Chenyan Lv

Chitinase III in pomegranate seeds (Punica granatum Linn.): a high-capacity calcium-binding protein in amyloplasts

Chitinases are a class of ubiquitous proteins that are widely distributed in plants. Defense is the major natural role for chitinases, primarily against fungal pathogens. Little is known regarding their non-defensive roles in seeds. In this study, a new class III chitinase from pomegranate seeds (pomegranate seed chitinase, PSC) was isolated and purified to homogeneity. The native state of PSC is a monomer with a molecular weight of approximately 30 kDa. This chitinase naturally binds calcium ions with high capacity and low affinity, suggesting that PSC is a calcium storage protein. Consistent with this idea, its amino acid sequence (inferred from cDNA) is rich in acidic amino acid residues, especially Asp, similar to reported calcium storage proteins. The presence of calcium considerably improves the stability of the protein but has little effect on its enzymatic activity. Transmission electron microscopy analyses indicate that, similar to phytoferritin, this enzyme is widely distributed in the stroma of amyloplasts of the embryonic cells, suggesting that amyloplasts in seeds could serve as an alternative plastid for calcium storage. Indeed, the transmission electron microscopy results showed that, within the embryonic cells, calcium ions are mainly distributed in the stroma of the amyloplasts, consistent with a role for PSC in calcium storage. Thus, the plant appears to have evolved a new plastid for calcium storage in seeds. During seed germination, the content of this enzyme decreases with time, suggesting that it is involved in the germination process.

NADH induces iron release from pea seed ferritin: A model for interaction between coenzyme and protein components in foodstuffs

Plant ferritin from legume seeds co-exists with coenzyme NADH (a reduced form of nicotinamide-adenine dinucleotide) in many foodstuffs. In the present study, the interaction of NADH with apo pea seed ferritin (PSF) was investigated by fluorescence resonance energy transfer (FRET), fluorescence titration, transmission electron microscope (TEM), and isothermal titration calorimetry (ITC). We found that NADH molecules bound on the outer surface of PSF close to the 4-fold channels, which was 1.58 nm from tryptophan residue (Trp). Consequently, such binding facilitates iron release from holo PSF, which might have a negative effect on PSF as an iron supplement, while NADH was oxidised into NAD(+). However, the binding of NADH to the protein does not affect the entry of toxic ferrous ions into the apo protein shell, where these ferrous ions were oxidised into less toxic ferric ions. Moreover, NADH binding markedly affects the tertiary structure around Trp residues of PSF. These findings advanced our understanding of the interactions between different naturally occurring components in a complex food system.

Four-Fold Channels Are Involved in Iron Diffusion into the Inner Cavity of Plant Ferritin

From an evolutionary point of view, plant and animal ferritins arose from a common ancestor, but plant ferritin exhibits different features as compared with the animal analogue. One major difference is that the 4-fold channels naturally occurring in plant ferritin are hydrophilic, whereas the 4-fold channels in animal ferritin are hydrophobic. Prior to this study, however, the function of the 4-fold channels in oxidative deposition of iron in phytoferritin remained unknown. To elucidate the role of the 4-fold channels in iron oxidative deposition in ferritin, three mutants of recombinant soybean seed H-2 ferritin (rH-2) were prepared by site-directed mutagenesis, which contained H193A/H197A, a 4-fold channel mutant, E165I/E167A/E171A, a 3-fold channel mutant, and E165I/E167A/E171A/H193A/H197A, where both 3- and 4-channels were mutated. Stopped-flow, electrode oximetry, and transmission electron microscopy (TEM) results showed that H193A/H197A and E165I/E167A/E171A exhibited a similar catalyzing activity of iron oxidation with each other, but a pronounced low activity compared to rH-2, demonstrating that both the 4-fold and 3-fold hydrophilic channels are necessary for iron diffusion in ferritin, followed by oxidation. Indeed, among all tested ferritin, the catalyzing activity of E165I/E167A/E171A/H193A/H197A was weakest because its 3- and 4- fold channels were blocked. These findings advance our understanding of the function of 4-fold channels of plant ferritin and the relationship of the structure and function of ferritin.

A novel strategy of natural plant ferritin to protect DNA from oxidative damage during iron oxidation.

