Cellular Mechanisms Controlling Surfacing of AICL Glycoproteins, Cognate Ligands of the Activating NK Receptor NKp80 This information is current as Sebastian Neuss, Yvonne Bartel, Christina Born, Sandra of September 28, 2021. Weil, Joachim Koch, Christian Behrends, Meike Hoffmeister and Alexander Steinle J Immunol 2018; 201:1275-1286; Prepublished online 6 July 2018; doi: 10.4049/jimmunol.1800059 Downloaded from http://www.jimmunol.org/content/201/4/1275 References This article cites 69 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/201/4/1275.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Cellular Mechanisms Controlling Surfacing of AICL Glycoproteins, Cognate Ligands of the Activating NK Receptor NKp80 Sebastian Neuss,* Yvonne Bartel,* Christina Born,* Sandra Weil,† Joachim Koch,†,1 Christian Behrends,‡,x Meike Hoffmeister,‡,{ and Alexander Steinle* AICL glycoproteins are cognate activation-induced ligands of the C-type lectin-like receptor NKp80, which is expressed on virtually all mature human NK cells, and NKp80–AICL interaction stimulates NK cell effector functions such as cytotoxicity and cytokine secretion. Notably, AICL and NKp80 are encoded by adjacent genes in the NK gene complex and are coexpressed by human NK cells. Whereas AICL is intracellularly retained in resting NK cells, exposure of NK cells to proinflammatory cytokines results in AICL surfacing and susceptibility to NKp80-mediated NK fratricide. In this study, we characterize molecular determinants of Downloaded from AICL glycoproteins that cause intracellular retention, thereby controlling AICL surface expression. Cys87 residing within the C-type lectin-like domain not only ensures stable homodimerization of AICL glycoproteins by disulfide bonding, but Cys87 is also required for efficient cell surface expression of AICL homodimers and essential for AICL–NKp80 interaction. In contrast, cytoplasmic lysines act as negative regulators targeting AICL for proteasomal degradation. One atypical and three conventional N-linked glycosylation sites in the AICL C-type lectin-like domain critically impact maturation and surfacing of AICL, which is strictly dependent on glycosylation of at least one conventional glycosylation site. However, although the extent of conventional http://www.jimmunol.org/ N-linked glycosylation positively correlates with AICL surface expression, the atypical glycosylation site impairs AICL surfacing. Stringent control of AICL surface expression by glycosylation is reflected by the pronounced interaction of AICL with calnexin and the impaired AICL expression in calnexin-deficient cells. Collectively, our data demonstrate that AICL expression and surfacing are tightly controlled by several independent cellular posttranslational mechanisms. The Journal of Immunology, 2018, 201: 1275–1286. atural killer cells are innate lymphocytes with cytotoxic (9–11). According to both the “missing-self” and the “induced- capacity toward virus-infected, transformed, or stressed self” hypotheses, NK cells preferentially react against cells with by guest on September 28, 2021 N cells (1, 2). Hence, there is continued interest to harness poor MHC class I expression but induced expression of ligands of NK cells for cancer immunotherapy (3–5). Furthermore, NK cells activating NKRs (2, 12–14). Such cell contact–dependent effector are important producers of cytokines, such as IFN-g and TNF, responses of NK cells are regulated by a variety of germline- thereby modulating immune responses and contributing to tissue encoded activating and inhibitory receptors (1, 3) that can be di- homeostasis (6–8). More recently, the potential of NK cells to vided into Ig-like and C-type lectin-like receptors (CTLR). The mediate certain memory responses and to develop into long-lived latter are homodimeric or heterodimeric type II transmembrane cells with enhanced effector functions has been appreciated glycoproteins with a single extracellular C-type lectin-like domain (CTLD) and are encoded in a certain genomic region, termed *Institute for Molecular Medicine, Goethe-University Frankfurt am Main, 60590 the NK gene complex (NKC) (15). In humans, NKC-encoded, Frankfurt am Main, Germany; †Institute for Tumor Biology and Experimental Ther- activating CTLR include NKG2D, CD94/NKG2C, NKp80, and