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Cited by 21Immune Cell Function and InteractionGlycosylation and Glycoproteins ResearchMonoclonal and Polyclonal Antibodies Research

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Natural Killer Cell Activation Receptor NKp30 Oligomerization Depends on Its N-Glycosylation

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Skořepa, Ondřej, Pazicky, Samuel, Kalousková, Barbora, Bláha, Jan, Abreu, Celeste, Ječmen, Tomáš, Rosůlek, Michal, Fish, Alexander, Sedivy, Arthur, Harlos, Karl, Dohnálek, Jan, Skálová, Tereza, Vaněk, Ondřej·2020·Cancers
DOI 10.3390/cancers12071998PMC PMC7409301
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Abstract

NKp30 is one of the main human natural killer (NK) cell activating receptors used in directed immunotherapy. The oligomerization of the NKp30 ligand binding domain depends on the length of the C-terminal stalk region, but our structural knowledge of NKp30 oligomerization and its role in signal transduction remains limited. Moreover, ligand binding of NKp30 is affected by the presence and type of N-glycosylation. In this study, we assessed whether NKp30 oligomerization depends on its N-glycosylation. Our results show that NKp30 forms oligomers when expressed in HEK293S GnTI− cell lines with simple N-glycans. However, NKp30 was detected only as monomers after enzymatic deglycosylation. Furthermore, we characterized the interaction between NKp30 and its best-studied cognate ligand, B7-H6, with respect to glycosylation and oligomerization, and we solved the crystal structure of this complex with glycosylated NKp30, revealing a new glycosylation-induced mode of NKp30 dimerization. Overall, this study provides new insights into the structural basis of NKp30 oligomerization and explains how the stalk region and glycosylation of NKp30 affect its ligand affinity. This furthers our understanding of the molecular mechanisms involved in NK cell activation, which is crucial for the successful design of novel NK cell-based targeted immunotherapeutics.

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Figure 1 Recombinant NKp30 and B7-H6 expression constructs. All constructs contain three amino acids which remain after the secretion signal is cleaved at the N-terminus and a histidine tag sequence at the C-terminus. Glycosylated asparagine residues and cysteines forming disulfide bridges are indicated, as well as the mutation of the odd cysteine C212S in B7-H6.
Figure 1 Recombinant NKp30 and B7-H6 expression constructs. All constructs contain three amino acids which remain after the secretion signal is cleaved at the N-terminus and a histidine tag sequence at the C-terminus. Glycosylated asparagine residues and cysteines forming disulfide bridges are indicated, as well as the mutation of the odd cysteine C212S in B7-H6.CC BY · PubMed Central
Figure 2 Recombinant B7-H6 is stabilized by the C212S mutation. (a) Size-exclusion chromatography (SEC) profiles of recombinantly expressed wild-type B7-H6 and its C212S mutant. (b) Fractions of the wild-type B7-H6 SEC peak were analyzed by 15% SDS-PAGE under non-reducing (left) and reducing (right) conditions. (c) Sedimentation analysis of wild-type B7-H6 at 0.5 mg/mL shown as continuous size distribution of the sedimenting species c(s). Dimeric species are marked with an asterisk in (b,c).
Figure 2 Recombinant B7-H6 is stabilized by the C212S mutation. (a) Size-exclusion chromatography (SEC) profiles of recombinantly expressed wild-type B7-H6 and its C212S mutant. (b) Fractions of the wild-type B7-H6 SEC peak were analyzed by 15% SDS-PAGE under non-reducing (left) and reducing (right) conditions. (c) Sedimentation analysis of wild-type B7-H6 at 0.5 mg/mL shown as continuous size distribution of the sedimenting species c(s). Dimeric species are marked with an asterisk in (b,c).CC BY · PubMed Central
Figure 3 SDS-PAGE analysis of deglycosylated NKp30_LBD and B7-H6 C212S mutant. Different glycosylation states are marked as WT for proteins expressed in HEK293T cells with wild-type (WT) N-glycosylation; GnTI− expressed in HEK293S GnTI− cells lacking N-acetylglucosaminyltransferase I activity which have, therefore, uniform Asn-GlcNAc2Man5 N-glycans; and DEG for the GnTI− proteins deglycosylated (DEG) with endoglycosidase Endo F1 (itself marked by an arrow).
