Human YKL-39 is a pseudo-chitinase with retained chitooligosaccharide-binding properties

Abstract

The chitinase-like proteins YKL-39 (chitinase 3-like-2) and YKL-40 (chitinase 3-like-1) are highly expressed in a number of human cells independent of their origin (mesenchymal, epithelial or haemapoietic). Elevated serum levels of YKL-40 have been associated with a negative outcome in a number of diseases ranging from cancer to inflammation and asthma. YKL-39 expression has been associated with osteoarthritis. However, despite the reported association with disease, the physiological or pathological role of these proteins is still very poorly understood. Although YKL-39 is homologous to the two family 18 chitinases in the human genome, it has been reported to lack any chitinase activity. In the present study, we show that human YKL-39 possesses a chitinase-like fold, but lacks key active-site residues required for catalysis. A glycan screen identified oligomers of N-acetylglucosamine as preferred binding partners. YKL-39 binds chitooligosaccharides and a newly synthesized derivative of the bisdionin chitinase-inhibitor class with micromolar affinity, through a number of conserved tryptophan residues. Strikingly, the chitinase activity of YKL-39 was recovered by reverting two non-conservative substitutions in the active site to those found in the active enzymes, suggesting that YKL-39 is a pseudo-chitinase with retention of chitinase-like ligand-binding properties.

INTRODUCTION

The human genome possesses two genes coding for active chitinases, hCHT (chitotriosidase) and the hAMCase (acidic mammalian chitinase), both from the CAZy GH18 (glycoside hydrolase 18) family [1]. hCHT is a phagocyte-specific chitinase that exists in two isoforms, a major 50 kDa and a C-terminally truncated 39 kDa isoform [2]. The C-terminal domain of the 39 kDa isoform has been shown to be involved in the binding of chitin [3], whereas the 50 kDa form is secreted from macrophages and the truncated form is not. Rather, it is routed to the lysosomes and can be used as a marker for measuring the progression of Gaucher’s disease [4–6]. AMCase has a similar domain structure, but a distinct expression pattern, being expressed in the gastrointestinal tract and lung by tissue macrophages and endothelial cells [7,8]. Expression is increased during Th2-driven pathologies, and consequently inhibition of enzymatic activity has been considered as a therapeutic strategy for allergic inflammation and asthma [8–10].

Along with active chitinases, a number of closely related proteins without detectable chitinase activity have been identified in mammalian genomes. These include YKL-40 (chitinase 3-like-1), YKL-39 (chitinase 3-like-2), Ym1/2 (chitinase 3-like-3/4), oviduct-specific glycoprotein and stabilin-1-interacting chitinase-like protein [11–17]. YKL-40 (also termed HC-gp39 or Chi3l1) was found to be secreted, along with chitotriosidase, from activated human macrophages and it was later detected in the culture supernatant of chondrocytes, synovial cells and osteoblasts [2,11]. In vitro experiments demonstrated YKL-40 induction through the cell-stress pathway when chondrocytes were exposed to LPS (lipopolysaccharide) [18]. This lectin has also been identified as a protein overexpressed in inflamed tissues in vivo [19,20]. Clinical research has shown that high levels of YKL-40 are found in the serum of patients suffering from chronic asthma and also in patients with severe arthritis [21–23]. Immune response studies have linked YKL-40 to a down-regulation of the inflammatory mediators MMP (matrix metalloprotease) 1 and MMP3 and IL-8 (interleukin-8), suggesting a protective influence under innate immune response conditions [24]. YKL-40 has been shown to have the ability to act as a growth factor for skin and fetal lung fibroblasts [25]. YKL-40 is also used as a disease marker in Type 1 Gaucher’s disease and in solid-state tumour progression (reviewed in [26]). Knockout studies of the mouse orthologue of YKL-40 [BRP-39 (breast regression protein 39)] revealed a significant reduction in the Th2 inflammatory response and an increase in cellular apoptosis under challenge with ovalbumin, which was rescued by supplementing the BRP-39 protein [27]. There is a paucity of information about the biological function of YKL-39; nevertheless, the protein has been suggested as a diagnostic marker for the diagnosis and management of osteoarthritis based on increased expression levels in osteoarthritic cartilage [28,29].

Despite a relatively high sequence identity and predicted structural similarity to the family 18 chitinases such as chitotriosidase and AMCase, chitinase-like proteins lack glycosyl hydrolase activity [30]. The loss of enzymatic activity is attributed to the substitution of the catalytic residues of the DxxDxDxE motif, which characterizes the active site of family 18 chitinases [13,31–34]. Although YKL-39 appears to have an active site incompatible with chitin hydrolysis, it may have retained the ability to bind chitin-like molecules, although the identity of the physiological ligand, if any, is currently unknown.

