C-Type Lectin Receptor Dectin-2 Binds to an Endogenous Protein β-Glucuronidase on Dendritic Cells

C-type lectin receptors (CLRs) recognize pathogen-derived ligands and abnormal self that trigger protective immune responses. However, the precise nature of self ligands recognized by CLRs remains to be determined. Here, we found that Dectin-2 recognizes bone marrow-derived dendritic cells (BMDCs) using Dectin-2-expressing reporter cells. This activity was inhibited by an excessive amount of mannose, and by the mutation of mannose-binding motif in Dectin-2. β-glucuronidase (Gusb) was identified as a protein bound to Dectin-2 and mutations of N-glycosylation sites in Gusb impaired the binding of Gusb to Dectin-2. Overexpression of Gusb in a macrophage cell line conferred an ability to stimulate Dectin-2-expressing reporter cells. Our study suggests that a glycosylated protein with mannose-related structure is recognized by Dectin-2.


Introduction
Innate immune responses are initiated by pattern recognition receptors (PRRs) such as Tolllike receptors (TLRs), NOD-like receptors, RIG-I like helicase and C-type lectin receptors (CLRs) that recognize pathogen-associated molecular patterns (PAMPs) [1]. C-type lectins are defined as a family of proteins that bind carbohydrates in the presence of calcium. CLRs comprise at least one carbohydrate recognition domain (CRD) that in most cases binds sugars [2]. After recognition of ligands, CLRs activate innate immune responses via an integral immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic tail or via association with ITAM-bearing adaptor molecules such as FcRγ and DAP-12 [3].
It is widely accepted that glycosylation is one of the major post-translational modifications in eukaryote including fungi and mice. Glycosylation can be occurred on Asn residue (Nlinked glycosylation) or Ser/Thr residues of polypeptide structures (O-linked glycosylation) and has an impact on characteristics of proteins such as half life, localization and structure [16]. In mice, particular glycan structures on pathogenic fungi are recognized by CLRs as PAMPs [4][5][6][7]9]. However, little is known about whether endogenous glycosylation status can be also sensed by CLRs.
In the present study, we searched for endogenous molecules by using Dectin-2-reporter cells [9] and Dectin-2-Ig-fusion proteins, and identified β-glucuronidase as an endogenous protein recognized by Dectin-2 in BMDCs.

Materials and Methods
Experimental animals C57BL/6 mice were obtained from Japan Clea (Tokyo, Japan). All mice were maintained in a filtered-air-laminar-flow enclosure and given standard laboratory food and water ad libitum. All animal experimental protocols were approved by the committee of Ethics on Animal Experiment, Faculty of Medical Sciences, Kyushu University.

Antibodies and flowcytometric analysis
Phycoerythrin-conjugated anti-CD11c mAb (HL3) was from BD Bioscience. Anti-Flag mAbs (1E6 and M2) were from Wako and Sigma-Aldrich, respectively. Anti-actin Ab (4970) was from Cell Signaling Technology. HRP-conjugated polyclonal anti-hIg antibody was from Jackson ImmunoResearch. Polyclonal anti-Gusb Ab was from proteintech. Protein A sepharose beads and streptavidin sepharose beads were from GE healthcare. For the flowcytometric analysis, stained cells with indicated mAbs were acquired on flow cytometer. Viable cells were analyzed after gating on cells negative for Propidium iodide (PI) (Sigma-Aldrich). The data were analyzed using FlowJo software version 10.0.7 (Tree Star).

Cells
For the development of BMDCs, bone marrow (BM) cells were collected from femurs, pelvises and fibulas and 1 × 10 6 cells of BM cells were cultured in RPMI 1640 medium with 10% FBS in the presence of GM-CSF. After the culture, cells were harvested on indicated days. 2B4-NFAT-GFP reporter cells expressing murine Dectin-2 or Dectin-2 QPD were prepared as described previously [9]. Gusb-overexpressing RAW264.7 cells were established by retrovirusmediated gene transfer.

