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.

Bioinova a s Prague Czech Republic

Department of Chemistry and Life Science Kogakuin University Hachioji Tokyo Japan

Department of Chemistry and Life Science Kogakuin University Hachioji Tokyo Japan; Research Fellow of Japan Society for the Promotion of Science Chiyoda ku Tokyo Japan

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Bueter C.L., Specht C.A., Levitz S.M. Innate sensing of chitin and chitosan. PLoS Pathog. 2013;9 PubMed PMC

Wysokowski M., Petrenko I., Stelling A.L., Stawski D., Jesionowski T., Ehrlich H. Poriferan chitin as a versatile template for extreme biomimetics. Polymers. 2015;7:235–265.

Van Dyken S.J., Locksley R.M. Chitins and chitinase activity in airway diseases. J. Allergy Clin. Immunol. 2018;142:364–369. PubMed PMC

Lee C.G., Da Silva C.A., Dela Cruz C.S., Ahangari F., Ma B., Kang M.J., et al. Role of chitin and chitinase/chitinase-like proteins in inflammation, tissue remodeling, and injury. Annu. Rev. Physiol. 2011;73:479–501. PubMed PMC

Hollak C.E., van Weely S., van Oers M.H., Aerts J.M. Marked elevation of plasma chitotriosidase activity. A novel hallmark gaucher Disease. J. Clin. Invest. 1994;93:1288–1292. PubMed PMC

Renkema G.H., Boot R.G., Muijsers A.O., Donker-Koopman W.E., Aerts J.M. Purification and characterization of human chitotriosidase, a novel member of the chitinase family of proteins. J. Biol. Chem. 1995;270:2198–2202. PubMed

Boot R.G., Renkema G.H., Strijland A., van Zonneveld A.J., Aerts J.M. Cloning of a cDNA encoding chitotriosidase, a human chitinase produced by macrophages. J. Biol. Chem. 1995;270:26252–26256. PubMed

Boot R.G., Blommaart E.F., Swart E., Ghauharali-van der Vlugt K., Bijl N., Moe C., et al. Identification of a novel acidic mammalian chitinase distinct from chitotriosidase. J. Biol. Chem. 2001;276:6770–6778. PubMed

Bussink A.P., Speijer D., Aerts J.M., Boot R.G. Evolution of mammalian chitinase(-like) members of family 18 glycosyl hydrolases. Genetics. 2007;177:959–970. PubMed PMC

Kawada M., Hachiya Y., Arihiro A., Mizoguchi E. Role of mammalian chitinases in inflammatory conditions. Keio J. Med. 2007;56:21–27. PubMed

Hakala B.E., White C., Recklies A.D. Human cartilage gp-39, a major secretory product of articular chondrocytes and synovial cells, is a mammalian member of a chitinase protein family. J. Biol. Chem. 1993;268:25803–25810. PubMed

Rehli M., Krause S.W., Andreesen R. Molecular characterization of the gene for human cartilage gp-39 (CHI3L1), a member of the chitinase protein family and marker for late stages of macrophage differentiation. Genomics. 1997;43:221–225. PubMed

Webb D.C., McKenzie A.N., Foster P.S. Expression of the Ym2 lectin-binding protein is dependent on interleukin (IL)-4 and IL-13 signal transduction: identification of a novel allergy-associated protein. J. Biol. Chem. 2001;276:41969–41976. PubMed

Ward J.M., Yoon M., Anver M.R., Haines D.C., Kudo G., Gonzalez F.J., et al. Hyalinosis and Ym1/Ym2 gene expression in the stomach and respiratory tract of 129S4/SvJae and wild-type and CYP1A2-null B6, 129 mice. Am. J. Pathol. 2001;158:323–332. PubMed PMC

Sun Y.J., Chang N.C., Hung S.I., Chang A.C., Chou C.C., Hsiao C.D. The crystal structure of a novel mammalian lectin, Ym1, suggests a saccharide binding site. J. Biol. Chem. 2001;276:17507–17514. PubMed

Chang N.C., Hung S.I., Hwa K.Y., Kato I., Chen J.E., Liu C.H., et al. A macrophage protein, Ym1, transiently expressed during inflammation is a novel mammalian lectin. J. Biol. Chem. 2001;276:17497–17506. PubMed