Figure 3 SDS-PAGE analysis of deglycosylated NKp30_LBD and B7-H6 C212S mutant. Different glycosylation states are marked as WT for proteins expressed in HEK293T cells with wild-type (WT) N-glycosylation; GnTI− expressed in HEK293S GnTI− cells lacking N-acetylglucosaminyltransferase I activity which have, therefore, uniform Asn-GlcNAc2Man5 N-glycans; and DEG for the GnTI− proteins deglycosylated (DEG) with endoglycosidase Endo F1 (itself marked by an arrow).CC BY · PubMed Central
Figure 4 B7-H6 is stabilized by glycerol and saccharose addition. Differential scanning fluorimetry was used to analyze changes in protein melting temperature when adding various reagents. The concentrations given in the graph correspond to the final concentrations present in the sample.
Figure 4 B7-H6 is stabilized by glycerol and saccharose addition. Differential scanning fluorimetry was used to analyze changes in protein melting temperature when adding various reagents. The concentrations given in the graph correspond to the final concentrations present in the sample.CC BY · PubMed Central
Figure 5 Glycosylation is necessary for NKp30 oligomerization. (a) SEC-MALS analysis of NKp30_Stalk and NKp30_LBD, confirming that the stalk region is required for oligomerization. (b) SEC-MALS analysis of recombinantly expressed NKp30_Stalk with wild-type glycosylation (black), uniform, simple glycans (blue), both showing non-covalent oligomers, and deglycosylated sample, which does not form oligomers (red). (c) Normalized continuous size distributions of sedimenting species for glycosylated and deglycosylated NKp30_Stalk oligomers and for its monomeric fraction. The main peak corresponds to the NKp30_Stalk monomer, whereas a broad distribution of oligomeric species is present in glycosylated NKp30_Stalk samples.
Figure 5 Glycosylation is necessary for NKp30 oligomerization. (a) SEC-MALS analysis of NKp30_Stalk and NKp30_LBD, confirming that the stalk region is required for oligomerization. (b) SEC-MALS analysis of recombinantly expressed NKp30_Stalk with wild-type glycosylation (black), uniform, simple glycans (blue), both showing non-covalent oligomers, and deglycosylated sample, which does not form oligomers (red). (c) Normalized continuous size distributions of sedimenting species for glycosylated and deglycosylated NKp30_Stalk oligomers and for its monomeric fraction. The main peak corresponds to the NKp30_Stalk monomer, whereas a broad distribution of oligomeric species is present in glycosylated NKp30_Stalk samples.CC BY · PubMed Central
Figure 6 NKp30_Stalk oligomers cannot be saturated by B7-H6. (a) Normalized SEC (Size Exclusion Chromatography) elution profiles of NKp30_Stalk, B7-H6 and their equimolar mixture with MALS detection. (b) Sedimentation analysis of complex formation for B7-H6 and NKp30_Stalk monomeric fraction and (c) of NKp30_Stalk oligomeric fraction with B7-H6 labeled with ATTO488 dye performed at two wavelengths. Letters S and T denote the type of protein glycosylation (HEK293S GnTI− or HEK293T N-glycans, respectively).
Figure 6 NKp30_Stalk oligomers cannot be saturated by B7-H6. (a) Normalized SEC (Size Exclusion Chromatography) elution profiles of NKp30_Stalk, B7-H6 and their equimolar mixture with MALS detection. (b) Sedimentation analysis of complex formation for B7-H6 and NKp30_Stalk monomeric fraction and (c) of NKp30_Stalk oligomeric fraction with B7-H6 labeled with ATTO488 dye performed at two wavelengths. Letters S and T denote the type of protein glycosylation (HEK293S GnTI− or HEK293T N-glycans, respectively).CC BY · PubMed Central
Figure 7 Characterization of the NKp30:B7-H6 interaction by SPR and ITC. Type of protein glycosylation: T—wild-type, S—uniform, D—deglycosylated, Mono—monomeric, Oligo—oligomeric fraction of NKp30_Stalk. Titles above individual graphs describe the given experiment: SPR or ITC, protein bound to SPR sensor or present in ITC cell + protein flown over the sensor or titrated into the cell, respectively. (a) B7-H6 glycosylation does not affect NKp30 binding. (b,d) Glycosylation of NKp30 weakens its interaction with B7-H6. (c,e) Stalk region and oligomerization of NKp30 enhance its affinity to B7-H6 in SPR but not in ITC. (f) Comparison of all thermodynamic parameters measured for the NKp30:B7-H6 interaction with different protein variants.
