Galactose-specific Protein Research Articles

Galactose-specific seed lectins from Cucurbitaceae.

Lectins, the carbohydrate binding proteins have been studied extensively in view of their ubiquitous nature and wide-ranging applications. As they were originally found in plant seed extracts, much of the work on them was focused on plant seed lectins, especially those from legume seeds whereas much less attention was paid to the lectins from other plant families. During the last two decades many studies have been reported on lectins from the seeds of Cucurbitaceae species. The main focus of the present review is to provide an overview of the current knowledge on these proteins, especially with regard to their physico-chemical characterization, interaction with carbohydrates and hydrophobic ligands, 3-dimensional structure and similarity to type-II ribosome inactivating proteins. The future outlook of research on these galactose-specific proteins is also briefly considered.

Purification and characterization of an N‐acetyl‐d‐galactosamine‐specific lectin from seeds of chickpea (Cicer arietinum L.)

A novel lectin (CAA-II) was isolated and purified from the seeds of Cicer arietinum by ammonium sulphate fractionation and affinity chromatography on an N-acetyl-D-galactosamine-linked agarose column. The lectin is composed of four identical subunits of 30 kDa and the molecular mass of the native lectin was estimated to be 120 kDa by gel filtration chromatography and confirmed by mass spectrometry. The lectin showed agglutination activity against rabbit erythrocytes (trypsin-treated and untreated) as well as against human erythrocytes. Haemagglutination inhibition assays showed that the lectin is a galactose-specific protein having a high affinity for N-acetyl-D-galactosamine. The molecular weight, haemagglutination pattern, carbohydrate specificity and N-terminal amino acid sequence indicated that the lectin is clearly distinct from the previously reported chickpea lectin CAA-I.

Synchronous modulation of cell surface lectin and its receptor in a homologous cell population: A novel mechanism of cellular regulation

Testicular immotile sperm undergo maturation during epididymal transit when these cells pass through caput, corpus, and cauda-epididymal regions. Maturing goat spermatozoa specifically at the distal corpus epididymal stage show head-to-head autoagglutination when incubated in vitro in a modified Ringer's solution. Here, we show the biochemical mechanism of autoagglutination event and its functional significance. A lectin-like molecule located on sperm surface specifically interacts with its receptor of the neighboring homologous cells to cause autoagglutination. Lectin is a Ca ++-dependent galactose-specific protein. Failure of the pre- and post-distal corpus sperm to show autoagglutination is due to lack of lectin-like molecule and its receptors, respectively. Maturing sperm at distal corpus stage acquire lectin-like molecule followed by sharp disappearance of its receptor, and this event is synchronously associated with the initiation of sperm forward motility that is essential for fertilization in vivo. Lectin and its receptor isolated from sperm plasma membrane showed high efficacy for blocking autoagglutination phenomenon. The data are consistent with the view that synchronous modulation of homologous cell surface lectin and their receptors constitutes a novel mechanism for cellular regulation by generating waves of signals by manipulating lectin–sugar-dependent “self-talk” and cell–cell “cross-talk”.

Ligand Density Effect on Biorecognition by PEGylated Gold Nanoparticles: Regulated Interaction of RCA120 Lectin with Lactose Installed to the Distal End of Tethered PEG Strands on Gold Surface

PEGylated gold nanoparticles (diameter: 20 nm) possessing various functionalities of lactose ligand on the distal end of tethered PEG ranging from 0 to 65% were prepared to explore the effect of ligand density of the nanoparticles on their lectin binding property. UV-visible spectra of the aqueous solution of the nanoparticles revealed that the strong steric stabilization property of the PEG layer lends the nanoparticles high dispersion stability even under the physiological salt concentration (ionic strength, I = 0.15 M). The number of PEG strands on a single particle was determined to be 520 from thermogravimetric analysis (TGA). Scanning electron microscopy (SEM) observation under controlled acceleration voltage revealed the thickness of the PEG layer on the nanoparticle to be approximately 7 nm. The area occupied by a single lactose molecule on the surface of PEGylated gold nanoparticles was then calculated based on TGA and SEM results and was varied in the range of 10-34 nm2 depending on the lactose functionality (65 approximately 20%). PEGylated gold nanoparticles with 40% and 65% lactose functionality showed a selective and time-dependent aggregation in phosphate buffer with the addition of Ricinus communis agglutinin (RCA120) lectin, a bivalent galactose-specific protein. The aggregates can be completely redispersed by adding an excess amount of galactose. Time-lapse monitoring of UV-visible spectra at 600-750 nm revealed that the aggregation of PEGylated gold nanoparticles was accelerated with an increase in both RCA120 concentration in the solution and the lactose density of the nanoparticles. Furthermore, the sensitivity of lectin detection could be controlled by the regulation of lactose density on the particle surface. Interestingly, there was a critical lactose density (>20%) observed to induce detectable particle aggregation, indicating that the interaction between the particles is triggered by the multimolecular bridging via lectin molecules.

The flocculation of wine yeasts: biochemical and morphological characteristics in Kloeckera apiculata.

The floc-forming ability of flocculent strains of Kloeckera apiculata, isolated from musts, was tested for susceptibility to proteinase and sugar treatments. Three different flocculation phenotypes were discriminated by protease digestion, whereas the inhibition of flocculation by sugars distinguished two definite patterns: one mechanism of flocculation involved a galactose-specific protein and the other a broad-specificity lectin. SEM and TEM observation of the cell surface of two different Kloeckera strains revealed fine fibrils and a diffuse structure at the point of contact in one strain, and thick masses of mucus on the cell wall of the other strain.

On the specificity of carbohydrate-lectin recognition. The interaction of a lectin from Ricinus communis beans with simple saccharides and concanavalin A.

A galactose‐specific protein (RC1) isolated from Ricinus communis beans was found to give a precipitin reaction with concanavalin A. Its carbohydrate content amounted to 8–9% of the total protein and was found to be rich in mannose. The interaction of RC1 with galactose and lactose was measured in 0.05 M phosphate buffer containing 0.2 M NaCl (pH 6.8) by the method of conventional equilibrium dialysis. From the analysis of the binding data according to Scatchard method the association constant (Ka) at 5°C was calculated as 3.8 mM−1 and 1.2 mM−1 for lactose and galactose, respectively. In both cases the number of binding sites per molecule of RC1 with molecular weight of 120000 was found to be 2. From the temperature‐dependent Ka values for the binding of lactose, the values of –5.7 kcal/mol and –4.3 cal × mol−1× K−1 were calculated for ΔH and ΔS, respectively.The addition of concanavalin A to RC1 or vice versa led to the formation of the insoluble complex RC1· ConA4 containing one molecule of RC1 and one molecule of tetrameric concanavalin A (ConA4) which could be dissociated upon addition of concanavalin A‐specific sugars. The complex formation results in a time‐dependent appearance of turbidity in the time range from 10s to 10 min. From the measurement of the time‐dependent appearance and disappearance of the turbidity the formation (kf) and dissociation (kd) rate constants were calculated as 3 mM−1× s−1 and 0.07 ks−1 respectively. The ratio kf/kd (43μM −1), that corresponds to the association constant of complex RC1· ConA4, is higher than that of mannoside · ConA4 and thereby suggests that protein‐protein interaction contributes significantly in stabilising glycoprotein · lectin complexes. The relevance of this finding to the understanding of the chemical specificities that are involved in a model cell‐lectin interaction is discussed.