YKL-40, also known as human cartilage glycoprotein-39 (HC-gp39) or CHI3L1, shares structural similarities with chitotriosidase (CHIT1), an active chitinase, but lacks chitinase activity. Despite being a biomarker for inflammatory disorders and cancer, the reasons for YKL-40's inert chitinase function have remained elusive. This study reveals that the loss of chitinase activity in YKL-40 has risen from multiple sequence modifications influencing its chitin affinity. Contrary to the common belief associating the lack of chitinase activity with amino acid substitutions in the catalytic motif, attempts to activate YKL-40 by creating two amino acid mutations in the catalytic motif (MT-YKL-40) proved ineffective. Subsequent exploration that included creating chimeras of MT-YKL-40 and CHIT1 catalytic domains (CatDs) identified key exons responsible for YKL-40 inactivation. Introducing YKL-40 exons 3, 6, or 8 into CHIT1 CatD resulted in chitinase inactivation. Conversely, incorporating CHIT1 exons 3, 6, and 8 into MT-YKL-40 led to its activation. Our recombinant proteins exhibited properly formed disulfide bonds, affirming a defined structure in active molecules. Biochemical and evolutionary analysis indicated that the reduced chitinase activity of MT-YKL-40 correlates with specific amino acids in exon 3. M61I and T69W substitutions in CHIT1 CatD diminished chitinase activity and increased chitin binding. Conversely, substituting I61 with M and W69 with T in MT-YKL-40 triggered chitinase activity while reducing the chitin-binding activity. Thus, W69 plays a crucial role in a unique subsite within YKL-40. These findings emphasize that YKL-40, though retaining the structural framework of a mammalian chitinase, has evolved to recognize chitin while surrendering chitinase activity.
- MeSH
- chitin * metabolismus chemie MeSH
- chitinasy metabolismus genetika chemie MeSH
- exony MeSH
- hexosaminidasy metabolismus chemie genetika MeSH
- katalytická doména MeSH
- lidé MeSH
- molekulární evoluce MeSH
- protein CHI3L1 * metabolismus genetika chemie MeSH
- sekvence aminokyselin MeSH
- substituce aminokyselin MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) have been implicated in food processing and various pathophysiological conditions such as chronic inflammatory diseases. By combination of the colorimetric analysis and fluorophore-assisted carbohydrate electrophoresis (FACE) method, we directly compared the chitinolytic properties of mouse Chit1 and AMCase and determined their combinatory effects in artificial and natural chitin substrates processing. Chit1 and AMCase display different dynamics of chitinolytic properties through acidic to neutral conditions. At pH2.0, the activity of AMCase was higher than that of Chit1 and stronger or comparable with that of Serratia marcescens chitinase B, a well-characterized bacterium chitinase. Changes of degradation products using different substrates indicate that AMCase and Chit1 have diverse properties under various pH conditions. Exposure of the chitin substrates to both Chit1 and AMCase did not indicate any mutual interference of these enzymes and showed no synergistic effect, in contrast to observations regarding some bacterial chitinases. Our results suggest that Chit1 and AMCase have no synergistic effect under physiological conditions.
- MeSH
- bakteriální proteiny chemie genetika MeSH
- chitin chemie MeSH
- chitinasy chemie genetika MeSH
- hexosaminidasy chemie MeSH
- hydrolýza MeSH
- kolorimetrie MeSH
- koncentrace vodíkových iontů MeSH
- molekulová hmotnost MeSH
- myši MeSH
- rekombinantní proteiny MeSH
- substrátová specifita MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
On the basis of a previous study performed in our laboratory, the use of organic and inorganic amendments can significantly modify the Hg mobility in soil. We have compared the effectiveness of organic and inorganic amendments such as digestate and fly ash, respectively, reducing the Hg mobility in Chernozem and Luvisol soils differing in their physicochemical properties. Hence, the aim of this work was to compare the impact of digestate and fly ash application on the chemical and biochemical parameters in these two mercury-contaminated soils in a model batch experiment. Chernozem and Luvisol soils were artificially contaminated with Hg and then incubated under controlled conditions for 21 days. Digestate and fly ash were applied to both soils in a dose of 10 and 1.5 %, respectively, and soil samples were collected after 1, 7, 14, and 21 days of incubation. The presence of Hg in both soils negatively affected to processes such as nitrification, provoked a decline in the soil microbial biomass C (soil microbial biomass C (MBC)), and the microbial activities (arylsulfatase, and β-glucosaminidase) in both soils. Meanwhile, the digestate addition to Chernozem and Luvisol soils contaminated with Hg improved the soil chemical properties (pH, dissolved organic carbon (DOC), N (Ntot), inorganic-N forms (N-NH4 (+) and N-NO3 (-))), as consequence of high content in C and N contained in digestate. Likewise, the soil MBC and soil microbial activities (dehydrogenase, arylsulfatase, and β-glucosaminidase) were greatly enhanced by the digestate application in both soils. In contrast, fly ash application did not have a remarkable positive effect when compared to digestate in Chernozem and Luvisol soil contaminated with mercury. These results may indicate that the use of organic amendments such as digestate considerably improved the soil health in Chernozem and Luvisol compared with fly ash, alleviating the detrimental impact of Hg. Probably, the chemical properties present in digestate may determine its use as a suitable amendment for the assisted-natural attenuation of mercury-polluted soils.
- MeSH
- arylsulfatasy chemie MeSH
- bakteriální proteiny chemie MeSH
- hexosaminidasy chemie MeSH
- koncentrace vodíkových iontů MeSH
- látky znečišťující půdu analýza chemie MeSH
- oxidoreduktasy chemie MeSH
- popel uhelný chemie MeSH
- půda chemie MeSH
- půdní mikrobiologie * MeSH
- regenerace a remediace životního prostředí MeSH
- rtuť analýza chemie MeSH
- znečištění životního prostředí MeSH
- Publikační typ
- časopisecké články MeSH