Jin H.M., Copeland N.G., Gilbert D.J., Jenkins N.A., Kirkpatrick R.B., Rosenberg M. Genetic characterization of the murine Ym1 gene and identification of a cluster of highly homologous genes. Genomics. 1998;54:316–322. PubMed

Lee C.G., Hartl D., Lee G.R., Koller B., Matsuura H., Da Silva C.A., et al. Role of breast regression protein 39 (BRP-39)/chitinase 3-like-1 in Th2 and IL-13-induced tissue responses and apoptosis. J. Exp. Med. 2009;206:1149–1166. PubMed PMC

Hu B., Trinh K., Figueira W.F., Price P.A. Isolation and sequence of a novel human chondrocyte protein related to mammalian members of the chitinase protein family. J. Biol. Chem. 1996;271:19415–19420. PubMed

Schimpl M., Rush C.L., Betou M., Eggleston I.M., Recklies A.D., van Aalten D.M. Human YKL-39 is a pseudo-chitinase with retained chitooligosaccharide-binding properties. Biochem. J. 2012;446:149–157. PubMed PMC

Ranok A., Wongsantichon J., Robinson R.C., Suginta W. Structural and thermodynamic insights into chitooligosaccharide binding to human cartilage chitinase 3-like protein 2 (CHI3L2 or YKL-39) J. Biol. Chem. 2015;290:2617–2629. PubMed PMC

Johansen J.S., Williamson M.K., Rice J.S., Price P.A. Identification of proteins secreted by human osteoblastic cells in culture. J. Bone Miner. Res. 1992;7:501–512. PubMed

Johansen J.S., Jensen B.V., Roslind A., Nielsen D., Price P.A. Serum YKL-40, a new prognostic biomarker in cancer patients? Cancer Epidemiol. Biomarkers Prev. 2006;15:194–202. PubMed

Connor J.R., Dodds R.A., Emery J.G., Kirkpatrick R.B., Rosenberg M., Gowen M. Human cartilage glycoprotein 39 (HC gp-39) mRNA expression in adult and fetal chondrocytes, osteoblasts and osteocytes by in-situ hybridization. Osteoarthritis Cartilage. 2000;8:87–95. PubMed

Volck B., Ostergaard K., Johansen J.S., Garbarsch C., Price P.A. The distribution of YKL-40 in osteoarthritic and normal human articular cartilage. Scand. J. Rheumatol. 1999;28:171–179. PubMed

Johansen J.S., Stoltenberg M., Hansen M., Florescu A., Horslev-Petersen K., Lorenzen I., et al. Serum YKL-40 concentrations in patients with rheumatoid arthritis: relation to disease activity. Rheumatology (Oxford) 1999;38:618–626. PubMed

Vos K., Steenbakkers P., Miltenburg A.M., Bos E., van Den Heuvel M.W., van Hogezand R.A., et al. Raised human cartilage glycoprotein-39 plasma levels in patients with rheumatoid arthritis and other inflammatory conditions. Ann. Rheum. Dis. 2000;59:544–548. PubMed PMC

Letuve S., Kozhich A., Arouche N., Grandsaigne M., Reed J., Dombret M.C., et al. YKL-40 is elevated in patients with chronic obstructive pulmonary disease and activates alveolar macrophages. J. Immunol. 2008;181:5167–5173. PubMed

Hector A., Kormann M.S., Mack I., Latzin P., Casaulta C., Kieninger E., et al. The chitinase-like protein YKL-40 modulates cystic fibrosis lung disease. PLoS One. 2011;6 PubMed PMC

Bernardi D., Podswiadek M., Zaninotto M., Punzi L., Plebani M. YKL-40 as a marker of joint involvement in inflammatory bowel disease. Clin. Chem. 2003;49:1685–1688. PubMed

Koutroubakis I.E., Petinaki E., Dimoulios P., Vardas E., Roussomoustakaki M., Maniatis A.N., et al. Increased serum levels of YKL-40 in patients with inflammatory bowel disease. Int. J. Colorectal Dis. 2003;18:254–259. PubMed

Vind I., Johansen J.S., Price P.A., Munkholm P. Serum YKL-40, a potential new marker of disease activity in patients with inflammatory bowel disease. Scand. J. Gastroenterol. 2003;38:599–605. PubMed

Chupp G.L., Lee C.G., Jarjour N., Shim Y.M., Holm C.T., He S., et al. A chitinase-like protein in the lung and circulation of patients with severe asthma. N. Engl. J. Med. 2007;357:2016–2027. PubMed