In the present study, we have investigated the ligand preferences of YKL-39 by screening a carbohydrate microarray, identifying chitooligosaccharides as the most likely ligands. Furthermore, YKL-39 showed micromolar binding affinity for chitooligosaccharides and chitinase inhibitors, but no measurable chitinase activity. The crystal structure of YKL-39 reveals the molecular basis for this affinity as well as for the lack of hydrolytic activity. Interestingly, the hydrolytic activity of YKL-39 can be generated by reconstructing the catalytic DxxDxDxE motif. Thus we show that YKL-39 is a pseudo-chitinase, having retained the ability to bind chitin, yet lost the ability to hydrolyse it.

MATERIALS AND METHODS

Molecular cloning

The coding sequence for YKL-39 residues 27–390 (lacking the signal peptide) was inserted into the pPIC9 Pichia pastoris expression vector. The following oligonucleotides were used as primers to amplify the 1145 bp fragment and introduce additional restriction sites (in bold letters and indicated): forward, 5′-CGGCAAGCTTACAAACTGGTTTGCTAC-3′ (HindIII) and reverse 5′-ACATACGCGTCATCTTGCCTGCTTCT-3′ (MluI). Point mutations were introduced by site-directed mutagenesis: N35Q (forward, 5′-GTTTGCTACTTTACCCAATGGTCCCAGGACCGG-3′ and reverse, 5′-CCGGTCCTGGGACCATTGGGTAAAGTAGCAAAC-3′) and S143D/I145E (forward, 5′-GATGATCTGGATGTAAGCTGGGAGTACCC-3′ and reverse 5′-CTACTAGACCTACATTCGACCCTCATGGG-3′).

The plasmid vectors were linearized with SacI before transforming into P. pastoris GS115 cells (Invitrogen) using the LiCl method according to the manufacturer’s instructions. Briefly, a 50-ml culture was grown to an D₆₀₀ of 0.8–1.0 at 30°C with shaking. The cells were pelleted at 3000 g, and washed with 25 ml of sterile water. The cell pellet was resuspended in 1 ml of 100 mM LiCl and 50 μl of cells were used per transformation. The 100 mM LiCl solution was removed by centrifugation at maximum speed for 12 s and the transformation solution was added in the following order: 240 μl sterile 50% PEG [poly(ethylene glycol)] 3000, 36 μl of 1 M LiCl, 25 μl of single-stranded salmon sperm DNA (2 mg/ml) and 5 μg of linearized plasmid DNA. After gentle resuspension the cultures were incubated at 37°C for 30 min, heat shocked at 42°C for 25 min and then plated on MD (minimal dextrose) selection plates. The plates were incubated for 2–4 days at 30°C.

Protein expression and purification

Expression cell lines were cultured in BMGY (buffered glycerol complex) medium at room temperature (22°C) with shaking until the D₆₀₀ value reached 40–80. The BMGY growth medium was removed by centrifugation and replaced by BMMY (buffered minimum methanol) medium to induce the overexpression and secretion of YKL-39. Protein expression was carried out over 72 h. Methanol was added on the second and third day to a final concentration of 1%. The supernatant was concentrated by cross-flow membrane filtration (25 kDa molecular-mass cut-off) and dialysed into 50 mM NaAc (sodium acetate; pH 5.5) for subsequent ion-exchange chromatography on CM (carboxymethyl)–Sepharose FF (fast flow). The protein eluted at approximately 30% of a 0–1 M NaCl elution gradient. Fractions containing YKL-39 were pooled and dialysed into 50 mM NaAc and 150 mM NaCl. Size-exclusion chromatography was used as a final purification step to obtain crystallization-grade protein. The protein was passed over a XK26/60 Superdex 75 column in 50 mM NaAc (pH 5.5) and 150 mM NaCl. Pure protein was dialysed into 50 mM NaAc (pH 5.5) and concentrated to 30 mg/ml.

