Ribosylation induced structural changes in Bovine Serum Albumin: understanding high dietary sugar induced protein aggregation and amyloid formation.

Non-enzymatic glycation of proteins is believed to be the root cause of high dietary sugar associated pathophysiological maladies. We investigated the structural changes in protein during progression of glycation using ribosylated Bovine Serum Albumin (BSA). Non enzymatic attachment of about 45 ribose molecules to BSA resulted in gradual reduction of hydrophobicity and aggregation as indicated by red-shifted tryptophan fluorescence, reduced ANS binding and lower anisotropy of FITC-conjugated protein. Parallely, there was a significant decrease of alpha helicity as revealed by Circular Dichroism (CD) and Fourier transformed-Infra Red (FT-IR) spectra. The glycated proteins assumed compact globular structures with enhanced Thioflavin-T binding resembling amyloids. The gross structural transition affected by ribosylation led to enhanced thermostability as indicated by melting temperature and Transmission Electron Microscopy. At a later stage of glycation, the glycated proteins developed non-specific aggregates with increase in size and loss of amyloidogenic behaviour. A parallel non-glycated control incubated under similar conditions indicated that amyloid formation and associated changes were specific for ribosylation and not driven by thermal denaturation due to incubation at 37 °C. Functionality of the glycated protein was significantly altered as probed by Isothermal Titration Calorimetry using polyphenols as substrates. The studies demonstrated that glycation driven globular amyloids form and persist as transient intermediates during formation of misfolded glycated adducts. To the best of our knowledge, the present study is the first systematic attempt to understand glycation associated changes in a protein and provides important insights towards designing therapeutics for arresting dietary sugar induced amyloid formation.

Trehalose mediated stabilisation of cellobiase aggregates from the filamentous fungus Penicillium chrysogenum.

Extracellular fungal cellobiases develop large stable aggregates by reversible concentration driven interaction. In-vitro addition of trehalose resulted in bigger cellobiase assemblies with increased stability against heat and dilution induced dissociation. In presence of 0.1 M trehalose, the size of aggregates increased from 344 nm to 494 nm. The increase in size was also observed in zymography of cellobiase. Activation energy of the trehalose stabilised enzyme (Ea = 220.9 kJ/mol) as compared to control (Ea = 257.734 kJ/mol), suggested enhanced thermostability and also showed increased resistance to chaotropes. Purified cellobiase was found to contain 196.27 µg of sugar/µg of protein. It was proposed that presence of glycan on protein's surface impedes and delays trehalose docking. Consequently, self-association of cellobiase preceded coating by trehalose leading to stabilisation of bigger cellobiase aggregates. In unison with the hypothesis, ribosylated BSA failed to get compacted by trehalose and developed into bigger aggregates with average size increasing from 210 nm to 328 nm. Wheat Germ Lectin, in presence of trehalose, showed higher molecular weight assemblies in DLS, native-PAGE and fluorescence anisotropy. This is the first report of cross-linking independent stabilisation of purified fungal glycosidases providing important insights towards understanding the aggregation and stability of glycated proteins.

Trehalose induced structural modulation of Bovine Serum Albumin at ambient temperature.

Trehalose is a well-known protein stabilizing osmolyte. The present study has been designed to understand the interaction of trehalose with BSA at ambient temperature. Steady state fluorescence and life-time analysis along with CD, DLS and ITC have been employed to show that trehalose causes surface-associated structural perturbation of BSA to promote its compaction. Trehalose at 0.1M concentration resulted in increased solvent exposure of one of the two tryptophans of BSA with a 5nm redshift in emission and enhanced susceptibility to acrylamide quenching with an increase in KSV from 2.61M-1to 5.16M-1. 0.5M trehalose resulted in reduced accessibility of tryptophan and destabilization of ANS binding (Forster radius increased from 24Å to 27.36Å for tryptophan-ANS FRET) indicating shielding of BSA in trehalose matrix. Simultaneously, there was compaction of BSA as shown by increased alpha-helicity from 45.85% to 48.81%, decreased thioflavin-T binding and reduction in hydrodynamic radius from 9.69nm to 6.59nm. Trehalose induced solution viscosity resulted in significant decrease in binding affinity of BSA towards curcumin and resveratrol. The results are in unison with the preferential exclusion and vitrification models to explain protein stabilization by trehalose and also points at the structure-function trade-off of proteins in presence of trehalose.