Figure 7 Characterization of the NKp30:B7-H6 interaction by SPR and ITC. Type of protein glycosylation: T—wild-type, S—uniform, D—deglycosylated, Mono—monomeric, Oligo—oligomeric fraction of NKp30_Stalk. Titles above individual graphs describe the given experiment: SPR or ITC, protein bound to SPR sensor or present in ITC cell + protein flown over the sensor or titrated into the cell, respectively. (a) B7-H6 glycosylation does not affect NKp30 binding. (b,d) Glycosylation of NKp30 weakens its interaction with B7-H6. (c,e) Stalk region and oligomerization of NKp30 enhance its affinity to B7-H6 in SPR but not in ITC. (f) Comparison of all thermodynamic parameters measured for the NKp30:B7-H6 interaction with different protein variants.CC BY · PubMed Central
Figure 8 Crystal structure of uniformly glycosylated NKp30_LBD in complex with deglycosylated B7-H6. The asymmetric unit contains two molecules of NKp30_LBD (chains LBD_A and B) and three molecules of B7-H6 (chains B7-H6_C, D and E). Interaction interface between chains LBD_A and B7-H6_C corresponds to the interaction surface observed in the previously published structure of this complex (PDB 3PV6) [20].
Figure 8 Crystal structure of uniformly glycosylated NKp30_LBD in complex with deglycosylated B7-H6. The asymmetric unit contains two molecules of NKp30_LBD (chains LBD_A and B) and three molecules of B7-H6 (chains B7-H6_C, D and E). Interaction interface between chains LBD_A and B7-H6_C corresponds to the interaction surface observed in the previously published structure of this complex (PDB 3PV6) [20].CC BY · PubMed Central
Figure 9 A dimer of the NKp30_LBD:B7-H6 complex is observed in the presented crystal structure. Within the NKp30:B7-H6 complex, both pairs of molecules bind to each other almost identically, and the binding mode of the two proteins is also very similar to that described based on the previously published structure of the complex (PDB 3PV6) [20]. However, the structure of the NKp30_LBD dimer is distinct to the previously published structure of NKp30_LBD itself (PDB 3NOI) [19]. The dimer has a two-fold rotation symmetry, and the N-glycosylation site at Asn42 of LBD_A (GlcNAc residue highlighted as spheres) is close to C-terminus of LBD_B (highlighted in red) and vice-versa.
Figure 9 A dimer of the NKp30_LBD:B7-H6 complex is observed in the presented crystal structure. Within the NKp30:B7-H6 complex, both pairs of molecules bind to each other almost identically, and the binding mode of the two proteins is also very similar to that described based on the previously published structure of the complex (PDB 3PV6) [20]. However, the structure of the NKp30_LBD dimer is distinct to the previously published structure of NKp30_LBD itself (PDB 3NOI) [19]. The dimer has a two-fold rotation symmetry, and the N-glycosylation site at Asn42 of LBD_A (GlcNAc residue highlighted as spheres) is close to C-terminus of LBD_B (highlighted in red) and vice-versa.CC BY · PubMed Central
Figure 10 Glycosylation-induced NKp30 dimerization positions the glycans at Asn42 residues near the C-terminal stalk regions. The NKp30_LBD dimers observed in the present crystal structure PDB 6YJP and the PDB 3NOI [19] were aligned using molecules on one side of the dimer only (grey color). On the left —the N-glycosylation site at Asn42 of LBD_B, highlighted as spheres, is near the C-terminus of LBD_A (highlighted in red), whereas the Asn42 residue in LBD_3NOI is buried within the dimer interface, interacting with Glu26 and Arg28 (all highlighted as spheres). On the right—side view of the dimer interface showing the difference in arrangement of the two dimers.
