期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 290, 期 5, 页码 2617-2629出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M114.588905
关键词
Biomarker; Carbohydrate-binding Protein; Crystal Structure; Glycobiology; Glycoside Hydrolase; Isothermal Titration Calorimetry (ITC); Protein Crystallization
资金
- Office of the Higher Education Commission through CHE333 PhD-THA-SUP Scholarship [CHE500307]
- Suranaree University of Technology, Thailand [SUT1-102-57-24-19]
- Biomedical Research Council of the Agency for Science, Technology, and Research (A*STAR)
Background: Human YKL-39 is currently recognized as a biomarker for osteoarthritis. Results: Crystal structures of YKL-39 reveal that chitooligosaccharide induces local conformational changes to stabilize sugarprotein complexes and that the protein contains five binding subsites for sugars. Conclusion: YKL-39 binds to chitooligosaccharide through enthalpic reactions. Significance: Our findings suggest how YKL-39 interacts with GlcNAc moieties of the natural ligands, which may possibly activate local tissue inflammation. Four crystal structures of human YKL-39 were solved in the absence and presence of chitooligosaccharides. The structure of YKL-39 comprises a major (/)(8) triose-phosphate isomerase barrel domain and a small + insertion domain. Structural analysis demonstrates that YKL-39 interacts with chitooligosaccharides through hydrogen bonds and hydrophobic interactions. The binding of chitin fragments induces local conformational changes that facilitate tight binding. Compared with other GH-18 members, YKL-39 has the least extended chitin-binding cleft, containing five subsites for sugars, namely (-3)(-2)(-1)(+1)(+2), with Trp-360 playing a prominent role in the sugar-protein interactions at the center of the chitin-binding cleft. Evaluation of binding affinities obtained from isothermal titration calorimetry and intrinsic fluorescence spectroscopy suggests that YKL-39 binds to chitooligosaccharides with K-d values in the micromolar concentration range and that the binding energies increase with the chain length. There were no significant differences between the K-d values of chitopentaose and chitohexaose, supporting the structural evidence for the five binding subsite topology. Thermodynamic analysis indicates that binding of chitooligosaccharide to YKL-39 is mainly driven by enthalpy.
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