Johansen J.S., Moller S., Price P.A., Bendtsen F., Junge J., Garbarsch C., et al. Plasma YKL-40: a new potential marker of fibrosis in patients with alcoholic cirrhosis? Scand. J. Gastroenterol. 1997;32:582–590. PubMed

Johansen J.S., Cintin C., Jorgensen M., Kamby C., Price P.A. Serum YKL-40: a new potential marker of prognosis and location of metastases of patients with recurrent breast cancer. Eur. J. Cancer. 1995;31A:1437–1442. PubMed

Cintin C., Johansen J.S., Christensen I.J., Price P.A., Sorensen S., Nielsen H.J. Serum YKL-40 and colorectal cancer. Br. J. Cancer. 1999;79:1494–1499. PubMed PMC

Dela Cruz C.S., Liu W., He C.H., Jacoby A., Gornitzky A., Ma B., et al. Chitinase 3-like-1 promotes Streptococcus pneumoniae killing and augments host tolerance to lung antibacterial responses. Cell Host Microbe. 2012;12:34–46. PubMed PMC

Schmidt I.M., Hall I.E., Kale S., Lee S., He C.H., Lee Y., et al. Chitinase-like protein Brp-39/YKL-40 modulates the renal response to ischemic injury and predicts delayed allograft function. J. Am. Soc. Nephrol. 2013;24:309–319. PubMed PMC

He C.H., Lee C.G., Dela Cruz C.S., Lee C.M., Zhou Y., Ahangari F., et al. Chitinase 3-like 1 regulates cellular and tissue responses via IL-13 receptor alpha2. Cell Rep. 2013;4:830–841. PubMed PMC

Xu N., Bo Q., Shao R., Liang J., Zhai Y., Yang S., et al. Chitinase-3-like-1 promotes M2 macrophage differentiation and induces choroidal neovascularization in neovascular age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 2019;60:4596–4605. PubMed

Lee C.M., He C.H., Nour A.M., Zhou Y., Ma B., Park J.W., et al. IL-13Ralpha2 uses TMEM219 in chitinase 3-like-1-induced signalling and effector responses. Nat. Commun. 2016;7 PubMed PMC

Zhou Y., He C.H., Yang D.S., Nguyen T., Cao Y., Kamle S., et al. Galectin-3 interacts with the CHI3L1 axis and contributes to Hermansky-Pudlak Syndrome lung disease. J. Immunol. 2018;200:2140–2153. PubMed PMC

Geng B., Pan J., Zhao T., Ji J., Zhang C., Che Y., et al. Chitinase 3-like 1-CD44 interaction promotes metastasis and epithelial-to-mesenchymal transition through beta-catenin/Erk/Akt signaling in gastric cancer. J. Exp. Clin. Cancer Res. 2018;37:208. PubMed PMC

Nishikawa K.C., Millis A.J. gp38k (CHI3L1) is a novel adhesion and migration factor for vascular cells. Exp. Cell Res. 2003;287:79–87. PubMed

Ngernyuang N., Yan W., Schwartz L.M., Oh D., Liu Y.B., Chen H., et al. A heparin binding motif rich in arginine and lysine is the functional domain of YKL-40. Neoplasia. 2018;20:182–192. PubMed PMC

Renkema G.H., Boot R.G., Au F.L., Donker-Koopman W.E., Strijland A., Muijsers A.O., et al. Chitotriosidase, a chitinase, and the 39-kDa human cartilage glycoprotein, a chitin-binding lectin, are homologues of family 18 glycosyl hydrolases secreted by human macrophages. Eur. J. Biochem. 1998;251:504–509. PubMed

Zhao T., Su Z., Li Y., Zhang X., You Q. Chitinase-3 like-protein-1 function and its role in diseases. Signal Transduct. Target. Ther. 2020;5:201. PubMed PMC

Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 1991;280:309–316. PubMed PMC

Cantarel B.L., Coutinho P.M., Rancurel C., Bernard T., Lombard V., Henrissat B. The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res. 2009;37:D233. PubMed PMC

Watanabe T., Kobori K., Miyashita K., Fujii T., Sakai H., Uchida M., et al. Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity. J. Biol. Chem. 1993;268:18567–18572. PubMed