Figure 10 Glycosylation-induced NKp30 dimerization positions the glycans at Asn42 residues near the C-terminal stalk regions. The NKp30_LBD dimers observed in the present crystal structure PDB 6YJP and the PDB 3NOI [19] were aligned using molecules on one side of the dimer only (grey color). On the left —the N-glycosylation site at Asn42 of LBD_B, highlighted as spheres, is near the C-terminus of LBD_A (highlighted in red), whereas the Asn42 residue in LBD_3NOI is buried within the dimer interface, interacting with Glu26 and Arg28 (all highlighted as spheres). On the right—side view of the dimer interface showing the difference in arrangement of the two dimers.CC BY · PubMed Central
Figure 11 Model of the possible position of the NKp30_LBD:B7-H6 dimeric complex, as observed in the crystal structure (PDB 6YJP), within the NK cell immune synapse. B7-H6 has a very short stalk sequence of several amino acids only, whereas NKp30 has the 15 amino acids long stalk region at its C-terminus (red lines). The stalk is long enough to confer flexibility to the NKp30 ligand binding domain. Such an arrangement would bring the membranes of both cells into very close contact, and such an effect could be further potentiated by NKp30 oligomerization. Local deformation of the NK cell plasma membrane caused by the conformational change of the stalk region induced by ligand binding might trigger signal transduction through the CD3ζ chains associated with the NKp30 transmembrane domain thanks to the interaction of CD3ζ Asp36 residue with NKp30 Arg143 residue occurring at plasma membrane which is required for NKp30 signaling [7].
Figure 11 Model of the possible position of the NKp30_LBD:B7-H6 dimeric complex, as observed in the crystal structure (PDB 6YJP), within the NK cell immune synapse. B7-H6 has a very short stalk sequence of several amino acids only, whereas NKp30 has the 15 amino acids long stalk region at its C-terminus (red lines). The stalk is long enough to confer flexibility to the NKp30 ligand binding domain. Such an arrangement would bring the membranes of both cells into very close contact, and such an effect could be further potentiated by NKp30 oligomerization. Local deformation of the NK cell plasma membrane caused by the conformational change of the stalk region induced by ligand binding might trigger signal transduction through the CD3ζ chains associated with the NKp30 transmembrane domain thanks to the interaction of CD3ζ Asp36 residue with NKp30 Arg143 residue occurring at plasma membrane which is required for NKp30 signaling [7].CC BY · PubMed Central

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Authors
Skořepa, O., Pazicky, S., Kalousková, B., Bláha, J., Abreu, C., Ječmen, T., Rosůlek, M., Fish, A., Sedivy, A., Harlos, K., Dohnálek, J., Skálová, T., & Vaněk, O.
Affiliations
  • Charles University (CZ)
  • Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, 12840 Prague, Czech Republic
  • Czech Academy of Sciences (CZ)
  • The Netherlands Cancer Institute (NL)
  • Vienna BioCenter Core Facilities (AT)
  • Centre for Human Genetics (GB)
Corresponding author
Vaněk, Ondřej
ORCID
Venue
Cancers
Publisher
MDPI AG
Published
2020-07-21
Volume
12
Issue
7
Pages
1998
ISSN
2072-6694 (ISSN-L)
DOI
10.3390/cancers12071998
PMC
PMC7409301
OpenAlex
W3044139241
Cited by
21
References
60
Citations by year
2026: 3 · 2025: 5 · 2024: 1 · 2023: 3 · 2022: 5 · 2021: 4
FWCI
1.05
Type
journal article
Language
en
Open access
Licence
Locations
  • Cancers (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Grantová Agentura České Republiky (18-10687S)
  • Ministerstvo Školství, Mládeže a Tělovýchovy (LTC17065, CZ.02.1.01/0.0/0.0/16_013/0001776, LM2015043)
  • European Regional Development Fund (CZ.1.05/1.1.00/02.0109)
  • Grantová Agentura, Univerzita Karlova (927916)
  • Univerzita Karlova v Praze (SVV 260427/2020)
  • Wellcome Trust (203141/Z/16/Z)
  • European Cooperation in Science and Technology (CA15126)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Cell Function and Interaction
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
  • Life Sciences › Immunology and Microbiology › Immunology › Monoclonal and Polyclonal Antibodies Research
Keywords
NKp30, receptor oligomerization, N-glycosylation, B7-H6, ligand binding, crystal structure, deglycosylation, NK cell activation, NK cell therapy, stalk region

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CoChat. (2026). Citation check: Natural Killer Cell Activation Receptor NKp30 Oligomerization Depends on Its N-Glycosylation. https://app.cochat.ai/papers/10.3390/cancers12071998
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Skořepa, O., Pazicky, S., Kalousková, B., Bláha, J., Abreu, C., Ječmen, T., Rosůlek, M., Fish, A., Sedivy, A., Harlos, K., Dohnálek, J., Skálová, T., & Vaněk, O. (2020). Natural Killer Cell Activation Receptor NKp30 Oligomerization Depends on Its N-Glycosylation. Cancers. https://doi.org/10.3390/cancers12071998