AkP from mushroom Termitomyces clypeatus is a proteoglycan specific protease with apoptotic effect on HepG2.

Termitomyces clypeatus is an edible mushroom, prized for its therapeutic values and as producer of industrially important enzymes. However, the biomedical efficacies of anticancer proteases have not been reported yet. The present study aimed to purify and characterize a serine protease (AkP) from T. clypeatus for investigating cytotoxic potency on HepG2, Hep3B, and compared the effect on normal hepatic L-02 cells. Purification and biochemical characterization of AkP were evaluated by three stage chromatography, 1D/2D-SDS-PAGE, 1D zymography, far-UV CD spectral analysis, N-terminal sequencing, MALDI-TOF/MS-MS analysis and enzyme kinetics studies. AkP could cleave the growth promoting cell surface proteoglycans of HepG2, corroborated by RP-HPLC analysis. AkP (IC50: 75±1.18nM) mediated anti-proliferative activity solely on HepG2 cells through the induction of apoptosis. Augmentation of apoptosis was attributed to up-regulation of p53 and Bax protein expression succeeded by caspase-3 activation. Serine protease inhibitor phenyl methane sulfonyl fluoride (PMSF) inhibited both its proteolytic activity and cytotoxicity on HepG2. These findings demonstrate that AkP could be an effective biomolecule for killing of cancer cells by p53 restoration and surface proteoglycans cleavage.

Purification and characterisation of κ-casein specific milk-clotting metalloprotease from Termitomyces clypeatus MTCC 5091.

Milk-clotting enzymes are valued as chymosin-like protease substitutes for cheese making industries. An extracellular metalloprotease (AcPs) with high milk-clotting activity was purified from edible mushroom Termitomyces clypeatus and characterised. AcPs was preferentially active towards κ-casein, analysed by Urea-PAGE and LC-ESI-MS, whereas the degradation of α and ß-casein components by AcPs proceeded slowly justifying its suitability for cheese making. RP-HPLC peptide profiling revealed that the AcPs activity on milk casein was similar to that of a commercial milk coagulant. The enzyme exhibited pH and temperature optima at 5.0 and 45 °C, respectively and showed a pI value of 4.6. One- and two dimensional zymographies revealed a single polypeptide band with proteolytic signal. The MALDI-TOF/MS followed by peptide mass fingerprinting revealed homology with a predicted protein of Populus trichocarpa. To our knowledge, this is the first report on a metalloprotease from T. clypeatus, and the results indicate that this enzyme can be considered as a potential substitute for chymosin in cheese manufacturing.

Enhancement of extracellular cellobiase activity by reducing agents in the filamentous fungus Termitomyces clypeatus.

Extracellular cellobiase activity of Termitomyces clypeatus increased from 2.9 U ml(-1) to 4.4 and 4.1 in presence of dithiothreitol (DTT) and ß-mercaptoethanol (ME), respectively, with a decrease in Km from 0.4 to 0.3 mM (DTT) and 0.35 mM (ME). Catalysis was further enhanced if the reduced enzyme was alkylated and activity increased from 11.4 U ml(-1) (control) to 15.2 (DTT+N-ethylmaleimide) and 15.3 (DTT+iodoacetamide) using p-nitrophenyl-ß-D-glucopyranoside and from 14.6 U ml(-1)(control) to 21.9 (DTT+N-ethylmaleimide) and 18.7 (DTT+iodoacetamide) using cellobiose. The reduced enzyme showed 17 % lesser glucose inhibition. CD and tryptophan fluorescence showed no change in secondary structure was caused by DTT up to 50 mM. Cysteine content of the enzyme was 24 %. It is postulated that reduction of disulphide bonds allows better substrate affinity for cellobiase. The studies describe a novel and simple method to increase cellobiase activity for industrial applications.

Increased enzyme secretion by 2-deoxy-D-glucose in presence of succinate by suppression of metabolic enzymes in Termitomyces clypeatus.

