Germin-like proteins (GLPs) have been identified in several plant tissues. However, only one work describes GLP in latex fluids. Therefore, the goal of this study was to investigate GLPs in latex and get new insights concerning the structural and functional aspects of these proteins. Two complete sequences with high identity (>50%) with other GLPs, termed CpGLP1 and CpGLP2, were obtained and consecutively presented 216 and 206 amino acid residues, corresponding to molecular masses of 22.7 and 21.7kDa, pI 6.8 and 6.5. The three-dimensional models revealed overall folding similar to those reported for other plant GLPs. Both deduced sequences were grouped into the GER 2 subfamily. Molecular docking studies indicated a putative binding site consisting of three highly conserved histidines and a glutamate residue, which interacted with oxalate. This interaction was later supported by enzymatic assays. Superoxide dismutase (common activity in GLPs) was not detected for CpGLP1 and CpGLP2 by zymogram. The two proteins were detected in the latex, but not in non-germinated or germinated seeds and calli. These results give additional support that germin-like proteins are broadly distributed in plants and they are tissue-specific. This particularity deserves further studies to better understand their functions in latex.
The latex from Thevetia peruviana is rich in plant defense proteins, including a 120 kDa cysteine peptidase with structural characteristics similar to germin-like proteins. More than 20,000 plant species produce latex, including Apocynaceae, Sapotaceae, Papaveraceae and Euphorbiaceae. To better understand the physiological role played by latex fluids, a proteomic analysis of Thevetia peruviana (Pers.) Schum latex was performed using two-dimensional gel electrophoresis and mass spectrometry. A total of 33 proteins (86 %) were identified, including storage proteins, a peptidase inhibitor, cysteine peptidases, peroxidases and osmotins. An unusual cysteine peptidase, termed peruvianin-I, was purified from the latex by a single chromatographic step involving gel filtration. The enzyme (glycoprotein) was inhibited by E-64 and iodoacetamide and exhibited high specific activity towards azocasein (K m 17.6 µM), with an optimal pH and temperature of 5.0-6.0 and 25-37 °C, respectively. Gel filtration chromatography, two-dimensional gel electrophoresis, and mass spectrometry revealed that peruvianin-I possesses 120 kDa, pI 4.0, and six subunits (20 kDa). A unique N-terminal amino acid sequence was obtained to oligomer and monomers of peruvianin-I (1ADPGPLQDFCLADLNSPLFINGYPCRNPALAISDDF36). High-resolution images from atomic force microscopy showed the homohexameric structure of peruvianin-I may be organized as a trimer of dimers that form a central channel similar to germin-like proteins. Peruvianin-I exhibited no oxalate oxidase and superoxide dismutase activity or antifungal effects. Peruvianin-I represents the first germin-like protein (GLP) with cysteine peptidase activity, an activity unknown in the GLP family so far.
Background:
Osmotin-Like Proteins (OLPs) have been purified and characterized from different plant tissues, including latex fluids. Besides its defensive role, tobacco osmotin seems to induce adiponectin-like physiological effects, acting as an agonist. However, molecular information about this agonistic effect on adiponectin receptors has been poorly exploited and other osmotins have not been investigated yet.
Objective and Methods:
The present study involved the characterization of three OLPs from Plumeria rubra latex and molecular docking studies to evaluate the interaction between them and adiponectin receptors (AdipoR1 and AdipoR2).
Results:
P. rubra Osmotin-Like Proteins (PrOLPs) exhibited molecular masses from 21 to 25 kDa and isoelectric points ranging from 4.4 to 7.7. The proteins have 16 cysteine residues, which are involved in eight disulfide bonds, conserved in the same positions as other plant OLPs. The three-dimensional (3D) models exhibited the three typical domains of OLPs, and molecular docking analysis showed that two PrOLP peptides interacted with two adiponectin receptors similarly to tobacco osmotin peptide.
Conclusion:
As observed for tobacco osmotin, the latex osmotins of P. rubra exhibited compatible interactions with adiponectin receptors. Therefore, these plant defense proteins (without known counterparts in humans) are potential tools to study modulation of glucose metabolism in type II diabetes, where adiponectin plays a pivotal role in homeostasis.
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