Glycan screen

YKL-39 was analysed for glycan binding by Core H of the Consortium of Functional Glycomics (http://www.functionalglycomics.org) using the Printed Array Version 2. The microarray contained 264 natural and synthetic glycans with amino linkers, printed on to chemically modified glass microscope slides in replicates of six. YKL-39 was labelled with AlexaFluor 488 (Invitrogen protein-labelling kit) and exposed to the glycan screen for 1 h at room temperature. Binding was determined fluorimetrically after washing steps in the following three solutions: (i) 20 mM Tris/HCl (pH 7.4), 150 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂ and 0.05% Tween 20; (ii) the same buffer without Tween 20; and (iii) deionized water. Slides were spun dry and then scanned on the ProScanArray Express from PerkinElmer (excitation at 495 nm and emission measured at 520 nm).

Structure solution

Wild-type YKL-39 protein expressed in P. pastoris cells contains a small proportion of N-glycosylated product. In the interest of obtaining a homogenous sample for crystallography, the single glycosylation site (Asn³⁵) was mutated (N35Q). YKL-39 N35Q protein crystals in complex with chitohexaose (GlcNAc)₆ were obtained through co-crystallization of 30 mg/ml protein with 1 mM (GlcNAc)₆ in conditions containing 23% PEG 3000 and 0.1 M sodium citrate (pH 6). Crystals were cryoprotected in 30% ethylene glycol, 23% PEG 3000 and 0.1 M sodium citrate (pH 6), cryocooled and diffraction data were collected at the ESRF BM14 (European Synchrotron Radiation Facility Bending Magnet beamline 14). They belonged to space group C2 and provided data sets to 1.95 Å (1 Å = 0.1 nm) resolution. The structure was solved by molecular replacement using a monomer of the YKL-40 structure (deposited amino acid sequence Swiss-Prot P36222, PDB code 1HJX) [34] using MolRep [35], where it was determined there were 12 monomers in the asymmetric unit. Following rigid-body refinement and restrained refinement using Refmac5 [36], model building was conducted in Coot [37] with refinements using non-crystallographic symmetry restraints. Water molecules were added and the chitooligosaccharide ligand was fitted into the positive density in the substrate-binding site of each monomer. Final refinement including a description of anisotropy with TLS (Translation–Libration–Screw-rotation) [38] resulted in an R factor of 0.191 (R_free = 0.239). Data collection and refinement statistics are summarized in Table 1. The co-ordinates and structure factors have been deposited in the PDB under accession code 4AY1.

Table 1. Details of data collection and structure refinement for YKL-39 in complex with (GlcNAc)₆.

Values in parentheses are for the highest resolution shell. R_merge, R_work and R_free were calculated according to the standard equations: R_merge= ∑∑_i|I_i – <I|/∑∑_i I_i, where I_i and <I> are the observed intensity and the average intensity of multiple observations of symmetry identical reflections respectively; Rwork = ∑∥Fobs| – |Fcalc∥/∑ |∑ |Fobs|, where F_obs and F_calc are the observed and calculated structure factors respectively; and R_free was computed as in R_work, but only for (2%) randomly selected reflections, which were omitted from refinement.

Parameter	Value
Data collection
Space group	C121
Cell dimensions
a, b, c (Å)	255.7, 152.5, 138.2
α, β, γ (°)	90.00, 94.62, 90.00
Resolution (Å)	20.00–1.95 (2.02–1.95)
R merge	0.055 (0.424)
I/σI	17.2 (2.2)
Completeness (%)	99.6
Redundancy	3.4 (3.2)
Refinement
Resolution (Å)	20.00–1.95
Number of unique reflections	383206
Rwork/Rfree (%)	19.1/23.9
Number of atoms
Protein	37103
Ligand/ion	2437
Water	1894
B-factors
Protein	26.3
Ligand/ion	25.9
Water	30.4
RMSD
Bond lengths (Å)	0.012
Bond angles (°)	1.36

Chitinase activity assay

Reactions were conducted in 0.2 M Na₂HPO₄/0.1 M citric acid (pH 5.5) with a final reaction volume of 50 μl. The final enzyme concentrations were 2 nM for hCHT and 20 nM for YKL-39. The fluorigenic substrate 4MU-(GlcNAc)₃ (4-methylumbelliferyl-β-D-N,N′,N′’-triacetylchitotriose; Sigma) was used. Reactions were quenched with 100 μl of 3 M glycine/NaOH (pH 10) after a 60 min incubation at 37°C. Product formation was quantified on a microtiterplate fluorescence reader, FL_X800 (Bio-Tek), at excitation/emission wavelengths of 360 nm and 460 nm respectively. All of the reactions were carried out in triplicate. The readings for samples containing no enzyme were subtracted to control for non-enzymatic substrate hydrolysis. Data were analysed and plotted using GraphPad Prism software.

