopy and cigarette smoking have been suggested as promoting factors of sensitization for several occupational allergens. OBJECTIVE: This study was carried out to assess the prevalence of allergic respiratory symptoms and of sensitization to both green coffee beans and castor bean in the whole workforce of a coffee manufacturing plant. Furthermore we wanted to ascertain both the presence of castor bean antigens in the settled dust of the green coffee beans warehouse and the possible crossreactivity between the two beans. Meanwhile, the effect of smoking and atopy was considered. METHOD: Two-hundred and eleven workers were examined. A questionnaire on oculorhinitis and asthma was administered and skin-prick tests for green coffee beans, castor bean and 15 common inhalant allergens were carried out. Isoelectric focusing, isoelectric focusing immunoblot and radioallergosorbent assay (RAST) inhibition were performed on samples of settled environmental dust from the green coffee area, as well as on castor bean and green coffee beans. RESULTS: Ten per cent of the workers complained of oculorhinitis alone and 16% of asthma (nearly always associated with oculorhinitis). The overall prevalence of skin-sensitization was: 15% for green coffee beans, 22% for castor bean, 22% for common allergens. Evidence of sensitization to occupational allergens was more common in smokers, with a more than twofold increase in relative risk. The strong association between skin positivity to common and occupational allergens suggests that atopy acts as an enhancing host factor towards occupational sensitization. The analysis of the dust confirmed the presence of castor bean antigens. CONCLUSION: Our findings indicate that castor bean is the major cause of occupational sensitization among coffee workers, whereas smoking and atopy act as enhancing factors.     

Differential intracellular regulation of cortical GABA(A) and spinal glycine receptors in cultured neurons

The N-terminal region of human cystatin C has been shown to be of crucial importance for the interaction of the inhibitor with cysteine proteinases. However, several studies have been unable to identify the corresponding region in bovine cystatin C, indicating that the binding of proteinases to the bovine inhibitor may not be dependent on this region. With the aim to resolve this apparent discrepancy and to elucidate the relation of bovine cystatin C to other cystatins, we have isolated a cDNA clone encoding bovine precystatin C. The sequence of this cDNA was similar to that of the human inhibitor and showed a putative signal peptidase cleavage site consistent with the N-terminal regions of the bovine and human inhibitors being of comparable size. This suggestion was verified by determination of the relative molecular mass of the mature bovine inhibitor isolated from cerebrospinal fluid under conditions minimising proteolysis. The N-terminal of the purified inhibitor was blocked, but the sequence of the N-terminal peptide produced by digestion with endopeptidase LysC could be unequivocally determined by tandem mass spectroscopy. Together, these results show that bovine cystatin C has 118 residues, in contrast with 110-112 residues reported previously, and has an N-terminal region analogous to that of human cystatin C. This region presumably is of similar importance for tight binding of target proteinases as in the human inhibitor.     

Antibody selection for immunohistochemical survey of equine tissue

A membrane-bound protease induced by sulfur mustard in cultured normal human epidermal keratinocytes (NHEK) was purified and partially characterized. Maximum enzyme stimulation occurred at 16 hr after normal human epidermal keratinocytes were exposed to 300 microM sulfur mustard. Purification to homogeneity of the protease was accomplished by Triton X-100 solubilization, ultracentrifugation, and dialysis, followed by ion-exchange chromatography through DEAE-cellulose and finally hydrophobic column chromatography through phenyl Sepharose. Analysis of the purified enzyme by SDS-PAGE revealed a single polypeptide at the 80 kDa region. Further investigation of biochemical properties showed that a synthetic serine-specific Chromozym TRY peptide and the physiological protein laminin were good substrates for this enzyme. Moreover, this enzyme was inhibited mostly by the serine-protease inhibitors leupeptin and di-isopropyl fluorophosphate and not by the cysteine protease inhibitor E-64 or the metalloprotease inhibitor 1,10-phenanthroline (Component H, CH), indicating the serine protease nature of this enzyme. This enzyme had a pH optimum in the range of 7.0 to 8.0. Amino acid sequencing of the purified enzyme revealed that this enzyme belongs to the endopeptidase family (serine protease), and is homologous with a mammalian-type bacterial serine endopeptidase that can preferentially cleave K-X, including K-P. These results suggest that serine-protease stimulation may be one of the mechanisms of mustard-induced skin blister formation, and that some specific serine-protease inhibitors may be useful for the treatment of this sulfur mustard toxicity.     