Tabata E., Itoigawa A., Koinuma T., Tayama H., Kashimura A., Sakaguchi M., et al. Noninsect-based diet leads to structural and functional changes of acidic chitinase in Carnivora. Mol. Biol. Evol. 2022;39 PubMed PMC

Okawa K., Ohno M., Kashimura A., Kimura M., Kobayashi Y., Sakaguchi M., et al. Loss and gain of human acidic mammalian chitinase activity by nonsynonymous SNPs. Mol. Biol. Evol. 2016;33:3183–3193. PubMed PMC

Kimura M., Watanabe T., Sekine K., Ishizuka H., Ikejiri A., Sakaguchi M., et al. Comparative functional analysis between human and mouse chitotriosidase: substitution at amino acid 218 modulates the chitinolytic and transglycosylation activity. Int. J. Biol. Macromol. 2020;164:2895–2902. PubMed

Fusetti F., von Moeller H., Houston D., Rozeboom H.J., Dijkstra B.W., Boot R.G., et al. Structure of human chitotriosidase. Implications for specific inhibitor design and function of mammalian chitinase-like lectins. J. Biol. Chem. 2002;277:25537–25544. PubMed

Fusetti F., Pijning T., Kalk K.H., Bos E., Dijkstra B.W. Crystal structure and carbohydrate-binding properties of the human cartilage glycoprotein-39. J. Biol. Chem. 2003;278:37753–37760. PubMed

Houston D.R., Recklies A.D., Krupa J.C., van Aalten D.M. Structure and ligand-induced conformational change of the 39-kDa glycoprotein from human articular chondrocytes. J. Biol. Chem. 2003;278:30206–30212. PubMed

Olland A.M., Strand J., Presman E., Czerwinski R., Joseph-McCarthy D., Krykbaev R., et al. Triad of polar residues implicated in pH specificity of acidic mammalian chitinase. Protein Sci. 2009;18:569–578. PubMed PMC

Yang Z. Paml 4: phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 2007;24:1586–1591. PubMed

Okawa K., Tabata E., Kida Y., Uno K., Suzuki H., Kamaya M., et al. Irreversible evolutionary loss of chitin-degrading ability in the chitinase-like protein Ym1 under positive selection in rodents. Protein Sci. 2023;32 PubMed PMC

Kashimura A., Okawa K., Ishikawa K., Kida Y., Iwabuchi K., Matsushima Y., et al. Protein A-mouse acidic mammalian chitinase-V5-His expressed in periplasmic space of Escherichia coli possesses chitinase functions comparable to CHO-expressed protein. PLoS One. 2013;8 PubMed PMC

Uehara M., Tabata E., Okuda M., Maruyama Y., Matoska V., Bauer P.O., et al. Robust chitinolytic activity of crab-eating monkey (Macaca fascicularis) acidic chitinase under a broad pH and temperature range. Sci. Rep. 2021;11 PubMed PMC

Uehara M., Takasaki C., Wakita S., Sugahara Y., Tabata E., Matoska V., et al. Crab-eating monkey acidic chitinase (CHIA) efficiently degrades chitin and chitosan under acidic and high-temperature conditions. Molecules. 2022;27:409. PubMed PMC

Kashimura A., Kimura M., Okawa K., Suzuki H., Ukita A., Wakita S., et al. Functional properties of the catalytic domain of mouse acidic mammalian chitinase expressed in Escherichia coli. Int. J. Mol. Sci. 2015;16:4028–4042. PubMed PMC

Tabata E., Wakita S., Kashimura A., Sugahara Y., Matoska V., Bauer P.O., et al. Residues of acidic chitinase cause chitinolytic activity degrading chitosan in porcine pepsin preparations. Sci. Rep. 2019;9 PubMed PMC

Jumper J., Evans R., Pritzel A., Green T., Figurnov M., Ronneberger O., et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596:583–589. PubMed PMC

Tunyasuvunakool K., Adler J., Wu Z., Green T., Zielinski M., Zidek A., et al. Highly accurate protein structure prediction for the human proteome. Nature. 2021;596:590–596. PubMed PMC

Pettersen E.F., Goddard T.D., Huang C.C., Meng E.C., Couch G.S., Croll T.I., et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 2021;30:70–82. PubMed PMC

A Simplified Method for Evaluating Chitin-Binding Activity Applied to YKL-40 (HC-gp39, CHI3L1) and Chitotriosidase