YKL-40 is structurally similar to chitotriosidase (CHIT1), an active chitinase, but it lacks chitin-degrading activity while retaining chitin-binding capability. Elevated YKL-40 levels are associated with inflammatory diseases and cancers, making it a valuable biomarker. We previously reported that the W69T substitution in YKL-40 significantly reduces its chitin-binding affinity, identifying W69 as a crucial binding site. In this study, we establish a novel chitin-binding affinity evaluation method using a three-step buffer system to assess the binding strength and specificity of chitin-binding proteins and apply it to characterize YKL-40's binding mechanism. Our findings confirm that YKL-40, through its key residue W69, exhibits highly specific and robust affinity to chitin. Unlike CHIT1, which has both a catalytic domain (CatD) and a chitin-binding domain (CBD) that allow for diverse binding and degradation activities, YKL-40 lacks a CBD and is specialized for specific chitin recognition without degrading it. Comparative analysis with YKL-39, which does not contain a corresponding W69 residue, highlights the unique role of this residue in YKL-40's chitin-binding activity that is potentially linked to immune and inflammatory responses. Our evaluation method clarifies YKL-40's binding properties and provides a versatile approach applicable to other chitin-binding proteins.

• Klíčová slova
• W69 residue, YKL-39, YKL-40, catalytic domain (CatD), chitin, chitin-binding activity, chitin-binding affinity assay, chitin-binding domain (CBD), chitinase-like proteins (CLPs), chitotriosidase (CHIT1),
• MeSH
• chitin * metabolismus   chemie   MeSH
• hexosaminidasy * metabolismus   chemie   MeSH
• lidé MeSH
• protein CHI3L1 * metabolismus   chemie   genetika   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• CHI3L1 protein, human MeSH Prohlížeč
• chitin * MeSH
• chitotriosidase MeSH Prohlížeč
• hexosaminidasy * MeSH
• protein CHI3L1 * MeSH

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.

• Klíčová slova
• YKL-40, asthma, biomarker, cartilage biology, chitin, chitin-binding, chitinase, enzyme inactivation, inflammation, tumor marker,
• 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
• práce podpořená grantem MeSH
• Názvy látek
• CHI3L1 protein, human MeSH Prohlížeč
• chitin * MeSH
• chitinasy MeSH
• chitotriosidase MeSH Prohlížeč
• hexosaminidasy MeSH
• protein CHI3L1 * MeSH

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.

• Klíčová slova
• chitin, chitinase, chitinase-like protein Ym1, enzyme inactivation, mouse,
• MeSH
• biologická evoluce MeSH
• chitin chemie   MeSH
• chitinasy * chemie   MeSH
• myši MeSH
• substituce aminokyselin MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Chil3 protein, mouse MeSH Prohlížeč
• chitin MeSH
• chitinasy * MeSH