Extracellular secretion of Escherichia coli heat-stable enterotoxin I across the outer membrane

Escherichia coli heat-stable enterotoxin Ip (STIp) is an extracellular toxin consisting of 18 amino acid residues that is synthesized as a precursor of pre (amino acid residues 1 to 19), pro (amino acid residues 20 to 54), and mature (amino acid residues 55 to 72) regions. The precursor synthesized in the cytoplasm is translocated across the inner membrane by the general export pathway consisting of Sec proteins. The pre region functions as a leader peptide and is cleaved during translocation. However, it remains unknown how the resulting peptide (pro-mature peptide) translocates across the outer membrane. In this study, we investigated the structure of the STIp that passes through the outer membrane to determine how it translocates through the outer membrane. The results showed that the pro region is cleaved in the periplasmic space. The generated peptide becomes the mature form of STIp, which happens to have disulfide bonds, which then passes through the outer membrane. We also showed that STIp with a carboxy-terminal peptide consisting of 3 amino acid residues passes through the outer membrane, whereas STIp with a peptide composed of 37 residues does not. Amino acid analysis of mutant STIp purified from culture supernatant revealed that the peptide composed of 37 amino acid residues was cleaved into fragments of 5 amino acid residues. In addition, analyses of STIps with a mutation at the cysteine residue and the dsbA mutant strain revealed that the formation of an intramolecular disulfide bond within STIp is not absolutely required for the mature region of STIp to pass through the outer membrane.     

Selective outgrowth and differential tropism of amacrine and photoreceptor axons to cell targets during early development in vitro

Plastids of nongreen tissues import carbon as a source of biosynthetic pathways and energy. Within plastids, carbon can be used in the biosynthesis of starch or as a substrate for the oxidative pentose phosphate pathway, for example. We have used maize endosperm to purify a plastidic glucose 6-phosphate/phosphate translocator (GPT). The corresponding cDNA was isolated from maize endosperm as well as from tissues of pea roots and potato tubers. Analysis of the primary sequences of the cDNAs revealed that the GPT proteins have a high degree of identity with each other but share only approximately 38% identical amino acids with members of both the triose phosphate/phosphate translocator (TPT) and the phosphoenolpyruvate/phosphate translocator (PPT) families. Thus, the GPTs represent a third group of plastidic phosphate antiporters. All three classes of phosphate translocator genes show differential patterns of expression. Whereas the TPT gene is predominantly present in tissues that perform photosynthetic carbon metabolism and the PPT gene appears to be ubiquitously expressed, the expression of the GPT gene is mainly restricted to heterotrophic tissues. Expression of the coding region of the GPT in transformed yeast cells and subsequent transport experiments with the purified protein demonstrated that the GPT protein mediates a 1:1 exchange of glucose 6-phosphate mainly with inorganic phosphate and triose phosphates. Glucose 6-phosphate imported via the GPT can thus be used either for starch biosynthesis, during which process inorganic phosphate is released, or as a substrate for the oxidative pentose phosphate pathway, yielding triose phosphates.     

Identification of three novel unique proteins in seed oil bodies of sesame

Plant seeds store triacylglycerols in discrete organelles called oil bodies. An oil body preserves a matrix of triacylglycerols surrounded by a monolayer of phospholipids embedded with abundant structural proteins termed oleosins and probably some uninvestigated minor proteins of higher molecular mass. Three polypeptides of 27, 37, and 39 kDa (temporarily denominated as Sop1, Sop2, and Sop3) were regularly co-purified with seed oil bodies of sesame. Comparison of amino acid composition indicated that they were substantially less hydrophobic than the known oleosins, and thus should not be aggregated multimers of oleosins. The results of immuno-recognition to sesame proteins extracted from subcellular fractions of mature seeds, various tissues, and oil bodies purified from different stages of seed formation revealed that these three polypeptides were unique proteins gathered in oil bodies, accompanying oleosins and triacylglycerols, during the active assembly of the organelles in maturing seeds. Both in vivo and in intro, immunofluorescence labeling using secondary antibodies conjugated with FITC (fluorescein isothiocyanate) confirmed the localization of these three polypeptides in oil bodies.     

Free pentosidine and neopterin as markers of progression rate in diabetic nephropathy. Collaborative Study Group

Stimulus-induced release of polyunsaturated fatty acids from membranes has been proposed to couple the processes of stimulus perception and oxylipin synthesis in the octadecanoid signaling pathway. This study investigated wound-induced changes in free fatty acids, diacylglycerol, and phospholipids at the site of wounding and at an unwounded area of the same wounded leaf in castor bean (Ricinus communis L.). Increases in free fatty acids and diacylglycerol and decreases in phospholipids were relatively large and continuous at the site of wounding. The changes at the unwounded area were selective and transient, suggesting a regulated activation of lipid turnover in response to wounding. In unwounded cells, the free fatty acids that increased in the early phase of wounding were linolenate and linoleate, which peaked within 5 min after wounding. Diacylglycerols that increased in unwounded cells were the species containing linolenate and linoleate, not those with oleate and stearate. Within 5 min of wounding, the levels of phosphatidylcholine and phosphatidylglycerol, but not other phospholipids, decreased in unwounded cells. These results provide evidence for the wound-induced selective increase in linolenate and linoleate in unwounded cells. The varied susceptibility of different phospholipids to hydrolysis after wounding indicates that phosphatidylcholine and phosphatidylglycerol may serve as substrates that lead to the increase in linolenate and linoleate in the early phase of wound response. The pattern of increases in polyunsaturated fatty acids, diacylglycerol, and phosphatidic acid and of decreases in phospholipids suggests the activation of a PLD-initiated signaling pathway in response to wounding in castor bean.     

