1
Dectin-1 Molecular Aggregation and Signaling is Sensitive to β-Glucan Structure 1
and Glucan Exposure on Candida albicans Cell Walls 2
3
Authors: 4
Eduardo U. Anaya
1
, Akram Etemadi Amin
1,2
, Michael J. Wester
3
, Michael E. 5
Danielson
4
, Kyle S. Michel
4
, Aaron K. Neumann
1,5
6
Affiliations: 7
1. Department of Pathology, University of New Mexico, School of Medicine, 8
Albuquerque, NM, USA, 87131 9
2. Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM, 10
USA 87131 11
3. Department of Mathematics and Statistics, University of New Mexico, Albuquerque, 12
NM, USA, 87131 13
4. ImmunoResearch Inc., Eagan, MN, USA, 55121 14
5. Corresponding author: akneumann@salud.unm.edu 15
16
Keywords: 17
Dectin-1, dimer, β-Glucan, Candida albicans, hemITAM, Syk, Calcium signaling, 18
(Förster Resonance Energy Transfer) FRET, Raster Image Correlation Spectroscopy 19
(RICS), Numbers & Brightness analysis (N&B) 20
21
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2
Abstract 22
Dectin-1A is a C-type Lectin innate immunoreceptor that recognizes β-(1,3;1,6)-glucan, 23
a structural component of Candida species cell walls. The higher order structure of β-24
glucans ranges from random coil to insoluble fiber due to varying degrees of tertiary 25
(helical) and quaternary structure. Model Saccharomyces cerevisiae β-glucans of 26
medium and high molecular weight (MMW and HMW, respectively) are highly 27
structured. In contrast, low MW glucan (LMW) is much less structured. Despite similar 28
affinity for Dectin-1A, the ability of glucans to induce Dectin-1A mediated calcium influx 29
and Syk phosphorylation positively correlates with their degree of higher order structure. 30
Chemical denaturation and renaturation of MMW glucan showed that glucan structure 31
determines agonistic potential, but not binding affinity, for Dectin-1A. We explored the 32
role of glucan structure on Dectin-1A oligomerization, which is thought to be required for 33
Dectin-1 signaling. Glucan signaling decreased Dectin-1A diffusion coefficient in inverse 34
proportion to glucan structural content, which was consistent with Dectin-1A 35
aggregation. Förster Resonance Energy Transfer (FRET) measurements revealed that 36
molecular aggregation of Dectin-1 occurs in a manner dependent upon glucan higher 37
order structure. Number and Brightness analysis specifically confirmed an increase in 38
the Dectin-1A dimer and oligomer populations that is correlated with glucan structure 39
content. Comparison of receptor modeling data with FRET measurements confirms that 40
in resting cells, Dectin-1A is predominantly in a monomeric state. Super Resolution 41
Microscopy revealed that glucan-stimulated Dectin-1 aggregates are very small (<15 42
nm) collections of a few engaged receptors. Finally, FRET measurements confirmed 43
increased molecular aggregation of Dectin-1A at fungal particle contact sites in a 44
.CC-BY 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted May 25, 2020. ; https://doi.org/10.1101/824995doi: bioRxiv preprint
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manner that positively correlated with the degree of exposed glucan on the particle 45
surface. These results indicate that Dectin-1A senses the solution conformation of β-46
glucans through their varying ability to drive receptor dimer/oligomer formation and 47
activation of membrane proximal signaling events. 48
49
Introduction 50
Overall, Candida infections have increased over the past 20 years in the United States 51
[1–5]. It is estimated that 46,000 cases of healthcare-associated invasive candidiasis 52
occur in the United States annually [6]. The fungal cell wall is composed of an inner 53
layer of chitin, a middle layer of β(1,3;1,6)-D-glucan and an outer layer of N- and O- 54
linked mannans [7]. During infection, the cell wall of Candida is an important and 55
relevant virulence factor, playing roles in adhesion, colonization and immune recognition 56
[8,9]. 57
Due to the abundant amount of mannan in the outer cell wall, β-glucan exhibits a very 58
limited, punctate pattern of nanoscale surface exposure. The extent of this glucan 59
masking is influenced by environmental conditions such as intestinal pH or lactate levels 60
[10,11]. In addition, interactions with neutrophils have been shown to “unmask” the 61
mannose layer through a neutrophil extracellular trap-mediated mechanism [12]. 62