Tryptophan fluorescence

Tryptophan fluorescence measurements were carried out in triplicate using 1.25 ml of protein solution per scan at a concentration of 1 μM in 50 mM Tris (pH 7.5). The final added volume of ligand stock was less than 1% of the total experimental sample. Control scans were conducted in which the ligand buffer (H₂O) was used to determine the effect of dilution. Peak scans were measured between 337 and 357 nm. All of the scans were taken in duplicate with a 5 min rest period in between scans. Controls were subtracted from the experimental measurements to normalize, and data were analysed and plotted using the GraphIt software (Gecces).

Preparation of chitinase inhibitors

General remarks

Chemical reagents were purchased from Sigma–Aldrich, Fluka, Acros and Lancaster. Anhydrous DMF (dimethylformamide) was obtained from Sigma–Aldrich. All of the other solvents were obtained from Sigma–Aldrich and used as received. Analytical TLC was performed using silica gel 60 F₂₅₄ pre-coated on aluminium sheets (0.25 mm thickness). Flash chromatography was performed on columns of silica gel 60 (35–70 μm) from Fisher Scientific. Melting points were determined with a Reichert–Jung apparatus and are uncorrected. 1H NMR spectra were recorded on a JEOL JMN GX-270 (270 MHz) or on a Bruker Avance III (500 MHz) spectrometer. ¹³C NMR spectra were recorded on a Varian Mercury VX 400 (100 MHz) or on a Bruker Avance III (125 MHz) spectrometer. Chemical shifts are reported in ppm (δ) and J values are in Hz. Mass spectra were recorded using a Bruker MicroTOF autospec electrospray ionization mass spectrometer. All of the compounds submitted to biological analysis had a purity >95%, as judged by analytical HPLC. Analytical HPLC was performed on a Dionex Ultimate 3000 Instrument (Dionex), equipped with a Phenomenex Gemini 5 μm C-18 (150 mm × 4.6 mm) column.

Bisdionin C [39] and F [10] were prepared as described previously. For the preparation of bisdionin G, 8-ethyl-3-methyl-1H-purine-2,6(3H, 7H)-dione [40] was first regioselectively alkylated at N-7 with 4-methoxybenzyl chloride, according to the method of van Muijlwijk-Koezen et al. [42]. This intermediate mono-xanthine was then alkylated at N-1 with 1-(3-bromopropyl)-3, 7-dimethyl-1H-purine-2,6(3H, 7H)-dione [41], followed by removal of the 4-methoxybenzyl group under acidic conditions [10], to give bisdionin G, which was characterized by ¹H and ¹³C NMR and HRMS (high-resolution MS). The purity of all three bisdionins was >95% as judged by analytical HPLC.

8-Ethyl-7-(4-methoxybenzyl)-3-methyl-1H-purine-2,6(3H, 7H)-dione (1)

A solution of 8-ethyl-3-methyl-1H-purine-2,6(3H, 7H)-dione [40] (0.98 g, 5.03 mmol) in anhydrous DMF (20 ml) was treated with anhydrous potassium carbonate (1.04 g, 7.55 mmol), and the resulting suspension was stirred at room temperature under nitrogen for 2 h. The mixture was treated with 4-methoxybenzyl chloride (0.88 ml, 6.50 mmol) and stirred overnight at room temperature. The reaction mixture was poured into H₂O (75 ml), extracted with dichloromethane (2×50 ml), and the organic extracts were dried (Na₂SO₄), filtered and evaporated to give a yellow solid. Purification by column chromatography (0–70% acetone in dichloromethane) gave 1 (Scheme 1) as a pale yellow solid (0.80 g, 50%). Mp > 230°C; δ_H (CDCl₃, 270 MHz): 1.25 (3H, t, J 7.4, CH₃CH₂), 2.71 (2H, q, J 7.4, CH₃CH₂), 3.53 (3H, s, 3H, N-3 CH₃), 3.77 (3H, s, OCH₃), 5.41 (2H, s, NCH₂Ar), 6.84 (2H, d, J 8.8, 2 × ArH), 7.14 (2H, d, J 8.8, 2× ArH), 7.86 (1H, s, H-1); δ_C (CDCl₃, 100 MHz): 11.62, 20.53, 28.88, 47.69, 55.20, 106.96, 113.90, 114.25, 128.06, 128.58, 150.08, 151.18, 154.66, 155.92, 159.37; found (ES⁺ ) 315.1449 [M + H]⁺ , C₁₆H₁₉N₄O₃ requires 315.1452.

