Structures of Xenopus Embryonic Epidermal Lectin Reveal a Conserved Mechanism of Microbial Glycan Recognition

Kittikhun Wangkanont,Darryl A Wesener,Jack A Vidani,Laura L Kiessling,Katrina T Forest

doi:10.1074/jbc.m115.709212

Kittikhun Wangkanont, Darryl A Wesener + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m115.709212

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Abstract

Intelectins (X-type lectins), broadly distributed throughout chordates, have been implicated in innate immunity. Xenopus laevis embryonic epidermal lectin (XEEL), an intelectin secreted into environmental water by the X. laevis embryo, is postulated to function as a defense against microbes. XEEL is homologous (64% identical) to human intelectin-1 (hIntL-1), which is also implicated in innate immune defense. We showed previously that hIntL-1 binds microbial glycans bearing exocyclic vicinal diol groups. It is unknown whether XEEL has the same ligand specificity. Also unclear is whether XEEL and hIntL-1 have similar quaternary structures, as XEEL lacks the corresponding cysteine residues in hIntL-1 that stabilize the disulfide-linked trimer. These observations prompted us to further characterize XEEL. We found that hIntL-1 and XEEL have similar structural features. Even without the corresponding intermolecular disulfide bonds present in hIntL-1, the carbohydrate recognition domain of XEEL (XEELCRD) forms a stable trimer in solution. The structure of XEELCRD in complex with d-glycerol-1-phosphate, a residue present in microbe-specific glycans, indicated that the exocyclic vicinal diol coordinates to a protein-bound calcium ion. This ligand-binding mode is conserved between XEEL and hIntL-1. The domain architecture of full-length XEEL is reminiscent of a barbell, with two sets of three glycan-binding sites oriented in opposite directions. This orientation is consistent with our observation that XEEL can promote the agglutination of specific serotypes of Streptococcus pneumoniae. These data support a role for XEEL in innate immunity, and they highlight structural and functional conservation of X-type lectins among chordates.

Highlights

Intelectins have been identified in diverse species from tunicates to humans [6]
Intelectins are produced by organisms ranging from tunicates to humans, and these soluble lectins have been suggested to participate in innate immunity
The x-ray crystal structures of XEELCRD and human intelectin-1 (hIntL-1) indicate that the intelectins are a discrete lectin class

Summary

Experimental Procedures

Expression and Purification of Xenopus laevis Embryonic Epidermal Lectin (XEEL)—The cDNA for XEEL (GenBankTM accession number BC087616) was purchased from Source BioScience and amplified with primer A (5Ј-TTTTTGCACTTGCATTTCCAGCAGGGCACGCTGGTTCATGGAGCCATCCGCAGTTTGAAAAGGGTTCATGTGAACAAGCTTCAATTTCTG-3Ј) and primer B (5Ј-GGTACCAAGCTCATTAACGGTAGAAGAGCATCACAGCTGCC-3Ј). X-ray Crystallography—Selenomethionine-labeled XEELCRD was redissolved in 20 mM bis-Tris (pH 6.7), 150 mM NaCl, 0.5 mM EDTA, and 5 mM CaCl2 at a concentration of 2 mg/ml, and crystallization (hanging drop vapor-diffusion) was achieved by mixing 1 ␮l of the protein solution and 1 ␮l of well. Sedimentation Equilibrium Analytical Ultracentrifugation— Recrystallized XEELCRD was dissolved in 20 mM bis-Tris (pH 6.7), 150 mM NaCl, 0.5 mM EDTA, and 5 mM CaCl2, and the concentration was determined using UV-visible absorbance spectroscopy. Full-length hexameric XEEL was expressed in High Five cells in a manner similar to the monomeric XEELCRD, except that 1% fetal bovine serum was included during expression and SIGMAFAST protease inhibitor (Sigma-Aldrich, S8820) was added after medium collection to suppress proteolysis. A scale bar was added using ImageJ, with a scale of 0.162 ␮m/pixel

Results

37 Monomer

Discussion