Deborah J. Siler

Hypoallergenicity of guayule rubber particle proteins compared to Hevea latex proteins

Abstract Hevea brasiliensis Muell. Arg. is currently the sole commercial source of natural rubber. However, products made from Hevea latex are responsible for causing allergies affecting an increasing number of people world-wide. In this paper we test the hypothesis that latex allergy can be circumvented by using rubber of low hypoallergenicity from other species. We also report studies to determine the feasibility of ameliorating latex allergy in Hevea. We show that antibodies raised against proteins extracted from films of ammoniated Hevea latex did not recognize latex proteins from two other rubber producing plant species, guayule (Parthenium argentatum Gray) and Ficus elastica Roxb., indicating that Hevea latex allergy can be circumvented using rubber from other species. The hypoallergenicity of guayule and F. elastica latex was confirmed in preliminary clinical trials (reported elsewhere). F. elastica rubber is mostly of low molecular weight and short chain, whereas guayule rubber is comparable in quality to that of Hevea. Guayule latex should be suitable for the manufacture of high-quality, hypoallergenic natural rubber products for the Hevea-hyperallergic individual. The Hevea latex protein antibodies recognized not only proteins from raw Hevea latex, but also rubber particle preparations from which the soluble latex proteins were removed. Hence extensive removal of rubber particle-bound proteins, in addition to the soluble latex proteins, would be required to produce a safe Hevea latex product. Latex samples from different commercial lines of H. brasiliensis reacted similarly to the antibodies, indicating that clonal selection is unlikely to be helpful in eliminating latex antigens.

A protein from Ficus elastica rubber particles is related to proteins from Hevea brasiliensis and Parthenium argentatum

Abstract Rubber particles, having high rubber transferase (RuT) activity, were isolated from Ficus elastica latex. The predominant protein in these rubber particles is a large hydrophobic glycoprotein (termed LPR for large protein from rubber particles), with an apparent monomeric M r of 376 000 on SDS-PAGE. A M r of 750 000 was determined with native PAGE, indicating that LPR probably exists as a dimer. Antibodies raised to purified LPR recognized blotted intact F. elastica rubber particles, as well as LPR, demonstrating that LPR is situated at or near the surface of the particles. Anti-LPR antibodies also recognized enzymatically active rubber particles and rubber particle proteins from two other rubber-producing plants, Hevea brasiliensis and Parthenium argentatum . This indicates that the rubber particles of all three rubber-producing species contain proteins with common antigenic determinants. Large proteins, similar in size to LPR, were found in preparations of rubber particles from H. brasiliensis and P. argentatum . These results are the first demonstration that rubber particles from different species contain similar proteins that may share common functions in rubber biosynthesis and/or rubber particle structure.

Effect of Different Allylic Diphosphates on the Initiation of New Rubber Molecules and on Cis-1,4-polyisoprene Biosynthesis in Guayule (Parthenium argentatum Gray)

Summary Natural rubber biosynthesis is the rubber-particle-bound rubber-transferase-catalyzed cis-1,4-polymerization of isoprene monomers derived from isopentenyl diphosphate (IPP). In addition to IPP, allylic diphosphate is required to initiate new rubber molecules. In this paper we describe, for the first time, detailed kinetic analyses of rubber molecule initiation and polymerization in Parthenium argentatum Gray . We compare the effects of four different allylic diphosphate initiators on the rate of rubber biosynthesis using [ 14 C]IPP. Also, we compare the rates of rubber molecule initiation and polymerization using [ 3 H]allylic diphosphates as well as [ 14 C]IPP. Under non-limiting substrate concentrations, new rubber molecules were initiated and terminated at a constant rate for each initiator. The rate of rubber biosynthesis increased with the chain length of the initiator up to C,s. Our results indicate that initiation regulates the overall rate of rubber biosynthesis. Therefore, strategies to enhance in vivo rubber yield by genetic manipulation should focus on rubber molecule initiation.

Characterization and Performance Testing of Guayule Latex

Abstract Physical and biological properties were determined for a preliminary production run of low-ammonia latex from guayule (GR latex). GR latex, and dipped films produced from compounded GR latex, contain none of the Hevea allergens known to elicit Type I systemic allergic reactions. The polymer phase of GR latex has a lower bulk viscosity and a higher resin content than that of NR. In addition, GR latex serum and rubber lack the activating components present in NR latex. As a result, GR latex cures more slowly and reaches a lower state of cure. The aged stress-strain properties of cured GR films are nonetheless similar to those of NR films.

Identification of Parthenium argentatum rubber particle proteins immunoprecipitated by an antibody that specifically inhibits rubber transferase activity

Abstract We have shown that an antibody raised to LPR ( M , 367 000) the predominant protein on Ficus elastica rubber particles, recognized three proteins from Parthenium argentatum (guayule) rubber particles, the glycoprotein RPP ( M r 52 000), and two smaller proteins ( M r 15 000 and 9000). Since anti-LPR IgG specifically inhibited the rubber particle-bound cis -prenyl transferase (rubber transferase) in both F. elastica and P. argentatum , our results indicate that these proteins probably play roles in rubber transferase activity. Although LPR and RPP differ considerably in size, we show that RPP can form a large protein complex. A M r of 450 000 was determined by electrophoresis under non-denaturing conditions. Thus rubber transferase activity appears to be associated with large proteins in both F. elastica and P. argentatum .

Immunoinhibition of rubber particle-bound cis-prenyl transferases in Ficus elastica and Parthenium argentatum

Abstract A cis -prenyl transferase (‘rubber transferase’) catalyses the polymerization of isoprene units, derived from isopentenyl diphosphate, into natural rubber ( cis -1,4-polyisoprene). Cis -prenyl transferase activity is firmly associated with cytoplasmic rubber particles but the membrane-bound enzyme has yet to be identified. We have compared two different species, Ficus elastica and Parthenium argentatum , which produce predominantly short chain and long chain rubber, respectively. The two species have disparate rubber particle protein complements. Antigenic similarities between the rubber particle proteins may include their cis -prenyl transferases, which likely contain some structural features in common. Cis -prenyl transferase activity in F. elastica rubber particles was specifically inhibited by affinity-purified polyclonal IgG raised against a protein (LPR) purified from F. elastica rubber particles. Thus, LPR appears to contain the cis -prenyl transferase active site. Anti-LPR IgG also specifically inhibited cis -prenyl transferase activity in rubber particles from P. argentatum . Thus, an anti-LPR IgG recognized similar epitopes at or near the active site of both the F. elastica and P. argentatum cis -prenyl transferases. This is the first time that an antibody to a rubber particle protein has been shown to specifically inhibit cis -prenyl transferase activity, and could lead to the isolation of the enzyme from P. argentatum .

Semisynthetic guayule latex with reduced allergenicity

Abstract Semisynthetic latices were prepared batchwise using solvent-swollen rubber extracted from guayule ( Parthenium argentatum ). Emulsification of the extracted rubber with anionic surfactants followed by vacuum desolventization yielded stable, low-resin products. The swollen guayule rubber had a low protein content (2.3 μ g/g dry rubber). As a consequence, the semisynthetic latices had protein contents (11–20 μ g/g dry rubber) significantly lower than that of native guayule latex (129 μ g/g dry rubber) isolated directly from the shrub. More importantly, these semisynthetic latices and the dipped films prepared from compounded latices contained none of the protein allergens associated with either Hevea brasiliensis latex or native guayule latex.