Irreversible evolutionary loss of chitin-degrading ability in the chitinase-like protein Ym1 under positive selection in rodents.

Ym1 (chitinase-like 3, Chil3) expressed in mice is a nonenzymatic chitinase-like protein, which shows 67% identity with mouse acidic chitinase (Chia). Similar to Chia, Ym1 is overexpressed in asthma and parasitic infections in mouse lungs. Due to the lack of chitin-degrading activity, the biomedical role of Ym1 under these pathophysiological conditions remains to be determined. In this study, we investigated what region and amino acid changes in Ym1 resulted in the loss of enzymatic activity. Replacing two amino acids at the catalytic motif to obtain a Chia-like sequence (N136D and Q140E; MT-Ym1) did not activate the protein. We conducted a comparative study of Ym1 and Chia. We found that three protein segments-(i) the catalytic motif residues, (ii) exons 6 and 7, and (iii) exon 10-are responsible for chitinase activity loss in Ym1. We show that replacing each of these three segments in Chia that are also involved in substrate recognition and binding by the Ym1 sequence can fully abolish the enzymatic activity. In addition, we show that there have been extensive gene duplication events at the Ym1 locus specific to the rodent lineages. Consistent with this result, Ym1 orthologs from the rodent genome were under positive selection when analyzed through the CODEML program. These data suggest that numerous amino acid substitutions in the regions involved in the chitin recognition, binding, and degradation ability of the ancestor Ym1 molecule lead to the irreversible inactivation of the protein.

Origin of enhanced boric acid adsorption in light-burned magnesium oxide.

Light-burned magnesium oxide (MgO) possesses a high surface area and has attracted interest as a promising candidate for boron adsorption materials; however, the detailed molecular structures decisive for enhancing the adsorption performance have not yet been elucidated. Here, the origin of enhanced boric acid adsorption for the light-burned MgO is studied by multiple probes, including positronium (Ps) annihilation spectroscopy, Fourier transform infrared spectroscopy, and sorption experiments coupled with molecular simulations. The state-of-the-art technique of open space analysis using Ps revealed the detailed structure of the interfaces between MgO nanograins: ∼10 Šand ∼30 Šopen spaces, participating in the chemisorption of B(OH)4 - and BO3 3- simultaneously with the physisorption of neutral B(OH)3 molecules. Furthermore, in addition to the fraction of open spaces, a proton quasi-layer formed on the interior surfaces of the above-mentioned angstrom-scale open spaces was identified to be attributable for enhancing both the chemisorption and physisorption.

Comparative functional analysis between human and mouse chitotriosidase: Substitution at amino acid 218 modulates the chitinolytic and transglycosylation activity.

Chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) have been attracting research interest due to their involvement in various pathological conditions such as Gaucher's disease and asthma, respectively. Both enzymes are highly expressed in mice, while the level of AMCase mRNA was low in human tissues. In addition, the chitinolytic activity of the recombinant human AMCase was significantly lower than that of the mouse counterpart. Here, we revealed a substantially higher chitinolytic and transglycosylation activity of human Chit1 against artificial and natural chitin substrates as compared to the mouse enzyme. We found that the substitution of leucine (L) by tryptophan (W) at position 218 markedly reduced both activities in human Chit1. Conversely, the L218W substitution in mouse Chit1 increased the activity of the enzyme. These results suggest that Chit1 may compensate for the low of AMCase activity in humans, while in mice, highly active AMCase may supplements low Chit1 activity.

Structural reconfigurations of nanosheet arrays in layered minerals caused by wave irradiation: desorption mechanism of Cs from nanosheet edges.

In layered minerals typically available in soil, there exists a high concentration of nanosheet edge sites formed by overlap among two-dimensional (2D) nanosheets, which act as heavily adhesive Cs chemisorption sites. Here, we propose the application of irradiation with ultrasonic waves in addition to microwaves employing ethylenediaminetetraacetic acid (EDTA) chelate to aid the decontamination of Cs from the nanosheet edges. The energy deposition by irradiation with both micro and ultrasonic waves is found to separate the overlap among the 2D nanosheets, succeeding in the desorption of Cs from the nanosheet edges. The decontamination of Cs became more prominent with ultrasonic irradiation owing to more efficient energy deposition than that with microwaves. The desorption mechanism is highlighted based on the results of systematic studies of the local structural reconfigurations of nanosheet arrays caused by wave irradiation. Surprisingly, not the aqueous solution nor interlayer water but constitution water triggers Cs desorption from the nanosheet edges, thus opening up a future decontamination strategy with a water saving system.

