title: Altered glycosylation of recombinant NKp30 hampers binding to heparan sulfate: a lesson for the use of recombinant immunoreceptors as an immunological tool.
abstract: NKp30 is a natural cytotoxicity receptor expressed by human NK cells and involved in NK lytic activity. We previously published that membranal heparan sulfate serves as a coligand for human NKp30. In the present study, we complement our results by showing direct binding of recombinant NKp30 to immobilized heparin. The heparan sulfate epitope(s) on target tumor cells and the heparin epitope(s) recognized by NKp30 share similar characteristics. Warren and colleagues (Warren HS, Jones AL, Freeman C, Bettadapura J, Parish CR. 2005. Evidence that the cellular ligand for the human NK cell activation receptor NKp30 is not a heparan sulfate glycosaminoglycan. J Immunol. 175:207-212) published that NKp30 does not bind to membranal heparan sulfate on target cells and that heparan sulfate is not involved in NKp30-mediated lysis. In the current study, we examine the binding of six different recombinant NKp30s to membranal heparan sulfate and conclude that NKp30 does interact with membranal heparan sulfate. Yet, two of the six recombinant NKp30s, including the commercially available recombinant NKp30 (employed by Warren et al.) did not show heparan sulfate-dependent binding. We demonstrate that this is due to an altered glycosylation of these two recombinant NKp30s. Upon removal of its N-linked glycans, heparan sulfate-dependent binding to tumor cells and direct binding to heparin were restored. Overall, our results emphasize the importance of proper glycosylation for analysis of NKp30 binding to its ligand and that membranal heparan sulfate could serve as a coligand for NKp30. At the cellular level, soluble heparan sulfate enhanced the secretion of IFNgamma by NK-92 natural killer cells activated with anti-NKp30 monoclonal antibody. We discuss the involvement of heparan sulfate binding to NKp30 in NKp30-mediated activation of NK cells.
authors.affiliation: The Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences and the Cancer Research Center, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
authors.forename: Oren
authors.initials: O
authors.lastname: Hershkovitz
authors.forename: Mostafa
authors.initials: M
authors.lastname: Jarahian
authors.forename: Alon
authors.initials: A
authors.lastname: Zilka
authors.forename: Ahuva
authors.initials: A
authors.lastname: Bar-Ilan
authors.forename: Guy
authors.initials: G
authors.lastname: Landau
authors.forename: Sergey
authors.initials: S
authors.lastname: Jivov
authors.forename: Yoram
authors.initials: Y
authors.lastname: Tekoah
authors.forename: Rachel
authors.initials: R
authors.lastname: Glicklis
authors.forename: John T
authors.initials: JT
authors.lastname: Gallagher
authors.forename: Sabrina C
authors.initials: SC
authors.lastname: Hoffmann
authors.forename: Hagit
authors.initials: H
authors.lastname: Zer
authors.forename: Ofer
authors.initials: O
authors.lastname: Mandelboim
authors.forename: Carsten
authors.initials: C
authors.lastname: Watzl
authors.forename: Frank
authors.initials: F
authors.lastname: Momburg
authors.forename: Angel
authors.initials: A
authors.lastname: Porgador
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D006367:HeLa Cells; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D055628:Natural Cytotoxicity Triggering Receptor 3; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis; D011971:Receptors, Immunologic / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006497:Heparan Sulfate; D055628:Natural Cytotoxicity Triggering Receptor 3; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; C431710:NCR3 protein, human