Crystal Structure of Human CD69:  A C-Type Lectin-Like Activation Marker of Hematopoietic Cells^,

Abstract

CD69 is a widely expressed type II transmembrane glycoprotein related to the C-type animal lectins that exhibits regulated expression on a variety of cells of the hematopoietic lineage, including neutrophils, monocytes, T cells, B cells, natural killer (NK) cells, and platelets. Activation of T lymphocytes results in the induced expression of CD69 at the cell surface. In addition, cross-linking of CD69 by specific antibodies leads to the activation of cells bearing this receptor and to the induction of effector functions. However, the physiological ligand of CD69 is unknown. We report here the X-ray crystal structure of the extracellular C-type lectin-like domain (CTLD) of human CD69 at 2.27 A resolution. Recombinant CD69 was expressed in bacterial inclusion bodies and folded in vitro. The protein, which exists as a disulfide-linked homodimer on the cell surface, crystallizes as a symmetrical dimer, similar to those formed by the related NK cell receptors Ly49A and CD94. The structure reveals conservation of the C-type lectin-like fold, including preservation of the two alpha-helical regions found in Ly49A and mannose-binding protein (MBP). However, only one of the nine residues coordinated to Ca(2+) in MBP is conserved in CD69 and no bound Ca(2+) is evident in the crystal structure. Surprisingly, electron density suggestive of a puckered six-membered ring was discovered at a site structurally analogous to the ligand-binding sites of MBP and Ly49A. This sugar-like density may represent, or mimic, part of the natural ligand recognized by CD69.

Human Neuroblastoma Cells Trigger an Immunosuppressive Program in Monocytes by Stimulating Soluble HLA-G Release

“…A, lysis of the SH-SY-5Y neuroblastoma cell line by activated NK cells following 1 h preincubation of the effector cells with medium (control), rHLA-G, or pooled sera from four neuroblastoma patients (sHLA-G range, 184-212 ng/mL), either unmanipulated or depleted of sHLA-G by immunomagnetic beads. Cytotoxicity was tested in a 51 Cr release assay. B, lysis of the IFN-g stimulated SH-SY-5Y neuroblastoma cell line by CTLs following 1 h preincubation of the effector cells with medium (control), rHLA-G or pooled sera from four neuroblastoma patients (sHLA-G range, 184-212 ng/mL), either unmanipulated or depleted of sHLA-G by immunomagnetic beads.…”

“…To investigate if the elevated production of sHLA-G by monocytes from neuroblastoma patients was related to cell activation, surface expression of CD69, a previously reported early activation marker (51,52), was assessed by flow cytometry on freshly isolated peripheral blood monocytes from four neuroblastoma patients and four healthy controls. As shown in Fig.…”

Mouse Clr-g, a Ligand for NK Cell Activation Receptor NKR-P1F: Crystal Structure and Biophysical Properties

“…The structure closely follows the fold of the CTL family immune receptors, and the receptor dimer mimics that of human CD69 (hCD69) (25,26). Thus, this study establishes the framework for subsequent detailed structural investigations of NKR-P1-Clr protein complexes and reveals the importance of electrostatics in the specificity of interaction of NK cell receptors with their ligands.…”

Src-Dependent Syk Activation Controls CD69-Mediated Signaling and Function on Human NK Cells

“…CD69 belongs to the family of C-type lectin receptors whose genes are clustered in the NK gene complex (18 -21) and bears strong similarity to the closely related NK receptors CD94 and Ly49A (22,23). Although constitutively expressed on few cell types, CD69 is rapidly acquired following in vitro activation of T, B, and NK cells, neutrophils, and eosinophils (24,25).…”