Scheme 1.

8-Ethyl-7-(4-methoxybenzyl)-3-methyl-1H-purine-2,6(3H, 7H)-dione (1)

1-[3-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)propyl]-8-ethyl-7-(4-methoxybenzyl)-3-methyl-1H-purine-2,6(3H, 7H)-dione (2)

A solution of 1 (0.19 g, 0.60 mmol) in anhydrous DMF (10 ml) was treated with anhydrous potassium carbonate (83 mg, 0.60 mmol), and the resulting suspension was stirred at 120°C under nitrogen for 2 h. The mixture was treated with 1-(3-bromopropyl)-3,7-dimethyl-1H-purine-2,6(3H, 7H)-dione [41] (0.88 ml, 6.50 mmol) and stirred overnight at this temperature. The cooled reaction mixture was poured into H₂O (15 ml), and the precipitate that formed was filtered off, washed with H₂O and dried in vacuo over NaOH pellets at 50°C. This gave 2 (Scheme 2) as a yellow solid (0.28 g, 87%) which was used without further purification. Mp > 230°C; δ_H (CDCl₃, 270 MHz): 1.22 (3H, t, J 7.7, CH₃CH₂), 2.00–2.05 (2H, m, CH₂CH₂CH₂), 2.67 (2H, q, J 7.7, CH₃CH₂), 3.49 (6H, s, 2× N-3 CH₃), 3.72 (3H, s, N-7 CH₃), 3.93 (3H, s, OCH₃), 4.06 (4H, t, J 6.9, CH₂CH₂CH₂), 5.43 (2H, s, NCH₂Ar), 6.80 (2H, d, J 8.8, 2× ArH), 7.11 (2H, d, J 8.8, 2× ArH), 7.46 (1H, s, H-8).

Scheme 2.

1-(3-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)propyl)-8-ethyl-7-(4-methoxybenzyl)-3-methyl-1H-purine-2,6(3H, 7H)-dione (2)

1-{3-[3,7-dimethyl-2,6-dioxo-2,3-dihydro-6H-purin-1(7H)-yl]propyl}-8-ethyl-3-methyl-1H-purine-2,6(3H, 7H)-dione (3)–Bisdionin G

A solution of 3 (Scheme 3; 0.25 g, 0.47 mmol) in trifluoracetic acid (1.5 ml) was treated with anisole (66 μl, 0.61 mmol) and concentrated sulfuric acid (2 drops). The mixture was heated at 75°C overnight, then the solvent was evaporated off. The residue was dissolved in dichloromethane and was washed with saturated aqueous NaHCO₃. The organic phase was dried (Na₂SO₄), filtered and evaporated. The crude product was purified by column chromatography (10–100% acetone in dichloromethane) to give 3 as a white solid (0.12 g, 61%). Mp > 230°C; δ_H (CDCl₃/CD₃OD, 1:1, 500 MHz): 1.33 (3H, t, J 7.8, CH₃CH₂), 2.02 (2H, quintet, J 7.3, CH₂CH₂CH₂), 2.79 (2H, q, J 7.8, CH₃CH₂), 3.52 (3H, s, N-3 CH₃), 3.53 (3H, s, N-3 CH₃), 3.96 (3H, s, N-7 CH₃), 4.05–4.10 (4H, m, CH₂CH₂CH₂), 7.76 (1H, s, H-8); δ_C (CDCl₃/CD₃OD, 1:1, 125 MHz): 12.69, 22.82, 27.60, 30.07, 30.39, 33.91, 40.05, 40.15, 108.50, 143.238, 149.44, 152.46, 156.10; found (ES⁺ ) 415.1833 [M + H]⁺ , C₁₈H₂₃N₈O₄ requires 415.1837.

Scheme 3.

1-(3-(3,7-dimethyl-2,6-dioxo-2,3-dihydro-6H-purin-1(7H)-yl)propyl)-8-ethyl-3-methyl-1H-purine-2,6(3H, 7H)-dione (3)–Bisdionin G

