Crystal structures of starch binding domain from Rhizopus oryzae glucoamylase in complex with isomaltooligosaccharide: insights into polysaccharide binding mechanism of CBM21 family.

Glucoamylases are responsible for hydrolysis of starch and polysaccharides to yield ß-D-glucose. Rhizopus oryzae glucoamylase (RoGA) is composed of an N-terminal starch binding domain (SBD) and a C-terminal catalytic domain connected by an O-glycosylated linker. Two carbohydrate binding sites in RoSBD have been identified, site I is created by three highly conserved aromatic residues, Trp47, Tyr83, and Tyr94, and site II is built up by Tyr32 and Phe58. Here, the two crystal structures of RoSBD in complex with only α-(1,6)-linked isomaltotriose (RoSBD-isoG3) and isomaltotetraose (RoSBD-isoG4) have been determined at 1.2 and 1.3 Å, respectively. Interestingly, site II binding is observed in both complexes, while site I binding is only found in the RoSBD-isoG4 complex. Hence, site II acts as the recognition binding site for carbohydrate and site I accommodates site II to bind isoG4. Site I participates in sugar binding only when the number of glucosyl units of oligosaccharides is more than three. Taken together, two carbohydrate binding sites in RoSBD cooperate to reinforce binding mode of glucoamylase with polysaccharides as well as the starch.

Two unique ligand-binding clamps of Rhizopus oryzae starch binding domain for helical structure disruption of amylose.

The N-terminal starch binding domain of Rhizopus oryzae glucoamylase (RoSBD) has a high binding affinity for raw starch. RoSBD has two ligand-binding sites, each containing a ligand-binding clamp: a polyN clamp residing near binding site I is unique in that it is expressed in only three members of carbohydrate binding module family 21 (CBM21) members, and a Y32/F58 clamp located at binding site II is conserved in several CBMs. Here we characterized different roles of these sites in the binding of insoluble and soluble starches using an amylose-iodine complex assay, atomic force microscopy, isothermal titration calorimetry, site-directed mutagenesis, and structural bioinformatics. RoSBD induced the release of iodine from the amylose helical cavity and disrupted the helical structure of amylose type III, thereby significantly diminishing the thickness and length of the amylose type III fibrils. A point mutation in the critical ligand-binding residues of sites I and II, however, reduced both the binding affinity and amylose helix disruption. This is the first molecular model for structure disruption of the amylose helix by a non-hydrolytic CBM21 member. RoSBD apparently twists the helical amylose strands apart to expose more ligand surface for further SBD binding. Repeating the process triggers the relaxation and unwinding of amylose helices to generate thinner and shorter amylose fibrils, which are more susceptible to hydrolysis by glucoamylase. This model aids in understanding the natural roles of CBMs in protein-glycan interactions and contributes to potential molecular engineering of CBMs.

Hydrophilic aromatic residue and in silico structure for carbohydrate binding module.

Carbohydrate binding modules (CBMs) are found in polysaccharide-targeting enzymes and increase catalytic efficiency. Because only a relatively small number of CBM structures have been solved, computational modeling represents an alternative approach in conjunction with experimental assessment of CBM functionality and ligand-binding properties. An accurate target-template sequence alignment is the crucial step during homology modeling. However, low sequence identities between target/template sequences can be a major bottleneck. We therefore incorporated the predicted hydrophilic aromatic residues (HARs) and secondary structure elements into our feature-incorporated alignment (FIA) algorithm to increase CBM alignment accuracy. An alignment performance comparison for FIA and six others was made, and the greatest average sequence identities and similarities were achieved by FIA. In addition, structure models were built for 817 representative CBMs. Our models possessed the smallest average surface-potential z scores. Besides, a large true positive value for liagnd-binding aromatic residue prediction was obtained by HAR identification. Finally, the pre-simulated CBM structures have been deposited in the Database of Simulated CBM structures (DS-CBMs). The web service is publicly available at http://dscbm.life.nthu.edu.tw/ and http://dscbm.cs.ntou.edu.tw/.

