The patatin-like protein from the latex of Hevea brasiliensis (Hev b 7) is not a vacuolar protein

Upon centrifugation, rubber latex is divided into a layer of rubber particles, the cytosol, and the lutoid-body fraction, which is of vacuolar origin. One of the proteins isolated from the lutoid-body fraction is a protein with a molecular mass of 43 kDa, which has esterase activity on p-nitrophenylpalmitate and which shows significant sequence similarity with patatin, a vacuolar protein with esterase activity from potato (Solanum tuberosum). This protein is a major allergen in rubber latex products (Hev b 7) and can also be isolated from the cytosol fraction of rubber latex. The mature protein isolated from lutoid-bodies has no structural features expected for a vacuolar protein: the N-terminal methionine in the cDNA-derived sequence is cleaved off, the second residue is N-acetylated, and the C-terminal sequence is identical to that in the cDNA-derived sequence. Thus the patatin-like protein in Hevea brasiliensis is not a vacuolar protein, but may be associated with not yet characterized particles in the cytoplasm, which either sediment with lutoid-bodies or remain in the cytosol fraction, depending on the centrifugation conditions.     

Hevein: an antifungal protein from rubber-tree (Hevea brasiliensis) latex

Several chitin-binding proteins were isolated from the bottom fraction of Hevea brasiliensis (Müll.) Arg. latex. One of these chitin-binding proteins is hevein, a small monomeric protein which strongly resembles the lectin from stinging nettle (Urtica dioica L.). Like the latter, hevein showed strong antifungal activity against several fungi in vitro. The possible involvement of this protein in the defense against invasion by potentially pathogenic fungi is discussed.Abbreviations FPLC fast protein liquid chromatography - M_r apparent molecular mass - SDS-PAGE Sodium dodecyl sulp-hatepolyacrylamide gel electrophoresis - UDA Urtica dioica agglutinin - WGA Wheat-germ agglutinin This work is supported in part by NIH grant and grants of the National Fund for Scientific Research (Belgium): W.J.P. is senior research associate, and W.F.B. senior research assistant of this fund. J.V.P. receives a fellowship of the Belgian Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw.     

Polyphenol oxidases from latex of Hevea brasiliensis: purification and characterization

Polyphenol oxidase (PPO) was isolated from the B-serum obtained after repetitive freeze-thawing of the bottom fraction isolated from ultracentrifuged fresh latex. The B-serum was subjected to acetone precipitation and CM-Sepharose chromatography, affording two PPOs, PPO-I and PPO-II, which, upon SDS-PAGE, were 32 and 34 kDa, respectively. Both PPOs possessed the same pI (9.2), optimum pH (7) and optimum temperature (35-45 degrees C). They are stable up to 60 degrees C and active at broad pH ranges from 4-9. The K(m) values of PPO-I for dopamine, L-dopa and catechol as substrates are 2.08, 8.33 and 9.09 mM, while those for PPO-II are 2.12, 4.76 and 7.14 mM, respectively. Among various PPO inhibitors tested, 4-hexylresorcinol was the most potent. Anionic detergents were among the most effective activators of the enzymes, while cationic and nonionic detergents showed little and no effect on the PPO activities, respectively.     

The isolation and composition of helical protein microfibrils from Hevea brasiliensis latex

1. The microfibrils contained within the lutoid particles of Hevea brasiliensis latex obtained from young tissue have been isolated by methods based on low-speed centrifugation, isoelectric precipitation and gel filtration. 2. The isolated microfibrils behave as a single protein having an isoelectric point of about 4 as determined by paper electrophoresis. 3. The only components so far detected in the microfibrils are protein and possibly carbohydrate; nucleic acid appears to be absent. 4. The amino acid composition of the microfibril protein shows no unusual features. 5. In latex from the more mature laticiferous tissues of H. brasiliensis, the lutoid particles appear to be devoid of microfibrils or their protein decomposition products.     

Hevains: Serine-centred proteases from the latex of Hevea brasiliensis

Hevains b and l, isolated respectively from the serum and lutoids of freeze-dried latex from Hevea brasiliensis, were purified to homogeneity and compared with hevain a from commercial, ammonia-treated latex. The M_rs of hevains a and b are 69 000 and 58 000, respectively, and both exist in several charged forms. The amino acid compositions of the two enzymes differ significantly, but the reactivities to a variety of ester and protein substrates are similar, as are the pH optima. Hevain l is a distinct protease of M_r 80 000 and unique amino acid composition. It displays esterolytic activity and will digest insulin B chain, but is not proteolytic to azocollagen, azocasein, bovine serum albumen or haemoglobin. The activities of all three enzymes are dependent on the presence of serine and histidine residues.     