Plant ferritin is a naturally occurring heteropolymer in plastids, where Fe(2+) is oxidatively deposited into the protein. However, the effect of this process on the coexistence of DNA and plant ferritin in the plastids is unknown. To investigate this effect, we built a system in which various plant ferritins and DNA coexist, followed by treatment with ferrous ions under aerobic conditions. Interestingly, naturally occurring soybean seed ferritin (SSF), a heteropolymer with an H-1/H-2 ratio of 1 to 1 in the apo form, completely protected DNA from oxidative damage during iron oxidative deposition into protein, and a similar result was obtained with its recombinant form, but not with its homopolymeric counterparts, apo rH-1 and apo rH-2. We demonstrate that the difference in DNA protection between heteropolymeric and homopolymeric plant ferritins stems from their different strategies to control iron chemistry during the above oxidative process. For example, the detoxification reaction occurs only in the presence of apo heteropolymeric SSF (hSSF), thereby preventing the production of hydroxyl radicals. In contrast, hydroxyl radicals are apparently generated via the Fenton reaction when apo rH-1 or rH-2 is used instead of apo hSSF. Thus, a combination of H-1 and H-2 subunits in hSSF seems to impart a unique DNA-protective function to the protein, which was previously unrecognized. This new finding advances our understanding of the structure and function of ferritin and of the widespread occurrence of heteropolymeric plant ferritin in nature.

Zn2 + rather than Ca2 + or Mg2 + used as a cofactor in non-muscular actin from the oyster to control protein polymerization

BACKGROUND The major cytoskeletal protein of most cells is actin, which polymerizes to form actin filaments (F-actin). Each actin monomer (G-actin) contains a divalent alkaline earth metal ion (in vivo Mg(2+); in vitro usually Ca(2+)) as a cofactor that is crucial for protein polymerization. Prior to this study, however, whether or not other types of metal ions can play the same role as Mg(2+) or Ca(2+) in actins remains unknown. METHODS A new actin from the gills of oyster (AGO) was prepared and characterized by protein purification techniques, SDS- and native-PAGE, and LC-MS\MS for the first time. The property of this protein was studied by CD, fluorescence and UV/vis spectroscopy, laser light scattering, and TEM. RESULTS AGO is a monomer with a MW of ~42kDa. AGO is unique among all known actins in that Zn(2+) is only a naturally binding metal in the protein, and that one native AGO molecule binds 8 zinc ions, which can be removed by EDTA treatment at pH7.2. The presence of zinc has a great effect on the secondary and tertiary structure of the protein. Correlated with such effect is that these zinc ions in native AGO facilitate protein polymerization, whereas removal of zinc ions from native AGO results in a loss of such polymerization property. CONCLUSIONS The present work demonstrates that AGO is a novel zinc-binding protein with high capacity, and high selectivity. GENERAL SIGNIFICANCE This work extends an understanding of the function of zinc and actin.

High-capacity calcium-binding chitinase III from pomegranate seeds (Punica granatum Linn.) is located in amyloplasts

We have recently identified a new class III chitinase from pomegranate seeds (PSC). Interestingly, this new chitinase naturally binds calcium ions with high capacity and low affinity, suggesting that PSC is a Ca-storage protein. Analysis of the amino acid sequence showed that this enzyme is rich in acidic amino acid residues, especially Asp, which are responsible for calcium binding. Different from other known chitinases, PSC is located in the stroma of amyloplasts in pomegranate seeds. Transmission electron microscopy (TEM) analysis indicated that the embryonic cells of pomegranate seeds are rich in calcium ions, most of which are distributed in the stroma and the starch granule of the amyloplasts, consistent with the above idea that PSC is involved in calcium storage, a newly non-defensive function.

The Interactions between Plant Proteins/enzymes and Other Food Components and Their Effects on Food Quality.

ABSTRACT Plant proteins are the main sources of dietary protein for humans, especially for vegetarians. There are a variety of components with different properties coexisting in foodstuffs, so the interactions between these components are inevitable to occur, thereby affecting food quality. Among these interactions, the interplay between plant proteins/enzymes from fruits and vegetables, cereals, and legumes and other molecules plays an important role in food quality, which recently has gained a particular scientific interest. Such interactions not only affect the appearances of fruits and vegetables and the functionality of cereal products but also the nutritive properties of plant foods. Non-covalent forces, such as hydrogen bond, hydrophobic interaction, electrostatic interaction, and van der Waals forces, are mainly responsible for these interactions. Future outlook is highlighted with aim to suggest a research line to be followed in further studies.

Identification of seven water-soluble non-storage proteins from pomegranate (Punica granatum Linn.) seeds.

As pomegranate (Punica granatum Linn.) processing is fast growing, the usage of pomegranate processing wastes containing seeds has been receiving great attention. The protein component accounts for 100–130 g/kg of the seeds in weight. However, so far, there is no information on the composition and function of the pomegranate seed proteins. In this study, a global view of water-soluble non-storage proteins isolated from mature pomegranate seeds were studied using two-dimensional polyacrylamide gel electrophoresis coupled with liquid chromatography–tandem mass spectrometry. With the two-dimensional polyacrylamide gel electrophoresis approach, over 120 protein spots were resolved, of which 7 abundant protein spots showing low molecular mass were identified. These identified proteins may be linked to seed development and metabolism, but more importantly, the occurrence of these proteins provides the possibility of conversion the pomegranate processing wastes into useful products or raw material for food industry.