Subunit association, and thermal and chemical unfolding of Cucurbitaceae phloem exudate lectins. A review.

Detailed characterization of protein (un)folding intermediates is crucial for understanding the (un)folding pathway, aggregation, stability and their functional properties. In recent years, stress-inducible lectins are being investigated with much interest. In plants phloem proteins PP1 and PP2 are major components of the phloem fluid. While PP1 is a structural protein, PP2 exhibits lectin activity, and was proposed to play key roles in wound sealing, anti-pathogenic activity, and transportation of various molecules including RNA within the plant. Cucurbitaceae fruits contain high concentrations of PP2 lectins, which recognize chitooligosaccharides with high specificity. Although the presence of PP2 lectins in the phloem exudate of Cucurbitaceae species was documented over 40 years ago, so far only a few proteins from this family have been purified and characterized in detail. This review summarizes the results of biophysical studies aimed at investigating the oligomeric status of these lectins, their thermal stability, structural perturbations caused by changes in pH and addition of chaotropic agents and characterization of intermediates observed in the unfolding process. The implications of these results in the functional roles played by PP2 type lectins in their native environment are discussed. Finally, perspectives for future biophysical research on these proteins are given.

Macromolecular Crowding Significantly Affects the Conformational Features and Carbohydrate Binding Properties of CIA17, a PP2-Type Lectin from Coccinia indica.

The effect of macromolecular crowding on the conformational features and carbohydrate binding properties of CIA17, a PP2-type lectin, was investigated employing polymeric dextrans D6, D40, and D70 (Mr ∼ 6, 40, and 70 kDa, respectively) as crowders. While the secondary structure of CIA17 was significantly affected by D6, with a considerable decrease in the number of ß-sheets and ß-turns with a corresponding increase in the number of unordered structures, relatively smaller changes were induced by D40 and D70. However, differential scanning calorimetry (DSC) studies revealed that the thermal stability of the protein remains unchanged in the presence of crowders. While the larger dextrans, D70 and D40, induced modest quenching (∼10%) of the protein fluorescence by a static pathway, the smaller D6 induced a higher degree of quenching (37%), which involved both static and collisional quenching processes. The results of fluorescence correlation spectroscopy measurements together with DSC studies suggested that CIA17 forms larger oligomers in the presence of D40 and D70 but D6 prevents the formation of higher-order oligomers. The association constant for the CIA17-chitooligosaccharide interaction increased by ∼30% and 160% in the presence of D40 and D70, respectively, but decreased by ∼30% in the presence of D6. The higher binding affinity can be attributed to the excluded volume effect, i.e., an increased effective concentration of the protein in the presence of D40 and D70, whereas D6, being smaller, possibly penetrates into the protein interior, disrupting the water structure around the protein and also inducing conformational changes, resulting in weaker binding. These observations demonstrate that molecular crowding significantly affects the carbohydrate binding characteristics of lectins, which can modulate their physiological function.

Cucurbitaceae phloem exudate lectins: Purification, molecular characterization and carbohydrate binding characteristics.

Much of the plant lectin research was focused on these proteins from seeds, whereas lectins from other plant tissues have been less investigated. Although presence of lectins in the phloem exudate of Cucurbitaceae species was reported over 40 years ago, only a few proteins from this family have been purified and characterized with respect to ligand binding properties, primary and secondary structures, while no 3D structure of a member of this family is known so far. Unlike lectins from other plant families and sources (e.g., seeds and tubers), which exhibit specificity towards different carbohydrate structures, all the Cucurbitaceae phloem exudate lectins characterized so far have been shown to recognize only chitooligosaccharides or glycans containing chitooligosaccharides. Interestingly, some of these proteins also bind various types of RNAs, suggesting that they may also play a role in the transport of RNA information molecules in the phloem. The present review gives an overview of the current knowledge of Cucurbitaceae phloem exudate lectins with regard to their purification, determination of primary and secondary structures, elucidation of thermodynamics and kinetics of carbohydrate binding and computational modeling to get information on their 3D structures. Finally, future perspectives of research on this important class of proteins are considered.

