Rubber Elongation Factor Research Articles

Is Rubber Elongation Factor the Major Allergen in Latex?

Is Rubber Elongation Factor the Major Allergen in Latex?

The rubber elongation factor of rubber trees ( Hevea brasiliensis) is the major allergen in latex

Background: Allergy to latex-containing articles is becoming more and more important because it can result in unexpected life-threatening anaphylactic reactions in sensitized individuals. Methods: A protein of 58 kd with an isoelectric point of 8.45 was purified from raw latex and from latex gloves and identified as the major allergen, completely blocking specific IgE antibodies in the serum of latex-sensitized subjects. The allergen is a noncovalent homotetramer molecule, in which the 14.6 kd monomer was identified, by amino acid composition and sequence homologies of tryptic peptides, to be the rubber elongation factor found in natural latex of the Malaysian rubber tree. Results: Competitive immunoinhibition tests showed that the starch powder covering the finished gloves is the airborne carrier of the allergen, resulting in bronchial asthma on inhalation. The purified allergen can induce allergic reactions in the nanogram range. Conclusion: The identification of the allergen ( Hev b I) may help to eliminate it during the production of latex-based articles in the future.

Cloning and Sequencing of the cDNA Encoding the Rubber Elongation Factor of Hevea brasiliensis

In Hevea brasiliensis, the rubber particle in the laticiferous vessel is the site of rubber (cis-1-4-polyisoprene) biosynthesis. A 14 kilodalton protein, rubber elongation factor (REF), is associated with the rubber particle in a ratio of one REF to one rubber molecule (Dennis M, Henzel W, Bell J, Kohr W, Light D [1989] J Biol Chem 264: 18618-18628; Dennis M, Light D [1989] J Biol Chem 264: 18608-18617). To obtain more information concerning the function of REF and its synthesis and assembly in the rubber particle, we isolated cDNA clones encoding REF. We used antibodies to REF to screen a Hevea leaf gammagt11 cDNA expression library and obtained several positive clones. Sequence analysis of the REF cDNA clones showed that the REF mRNA contains 121 nucleotides of 5'-nontranslated sequences and a 205 nucleotide 3'-nontranslated region. The open reading frame encodes the entire 14 kilodalton REF protein without any extra amino acids (Dennis M, Henzel W, Bell J, Kohr W, Light D [1989] J Biol Chem 264: 18618-18628). The REF cDNA was subcloned in pGEM-3Z/-4Z and expressed in vitro. The translation product is a 14 kilodalton protein that can be immunoprecipitated with antibodies to REF. Addition of microsomal membranes to the in vitro translation product did not alter the mobility of the REF protein. This, and the sequence data, indicate that REF is not made as a preprotein. Our results suggest that REF is synthesized on free polysomes in the laticifer cytoplasm and that assembly of the rubber particles is likely to occur in the cytosol.

Molecular cloning and nucleotide sequencing of the rubber elongation factor gene from Hevea brasiliensis

Molecular cloning and nucleotide sequencing of the rubber elongation factor gene from Hevea brasiliensis

Rubber elongation factor from Hevea brasiliensis: Identification, characterization, and role in rubber biosynthesis.

The presence of a protein, rubber elongation factor (REF), which is tightly bound to serum-free rubber particles purified from Hevea brasiliensis latex, is necessary for prenyltransferases from a number of sources to add multiple cis-isoprene units to rubber molecules. These prenyltransferases show normal farnesyl pyrophosphate synthase activity (two trans additions of isopentenyl pyrophosphate to dimethylallyl pyrophosphate) in the absence of REF bound to rubber particles. REF bound to rubber molecules can be highly purified from all other proteins in whole latex by treatment of rubber particles with low concentrations of detergent. Treatment of rubber particles with trypsin which hydrolyzes bound REF, removal of REF with high concentrations of various detergents, or treatment of whole latex with polyclonal antibodies specific for REF all prevent prenyltransferase from adding [14C]isopentenyl pyrophosphate to rubber molecules. However, we have not been successful using detergent-solubilized REF in the reconstitution of in vitro rubber biosynthesis with either REF-depleted rubber particles or allylic pyrophosphate primers. REF has a molecular mass of 14,600 Da and is associated specifically with rubber particles in whole latex. It makes up between 10-60% of the total protein in whole latex but is absent in C-serum, the supernatant fluid obtained when rubber particles are removed by centrifugation. The amount of REF in whole latex is proportional to the rubber content. Based on a number average molecular mass of 500,000 Da for rubber and the content of rubber and REF in whole latex or serum-free rubber particles, the stoichiometry of REF molecules to rubber molecules is 1:1 in both cases. There is sufficient REF to form a monomolecular protein layer coating large rubber particles (700-1,000 nm). In the electron microscope, serum-free rubber particle preparations contain particles with diameters from 800 to as small as 10 nm. In the presence of 1% sodium dodecyl sulfate no particles smaller than 100 nm are observed. We suggest that the smaller particles may be mainly composed of REF molecules.

Amino Acid Sequence of Rubber Elongation Factor Protein Associated with Rubber Particles in Hevea Latex

The amino acid sequence of rubber elongation factor, a recently discovered protein tightly bound to rubber particles isolated from the commercial rubber tree Hevea brasiliensis, is presented. The role of this protein in rubber elongation and its interaction with prenyltransferase and rubber particles have been discussed in the preceding paper in this series (Dennis, M. S., and Light, D. R. (1989) J. Biol. Chem. 264, 18608-18617). Trypsin, Staphylococcus protease, chymotrypsin, acetic acid, and hydroxylamine cleavage were used to generate peptide fragments that were isolated by reverse phase high pressure liquid chromatography and analyzed by amino acid composition and automated Edman degradation. Each digest contained one blocked peptide identified as the amino terminus. The blocked amino-terminal peptide from the tryptic digest was analyzed by amino acid composition, fast atom bombardment mass spectrometry (molecular ion 1659.9), subdigested with Staphylococcus protease for partial sequence analysis, and finally deblocked with bovine liver acyl-peptide hydrolase removing an acetylalanine to allow analysis by Edman degradation. Rubber elongation factor is 137 amino acids long, has a molecular mass of 14,600 daltons, and lacks four amino acids: cysteine, methionine, histidine, and tryptophan. The NH2 terminus is highly charged and contains only acidic residues (5 of the first 12 amino acids). The first four amino acids are highly represented in other known NH2-terminally acetylated proteins. Comparison of the sequence of rubber elongation factor with other known sequences does not reveal significant sequence similarities that would suggest an evolutionary relationship.