Euthyroid sick syndrome and nutritional status are correlated with hyposelenemia in hemodialysis patients.

BACKGROUND: Maintenance hemodialysis (HD) patients have altered levels of thyroid hormone (TH) in euthyroid sick syndrome, along with low T3 levels and several nutritional metabolic disturbances. Selenium (Se) is an essential trace element for living organisms, which has been shown to play a major role in thyroid hormone levels and the nutritional metabolism. The aims of the present study were to assess the changes in serum levels of selenium and their correlation with disorders of the endocrine and nutritional metabolism in HD patients. METHODS: Fifty-three uremic patients with hemodialysis were evaluated; 30 healthy volunteers served as controls. Baseline serum concentrations of total triiodothyronine (TT3), free triiodothyronine (FT3), total thyroxine (TT4), and free thyroxine (FT4) were determined by radioimmunoassay (RIA). Serum levels of thyroid-stimulating hormone (TSH) and intact parathyroid hormone (iPTH) were measured by a sensitive immunoradiometric assay (IRMA). Serum selenium was analyzed using Hitachi Z- 2000 polarized Zeeman atomic absorption spectrometry. Other metabolic variables were measured in all patients and control subjects. Multiple correlation analysis was performed among variables. RESULTS: Higher serum triglyceride, LDL-C, ApoB and lower albumin, HDL-C levels were found in subjects with HD. Mean serum selenium concentration was significantly lower in the HD group than in the control group (p<0.01). The levels of serum TT3 and FT3 in HD patients were significantly lower than in healthy control subjects (p<0.01; p<0.05, respectively), but TT4, FT4 and TSH were not different. However, serum iPTH levels were significantly higher in patients than in controls (p<0.01). In the group of HD patients, serum selenium levels were significantly positively correlated with albumin, HDL-C, TT3 and FT3 ; and negatively correlated with triglyceride (TG), LDL-C, ApoB and iPTH. Both serum TT3 and FT3 levels were significantly positively correlated with HDL-C; and negatively correlated with TG, LDL-C and ApoB. CONCLUSIONS: These data suggest that hyposelenemia in HD patients correlated with euthyroid sick syndrome with low T3 levels, and nutritional status with hyperlipidemia and hypoalbuminemia which might be involved in dysfunction in the endocrine and nutrition metabolism in dialysis patients.

Feature-incorporated alignment based ligand-binding residue prediction for carbohydrate-binding modules.

MOTIVATION: Carbohydrate-binding modules (CBMs) share similar secondary and tertiary topology, but their primary sequence identity is low. Computational identification of ligand-binding residues allows biologists to better understand the protein-carbohydrate binding mechanism. In general, functional characterization can be alternatively solved by alignment-based manners. As alignment accuracy based on conventional methods is often sensitive to sequence identity, low sequence identity among query sequences makes it difficult to precisely locate small portions of relevant features. Therefore, we propose a feature-incorporated alignment (FIA) to flexibly align conserved signatures in CBMs. Then, an FIA-based target-template prediction model was further implemented to identify functional ligand-binding residues. RESULTS: Arabidopsis thaliana CBM45 and CBM53 were used to validate the FIA-based prediction model. The predicted ligand-binding residues residing on the surface in the hypothetical structures were verified to be ligand-binding residues. In the absence of 3D structural information, FIA demonstrated significant improvement in the estimation of sequence similarity and identity for a total of 808 sequences from 11 different CBM families as compared with six leading tools by Friedman rank test.

Early change of plasma and cerebrospinal fluid arginine vasopressin in traumatic subarachnoid hemorrhage.

