Interaction of C-type lectin-like receptors NKp65 and KACL facilitates dedicated immune recognition of human keratinocytes

Abstract

Many well-known immune-related C-type lectin-like receptors (CTLRs) such as NKG2D, CD69, and the Ly49 receptors are encoded in the natural killer gene complex (NKC). Recently, we characterized the orphan NKC gene CLEC2A encoding for KACL, a further member of the human CLEC2 family of CTLRs. In contrast to the other CLEC2 family members AICL, CD69, and LLT1, KACL expression is mostly restricted to skin. Here we show that KACL is a nondisulfide-linked homodimeric surface receptor and stimulates cytotoxicity by human NK92MI cells. We identified the corresponding activating receptor on NK92MI cells that is encoded adjacently to the CLEC2A locus and binds KACL with high affinity. This CTLR, termed NKp65, stimulates NK cytotoxicity and release of proinflammatory cytokines upon engagement of cell-bound KACL. NKp65, a distant relative of the human activating NK receptor NKp80, possesses an amino-terminal hemITAM that is required for NKp65-mediated cytotoxicity. Finally, we show that KACL expression is mainly restricted to keratinocytes. Freshly isolated keratinocytes express KACL and are capable of stimulating NKp65-expressing cells in a KACL-dependent manner. Thus, we report a unique NKC-encoded receptor-ligand system that may fulfill a dedicated function in the immunobiology of human skin.CLEC2 family | natural killer cells | NKRP1 receptors | skin

Crystal structure of extracellular domain of human lectin‐like transcript 1 (LLT1), the ligand for natural killer receptor‐P1A

“…Next, we further considered the bivalent mode, since both LLT1 and NKRP1A are believed to form disulfide-bonded homodimers on the cell surface [14][15][16]. Furthermore, the present analytical ultracentrifugation clearly showed the dominant existence of the dimers of LLT1.…”

“…Some members of the NKRP1 subfamily contain cysteine residues within the stalk region and form disulfide-linked homodimers [14][15][16]. The CTLDs are responsible for ligand recognition.…”

“…Although a slight rotation and/or transformation may be required to reflect the true complex structure, the updated LLT1-NKRP1A model sufficiently represents the binding mode for understanding LLT1-NKRP1A signaling. Next, we further considered the bivalent mode, since both LLT1 and NKRP1A are believed to form disulfide-bonded homodimers on the cell surface [14][15][16]. Furthermore, the present analytical ultracentrifugation clearly showed the dominant existence of the dimers of LLT1.…”

“…The binding affinities between the ligands and the receptors were quite diverse. LLT1 and AICL bind their ligands, NKRP1A and NKp80, respectively, with low affinity (LLT1-NKRP1A: Kdß50 μM [23], AICL-NKp80: Kdß4 μM [14]), whereas NKp65 binds to KACL with significantly higher affinity (Kdß0.01 μM) [15]. Since these differences are strongly correlated with the recognition mechanisms, further structural studies at atomic resolution will be required for their clarification.…”

“…The NKRP1 subfamily molecules are type II transmembrane glycoproteins, which contain an amino-terminal cytoplasmic domain, a single transmembrane domain followed by a stalk region, and a single extracellular C-type lectin-like domain (CTLD) [13]. Some members of the NKRP1 subfamily contain cysteine residues within the stalk region and form disulfide-linked homodimers [14][15][16]. The CTLDs are responsible for ligand recognition.…”