Mouse acidic mammalian chitinase exhibits transglycosylation activity at somatic tissue pH.

Mouse acidic mammalian chitinase (AMCase) degrades chitin with highest efficiency at pH 2.0 and is active up to pH 8.0. Here, we report that mouse AMCase also exhibits transglycosylation activity under neutral conditions. We incubated natural and artificial chitin substrates with mouse AMCase at pH 2.0 or 7.0 and analyzed the resulting oligomers using an improved method of fluorescence-assisted carbohydrate electrophoresis. Mouse AMCase produces primarily dimers of N-acetyl-d-glucosamine [(GlcNAc)2 ] under both pH conditions while generating transglycosylated (GlcNAc)3 primarily at pH 7.0 and at lower levels at pH 2.0. These results indicate that mouse AMCase catalyzes hydrolysis as well as transglycosylation and suggest that this enzyme can play a novel role under physiological conditions in peripheral tissues, such as the lungs.

Loss and Gain of Human Acidic Mammalian Chitinase Activity by Nonsynonymous SNPs.

Acidic mammalian chitinase (AMCase) is implicated in asthma, allergic inflammation, and food processing. Little is known about genetic and evolutional regulation of chitinolytic activity of AMCase. Here, we relate human AMCase polymorphisms to the mouse AMCase, and show that the highly active variants encoded by nonsynonymous single-nucleotide polymorphisms (nsSNPs) are consistent with the mouse AMCase sequence. The chitinolytic activity of the recombinant human AMCase was significantly lower than that of the mouse counterpart. By creating mouse-human chimeric AMCase protein we found that the presence of the N-terminal region of human AMCase containing conserved active site residues reduced the enzymatic activity of the molecule. We were able to significantly increase the activity of human AMCase by amino acid substitutions encoded by nsSNPs (N45, D47, and R61) with those conserved in the mouse homologue (D45, N47, and M61). For abolition of the mouse AMCase activity, introduction of M61R mutation was sufficient. M61 is conserved in most of primates other than human and orangutan as well as in other mammals. Orangutan has I61 substitution, which also markedly reduced the activity of the mouse AMCase, indicating that the M61 is a crucial residue for the chitinolytic activity. Altogether, our data suggest that human AMCase has lost its chitinolytic activity by integration of nsSNPs during evolution and that the enzyme can be reactivated by introducing amino acids conserved in the mouse counterpart.

Quantification of Chitinase mRNA Levels in Human and Mouse Tissues by Real-Time PCR: Species-Specific Expression of Acidic Mammalian Chitinase in Stomach Tissues.

Chitinase hydrolyzes chitin, which is an N-acetyl-D-glucosamine polymer that is present in a wide range of organisms, including insects, parasites and fungi. Although mammals do not contain any endogenous chitin, humans and mice express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Because the level of expression of these chitinases is increased in many inflammatory conditions, including Gaucher disease and mouse models of asthma, both chitinases may play important roles in the pathophysiologies of these and other diseases. We recently established a quantitative PCR system using a single standard DNA and showed that AMCase mRNA is synthesized at extraordinarily high levels in mouse stomach tissues. In this study, we applied this methodology to the quantification of chitinase mRNAs in human tissues and found that both chitinase mRNAs were widely expressed in normal human tissues. Chit1 mRNA was highly expressed in the human lung, whereas AMCase mRNA was not overexpressed in normal human stomach tissues. The levels of these mRNAs in human tissues were significantly lower than the levels of housekeeping genes. Because the AMCase expression levels were quite different between the human and mouse stomach tissues, we developed a quantitative PCR system to compare the mRNA levels between human and mouse tissues using a human-mouse hybrid standard DNA. Our analysis showed that Chit1 mRNA is expressed at similar levels in normal human and mouse lung. In contrast, the AMCase expression level in human stomach was significantly lower than that expression level observed in mouse stomach. These mRNA differences between human and mouse stomach tissues were reflecting differences in the chitinolytic activities and levels of protein expression. Thus, the expression level of the AMCase in the stomach is species-specific.