OBJECTIVE: To investigate the changes and effects of arginine vasopressin (AVP) in patients with acute traumatic subarachnoid hemorrhage (tSAH). METHODS: The plasma and cerebrospinal fluid (CSF) level of AVP, and intracranial pressure (ICP) were measured in a total of 21 patients within 24 hours after tSAH. The neurological status of the patients was evaluated by Glasgow Coma Scale (GCS). Correlation between AVP and ICP, GCS was analyzed respectively. Meanwhile, 18 healthy volunteers were recruited as control group. RESULTS: Compared with control group, the levels (pg/ml) of AVP in plasma and CSF (x+/-s) in tSAH group were significantly increased within 24 hours (38.72+/-24.71 vs 4.54+/-1.38 and 34.61+/-21.43 vs 4.13+/-.26, P less than 0.01), and was remarkably higher in GCS less than or equal to 8 group than GCS larger than 8 group (50.96+/-36.81 vs 25.26+/-12.87 and 44.68+/-31.72 vs 23.53+/-10.94, P less than 0.05). The CSF AVP level was correlated with ICP (r eqaul to 0.46, P less than 0.05), but no statistically significant correlation was found between plasma AVP, CSF AVP and initial GCS (r equal to -0.29, P larger than 0.05 and r equal to -0.32, P larger than 0.05, respectively). The ICP (mm Hg) in tSAH patients was elevated and higher in GCS less than or equal to 8 group than in GCS larger than 8 group (25.9+/-9.7 vs 17.6+/-5.2, P less than 0.05). CONCLUSION: Our research suggests that AVP is correlated with the severity of tSAH, and may be involved in the pathophysiological process of brain damage in the early stage after tSAH. It seems that compared with the plasma AVP concentration, CSF AVP is more related to the severity of tSAH.

Th1 polarization and low-T3 syndrome are correlated with hyperleptinemia in hemodialysis patients.

BACKGROUND: Patients undergoing chronic hemodialysis (HD) have an impaired immune response with a dysregulated Th1/Th2 cytokine network and altered the levels of thyroid hormone (TH) in euthyroid sick syndrome. Leptin, an adipocyte-secreted hormone, is considered to be a proinflammatory adipocytokine, with multiple effects on several tissues acting on the intermediate and energy metabolism. The aims of the present study were to assess the changes in serum levels of leptin and their correlation with Th1/Th2 cytokine and TH production in HD patients. METHODS: Fifty-three uremic patients with hemodialysis were evaluated; 30 healthy volunteers served as controls. Baseline serum concentrations of interleukin-2 (IL-2), sIL-2R, interferon-gamma (IFN-gamma), IL-4 and IL-10 were analyzed using ELISA. Serum levels of leptin, total triiodothyronine (TT3), free triiodothyronine (FT3), total thyroxine (TT4), free thyroxine (FT4) and thyroid-stimulating hormone (TSH) were determined by radioimmunoassay (RIA). Other metabolic variables were measured in all patients and control subjects. Multiple correlation analysis was performed among variables. RESULTS: Mean serum leptin concentration was significantly higher in HD patients than that in controls (p<0.01), especially in women (p<0.001). While the fasting serum levels of sIL-2R and Th1-type cytokines including IL-2 and IFN-gamma were significantly higher in HD patients compared with controls, Th2-type cytokine, including IL-4 and IL-10, levels did not differ between patients and controls. The serum TT3 and FT3 levels were lower in patients than controls, but TT4, FT4 and TSH were no different. Serum leptin levels in HD patients were significantly positively correlated with IL-2, IFN-gamma, sIL-2R and TSH; and negatively correlated with IL-4, IL-10, TT3 and FT3. Serum IL-2 levels correlated positively with serum IL-4, sIL-2R, TT3 and FT3. A negative correlation was observed between serum IFN-gamma and IL-4 levels in the patients. CONCLUSIONS: These data suggest that hyperleptinemia in HD patients correlated with cytokine dysregulation with a high level of Th1-type cytokines, and euthyroid sick syndrome with low T3 levels which might be involved in Th1 polarization and low-T3 syndrome in dialysis patients.