Furthermore, our lab and others have determined that anti-fungal drugs “unmask” the 63
fungal cell wall, which leads to increases in nanoscale regions of glucan exposure and 64
correlates with enhanced host defense [13–15]. Therefore, fungal species use masking 65
as a way to evade immune recognition of β-glucan by the host’s immune system [16]. 66
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β-glucans consist of a β-1,3-linked backbone with side chains of β-1,6-linked units that 67
vary in length and degree of branching [17]. β-glucan forms triple-helical structures 68
through intermolecular hydrogen bonds with two other strands [17–21]. This triple helix 69
conformation is shown to form with just the β-1,3-linked backbone, however β-1,6-linked 70
side chains play an important role in determination of the triple helix cavity formation 71
through side chain/side chain interactions [21]. 72
β-glucans are known for their biological activities such as enhancing anti-tumor, anti-73
bacterial, and anti-viral immunity as well as wound healing [22–25]. The biological 74
activity of glucan is affected by its structure, size, structural modification, conformation 75
and solubility [26]. Research has found that branching is not required to observe 76
biological activity, but branching has been shown to enhance binding to the Dectin-1 77
receptor [27]. In contrast, β-glucan size is thought to play a major role in biological 78
activity with glucans that are shorter than 10,000 Da being generally inactive in vivo 79
[28,29]. However, despite having similar sizes, glucans can display differences in their 80
biological activities [30–32]. For example, studies have demonstrated that the 81
immunoregulatory activity of variously sourced laminarin ranges from agonistic to 82
antagonistic depending on its physicochemical properties, purity and structure [33]. 83
Furthermore, β-glucans that have a triple helical conformation are more potent agonists 84
of host immune response than single helical glucan [27,34–36]. We propose that the β-85
glucan triple-helix conformation plays an important role in determining the biological 86
activity of the β-glucan through modulating the degree of receptor oligomerization upon 87
ligation. 88
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During innate immune recognition of Candida, the organization of cell wall ligands and 89
pattern recognition receptors is an important determinant of successful immune 90
activation [8]. The fungi-responsive C-type lectin (CTL) anti-fungal immunoreceptors 91
play a central role in the detection of Candida [37]. Human Dectin-1A is the main CTL 92
that recognizes the β-glucan found in the fungal cell wall [38–40]. Dectin-1A is found in 93
myeloid lineage cells, and once activated, it stimulates phagocytic activity, the 94
production of reactive oxygen intermediates and inflammatory mediators. Dectin-1A 95
contains a CTL-like domain, separated from the cell membrane by a glycosylated stalk 96
region, a transmembrane domain and an intracellular cytosolic domain. Dectin-1 97
contains half an Immunoreceptor Tyrosine-based Activation Motif (a YXXL sequence 98
with an upstream stretch of acidic amino acids) in its cytoplasmic tail, which is termed a 99
(hem)ITAM domain [41,42]. Monophosphorylated ITAM domains, which are anticipated 100
to approximate the structure of phosphorylated (hem)ITAM domains, poorly recruit and 101
activate Syk for downstream signaling because they cannot support bivalent 102
engagement of both of Syk’s SH2 domains [43]. Another (hem)ITAM bearing receptor, 103
CLEC-2, is reported to require dimerization for its signaling [44]. By analogy to this and 104
other (hem)ITAM receptors, it is speculated that Dectin-1A must oligomerize to 105
recapitulate a multivalent binding site for Syk to facilitate signal transduction [8,44,45]. 106
However, this prediction has not been directly explored for Dectin-1A in live cells at the 107
molecular level with relation to signaling outcomes. 108
In this study, we propose that factors that induce an aggregated membrane organization 109
of Dectin-1A during activation are very important for determining signaling outcomes of 110
Dectin-1A engagement by β-glucan [44,45]. We hypothesize that ligand structure, at the 111
.CC-BY 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted May 25, 2020. ; https://doi.org/10.1101/824995doi: bioRxiv preprint