Isolation and characterization of isoinhibitors of the potato protease inhibitor I family from the latex of the rubber trees, Hevea brasiliensis

Three isoinhibitors have been isolated to homogeneity from the C-serum of the latex of the rubber tree, Hevea brasiliensis clone RRIM 600, and named HPI-1, HPI-2a and HPI-2b. The three inhibitors share the same amino acid sequence (69 residues) but the masses of the three forms were determined to be 14,893+/-10, 7757+/-5, and 7565+/-5, respectively, indicating that post-translational modifications of the protein have occurred during latex collection. One adduct could be removed by reducing agents, and was determined to be glutathione, while the other adduct could not be removed by reducing agents and has not been identified. The N-termini of the inhibitor proteins were blocked by an acetylated Ala, but the complete amino acid sequence analysis of the deblocked inhibitors by Edman degradation of fragments from endopeptidase C digestion and mass spectrometry confirmed that the three isoinhibitors were derived from a single protein. The amino acid sequence of the protein differed at two positions from the sequence deduced from a cDNA reported in GenBank. The gene coding for the inhibitor is wound-inducible and is a member of the potato inhibitor I family of protease inhibitors. The inhibitor strongly inhibited subtilisin A, weakly inhibited trypsin, and did not inhibit chymotrypsin. The amino acid residues at the reactive site P(1) and P(1)(') were determined to be Gln45 and Asp46, respectively, residues rarely reported at the reactive site in potato inhibitor I family members. Comparison of amino acid sequences revealed that the HPI isoinhibitors shared from 33% to 55% identity (50-74% similarity) to inhibitors of the potato inhibitor I family. The properties of the isoinhibitors suggest that they may play a defensive role in the latex against pathogens and/or herbivores.     

Crystallization of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex

Hevamine, an enzyme with both lysozyme and chitinase activity, was isolated and purified from Hevea brasiliensis (rubber tree) latex. The enzyme (molecular weight 29,000) is homologous to certain "pathogenesis-related" proteins from plants, but not to hen egg-white or phage T4 lysozyme. To investigate the atomic details of the substrate specificity and the cause for hevamine's low pH optimum (pH 4.0), we have crystallized two hevamine isozymes as a first step towards a high-resolution X-ray structure determination. Suitable crystals were obtained at room temperature from hanging drop experiments by vapor diffusion against 1.7 M to 3.4 M-NaCl (pH 5.0 to 9.0) for the major isozyme, and by vapor diffusion against 2.5 M to 4.3 M-NaCl (pH 5.0 to 8.0) for the minor one. Both isozymes give the same crystal morphology and space group. Their space group is P2(1)2(1)2(1) with cell dimensions a = 82.3 A, b = 58.1 A and c = 52.5 A (1 A = 0.1 nm). The crystals diffract to at least 2.0 A resolution.     

Electron transport in the membrane of lutoids from the latex of Hevea brasiliensis.

1. An antimycin-insensitive NADH-cytochrome c oxidoreductase (E.C. 1.6.99.3) activity can be demonstrated in the membrane of lutoids isolated from the latex of Hevea brasiliensis. This electron transport system can also use ferricyanide as an electron acceptor, but is unable to oxidize NADPH. 2. Two beta-type cytochromes are present in the membranes. Cytochrome beta563 is partially reduced by NADH and ascorbate, but is not reducible by NADPH. It shows a double peak at 555 and 561 nm at 77 degrees K. A second cytochrome, cytochrome beta561, seems to be reducible by hydrosulfite only. 3. In the reduced state, these cytochromes do not combine with CO. The occurrence of cytochrome P-450 could not be demonstrated. 4. The role of the NADH oxidation system is considered in relation to the biosynthesis of polyisoprene compounds in the latex.     