RESULTS AND DISCUSSION

YKL-39 selectively binds chitooligosaccharides with micromolar affinity

In an attempt to discover potential carbohydrate ligands for the chitinase-like protein YKL-39, a glycan array screen was carried out by Core H of the Consortium of Functional Glycomics. The screen revealed several potential binding partners for YKL-39 (Figure 1A). A common characteristic of these glycan ligands was the presence of β(1,4) glycosidic bonds, and the strongest binders were the chitooligosaccharides (GlcNAc)₅ and (GlcNAc)₆. To quantify the strength of this interaction, intrinsic tryptophan fluorescence was used, exploiting the presence of several conserved tryptophan residues (Figure 1B), which have been shown to play a role in substrate binding by family 18 chitinase [31,43,44]. Indeed, dose-dependent changes in intrinsic tryptophan fluorescence were observed in the presence of chitooligosaccharides (Figure 1C). This allowed determination of the dissociation constants for these ligands, indicating that YKL-39 has a higher affinity for (GlcNAc)₆ (K_d 0.6 ± 0.1 μM) than for the shorter ligand (GlcNAc)₃ (32 ± 4 μM).

Figure 1. Ligand binding and sequence conservation of YKL-39.

(A) The carbohydrate specificity of YKL-39 was investigated by screening an array of 264 glycans (Consortium of Functional Glycomics, Printed Array Version 2). YKL-39 was labelled with AlexaFluor 488 and binding was determined fluorimetrically. Relative fluorescence units (means ± S.D. for four observations) are shown for the top 25 synthetic carbohydrate structures. (B) Sequence alignement of the active chitinases hCHT and the chitinase-like proteins YKL-40 and YKL-39. Identical residues are shaded in grey and conserved tryptophan residues are marked with ▾. The active sitemotif DxxDxDxE is highlighted in black shading. (C) Changes in intrinsic tryptophan flourescence for YKL-39 and (GlcNAc)₃ and (GlcNAc)₆ fitted to a single-site-binding isotherm. Results are means ± S.D. for three independent experiments.

YKL-39 adopts a chitinase-like fold but does not possess a catalytically competent active site

To investigate how YKL-39 binds chitooligosaccharide ligands without catalysing their hydrolysis, we determined the structure of a YKL-39–chitooligosaccharide complex by X-ray crystallography. A recombinant expression system of an N-glycosylation-deficient YKL-39 mutant was developed in P. pastoris, producing 3–5 mg of protein per litre of culture. Crystals of a YKL-39–(GlcNAc)₆ complex were obtained through co-crystallisation and diffracted to 1.95 Å (Table 1). The YKL-39 structure was solved by molecular replacement, and refined to an R factor of 0.191 (R_free = 0.239) with good geometry (Table 1). The asymmetric unit contains 12 monomers with very similar conformations [RMSD (root mean square deviation) 0.2 352 = Å over C_α atoms]. The structure (Figure 2A) reveals a (β/α)₈ barrel, similar to YKL-40 (RMSD = 1.1 Å for 352 C_αs) and the catalytic domain of hCHT (RMSD = 1.1 Å for 364 C_αs), with well-defined electron density for sugars in the −2 to +2 subsites (Figure 2A). The short α/β domain inserted after strand β7, which forms the substrate-binding groove in family 18 chitinases, is likewise conserved in YKL-39 (Figure 2A).

Figure 2. Structure of the YKL-39 chitooligosaccharide complex.

(A) Stereo image of a cartoon representation of the structure of YKL-39 in complex with (GlcNAc)₆, showing four monosaccharide units of the ligand (cyan carbon atoms) with ordered electron density. Unbiased F_o-F_c, Φ_calc electron density for the ligand is shown in green (contoured at 2.25 σ). (B) Surface representation of the YKL-39 chitooligosaccharide complex, coloured by sequence conservation with hCHT (light grey = similarity, grey identity). The side chains of key solvent exposed aromatic residues are shown as sticks with = pink carbon atoms. The ligand is shown as a sticks model with cyan carbon atoms. (C) Surface representation of the hCHT chitobiose complex (PDB code 1LQ0 [31]), coloured by sequence conservation with YKL-39 (light grey = similarity, grey = identity). The side chains of key solvent exposed aromatic residues are shown as sticks with pink carbon atoms. The ligand is shown as a sticks model with cyan carbon atoms. (D) and (E) Stereo images of the ligand binding site of YKL-39 (D) and the active site of hCHT (E). The sugar ligand is shown as sticks with cyan carbon atoms. Catalytic residues in hCHT and their equivalents in YKL-39 are shown with dark grey carbon atoms. The side chains of key solvent exposed aromatic residues are shown as sticks with pink carbon atoms.