apy, Georg-Speyer-Haus, 60596 Frankfurt am Main, Germany; ‡Institute of Bio- chemistry II, Goethe-University Frankfurt am Main, 60590 Frankfurt am Main, NKp65, whereas CD94/NKG2A and NKR-P1A (CD161) are Germany; xMunich Cluster for Systems Neurology, Ludwig Maximilian University ITIM-bearing inhibitory receptors (16). Among these, NKR-P1A, { of Munich, 80539 Munich, Germany; and Institute of Biochemistry, Brandenburg NKp80, and NKp65 are peculiar because they engage cognate Medical School Theodor Fontane, 16816 Neuruppin, Germany 1 ligands of the CLEC2 family that are encoded in the NKC in close Current address: Affimed GmbH, Heidelberg, Germany. proximity to their respective receptors (16, 17). Like their recep- ORCIDs: 0000-0003-4439-4307 (C. Born); 0000-0001-5081-8503 (A.S.). tors, CLEC2 family members are dimeric type II glycoproteins Received for publication January 16, 2018. Accepted for publication June 8, 2018. with an amino-terminal cytosolic domain, a transmembrane do- This work was supported in part by a grant to A.S. from the German Research main, a short stalk region, and a single C-terminal CTLD, which is Council (DFG STE 828/6-2). stabilized by two or three intramolecular disulfide bonds (15, 18). Address correspondence and reprint requests to Dr. Alexander Steinle, Institute for Molecular Medicine, Goethe-University Frankfurt am Main, Theodor-Stern-Kai 7, NKR-P1A binds CLEC2D-encoded LLT1 glycoproteins expressed 60590 Frankfurt am Main, Germany. E-mail address: [email protected] on activated B cells, thereby inhibiting cytotoxicity and IFN-g se- Abbreviations used in this article: AICL-C87S, cysteine at position 87 of AICL cretion of NKR-P1A–expressing NK cells (19, 20). The activating substituted for serine; CCMA, concanamycin A; CHX, cycloheximide; CTLD, receptor NKp65 on the human NK cell line NK-92 engages C-type lectin-like domain; CTLR, C-type lectin-like receptor; ED, ectodomain; eGFP, enhanced GFP; Endo H, endoglycosidase H; ER, endoplasmic reticulum; CLEC2A-encoded KACL molecules with high affinity–stimulating FH-tag, C-terminal FLAG and hexahistidine sequence; KSHV, Kaposi sarcoma–associated cytotoxicity and IFN-g secretion (21–23). In contrast to NKp65, herpesvirus; MS, mass spectrometry; NKC, NK gene complex; PFA, paraformaldehyde; NKp80 is expressed on virtually all mature human NK cells (24, 25) PNGase F, peptide:N-glycosidase F; wt, wild-type. but also on subsets of effector memory T cells (26) and binds to Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 CLEC2B-encoded AICL glycoproteins which are inducibly expressed www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800059 1276 POSTTRANSLATIONAL CONTROL OF AICL EXPRESSION on activated myeloid cells and NK cells (25, 27). Engagement of Immunoblotting NKp80byAICLpromotesanactivatingcross-talkbetweenmono- Cells were washed twice with PBS and cell pellets lysed with ice-cold lysis cytes and NK cells (25) and between macrophages and effector buffer (1% Triton X-100, 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, and T cells (26) in the presence of inflammatory cytokines, leading to the 50 mM octyl glucopyranoside, Complete Protease Inhibitor [Roche, Mann- secretion of IFN-g and TNF. Further, NKp80 ligation contributes to heim, Germany]). Cell lysates containing 25–50 mg of total protein were then the cytolysis of malignant AICL-expressing myeloid cells, suggesting subjected to SDS-PAGE. For protein deglycosylation, cell lysates were treated with endoglycosidase H (Endo H) or peptide:N-glycosidase F (PNGase F) a role of NKp80–AICL interaction in NK-mediated surveillance of prior to SDS-PAGE (both from New England Biolabs, Ipswich, MA) myeloid leukemia cells (25, 28). according to the manufacturer’s protocol. After blotting onto a Hybond ECL Resting human NK cells were shown to contain intracellular nitrocellulose membrane (GE Healthcare Europe, Freiburg, Germany) or stores of AICL molecules (27). Therefore, NK cells simulta- polyvinylidene difluoride membrane (Carl Roth, Karlsruhe, Germany), the membrane was either probed with anti-AICL mAb 7G4 (25) or anti-FLAG neously express both NKp80 and its ligand AICL. Intracellular mAb (clone M2; Sigma-Aldrich) followed by detection of the unconjugated retention in resting lymphocytes has also been shown for the primary Ab