Regulatory mode of secretion of proteins was detected for the industrial glycosidase, cellobiase, under secreting conditions (in presence of TCA cycle intermediates like succinate etc.) in the filamentous fungus Termitomyces clypeatus. The titers of key metabolic enzymes were investigated under secreting and non-secreting conditions of growth and compared to the corresponding production of intra and extracellular levels of cellobiase. Results were compared in presence of 2-deoxy-D-glucose, a potent glycosylation inhibitor in the secreting media. Inclusion of 2-deoxy-D-glucose in presence of succinate caused about 10 to 100 times decrease in titers of the metabolic enzymes hexokinase, fructose-1,6-bisphosphatase, isocitrate lyase and malate dehydrogenase leading to increased secretion of cellobiase by more than 100 times. The intracellular concentration of cAMP (86-fold decrease in presence of 2-deoxy-D-glucose under secreting conditions) and turnover rate of proteins also dropped significantly. In this suppressed metabolic state, a 10-fold increase in the titer of the secreted cellobiase was noticed. The results indicated elucidation of carbon catabolite repression like phenomenon in the fungus under secreting conditions which was more pronounced by 2-deoxy-D-glucose. The interdependence between secretion and regulation of metabolic enzymes will help in better understanding of the physiology of these highly adapted organisms for increasing their secretion potential of glycosidases like cellobiase with high industrial value.

Enhanced activity and stability of cellobiase (beta-glucosidase: EC 3.2.1.21) produced in the presence of 2-deoxy-D-glucose from the fungus Termitomyces clypeatus.

Generally less glycosylation or deglycosylation has a detrimental effect on enzyme activity and stability. Increased production and secretion of cellobiase was earlier obtained in the presence of the glycosylation inhibitor 2-deoxy-d-glucose in filamentous fungus Termitomyces clypeatus [Mukherjee, S.; Chowdhury, S.; Ghorai, S.; Pal, S.; Khowala, S. Biotechnol. Lett.2006, 28, 1773-1778]. In this study the enzyme was purified from the culture medium by ultrafiltration and gel-permeation, ion-exchange and high-performance liquid chromatography, and its catalytic activity was six times higher compared to the control enzyme. K(m) and V(max) of the purified enzyme were measured as 0.187 mM and 0.018 U mg(-1), respectively, using pNPG as the substrate. The enzyme had temperature and pH optima at 45 degrees C and pH 5.4, respectively, and retained full activity in a pH range of 5-8 and temperatures of 30-60 degrees C. Interestingly less glycosylated cellobiase was resistant towards proteolytic as well as endoglycosidase-H digestion and showed higher stability than native enzyme due to increased aggregation of the protein. The enzyme also showed higher specific activity in the presence of cellobiose and pNPG and less susceptibility towards salts and different chemical agents. The beta-glucosidase can be considered as a potentially useful enzyme in various food-processing, pharmaceutical and fermentation industries.

Purification and characterization of a thermostable intra-cellular beta-glucosidase with transglycosylation properties from filamentous fungus Termitomyces clypeatus.

An intra-cellular beta-glucosidase was purified to homogeneity by gel filtration, ion exchange chromatography and HPGPLC from mycelial extract of Termitomyces clypeatus in the presence of the glycosylation inhibitor 2-deoxy-d-glucose. CD spectroscopy demonstrated that the purified enzyme exhibited alpha-helical conformation. MALDI-TOF identified the enzyme's molecular weight as 6688Daltons, but SDS-PAGE and immunoblotting indicated that the enzyme formed aggregates. The enzyme also showed unique properties of co-aggregation with sucrase in the fungus. The enzyme showed around 80% stability up to 60 degrees C and residual activity was 80-100% between pH ranges 5-8. The enzyme had higher specific activity against p-nitrophenyl-d-glucopyranoside than cellobiose and HPLC showed that the enzyme possesses transglycosylation activity and synthesizes cello-oligosaccharides by addition of glucose. The enzyme will be useful in synthetic biology to produce complex bioactive glycosides and to avoid chemical hazards. This is the first report of a beta-glucosidase enzyme with such a low monomeric unit size.

Interference of sugars in the Coomassie Blue G dye binding assay of proteins.

The presence of sugars causes significant deviation from the actual absorbance of proteins in the Bradford protein assay. In these studies, polysaccharides and disaccharides at milligram levels mimicked proteins in microgram equivalents. Monosaccharides, which individually did not show any absorbance, interfered significantly by sequestering the dye species. The studies demonstrated that in a mixture of sugars and proteins, sugar interference was much higher than expected from sugar molecules' individual contribution. Estimated protein values were increased 2 to 4 times after precipitation from fungal culture broths. Thus, in carbohydrate-rich samples, protein concentrations should be ascertained by precipitation from crude extracts and resolubilization in a noninterfering buffer.