Low-pH Molten Globule-Like Form of CIA17, a Chitooligosaccharide-Specific Lectin from the Phloem Exudate of Coccinia indica, Retains Carbohydrate-Binding Ability.

pH-induced changes in the conformation, structural dynamics, and carbohydrate-binding activity of Coccinia indica agglutinin (CIA17), a PP2-type lectin, were investigated employing biophysical approaches. The secondary structure of CIA17 remains nearly unaltered over a wide pH range (2.0-8.5), while the tertiary structure of the protein exhibits considerable changes. A decrease in the fluorescence intensity and excited-state lifetime at low pH indicated perturbation in the local conformation (near Trp residues) of CIA17, which was further supported by enhancement in the Trp accessibility toward charged quenchers under acidic conditions. Fluorescence correlation spectroscopic studies indicated that at pH 2.0, CIA17 exists as a monomer over the concentration range of 10-200 nM and forms dimers at higher concentrations (KD ∼ 387 nM) but could not form higher oligomers even at ∼150-fold higher concentrations, unlike under native conditions at pH 7.4. Thermal unfolding of the low pH intermediate involves two distinct steps: dissociation of a dimer to a monomer, followed by the unfolding of the monomer. These results strongly suggest that the acid-induced unfolding pathway of CIA17 involves the formation of a monomeric molten globule-like intermediate, which retains appreciable carbohydrate-binding ability. These observations are of great physiological significance since the PP2 proteins are involved in plant defense responses.

DSC and FCS Studies Reveal the Mechanism of Thermal and Chemical Unfolding of CIA17, a Polydisperse Oligomeric Protein from Coccinia Indica.

The mechanism of thermal and chemical unfolding of Coccinia indica agglutinin (CIA17), a chitooligosacharide-specific phloem exudate lectin, was investigated by biophysical approaches. DSC studies revealed that the unfolding thermogram of CIA17 consists of three components (Tm ∼ 98, 106, and 109 °C), which could be attributed to the dissociation of protein oligomers into constituent dimers, dissociation of the dimers into monomers, and unfolding of the monomers. Intrinsic fluorescence studies on the chemical denaturation by guanidinium thiocyanate and guanidinium chloride indicated the presence of two distinct steps in the unfolding pathway, which could be assigned to dissociation of the dimeric protein into monomers and unfolding of the monomers. Results of fluorescence correlation spectroscopic studies could be interpreted in terms of the following model: CIA17 forms oligomeric structures in a concentration dependent manner, with the protein existing as a monomer below 1 nM concentration but associating to form dimers at higher concentrations (KD ≈ 2.9 nM). The dimers associate to yield tetramers with a KD of ∼50 µM, which further associate to form higher oligomers with further increase in concentration. These results are consistent with the proposed role of CIA17 as a key player in the defense response of the plant against microbes and insects.

Purification, biochemical/biophysical characterization and chitooligosaccharide binding to BGL24, a new PP2-type phloem exudate lectin from bottle gourd (Lagenaria siceraria).

Phloem Protein 2 (PP2), highly abundant in the sieve elements of plants, plays a significant role in wound sealing and anti-pathogenic responses. In this study, we report the purification and characterization of a new PP2-type lectin, BGL24 from the phloem exudate of bottle gourd (Lagenaria siceraria). BGL24 is a homodimer with a subunit mass of ~24 kDa and exhibits high specificity for chitooligosaccharides. The isoelectric point of BGL24 was estimated from zeta potential measurements as 5.95. Partial amino acid sequence obtained by mass spectrometric studies indicated that BGL24 exhibits extensive homology with other PP2-type phloem exudate lectins. CD spectroscopic measurements revealed that the lectin contains predominantly ß-sheets, with low α-helical content. CD spectroscopic and DSC studies showed that BGL24 exhibits high thermal stability with an unfolding temperature of ~82 °C, and that its secondary structure is essentially unaltered between pH 3.0 and 8.0. Fluorescence titrations employing 4-methylumbelliferyl-ß-D-N,N',N″-triacetylchitotrioside as an indicator ligand revealed that the association constants for BGL24-chitooligosaccharide interaction increase considerably when the ligand size is increased from chitotriose to chitotetraose, whereas only marginal increase was observed for chitopentaose and chitohexaose. BGL24 exhibited moderate cytotoxicity against MDA-MB-231 breast cancer cells, whereas its effect on normal splenocytes was marginal.

Purification and biochemical/biophysical characterization of two hexosaminidases from the fresh water mussel, Lamellidens corrianus.

