Abstract

C-type lectin receptors (CLRs) comprise a large family of immunoreceptors that recognize polysaccharide ligands exposed on pathogen surfaces and are conserved among mammals. However, interspecies differences in their ligand spectrums are not fully understood. Dectin-1 is a well-characterized CLR that recognizes β-glucan. We report here that seaweed-derived fucan activates cells expressing human Dectin-1 but not mouse Dectin-1. Low-valency β-glucan components within fucan appeared to be responsible for this activation, as the ligand activity was eliminated by β-glucanase treatment. The low-valency β-glucan laminarin also acted as an agonist for human Dectin-1 but not for mouse Dectin-1, whereas the high-valency β-glucan curdlan activated both human and mouse Dectin-1. Reciprocal mutagenesis analysis revealed that the ligand-binding domain of human Dectin-1 does not determine its unique sensitivity to low-valency β-glucan. Rather, we found that its intracellular domain renders human Dectin-1 reactive to low-valency β-glucan ligand. Substitution with two amino acids, Glu2 and Pro5, located in the human Dectin-1 intracellular domain was sufficient to confer sensitivity to low-valency β-glucan in mouse Dectin-1. Conversely, the introduction of mouse-specific amino acids, Lys2 and Ser5, to human Dectin-1 reduced the reactivity to low-valency β-glucan. Indeed, low-valency ligands induced a set of proinflammatory genes in human but not mouse dendritic cells. These results suggest that the intracellular domain, not ligand-binding domain, of Dectin-1 determines the species-specific ligand profile.

Highlights

  • C-type lectin receptors (CLRs) comprise a large family of immunoreceptors that recognize polysaccharide ligands exposed on pathogen surfaces and are conserved among mammals

  • We found that two intracellular amino acids, which are conserved in primates, play a critical role for enhancing the sensitivity of Dectin-1 independently of the hemITAM

  • We found a seaweed-derived, fucose-containing polysaccharide called fucan activated reporter cells expressing human Dectin-1 (Fig. 1)

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Summary

Results

To search for novel CLRs that recognize natural polysaccharides, we employed nuclear factor of activated T-cells (NFAT)GFP reporter cells expressing various CLRs. Contrary to our initial assumption, the reporter cells expressing the mDectin-1hCRD chimera were not activated by laminarin similar to mDectin-1-bearing cells (Fig. 4B, mD1hCRD), they showed substantial activity upon stimulation with high-valency curdlan (Fig. 4C) These results suggest that the direct ligand-binding domain, CRD, is not responsible for determining laminarin sensitivity to hDectin-1. The introduction of mouse-specific Lys and Ser substitutions into hDectin-1 (hDectin-1E2K/P5S) resulted in the reduction of its reporter activity against laminarin (supplemental Fig. S4) Taken together, these results suggest that two amino acids derived from mDectin-1, Lys and Ser, are critical for “desensitizing” Dectin-1 to low-valency ␤-glucan. Note that mBMDCs constitutively expressed Dectin-1 (see GSE98814 and GSE98825), and responded normally to other stimuli, such as LPS (supplemental Fig. S5) These data support the idea that hDectin-1, but not mDectin-1, is an activating receptor for low-valency ␤-glucan

Discussion
Reagents and antibodies
Microarray analysis
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