Myeloid cells express molecule(s) recognized by Dectin-2
To search for molecules recognized by CLRs, Dectin-2-expressing reporter cells were cultured with BMDCs or bone marrow-derived macrophages (BMDMs) and then GFP expression was analyzed as a read-out [9]. As a negative control, FcRγ-expressing reporter cells were used. As shown in Fig 1A, GFP expressions on Dectin-2-reporter cells were induced after the co-culture with both BMDCs and BMDMs. BMDCs had a stronger activity to induce the GFP expression than those in BMDMs (Fig 1A). The GFP expression levels on Dectin-2-reporter cells correlated with the number of BMDCs (Fig 1B). Culture supernatants of BMDCs also had an activity to induce the GFP expression, implying that Dectin-2 might recognize molecules released into the culture medium ( Fig 1C). We next asked whether molecules recognized by Dectin-2 were induced during differentiation of BMDCs. Before the differentiation of BMDCs, bone marrow cells had relatively weak activity to induce GFP expression on Dectin-2 reporter cells. After addition of GM-CSF, the GFP expression was enhanced as the percentage of CD11c + DCs increased (Fig 1D). These results suggest that Dectin-2 recognizes molecules which are mainly expressed on BMDCs.
Since CLRs are defined as carbohydrates-binding proteins, we next asked whether carbohydrates were involved in Dectin-2 recognition of molecules in BMDCs. EPN motif, which is mannose-binding motif for C-type lectin, is crucial for Dectin-2 recognition to α-mannan [9,17]. To test whether the motif was involved in recognition of molecules on BMDCs, we set out two experiments. First, we generated reporter cells harboring mutated EPN motif to galactosebinding motif in Dectin-2 (Dectin-2 QPD ). After the co-culture of Dectin-2 QPD with BMDCs, GFP expression was not induced and the expression level was comparable to that of FcRγexpressing control cells (Fig 1E). Second, to examine whether blocking of mannose binding in EPN motif had an impact on interaction between Dectin-2 and the BMDCs, an excessive amount of mannose was added during the co-culture. We found that blocking of EPN motif by addition of mannose, but not other monosaccharides such as glucose and galactose, greatly reduced Dectin-2-mediated ligand recognition activity as judged by GFP expressions (Fig 1F). These results suggest that Dectin-2 may recognize BMDCs through mannose-related structure.

β-glucuronidase is an endogenous molecule bound to Dectin-2
To search for the candidate molecules recognized by Dectin-2, we generated a soluble protein harboring extracellular domain of Dectin-2 or Dectin-2 QPD , which was fused to the carboxyl terminus of the human immunoglobulin Fc region (Dectin-2-Ig or Dectin-2 QPD -Ig). By using Dectin-2-Ig as a probe, Dectin-2-binding molecules were purified from lysates of BMDCs. The candidate proteins specifically bound to Dectin-2-Ig, but not beads itself, Ig only and Dectin-2 QPD -Ig, were detected around 75 kilo-Dalton (kD) (Fig 2A). We observed similar size of the bands when Dectin-2-binding proteins were eluted by addition of an excessive amount of mannose ( Fig 2B). The band at 75 kD was excised and analyzed by Mass spectrometry. Further analysis of the band with Mascot search engine (http://www.matrixscience.com) revealed that this fraction contained β-glucuronidase (Gusb), Hspa9, Hspa8, Lgals3bp, Krt10 and, based on the Mascot score, Gusb had the highest probability among the detected proteins (Fig 2A and  2B). Hspa9, Hspa8, Lgals3bp and Krt10 were also detected in the band at 75kD from samples purified with Ig. These results suggest that Gusb was the most probable candidate protein bound to Dectin-2. Indeed, Gusb is highly expressed in BMDCs, and was precipitated by Dectin-2-Ig, but not Ig (Fig 2C). Consistent with the observation that culture supernatants of BMDCs activated Dectin-2-expressing cells (Fig 1C), Gusb was also detected in the supernatants by Dectin-2-Ig (Fig 2D). In order to examine whether Gusb expression was sufficient for being recognized by Dectin-2, Gusb was transduced into macrophage cell line RAW264.7 cells and co-cultured with Dectin-2-expressing reporter cells. GFP expression in Dectin-2-expressing reporter cells was significantly enhanced after the co-culture with Gusb-overexpressing RAW264.7 cells as compared with parental cells (Fig 2E) and this activity was reduced in the presence of an excess amount of mannose (Fig 2F). These results indicate that Gusb is an endogenous protein recognized by Dectin-2.