Previous studies have reported the structures of chitooligosaccharide complexes of chitinases and chitinase-like proteins (e.g. [31,34,44]). In YKL-39, the −2 to +2 sugars of the chitooligosaccharide ligand adopt a similar conformation to that observed in the YKL-40 complex (RMSD of 0.3 Å over 57 atoms). Binding of the oligosaccharide is achieved through a combination of hydrogen-bonding interactions and hydrophobic stacking between the sugar rings and surface-exposed aromatic amino acids, similar to that reported in previous chitinase–oligosaccharide complexes (Figure 2). These aromatic residues are conserved between hCHT, YKL-40 and YKL-39, except for a single tryptophan-to-tyrosine substitution in YKL-39 (Tyr¹⁰⁴ in Figures 1B and 2). Stacking between sugar ligands and tryptophans in their cognate binding proteins/enzymes is known to be an important contribution to binding affinity in protein–glycan complexes [33,45,46]. Previous mutational studies of the conserved tryptophans in active chitinases have shown a reduction or complete loss of activity [45,47,48]. Owing to the conservation of the chitinase solvent-exposed/ligand stacking tryptophans in YKL-39, and their extensive interactions with the co-crystallized chitooligosaccharide ligand, it is likely that these residues are important for chitooligosaccharide binding.

Analysis of the active site region reveals the reason for the lack of chitinase activity: the catalytic machinery is severely disrupted (Figures 2D and 2E). The active site motif DxxDxDxE, essential for the activity of GH18 chitinases [31,33,34], is not conserved in YKL-39 (Figure 1B). Catalysis involves participation of the acetamido group of N-acetylglucosamine and proceeds via a bicyclic oxazoline intermediate [31,33,44]. The last aspartate residue in the DxxDxDxE motif (Asp¹³⁸ in hCHT) positions the acetamido group for nucleophilic attack, whereas the glutamate residue (Glu¹⁴⁰) performs general acid/base catalysis (Figure 2E). Notably, in YKL-39 these catalytic residues are substituted to a serine (Ser¹⁴³) and an isoleucine (Ile¹⁴⁵) residue (Figure 2D). Strikingly, although the hCHT oligosaccharide complex shows the sugar in the −1 subsite to be in the boat conformation with the N-acetyl group aligned for nucleophilic attack, in the YKL-39–(GlcNAc)₆ complex the N-acetyl group assumes a catalytically incompetent conformation, instead occupying a pocket created by the hydrophobic substitutions in the DxxDxDxE motif (Figures 2D and 2E).

Two point mutations suffice to generate catalytically competent YKL-39

To confirm that the substitutions within the DxxDxDxE motif are the sole cause for the lack of hydrolytic activity, Ser¹⁴³ and Ile¹⁴⁵ were mutated to the corresponding active site residues (aspartate and glutamate respectively) in active chitinases. Strikingly, the S143D/I145E double mutant showed significant chitinase activity (Figure 3). Both human chitotriosidase and YKL-39 S143D/I145E showed similar affinity for the fluorigenic pseudosubstrate 4MU-(GlcNAc)₃ with a Michaelis constant (K_m) of 53 ± 3 μM for hCHT and 9 ± 1 μM for the active YKL-39 mutant. However, substrate turnover by the active YKL-39 mutant is significantly slower, with a k_cat value of 0.02 ± 0.0002 s⁻¹, as compared with 15.7 ± 0.3 s⁻¹ for hCHT (Figure 3). Slight conformational differences induced by mutations in the vicinity of the active site may account for the difference in turnover rate. Nevertheless, the implications of this experiment are that YKL-39 is a pseudo-chitinase, that is, in the course of evolution, YKL-39 appears to have retained the ability to bind to chitooligosaccharides, but lost the ability to hydrolyse them. Although chitooligosaccharides are the tightest YKL-39-binding ligands identified to date, it is not yet clear whether these represent physiological ligands for this protein.

Figure 3. Steady-state kinetics of hCHT and an active YKL–39 mutant.

Initial velocity measurements of chitinase activity of YKL-39 active mutant (S143D/I145E) and hCHT on the fluorigenic pseudosubstrate 4MU-(GlcNAc)₃, fitted to the standard Michaelis–Menten equation. Results are means ± S.E.M. for three independent experiments. From the fit the following steady-state kinetics parameters were obtained: K_m 53 ± 3 μM and k_cat = 15.7 ± 0.3 s⁻¹ for hCHT, and K_m = 9 ± 1 μM and k_cat 0.0200 ± 0.0002 s⁻¹ for the active YKL-39 mutant.

