A chitotetrose specific lectin from Luffa acutangula :Physico-chemical properties and the assignment of orientation of sugars in the lectin binding site

A chitooligosaccharide specific lectin (Luffa acutangula agglutinin) has been purified from the exudate of ridge gourd fruits by affinity chromatography on soybean agglutininglycopeptides coupled to Sepharose-6B. The affinity purified lectin was found homogeneous by polyacrylamide gel electrophoresis, in sodium dodecyl sulphate-polyacrylamide gels, by gel filtration on Sephadex G-100 and by sedimentation velocity experiments. The relative molecular weight of this lectin is determined to be 48,000 ± 1,000 by gel chromatography and sedimentation equilibrium experiments. The sedimentation coefficient (S20, w) was obtained to be 4·06 S. The Stokes’ radius of the protein was found to be 2·9 nm by gel filtration. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis the lectin gave a molecular weight of 24,000 in the presence as well as absence of 2-mercaptoethanol. The subunits in this dimeric lectin are therefore held by non-covalent interactions alone. The lectin is not a glycoprotein and circular dichroism spectral studies indicate that this lectin has 31% α-helix and no ß-sheet. The lectin is found to bind specifically to chitooligosaccharides and the affinity of the lectin increases with increasing oligosaccharide chain length as monitored by near ultra-violetcircular dichroism and intrinsic fluorescence titration. The values of ΔG, ΔΗ and ΔS for the binding process showed a pronounced dependence on the size of the oligosaccharide. The values for both ΔΗ and ΔS show a significant increase with increase in the oligosaccharide chain length showing that the binding of higher oligomers is progressively more favoured thermodynamically than chitobiose itself. The thermodynamic data is consistent with an extended binding site in the lectin which accommodates a tetrasaccharide. Based on the thermodynamic data, blue shifts and fluorescence enhancement, spatial orientation of chitooligosaccharides in the combining site of the lectin is assigned.

Studies on a chitooligosaccharide-specific lectin from Coccinia indica. Thermodynamics and kinetics of umbelliferyl glycoside binding.

Coccinia indica agglutinin (CIA) is a chitooligosaccharide-specific lectin with two binding sites/homodimer of M(r) 32,000. Quenching studies implied tryptophan involvement in binding activity, which was confirmed by chemical modification experiments (A. R. Sanadi and A. Surolia, submitted for publication). Binding of 4-methylumbelliferyl chitooligosaccharides has been carried out to study their binding by CIA. Reversal experiments confirm the validity of the data previously obtained (A. R. Sanadi and A. Surolia, submitted for publication) from intrinsic fluorescence studies. Surprisingly, unlike wheat germ agglutinin, there is no consistent thermodynamic effect of the chromophoric label on binding activities as compared with the native sugars. From the changes in the optical properties of the chromophoric group upon binding to CIA, it has been possible to confirm that the tryptophan located in the binding site is closest to the fourth subsite. Thermodynamic analysis shows that the binding of the labeled tetrasaccharide is very strongly entropically driven, with the terminal, nonreducing sugar residue protruding from the binding pocket. The results of stopped-flow kinetic studies on the binding of the chromophoric trisaccharide by CIA show that the mechanism of binding is a one-step process.

The chbG Gene of the Chitobiose (chb) Operon of Escherichia coli Encodes a Chitooligosaccharide Deacetylase

The chb operon of Escherichia coli is involved in the utilization of the beta-glucosides chitobiose and cellobiose. The function of chbG (ydjC), the sixth open reading frame of the operon that codes for an evolutionarily conserved protein is unknown. We show that chbG encodes a monodeacetylase that is essential for growth on the acetylated chitooligosaccharides chitobiose and chitotriose but is dispensable for growth on cellobiose and chitosan dimer, the deacetylated form of chitobiose. The predicted active site of the enzyme was validated by demonstrating loss of function upon substitution of its putative metal-binding residues that are conserved across the YdjC family of proteins. We show that activation of the chb promoter by the regulatory protein ChbR is dependent on ChbG, suggesting that deacetylation of chitobiose-6-P and chitotriose-6-P is necessary for their recognition by ChbR as inducers. Strains carrying mutations in chbR conferring the ability to grow on both cellobiose and chitobiose are independent of chbG function for induction, suggesting that gain of function mutations in ChbR allow it to recognize the acetylated form of the oligosaccharides. ChbR-independent expression of the permease and phospho-beta-glucosidase from a heterologous promoter did not support growth on both chitobiose and chitotriose in the absence of chbG, suggesting an additional role of chbG in the hydrolysis of chitooligosaccharides. The homologs of chbG in metazoans have been implicated in development and inflammatory diseases of the intestine, indicating that understanding the function of E. coli chbG has a broader significance.

Luffa acutangula agglutinin: Primary structure determination and identification of a tryptophan residue involved in its carbohydrate-binding activity using mass spectrometry

A lectin from phloem exudates of Luffa acutangula (ridge gourd) was purified on chitin affinity chromatography and characterized for its amino acid sequence and to study the role of tryptophan in its activity. The purified lectin was subjected to various proteolytic digestions, and the resulting peptides were analyzed by liquid chromatography coupled electrospray ionization ion trap mass spectrometer. The peptide precursor ions were fragmented by collision-induced dissociation or electron transfer dissociation experiments, and a manual interpretation of MS/MS was performed to deduce amino acid sequence. This gave rise to almost complete sequence coverage of the lectin which showed high-sequence similarity with deduced sequences of phloem lectins present in the database. Chemical modification of lysine, tyrosine, histidine, arginine, aspartic acid, and glutamic acid residues did not inhibit the hemagglutinating activity. However, the modification of tryptophan residues using N-bromosuccinimide showed the loss of hemagglutinating activity. Additionally, the mapping of tryptophan residues was performed to determine the extent and number of residues modified, which revealed that six residues per molecule were oxidized suggesting their accessibility. The retention of the lectin activity was seen when the modifications were performed in the presence of chitooligosaccharides due to protection of a tryptophan residue (W-102) in the protein. These studies taken together have led to the identification of a particular tryptophan residue (W-102) in the activity of the lectin. (c) 2015 IUBMB Life, 67(12):943-953, 2015

Negative Cooperativity and High Affinity in Chitooligosaccharide Binding by a Mycobacterium smegmatis Protein Containing LysM and Lectin Domains

LysM domains have been recognized in bacteria and eukaryotes as carbohydrate-binding protein modules, but the mechanism of their binding to chitooligosaccharides has been underexplored. Binding of a Mycobacterium smegmatis protein containing a lectin (MSL) and one LysM domain to chitooligosaccharides has been studied using isothermal titration calorimetry and fluorescence titration that demonstrate the presence of two binding sites of nonidentical affinities per dimeric MSL-LysM molecule. The affinity of the molecule for chitooligosaccharides correlates with the length of the carbohydrate chain. Its binding to chitooligosaccharides is characterized by negative cooperativity in the interactions of the two domains. Apparently, the flexibility of the long linker that connects the LysM and MSL domains plays a facilitating role in this recognition. The LysM domain in the MSL-LysM molecule, like other bacterial domains but unlike plant LysM domains, recognizes equally well peptidoglycan fragments as well as chitin polymers. Interestingly, in the case presented here, two LysM domains are enough for binding to peptidoglycan in contrast to the three reportedly required by the LysM domains of Bacillus subtilis and Lactococcus lactis. Also, the affinity of the MSL-LysM molecule for chitooligosaccharides is higher than that of LysM-chitooligosaccharide interactions reported so far.