Rubber elongation factor (REF), a major allergen component in Hevea brasiliensis latex has amyloid properties

Abstract

REF (Hevb1) and SRPP (Hevb3) are two major components of Hevea brasiliensis latex, well known for their allergenic properties. They are obviously taking part in the biosynthesis of natural rubber, but their exact function is still unclear. They could be involved in defense/stress mechanisms after tapping or directly acting on the isoprenoid biosynthetic pathway. The structure of these two proteins is still not described. In this work, it was discovered that REF has amyloid properties, contrary to SRPP. We investigated their structure by CD, TEM, ATR-FTIR and WAXS and neatly showed the presence of β-sheet organized aggregates for REF, whereas SRPP mainly fold as a helical protein. Both proteins are highly hydrophobic but differ in their interaction with lipid monolayers used to mimic the monomembrane surrounding the rubber particles. Ellipsometry experiments showed that REF seems to penetrate deeply into the monolayer and SRPP only binds to the lipid surface. These results could therefore clarify the role of these two paralogous proteins in latex production, either in the coagulation of natural rubber or in stress-related responses. To our knowledge, this is the first report of an amyloid formed from a plant protein. This suggests also the presence of functional amyloid in the plant kingdom.

Figure 1. Phylogenetic analysis of REF and SRPP protein family.

A BlastP was realized on REF P15252 at http://www.phylogeny.fr. The accession numbers of each 24 aligned sequences with ClustalW (http://www.genome.jp/tools/clustalw/program) were as follows: REF Hevea brasiliensis P15252, REF Hevea brasiliensis AEH05970 (2), REF Hevea brasiliensis AAR11448 (3), SRPP Hevea brasiliensis O82803, SRPP Hevea brasiliensis AAO66432 (2), RLP1 Hevea brasiliensis AAP46159, RLP2 Hevea brasiliensis AAP46160, REF Ricinus communis XP_002512427, REF Arabidopsis thaliana NP_187201, SRP Vitis riparia Q9SW70, REF Morus alba ACV90044, REF Amblyomma maculatum AEO33677, SRP Ipomoea batatas ABP35522, SRP Oryza sativa AAO72547, SRP Zea mays ACG39345, REF Selaginella moellendorffii XP_002969776, SRPP Parthenium argentatum AAQ11374, SRP Capsicum annuum ADI60300, SRPP Ricinus communis XP_002514917, REF Populus trichocarpa XP_002319520, REF Arabidopsis lyrata XP_002882419, SRP Glycine max XP_003543052, REF/SRPP-like protein Medicago truncatula XP_003593563. Evolutionary analyses were conducted in MEGA5 using the Neighbor-Joining method, the bootstrap test (1000 replicates) and the p-distance method. All positions containing gaps and missing data were eliminated. On the right, characterized proteins are presented. SRPP: Small Rubber Particle Protein; REF: Rubber Elongation Factor; SRP: Stress-related protein; RLP: REF-like stress related protein. Both REF and SRPP proteins used in this study are framed. Hevea brasiliensis protein family is framed by a gray dashed box.

Figure 2. REF and SRPP are major latex proteins with homology but distinct aggregation properties. A.

Sequence homology of REF and SRPP after alignment with ClustalW (http://www.genome.jp/tools/clustalw/program) and shading by using GeneDoc 2.7.0 (http://www.nrbsc.org/downloads/). Cons: consensus sequence for homology in proteins found in Hevea brasiliensis and showed in Supplementary Figure S1. B. Protein content of 60% latex by 15% SDS-PAGE. C. Aggregation state of REF and SRPP at 50 µM in PBS buffer 1 X after 3 h at 37°C. D. Differential Proteinase K resistance of REF and SRPP after incubation with Proteinase K, 30 min at 37°C and visualization on 15% SDS-PAGE.

Figure 3. Amyloid dye-binding properties of REF and SRPP.

A. Samples (top image) and absorbance spectra in presence of Congo red (CR). B. Birefringence of REF red aggregates under visible light (top) and cross-polarized light (bottom). C. ThT binding spectra. D. Bis-ANS binding spectra.

Figure 4. Amyloid structural properties of REF aggregates.

A. Circular dichroism spectra of REF aggregated protein compared to soluble SRPP protein. B. ATR-FTIR spectra of air-dried aggregated REF and soluble SRPP. Deconvolutions of the amide I band are presented for each protein. C. TEM image of REF amorphous aggregates obtained at the end of the polymerization. D. Analysis by WAXS of REF lyophilized aggregates. The diffraction rays at about 4.7 Å/10 Å, characteristic of the amyloid cross-beta core are showed by the arrows. Image has a 345 mm diameter corresponding to 2300 p×2300 p (0.15 mm/p). In these experiments aggregates were grown 3 h at 37°C and 20 µM and kept at 4°C.

Figure 5. REF self-assembles rapidly as amyloid fibers and amorphous aggregates.

In order to visualize early step of fibrillization, we incubated the sample at 20°C, 20 µM and 20 min. The sample was composed of large (about 10 nm) and µM-long fibers assembling laterally and a lot of amorphous aggregates.

Figure 6. REF and SRPP interact differently with membrane lipids.

A. Dot blots of DMPC, asolectin, cholesterol have been incubated overnight at 20°C with 20 µM of each protein. Proteins interacting with lipids were revealed with mouse anti-histidine antibody and then anti-mouse-phosphatase-alkaline antibody followed by a NBT/BCIP staining. B. Ellipsometric images of REF and SRPP interacting with DMPC and asolectin monolayers at the air/water or lipid interface.

Figure 7. Proposed model of REF and SRPP interaction with the monolayer membrane of the rubber particle.

SRPP is bound to the surface of the Small Rubber Particle (SRP) while REF is inserted into the membrane of the Large Rubber Particle (LRP). Other proteins are also transmembrane proteins such as cis-prenyl transferases (CPT) or rubber transferases able to condensate and elongate natural rubber from isopentenyl and dimethylallyl diphosphates.