Alternative splicing in the extracellular ligand binding domain of the AMPA receptors generates two variants, i.e., flip and flop. The flop variant of the GluR2 AMPA receptor is known to desensitize faster than the flip counterpart, whereas the GluR1 flip and flop variants exhibit the same rate of desensitization. However, whether the alternative splicing affects the channel opening kinetic properties of these receptors is unknown. Using a laser-pulse photolysis technique, we have characterized the channel opening kinetic mechanism for the flip and flop channels of GluR1 and GluR2, respectively. We find that the flop variant of GluR2 opens the channel, following the binding of glutamate, with the same rate as the flip channel, but closes its channel more rapidly. The difference in the kinetic properties between the two receptor isoforms can be described by a model we proposed previously in which the channel closing rate, a measure of the stability of the open channel state, controls an apparent tendency of the channel to desensitize, most likely, through the closed channel state. Specifically, the flop sequence of GluR2 promotes the channel to close more rapidly and consequently to desensitize with a faster rate than the flip sequence. For GluR1, the alternative splicing does not seem to affect the channel opening kinetics, since the flip and flop variants of GluR1 have the same channel opening rate, and the same channel closing rate. As expected and indeed observed, the flop variant desensitizes with the same rate as the flip variant does. On the basis of these results, we hypothesize that the flip/flop sequence cassette of AMPA receptors, in a sequence-dependent manner, regulates the rate of the channel closing process, in the microsecond time domain, through which it further regulates the channel desensitization in the millisecond time region.
The Clostridium cellulovorans cellulosome is comprised of a large, nonenzymatic scaffolding protein called the cellulose binding protein A (CbpA) and a number of endoglucanases/xylanases. The CbpA contains several functional domains, including a signal peptide, a cellulose binding domain (CBD), a hydrophilic domain (HLD) present four times, and a hydrophobic domain (HBD) present nine times. The functions of the domains were studied by the construction of minigenes containing the putative functional domains and by expression of the minigenes in Escherichia coli. The purified product of the CBD was able to bind to various crystalline forms of cellulose and chitin with a Kd of 1 microM. The binding capacity for CBD was a function of the crystallinity of the cellulose sample. Furthermore, the binding of CBD to Avicel was not inhibited by cellobiose or carboxymethylcellulose, suggesting that the CBD binding target was a three-dimensional structure found only in crystalline forms of cellulose. The HBD was tested for its ability to bind endoglucanases by an interaction Western as well as a sandwich enzyme immunoassay technique. The HBD was able to bind both EngB and EngD, indicating that the HBD contained an endoglucanase binding domain (EBD). Because there are nine EBD domains, it is possible that CbpA can bind up to nine endoglucanases. The role of the HLDs remains elusive. The data indicate that the cellulosome is a complex enzyme containing a scaffolding protein (CbpA) to which is attached a number of endoglucanase molecules. This arrangement allows the complex to bind and degrade crystalline cellulose, which resists degradation by the free forms of cellulosomal endoglucanases.
Neoagarooligosaccharides (NAOs), mainly comprising neoagarotetraose and neoagarohexaose, were prepared by hydrolyzing agar with β-agarase DagA from Streptomyces coelicolor, and the anti-obesity and anti-diabetic effects of NAOs on high-fat diet (HFD)-induced obesity in mice were investigated after NAOs-supplementation for 64 days. Compared to the HFD group, the HFD-0.5 group that was fed with HFD + NAOs (0.5%, w/w) showed remarkable reduction of 36% for body weight gain and 37% for food efficiency ratios without abnormal clinical signs. Furthermore, fat accumulation in the liver and development of macrovesicular steatosis induced by HFD in the HFD-0.5 group were recovered nearly to the levels found in the normal diet (ND) group. NAOs intake could also effectively reduce the size (area) of adipocytes and tissue weight gain in the perirenal and epididymal adipose tissues. The increased concentrations of total cholesterol, triglyceride, and free fatty acid in serum of the HFD group were also markedly ameliorated to the levels found in serum of the ND group after NAOs-intake in a dose dependent manner. In addition, insulin resistance and glucose intolerance induced by HFD were distinctly improved, and adiponectin concentration in the blood was notably increased. All these results strongly suggest that intake of NAOs can effectively suppress obesity and obesity-related metabolic syndromes, such as hyperlipidemia, steatosis, insulin resistance, and glucose intolerance, by inducing production of adiponectin in the HFD-induced obese mice.
This paper reviews the properties of the cellulosome and noncellulosome cellulases produced by Clostridium cellulovorans, an anaerobic, mesophilic, spore-forming microorganism that produces copious amounts of cellulase. The three major subunits of the cellulosome, CbpA, exoglucanase S (ExgS), and P100, are described, as well as the properties of the functional domains of CbpA. The properties of two noncellulosomal endoglucanases, EngD and EngF, are compared. The functions of the cellulose-binding domain (CBD) of CbpA indicate its potential uses in biotechnology.
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