In vitro protein anti-aggregation function
A protein stabilisation function has been demonstrated using a recombinant form of the nematode Group 3 LEA protein, AavLEA1, which prevents aggregation of desiccation-sensitive proteins such as citrate synthase (CS) (Fig. 1). When subjected to repeated rounds of vacuum drying and rehydration (“drying cycles”), CS shows marked aggregation and associated loss of activity. Aggregation is maximal after two drying cycles, as shown by apparent increase in A340 due to light scattering; with increasing numbers of desiccation cycles, the A340 of the CS sample decreases, but this is due to the aggregates becoming so large that they no longer scatter light and are not maintained in suspension, depositing on the vessel walls. Addition of nematode LEA protein reduces CS aggregation to negligible levels, even after four cycles of desiccation. Protection against aggregation by commonly-used stabilisers such as bovine serum albumin (BSA) is not limited in this assay. The LEA protein also preserves CS activity, as does BSA, although to a lesser extent, suggesting that aggregate formation does not always result in complete loss of CS activity.
|Fig 1: Aggregation and activity of citrate synthase after desiccation, in the presence of nematode LEA protein or BSA. Aggregation is measured by light scattering (apparent absorbance at A340); enzyme activity is assayed according to standard methods and results expressed as percentage of control activity. One drying cycle corresponds to vacuum drying (without freezing) for 1 h followed by immediate rehydration in water to the original volume. Results after two and four drying cycles are shown. See Goyal et al. (2005) for more detail.|
In vivo protein anti-aggregation function
Huntington's disease (HD) is a neurodegenerative disorder characterised by expansion of a glutamine-encoding trinucleotide (CAG) repeat in the huntingtin gene beyond the asymptomatic limit of 35 repeat units. Most adult-onset HD patients have CAG repeats in the range 40-50, while in the juvenile form of HD, a CAG repeat number in excess of 55 is frequently observed. These higher order repeat lengths result in aggregation of N-terminal fragments of huntingtin which form inclusion bodies in neural tissue. In collaboration with Prof David Rubinsztein's laboratory at CIMR, Cambridge, we produced a fluorescent version of a spontaneously aggregating polyglutamine (polyQ) expansion protein derived from huntingtin and co-expressed nematode LEA protein in the same human cell line. We found that the LEA protein reduces the propensity of the polyQ protein to form aggregates, and similar results were obtained with a polyalanine expansion protein, demonstrating in vivo function as a protein stabiliser (Fig. 2).
|Fig 2: LEA protein reduces protein aggregation in vivo. Confocal microscopy of human cells after introduction of HD polyQ ("EGFP-HDQ74") expression construct, with (induced) or without (uninduced) expression of LEA protein. Nematode LEA protein AavLEA1 is immunostained red, polyQ protein is green and nuclei are stained blue with DAPI. Indicative polyQ aggregates are shown by arrowheads; fewer aggregates are observed in the presence of LEA protein. See Chakrabortee et al. (2007) for more detail.|
One hypothesis we are considering to explain this protein stabilisation function is that LEA proteins, and other hydrophilic proteins, behave as “molecular shields”. We define a molecular shield as a macromolecule which sterically or electrosterically prevents interaction between partially unfolded proteins which might otherwise form aggregates. A molecular shield has some similarity to a molecular chaperone, but does not necessarily have specific binding sites for unfolded proteins. By extension, a molecular shield is not itself susceptible to aggregation under the stress conditions studied, i.e. desiccation, in our case.
LEA Protein Research Topics:
Chakrabortee, S., Boschetti, C., Walton, L. J., Sarkar, S., Rubinsztein, D. C. and Tunnacliffe, A. (2007) Hydrophilic protein associated with desiccation tolerance exhibits broad protein stabilization function. Proc. Natl. Acad. Sci. USA 104: 18073-18078.
Goyal, K., Walton, L. and Tunnacliffe, A. (2005) LEA proteins prevent aggregation due to water stress. Biochem J. 388: 151-157.