A novel bisdionin-based compound binds YKL-39 with micromolar affinity

To enable cell biological studies towards the role of YKL-39, a potent antagonist that competes with glycan binding would be a useful chemical biological tool. Rationally designed dixanthine derivatives, bisdionins, have been reported as micromolar range inhibitors of family 18 chitinases [49] (Figure 4). Structure-guided modifications to either xanthine ring can be readily incorporated to fine-tune the affinity and selectivity of these ligands, as illustrated by the recently reported bisdionin F derivative (Figure 4), that possesses submicromolar activity against hAMCase with selectivity over hCHT [10]. The bisdionins have been shown to tightly bind the active site of family 18 chitinases by forming extensive stacking interactions with the solvent-exposed tryptophan residues lining the chitooligosaccharide-binding site. To investigate whether the bisdionins would also potently bind YKL-39, we conducted tryptophan fluorescence binding assays (Figure 4). Both bisdionin C and F bind YKL-39 with K_d values in the high micromolar range, K_d = 500 ± 2 and 100 ± 7 μM respectively (Figure 4). By screening a small library of synthetic bisdionin derivatives, we identified bisdionin G, a derivative of bisdionin F carrying an ethyl substituent on the 8-position of the second xanthine moiety, that binds YKL-39 with a K_d value of 500 ± 4 μM (Figure 4). The binding mode for this compound was further confirmed by inhibition studies with the active mutant (S143D/I145E) of YKL-39. Competitive inhibition with a K_i of 120 ± 2 μM was observed (Figure 4). The structural information provided here will aid the development of YKL-39 targeted bisdionin derivatives as cell biological tools to study the function of these proteins.

Figure 4. The bisdionins and their affinity for YKL-39.

The chemical structures of the bisdionin family of chitinase inhibitors are shown, together with bisdionin-induced changes in YKL-39 intrinsic tryptophan fluorescence fitted to a single-site-binding isotherm. Results are means ± S.D. for three independent experiments. The lower right-hand panel shows inhibition of the active mutant of YKL-39, S143D/I145E, by bisdionin G. A double-reciprocal plot is shown to visualize the competitive nature of the inhibition. The K_i value was determined by non-linear regression as described in the Materials and methods section.

Conclusions

YKL-39 shares sequence homology with mammalian chitinases, but differences in expression pattern and the absence of any hydrolytic activity suggest an independent function. A glycan microarray, performed at the Consortium of Functional Glycomics, identified chitooligosaccharides as the best ligands among a selection of 264 synthetic carbohydrates. They are somewhat unlikely physiological ligands: chitin is not synthesized by mammals, and the tissue expression of YKL-39 largely precludes contact with nutrition or pathogens. Nevertheless, chitin and short fractions of the polymer remain the only binding partners of YKL-39 reported to date, and we proceeded to study the binding mode by determining the structure of a complex between YKL-39 and chitohexaose. The conformation of the ligand is strikingly similar to that observed in structures of active chitinases, including the distortion of the sugar in the −1 position, which assumes the boat conformation characteristic of enzymes utilizing a substrate-assisted catalysis mechanism involving neighbouring group participation. The results of the present study demonstrate that the lack of enzymatic activity is attributable to substitutions in the DxxDxDxE motif, which characterizes the active site of mammalian chitinases. Indeed, restoration of this motif by site-directed mutagenesis at just two positions resulted in recovery of chitinase activity.

A distinctive feature of the chitinase active site groove is a series of solvent-exposed aromatic side chains acting as hydrophobic stacking-interfaces for sugar binding. These are conserved without exception in YKL-39. In contrast with hydrogen bonding, such interactions are less specific, suggesting that other glycans possessing β-1,4 linkages could potentially be accommodated in a similar manner. The bisdionin family of chitinase inhibitors was identified as micromolar binders and may be useful tools in functional studies to compete with ligand binding. Such studies will reveal more of the enigmatic function of the chitinase-like proteins. From the conservation of glycan binding and loss of hydrolase activity of YKL-39, we may conclude that its physiological role is that of a pseudo-chitinase, whose physiological ligand is yet to be identified.

Abbreviations used

AMCase

acidic mammalian chitinase

BMGY

buffered glycerol complex

BRP-39

breast regression protein 39

DMF

dimethylformamide

GH18

glycoside hydrolase 18

hAMCase

human AMCase

hCHT

human chitotriosidase

MMP

matrix metalloprotease

NaAc

sodium acetate

PEG

poly(ethylene glycol)

RMSD

root mean square deviation

YKL-39

chitinase 3-like-2

YKL-40

chitinase 3-like-1