The clinical evaluation of hepatic arterial infusion chemotherapy in patients with liver metastases from colorectal cancer employing an implanted port system

The isolation and sequence determination of a new 2S albumin storage protein from Ricinus communis seeds denoted 2S ASP-Ib are described. The fragment approach using selective enzymatic cleavage, Edman degradation, and mass spectrometry was used to demonstrate that the 11-kDa heterodimer protein linked by disulfide bridges has the following structure: short chain, GEREGSSSQQCRQEVQRKDLSSCERYLRQSSS; long chain, 相似文献   

A new method for seed oil body purification and examination of oil body integrity following germination

Plant seeds store triacylglycerols as energy sources for germination and postgerminative growth of seedlings. The triacylglycerols are preserved in small, discrete, intracellular organelles called oil bodies. A new method was developed to purify seed oil bodies. The method included extraction, flotation by centrifugation, detergent washing, ionic elution, treatment with a chaotropic agent, and integrity testing by use of hexane. These processes subsequently removed non-specifically associated or trapped proteins within the oil bodies. Oil bodies purified by this method maintained their integrity and displayed electrostatic repulsion and steric hindrance on their surface. Compared with the previous procedure, this method allowed higher purification of oil bodies, as demonstrated by SDS-PAGE using five species of oilseeds. Oil bodies purified from sesame were further analyzed by two-dimensional gel electrophoresis and revealed two potential oleosin isoforms. The integrity of oil bodies in germinating sesame seedlings was examined by hexane extraction. Our results indicated that consumption of triacylglycerols reduced gradually the total amount of oil bodies in seedlings, whereas no alteration was observed in the integrity of remaining oil bodies. This observation implies that oil bodies in germinating seeds are not degraded simultaneously. It is suggested that glyoxisomes, with the assistance of mitochondria, fuse and digest oil bodies one at a time, while the remaining oil bodies are preserved intact during the whole period of germination.     

Cloning and sequencing of the gene encoding a novel lysine-specific cysteine proteinase (Lys-gingipain) in Porphyromonas gingivalis: structural relationship with the arginine-specific cysteine proteinase (Arg-gingipain)

Lys-gingipain (KGP), so termed due to its peptide cleavage specificity for lysine residues, is a cysteine proteinase produced by the Gram-negative anaerobic bacterium Porphyromonas gingivalis. Mixed oligonucleotide primers designed from the NH2-terminal sequence of the purified enzyme were used to clone the KGP-encoding gene (kgp) from the organism. The nucleotide sequence of kgp had a 5,169-bp open reading frame encoding 1,723 amino acids with a calculated molecular mass of 218 kDa. As the extracellular mature enzyme had an apparent molecular mass of 51 kDa in gels, the precursor of KGP was found to comprise at least four domains, the signal peptide, the NH2-terminal prodomain, the mature proteinase domain, and the COOH-terminal hemagglutinin domain, and to be proteolytically processed during its transport. Importantly, the COOH-terminal region contained three direct repeats of two different amino acid sequences, LKWD(or E)AP and YTYTVYRDGTKI, and the subdomains located between the two repeats exhibited strong similarity to those of Arg-gingipain (RGP), another major cysteine proteinase produced by the organism and having cleavage specificity for arginine residues, although the arrangement of the subdomains was not necessarily identical in the two enzymes. Since the KGP activity was greatly decreased in RGP-deficient mutants and since the most probable site of the propeptide cleavage was present in the homologous sequence highly susceptible to proteolysis by RGP, the precursor of KGP is likely to be processed by RGP to form the mature enzyme.     

Isolation and characterization of a dibasic selective metalloendopeptidase from rat testes that cleaves at the amino terminus of arginine residues

A metalloendopeptidase that selectively cleaves doublets of basic amino acids on the amino-terminal side of arginine residues was purified to homogeneity from rat testes and analyzed further. Two catalytically active forms with apparent relative molecular masses of 110,000 and 140,000 Da, respectively, were present in the purified preparation of the enzyme. Antibodies raised against the purified testis endopeptidase revealed by immunoblot both the 110- and 140-kDa forms in both rat testis and brain cortex extracts. The isolated enzyme was inhibited by metal chelators and divalent cations. Its activity, lost after preincubation with EDTA, was restored by low concentrations of Zn2+ and Mn2+, thus demonstrating the metallopeptidase nature of the enzyme. This endopeptidase also exhibited a high sensitivity to amastatin (100% inhibition at 20 microM), an aminopeptidase inhibitor. A substrate specificity study using physiologically important or synthetic peptides containing a processing dibasic site indicated that cleavage occurred selectively at the amino-terminal side of an arginine residue, independent of the nature of the basic doublet. The enzyme produced such a cleavage at the Arg-Lys doublet of somatostatin 28 (Km = 43 microM), at the Arg-Arg doublet of dynorphin A (Km = 6.45 microM) and atrial natriuretic factor (Km = 6.25 microM), and at the Lys-Arg doublet of preproneurotensin-(154-170) (Km = 17.3 microM). Moreover, cleavage efficiency was found to be higher for the larger substrates. The distinctive properties of this endopeptidase imply that this protein is a member of a novel class of proteolytic enzymes that may be involved in the endoproteolytic maturation of hormonal precursors.