Biochemical and Biophysical Research Communications
Comparative study of 7S globulin from Corylus avellana and Solanum lycopersicum revealed importance of salicylic acid and Cu-binding loop in modulating their function
Introduction
Globulins constitute a major part of the seed proteome and are classified into 7S globulins and 11S globulins on the basis of their sedimentation rates [1]. The 7S globulins are also termed as vicilins and are distributed widely in the plant kingdom. They were classically known to be an important source of nutrition during the growth and development of the seeds [1], but further studies revealed that they have diverse activities. Vicilins from legumes are reported to bind to sugars and chitin, thus inhibiting the growth of fungi and pests that infect plants [2]. Vicilin from Pisum sativum indicated its role in desiccation tolerance [3]. A more recent study of vicilin from bell pepper (Capsicum annuum) showed that it has superoxide dismutase (SOD) function and its role has been associated with managing oxidative stress [4].
Interestingly, not all vicilins share common function. Of course, structural similarity does not imply same function and various plant proteins (germins, germin like proteins, fatty acid multifunctional proteins, ferredoxin:sulfite reductase, etc.) are already known to exhibit different functions in different species [5]. Similar to these proteins, the ability of vicilins to bind to chitin has only been reported in leguminous plants [6] and the SOD activity of C. annuum vicilin was absent in vicilins isolated from okra (A. esculentus) and cowpea (V. unguiculata) [4]. However, it is not yet clear what makes these proteins functionally diverse. The differences in their function could be possibly because of small differences in their structures, sequences and the environments in which they act [7].
In the present study, we have carried out functional and structural analyses of vicilins from tomato (S. lycopersicum; SL80.1) and hazelnut (C. avellana; HZ.1) to understand the basis for their functional diversity. The SOD activities of HZ.1 and SL80.1 were assayed. It was realized that while SL80.1 exhibits the SOD activity, HZ.1 lacks the same [4]. Structural comparison was further done to understand the underlying reasons behind the differential activities of vicilins. HZ.1 and SL80.1 were purified, crystallized and their structures were determined. The detailed analyses of the three-dimensional structures accompanied with biophysical characterization of bound ligand were aimed to establish a meaningful correlation with their enzymatic activities. Our findings provide key insights about the possible role of salicylic acid (SA) and copper-binding loop in regulating the SOD activity of vicilins.
Section snippets
Fractionation of seed proteome
Seeds of S. lycopersicum and C. avellana were washed with 70% alcohol and were grounded to powder. This was followed by delipidification in petroleum ether. The dried delipidified powder was homogenized in 50 mM Tris-Cl (pH 8 and pH 7.5 for S. lycopersicum and C. avellana, respectively) containing 150 mM NaCl along with protease inhibitor cocktail (Sigma). The extract in homogenization buffer was subjected to salt fractionation over a concentration range of 0–95% (w/v) ammonium sulfate. This
Results
The ammonium sulfate fractionation yielded an array of proteins. The precipitated proteins in each fraction in both the cases of S. lycopersicum and C. avellana could be visualised in Supplementary Figs. S1 and S2. For targeting globulins from S. lycopersicum and C. avellana seeds, we characterized all the fractions of ammonium sulfate. Two major abundantly present proteins in ammonium sulfate fractions of S. lycopersicum were identified as 7S vicilin (SL80.1) and pathogenesis-related protein
Discussion
Vicilins are important plant proteins that structurally belong to cupin superfamily which include enzymatically diverse proteins from all kingdoms. Vicilins were considered as enzymatically inactive cupins, but a recent structural study of a vicilin from C. annuum revealed that it has SOD activity. Reactive oxygen species are generated in plants during various biotic and abiotic stress conditions such as drought, desiccation as well as bacterial and fungal infections. Vicilins continue to be of
Authors’ contribution
AJ, DMS and MS conceived and designed the experiments. AJ and MS solved the structure. MS performed the biochemical experiments. AJ, DMS and MS wrote the manuscript. The authors declare no competing interests.
Acknowledgements
We thank the EMBL staff and EMBL-DBT for providing access to the BM14 beamline at the ESRF. We thank mass spectrometry facility and staff at RCB and SCIEX. The financial support from the Department of Biotechnology, Government of India through core grants is gratefully acknowledged. MS thanks CSIR for fellowship. PDB: 6L4M and 6L4C.
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Both the authors equally contributed to the work.