The crystal structure of Ym1 at 1.31 Å resolution☆
Introduction
Ym1, an inducible and crystallizable protein secreted by activated peritoneal macrophages upon nematode infection, has gained credence as a prominent marker for alternative macrophage activation (Chang et al., 2001, Gordon, 2003, Hung et al., 2002, Welch et al., 2002). This type of activation is triggered by TH2 cytokines and is often STAT-6 dependent. Genomic organization and chromosomal mapping studies of Ym1 reveal the existence of a multigene family (Chang et al., 2001, Jin et al., 1998). Ym2 has been identified as a novel allergy-associated protein, and its expression is also dependent on IL-4 and IL-13 signaling (Webb et al., 2001). It should be noted, however, there is no supportive evidence to indicate that Ym1 is a chemotactic factor for eosinophils (Owhashi et al., 2000), since neither a cellular receptor mediating this effect has been identified, nor does any cytokine bear a structural resemblance to Ym1.
Several secretory proteins including Ym1, a 39-kDa human cartilage glycoprotein (hCGP39), a 40-kDa goat mammary gland glycoprotein (gMGP40), and Drosophila melanogaster imaginal disc growth factor-2 (DmIDGF2) share significant structural similarity to the family 18 chitinases (Fusetti et al., 2003, Houston et al., 2003, Mohanty et al., 2003, Synstad et al., 2004, Sun et al., 2001, Varela et al., 2002). However, these proteins do not possess the essential catalytic residues for the glycosyl hydrolysis, and are thought to be involved in tissue remodeling and/or immune response through their interactions with carbohydrate ligands and hence are defined as chi-lectins. Co-crystallization trials and intrinsic tryptophan fluorescence measurements have shown that hCGP39 binds N-acetylglucosamine (NAG)1 oligomers with micromolar affinity (Houston et al., 2003). In contrast, results using similar methods suggest a lack of carbohydrate-interacting ability for gMGP40 and DmIDGF2, even though gMGP40 shares 83% sequence identity with hCGP39 (Mohanty et al., 2003, Varela et al., 2002).
Previous characterization of Ym1 for chitinase activity and carbohydrate-binding affinity has given conflicting results. Guo et al. (2000) demonstrated that Ym1 could cleave the β-d-linkage of 4-methylumbelliferone (4-MU) NAG, NAG2, and NAG3, whereas Harbord et al. (2002) showed that isolated Ym1 could only cleave 4-MU NAG but not 4-MU NAG2 or NAG3, thus the protein functions as a weak β-N-acetylglucosaminidase rather than a chitinase. On the other hand, Chang et al. (2001) displayed that Ym1 contains no measurable chitinase activity, but preferentially interacts with N-unsubstituted hexosamines such as glucosamine oligomers and heparin. Although no oligosaccharides were added during protein isolation and crystallization, a notable electron density in the difference Fourier map at 2.5 Å resolution was observed at the “active-site” cleft, and proposed to be a monoglucosamine (Sun et al., 2001). To understand the detailed interaction between Ym1 and NAG or glucosamine, various oligomers were added for co-crystallization trials or soaked into the protein crystals. The crystal structure of Ym1 at 1.31 Å resolution was refined and compared with the other known members of the family 18 glycosidase (-like) structures.
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Experimental procedures
Mature Ym1 was purified and crystallized as described previously (Chang et al., 2001). To obtain Ym1 crystals of high quality, the isolated Ym1 crystals were solubilized using 10 mM sodium bicarbonate buffer (pH 9.0) and dialyzed against distilled water at 4 °C. Nice thin hexagonal crystals of dimensions 0.2 × 0.2 × 0.02 mm3 appeared after a period of 2–3 months. The different carbohydrate derivatives were prepared by soaking crystals in a solution containing 20 μl of mono-, di-, tri-, and
The refined Ym1 structure
All the family 18 chitinase-like proteins of known structures possess a 20- or 21-residue signal peptide, the residues are numbered as here according to the mature proteins. Similar to many other family 18 chitinase (-like) structures solved to date, the Ym1 structure contains a TIM-barrel domain with an additional small α + β domain inserted between strand β7 and helix α7, which makes up one of the sides of the substrate-binding groove (Fig. 1). Some other family 18 glycosidase (-like) proteins
Acknowledgments
The authors thank Dr. S.I. Hung for preparation of the mouse Ym1, Drs. R. Kirby and K.Y. Hwa for critical reading of the manuscript. This work was supported by National Science Council, NSC 91-3112-B-010-012 to S.H.L. and NSC91-3112-B-010-014 to N.C.C.
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The atomic coordinates and structure factors have been deposited in the Protein Data Bank (1VF8).