Two thermostable isoforms of a hexosaminidase were purified to homogeneity from the soluble extract of fresh water mussel Lamellidens corrianus, employing a variety of chromatographic techniques. Hexosaminidase A (HexA) is a heterodimer with subunit masses of ~80 and 55 kDa. Hexosaminidase B (HexB) is a homodimer with a subunit mass of 55-60 kDa. Circular dichroism spectroscopic studies indicated that both HexA and HexB contain ß-sheet as the major secondary structural component with considerably lower content of α-helix. The temperature and pH optima of both the isoforms were found to be 60 °C and 4.0, respectively. The IC50 values for HexA with N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, d-galactosamine, d-glucosamine, methyl α-d-mannopyranoside and d-mannose are 3.7, 72.8, 307, 216, 244 and 128 mM, respectively, whereas the corresponding IC50 values for HexB were estimated as 5.1, 61, 68, 190, 92 and 133 mM, respectively. Kinetic parameters KM and Vmax for HexA and B with p-nitrophenyl N-acetyl-ß-d-glucosaminide are 4 mM, 0.23 µmol·min-1·mL-1 and 2.86 mM, 0.29 µmol·min-1·mL-1, respectively, and with p-nitrophenyl N-acetyl-ß-d-galactosaminide are 4.5 mM, 0.054 µmol·min-1·mL-1 and 1.4 mM, 0.14 µmol·min-1·mL-1, respectively. GalNAc inhibited both isoforms in a non-competitive manner, whereas a mixed mode of inhibition was observed with GlcNAc with both forms.

Macromolecular properties and partial amino acid sequence of a Kunitz-type protease inhibitor from okra (Abelmoschus esculentus) seeds.

A Kunitz-type protease inhibitor (OPI, okra protease inhibitor) has been purified from okra (Abelmoschus esculentus) seeds by a combination of ammonium sulfate precipitation, anion-exchange chromatography and reverse-phase high-performance liquid chromatography. The protein shows an apparent mass of 21 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition. OPI exhibits inhibitory activity against trypsin. Analysis of the far-UV circular dichroism spectrum showed that the protein contains approx. 39% beta-sheets but only approx. 5% alpha-helices. The protein is thermally quite stable, and exhibits a cooperative thermal unfolding transition at approx. 70 degree C, as determined by circular dichroism spectroscopy and differential scanning fluorimetry. De novo sequencing of OPI by nanoESI-Q-ToF mass spectrometry (MS) allowed the assignment of about 83% of its primary structure, which indicated that the protein shares 43% sequence identity with a putative 21 kDa trypsin inhibitor from Theobroma bicolor. An intramolecular disulfide linkage between Cys149 and Cys156 was also detected. The protein showed approx 24 and approx 25% sequence identity with alpha-amylase/subtilisin inhibitor from barley and soybean (Kunitz) trypsin inhibitor, respectively. Comparative structure modeling of OPI revealed a structural fold similar to other Kunitz-type TIs. The presence of Cys149-Cys156 disulfide bond as detected by MS and a second disulfide bond connecting Cys44-Cys91, conserved in all Kunitz-type TIs, is also identified in the model.

Purification, chitooligosaccharide binding properties and thermal stability of CIA24, a new PP2-like phloem exudate lectin from ivy gourd (Coccinia indica).

PP2-like chitin binding phloem exudate lectins, abundant in the sieve tube of cucurbits, have been implicated to play key roles in wound sealing and antipathogenic responses of the plant. Here we report the affinity purification, macromolecular characterization and carbohydrate binding properties of a new chitooligosaccharide-specific lectin from the phloem exudate of ivy gourds (Coccinia indica). The protein, CIA24, has a subunit mass of 24 kDa. Partial sequence analysis indicated that CIA24 exhibits high homology with CIA17 and other Cucurbitaceae PP2 proteins whereas CD spectroscopic studies suggested that ß-sheets constitute the predominant secondary structure. Temperature dependent CD spectroscopic and differential scanning calorimetric studies revealed that CIA24 is a highly thermostable protein, which undergoes complete unfolding at ∼105 °C. Isothermal titration calorimetric studies suggested that binding of chitooligosaccharides to CIA24 is a highly exothermic process. The lectin combining site can accommodate upto a tetrasaccharide with the binding stoichiometry (n) close to unity with respect to each protein subunit, whereas for chitohexaose a sharp decrease in the binding stoichiometry (n) to ∼1:0.5 was observed. This suggests that the protein probably undergoes dimerisation in presence of chitohexaose, wherein two protein molecules bind to the oligosaccharides from the reducing and non-reducing end, respectively.