Discussion
In the present study, we identified an endogenous protein Gusb as a Dectin-2-binding protein.
However, the specificity of protein ligands for Dectin-2 is still an open question. As Gusb-deficient BMDCs were capable of activating Dectin-2-reporter cells, other protein(s) are likely to provide the same particular glycans to be recognized by Dectin-2. Given that Gusb is one of the most abundant proteins in myeloid cells [22] but not lymphocytes (data not shown), more detail analysis using sensitive Mass spectrometry may identify other "career" proteins which are less abundantly expressed. Although the precise glycan structure on Gusb has not been determined yet, our data suggest that mannose is likely to be involved in the recognition by Dectin-2 ( Fig 1F).
It has been reported that Dectin-2 recognizes terminal mannose of N-linked glycans in fungi [5,7]. However, recent studies showed that Dectin-2 also recognizes lipomannan, mannoprotein and mannosylated O-antigen that contain terminal mannose [9,11,23]. Interestingly, in vertebrates, high mannose-type N-linked glycans are also found on particular type of DC [24]. Gusb is known to contain high mannose type glycans [18]. These observations suggest that, irrespective of pathogens and host tissues, mannose moieties which are found in proteins, glycans, O-antigens or any kinds of scaffold, could be recognized by Dectin-2.
Glycosylation is one of major post-translational modifications [16,25]. Our results showed that Dectin-2 binding of Gusb was not completely abrogated if all of N-glycosylation sites were mutated. We found that Gusb has putative O-glycosylation sites which might be recognized by Dectin-2 as previously described (http://www.cbs.dtu.dk/services/NetOGlyc/) [9]. This observation suggests that Dectin-2 may bind to O-linked glycan on Gusb. We also could not exclude the possibility that Gusb protein itself is involved in Dectin-2 binding.
Accumulating evidence has revealed that Dectin-2 contributes to host defense against pathogens. In addition to protective effects against pathogens, it is proposed that Dectin-2 plays a role in protecting the host by dampening the excessive inflammatory responses. It was reported that putative Dectin-2 ligand was expressed on regulatory T cells and blockage of Dectin-2-mediated signaling broke the immune tolerance [26]. We previously reported that Dectin-2 is essential for production of anti-inflammatory cytokine, IL-10 in BMDCs [11]. Gusb -/mice spontaneously develop Mucopolysaccharidoses (MPS) accompanied by overexpression of inflammatory cytokines [27][28][29]. It is interesting to see whether the lack of such Dectin-2-mediated inhibitory effects is involved in the development of the disease in Gusb -/mice.
Some N-glycan on Gusb contains a terminal mannose 6-phosphate (M6P), which is important for lysosomal enzymes to localize in lysosome. Gusb is also known to be released into culture supernatants [19,30] and we also confirmed this in BMDCs (Fig 2D). Although the exact location in which Gusb is sensed by Dectin-2 are yet uncharacterized, Dectin-2 might sense Gusb both on cell surface and in the extracellular milieu. The possibility that Gusb reflects cellular damages would be an intriguing issue to be addressed in the future.  Recently, it is widely accepted the idea of immune surveillance-normal and abnormal self are continuously monitored by our immune systems. In line with this concept, Dectin-2 may monitor abnormal glycosylation status of several proteins exposed on the cell surface. This "status" may include the exposure of terminal mannose due to aberrant loss of sialic acid or high-mannose structure which should be only present in intracellular organelles. Interestingly, fungus and yeast normally express high-mannose glycan on the cell surface. Thus, it is tempting to speculate that Dectin-2, and presumably other CLRs, may sense "danger" derived from self and non-self through similar criteria, which warrants further extensive investigation.