title: Key residues at the membrane-distal surface of KACL, but not glycosylation, determine the functional interaction of the keratinocyte-specific C-type lectin-like receptor KACL with its high-affinity receptor NKp65.
abstract: Keratinocyte-associated C-type lectin (KACL) is a peculiar C-type lectin-like receptor (CTLR) due to its selective expression by human keratinocytes and cognate interaction with the genetically coupled CTLR NKp65. KACL and NKp65 are members of the CLEC2 and NKRP1 subfamilies of natural killer gene complex (NKC)-encoded CTLR, respectively. Most NKRP1 molecules are expressed on NK cells and T cells and act as receptors of CLEC2 glycoproteins with their genes being intermingled in a certain sub-region of the mammalian NKC. The reasons for the tight genetic linkage of these dedicated receptor/ligand pairs are unknown, as is the physiological expression of NKp65. Recently, we reported that the CTLR NKp65 and KACL interact with high affinity, resulting in activation of NKp65-expressing NK-92MI cells. Here, we address the molecular basis of this high-affinity interaction by analysing KACL mutants with KACL-specific monoclonal antibodies (mAb), soluble NKp65 (sNKp65) and NK-92MI-NKp65 cells. We find that none of the three N-linked carbohydrates of KACL glycoproteins significantly contributes to KACL surface expression and NKp65 interaction. However, KACL mutants with non-conservative amino acid substitutions of arginine 158 or isoleucine 161 abrogated binding of both KACL-specific mAb OMA1 and sNKp65, well in line with the blockade of NKp65-KACL interaction by OMA1. Accordingly, functional recognition of these KACL mutants by NK-92M-NKp65 cells was completely abolished. Arginine 158 and isoleucine 161 located at the membrane-distal surface of KACL were defined as residues, decisively determining functional KACL-NKp65 interaction that is independent of KACL glycosylation.
authors.affiliation: Institute for Molecular Medicine, Goethe-University Frankfurt am Main, Frankfurt am Main, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Bauer
authors.forename: Jessica
authors.initials: J
authors.lastname: Spreu
authors.forename: Christina
authors.initials: C
authors.lastname: Rohe
authors.forename: Isabel
authors.initials: I
authors.lastname: Vogler
authors.forename: Alexander
authors.initials: A
authors.lastname: Steinle
mesh_terms: D019943:Amino Acid Substitution; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry; D002478:Cells, Cultured; D006031:Glycosylation; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000276:immunology*; D037181:Lectins, C-Type / Q000235:genetics / Q000276:immunology*; D020125:Mutation, Missense; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D055630:Receptors, NK Cell Lectin-Like / Q000235:genetics / Q000276:immunology*
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; C526358:CLEC2A protein, human; C548774:KLRF2 protein, human; D037181:Lectins, C-Type; D055630:Receptors, NK Cell Lectin-Like
keywords: epitope mapping; immune receptors; natural killer gene complex; skin