A molecular and in silico characterization of Hev b 4, a glycosylated latex allergen

Hev b 4 is a heavily glycosylated latex allergen with seven attached N-glycans, comprising of both oligomannose and complex type structures. Treatment with a mixture of N-glycosidase A and N-glycosidase F resulted in lowering Hev b 4 protein on SDS-gel from 53 to 55kDa to circa 40kDa, this being comparable to the 38.53kDa mass predicted by its cDNA. In Western-immunoblots, the enzymatically deglycosylated Hev b 4 showed negligible binding to IgE from latex allergic patients; the results indicated that IgE essentially binds to Hev b 4 via its N-glycan moiety. Structural modelling of the Hev b 4 was carried out based on the template protein and carbohydrate crystal coordinates of rhamnogalacturonan acetylesterase (PDB ID 1DEO). We managed to link four N-glycan structures on to the Hev b 4 model; the glycans were scattered over the surface of the model. The structural and functional features of Hev b 4 could prove useful to elucidate its exposed epitopes which are important for IgE binding.     

Demonstration of a pyrophosphate-fructose 6-phosphate 1-phosphotransferase in the latex from Hevea brasiliensis

A pyrophosphate: fructose-6-phosphate 1-phosphotransferase activity (EC 2.7.1.90) has been characterized in cytosol from Hevea brasiliensis latex. It is Mg+ dependent enzyme, and the cation has an optimal effect between 2.5 to 3 mM for a concentration of 1 mM of pyrophosphate and 10 mM of fructose-6-phosphate. It is activated by catalytic content of fructose-2,6-diphosphate. Its potential activity is higher than 40% of that of ATP dependent phosphofructokinase (EC 2.7.1.11). Its optimum pH is between 7.5-7.6; then, the enzyme affinity is 0.3 mM for pyrophosphate and 3.5 mM for fructose-6-phosphate. It is suggested that the transferase plays a role in the pyrophosphate metabolism and the increasing of the energetic efficiency of glycolysis and so takes a significant part in the biochemical mechanisms involved in the latex yield.     

Purification of a prenyltransferase that elongates cis-polyisoprene rubber from the latex of Hevea brasiliensis

We have purified "rubber transferase" from latex of the commercial rubber tree Hevea brasiliensis and find that it is a dimer with a monomeric molecular mass of 38,000 Da, requires Mg2+, and is stabilized by thiols in agreement with studies of a partially purified preparation previously described (Archer, B. L., and Cockbain, E. G. (1969) Methods Enzymol. 15, 476-480). Greater than 90% of the [1-14C]isopentenyl pyrophosphate which is incorporated into deproteinated rubber particles by the purified prenyltransferase is added to high molecular mass polyisoprene (greater than 20,000 Da). Purified prenyltransferase and deproteinated rubber particles reconstitute 40-60% of the biosynthetic activity of whole latex in samples matched for rubber content. Incorporation is linear with added rubber particles up to at least 10 mg/ml rubber or 20 microM rubber molecules (based on a number average molecular mass of 500,000 Da). Prenyltransferase concentrations estimated in whole latex (0.37% or 160 nM) are sufficient to saturate all elongation sites in whole latex, and addition of purified prenyltransferase does not increase [1-14C]isopentenyl pyrophosphate incorporation. Deproteinated rubber particles can be titrated with the pure enzyme (Kd = 9 nM) demonstrating that the fraction of rubber molecules available for addition is low (approximately 0.01%). An estimated 7,000 isoprene units are added per complex at a rate of 1/s in a typical assay. Hevea prenyltransferase catalyzes the formation of cis-isoprene in the presence of rubber particles. However, in the absence of rubber particles and in the presence of dimethylallyl pyrophosphate, the purified prenyltransferase catalyzes the formation of geranyl pyrophosphate and all trans-farnesyl pyrophosphate as demonstrated by thin layer chromatography, gas chromatography, and molecular exclusion chromatography.     

The primary structure of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex

The primary structure of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex, has been determined predominantly with conventional non-automatic methods. The positions of three disulfide bridges have been determined. The sequence has about 60% identity with that of a chitinase from cucumber and 95% with the N-terminal sequence of the lysozyme/chitinase of Parthenocissus quinquefolia. The half-cystine residues in hevein and cucumber chitinase are located at identical positions. Hevamine is a basic protein from the lutoids (vacuoles) of rubber latex and may have a role in plugging the latex vessels and cessation of latex flow. The differences in cellular location, charge properties and sequence between hevamine and cucumber chitinase are similar to those between class I and class II chitinases from tobacco and other plant species.