Characterization of the heparin/heparan sulfate binding site of the natural cytotoxicity receptor NKp46.
NKp46 is a member of a group of receptors collectively termed natural cytotoxicity receptors (NCRs) that are expressed by natural killer (NK) cells. NCRs are capable of mediating direct killing of tumor and virus-infected cells by NK cells. We have recently shown that NKp46 recognizes the heparan sulfate moieties of membranal heparan sulfate proteoglycans (HSPGs), thus enabling lysis of tumor cells by NK cells. In the current study, we further examined the residues in NKp46 that may be involved in heparan sulfate binding on tumor cells. On the basis of both the electrostatic potential map and comparison to the heparin binding site on human fibronectin, we predicted a continuous region containing the basic amino acids K133, R136, H139, R142, and K146 to be involved in NKp46 binding to heparan sulfate. Mutating these amino acids on NKp46D2 to noncharged amino acids retained its virus binding capacity but reduced its binding to tumor cells with a 10-100 fold lower K(D) when tested for direct binding to heparin. The minimal length of the heparin/heparan sulfate epitope recognized by NKp46 was eight saccharides as predicted from the structure and proven by testing heparin oligomers. Testing selectively monodesulfated heparin oligomers emphasized the specific contributions of O-sulfation, N-sulfation, and N-acetylation to epitope recognition by NKp46. The characterization of heparan sulfate binding region in NKp46 offers further insight into the identity of the ligands for NKp46 and the interaction of NK and cancers.
Zilka
Alon
A
Department of Microbiology and Immunology, Faculty of Health Sciences, and Cancer Research Center, Ben Gurion University of the Negev, Beer Sheva 84105, Israel.
Landau
Guy
G
Hershkovitz
Oren
O
Bloushtain
Noga
N
Bar-Ilan
Ahuva
A
Benchetrit
Fabrice
F
Fima
Eyal
E
van Kuppevelt
Toin H
TH
Gallagher
John T
JT
Elgavish
Sharona
S
Porgador
Angel
A
D000595:Amino Acid Sequence; D000925:Anticoagulants / Q000378:metabolism*; D001665:Binding Sites; D045744:Cell Line, Tumor; D006493:Heparin; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D055627:Natural Cytotoxicity Triggering Receptor 1; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D000925:Anticoagulants; D006493:Heparin; D006497:Heparan Sulfate; D008562:Membrane Glycoproteins; D055627:Natural Cytotoxicity Triggering Receptor 1; D011971:Receptors, Immunologic; C112260:NCR1 protein, human