Innate Antifungal Immune Receptor, Dectin-1, Undergoes Ligand-Induced Oligomerization with Highly Structured β-Glucans and at Fungal Cell Contact Sites

TL;DR: The results indicate that Dectin-1A senses the solution conformation of β-glucans through their varying ability to drive receptor dimer/oligomer formation and activation of membrane proximal signaling events.

Abstract: Dectin-1A is a C-type Lectin innate immunoreceptor that recognizes {beta}-(1,3:1,6)-glucan, a structural component of Candida species cell walls. The higher order structure of {beta}-glucans ranges from random coil to insoluble fiber due to varying degrees of tertiary (helical) and quaternary structure. Model Saccharomyces cerevisiae {beta}-glucans of medium and high molecular weight (MMW and HMW, respectively) are highly structured. In contrast, low MW glucan (LMW) is much less structured. Despite similar affinity for Dectin-1A, the ability of glucans to induce Dectin-1A mediated calcium influx and Syk phosphorylation positively correlates with their degree of higher order structure. Chemical denaturation and renaturation of MMW glucan showed that glucan structure determines agonistic potential, but not binding affinity, for Dectin-1A. We explored the role of glucan structure on Dectin-1A oligomerization, which is thought to be required for Dectin-1 signaling. Glucan signaling decreased Dectin-1A diffusion coefficient in inverse proportion to glucan structural content, which was consistent with Dectin-1A aggregation. Forster Resonance Energy Transfer (FRET) measurements revealed that molecular aggregation of Dectin-1 occurs in a manner dependent upon glucan higher order structure. Number and Brightness analysis specifically confirmed an increase in the Dectin-1A dimer and oligomer populations that is correlated with glucan structure content. Receptor modeling data confirms that in resting cells, Dectin-1A is predominantly in a monomeric state. Super Resolution Microscopy revealed that glucan-stimulated Dectin-1 aggregates are very small (<15 nm) collections of engaged receptors. Finally, FRET measurements confirmed increased molecular aggregation of Dectin-1A at fungal particle contact sites in a manner that positively correlated with the degree of exposed glucan on the particle surface. These results indicate that Dectin-1A senses the solution conformation of {beta}-glucans through their varying ability to drive receptor dimer/oligomer formation and activation of membrane proximal signaling events.nnAuthor SummaryCandidemia is the most common bloodstream infection in the United States. During infection, the fungal cell wall is an important virulence factor, playing roles in adhesion, immune recognition and colonization. The human innate immune system recognizes {beta}-glucan, a highly immunogenic component of the fungal cell wall. During innate immune recognition of Candida, the organization of cell wall {beta}-glucan is an important determinant of a successful immune activation. However, there have been many reports showing conflicting biological activities of {beta}-glucans with different size, branching and structure. Here, using quantitative fluorescence imaging techniques, we investigate how differential size and structure of {beta}-glucan impacts activation of the innate immune receptor, Dectin-1A. Our results indicate a positive correlation between highly structured glucans and Dectin-1A activation. Furthermore, we determined this is due to the higher ordered {beta}-glucan causing Dectin-1A receptors to form aggregates that are below 15 nm in size. Finally, Dectin-1A receptor aggregation has also been shown to form at fungal particle contact sites with high {beta}-glucan exposure.

Summary

Results

• Dectin-1A activation is dependent on the molecular weight of the soluble βglucan.
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• To accomplish this, the authors used high molecular weight (HMW 450 kDa), medium molecular weight (MMW 145 kDa), and low molecular weight (LMW 11 kDa) soluble glucans, in decreasing order of tertiary and quaternary structures, derived from S. cerevisiae cell walls.
• Cells stimulated with HMW glucan exhibited a more heterogeneous response, with some cells achieving comparable maximum amplitudes as with MMW and others exhibiting little change from basal calcium levels (Fig. 1A, B ).
• These results indicate that glucans with higher order structure are better able to activate Dectin-1A-mediated Ca 2+ signaling and that this is a Syk dependent process.

β-glucan denaturation abrogates its potential for Dectin-1A activation

• To determine if the glucan structure affects signaling outcomes, the authors denatured MMW (highly stimulatory glucan) using DMSO, a chaotrope that promotes a reduction in glucan tertiary structure, thus shifting MMW's triple helix structure to a more single helical or random coil structure [17] .
• The results showed that when the authors denatured MMW .
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• In addition, the authors confirmed the loss of helical structure via a Congo Red assay.
• Similarly, their results show that cells lose the ability to activate Dectin-1A calcium signaling when stimulated with denatured MMW glucan but regain the ability to stimulate Dectin-1A activation when the glucan is renatured (Fig. 2 E,F).

β-glucan structure variation and manipulation does not alter affinity for Dectin-1

• The authors considered the possibility that these soluble glucans might have different affinities for Dectin-1A, resulting in differential receptor activation.
• This was accomplished using a chimeric fusion protein of the carbohydrate recognition domain of Dectin-1A and the human IgG Fc region.
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• The results shown in Fig. 3A indicate that all the glucans have approximately nanomolar dissociation constants for Dectin-1A carbohydrate recognition domain despite the previously described differences in signaling [27] .
• The authors results show that the amount of glucan tertiary structure scales with molecular weight as measured by the concentration of NaOH required to reduce Congo Red binding to glucan (Fig. 3B ), suggesting that the size of the glucans is correlated with their higher-order structure.

Dectin-1A decreases in diffusion coefficient when stimulated with highly structured β-glucans

• The Stokes-Einstein equation predicts that if a diffusing object increases in hydrodynamic radius, it will slow down proportionally to that change.
• The authors measured the diffusion coefficient of Dectin-1A pre/post glucan stimulation to determine whether the .
• The authors obtained average diffusion coefficients and spatial number density of their receptor using Raster Image Correlation Spectroscopy (RICS).
• RICS allowed us to survey multiple areas of the cell for molecular parameters such as diffusion coefficient and receptor density.
• The authors observed fluorescent molecules (i.e., Dectin-1A-mEmerald) diffusing in and out of this excitation volume through fluctuation in the number of photons obtained.

Dectin-1A forms dimers/oligomers when stimulated with highly structured βglucans

• The results shown above indicate that the β-glucan structure is an important factor in signaling outcomes.
• The occurrence of FRET causes rapid quenching of donor fluorescence, so FRET can be determined by measuring the shortening of donor fluorescence lifetime when in proximity to acceptor.
• Data from co-expression of cytosolic donor and acceptor-tagged receptor was fit bi-exponentially and the lifetime of both components is shown (Fig. 5B ).
• These results suggest that the highly structured soluble glucans allow for an increase in Dectin-1A dimerization or oligomerization to occur, which directly correlates with the amount of receptor activation and signaling observed.
• Consistent with a Dectin-1 distribution of predominantly monomers or low order oligomers (likely unresolvable by dSTORM), singlet exposures greatly outnumber multiple exposures on the cell wall surface (Fig. 7C ).

Dectin-1A is predominantly monomeric in resting cell membranes

• FRET-based measurements and N&B analysis reported that the large majority of Dectin-1 is distributed as monomers in unstimulated cells.
• The authors compared simulated and experimental FRET efficiencies and used maximum radii for simulations that yielded FRET efficiency in best agreement with observed FRET efficiency on resting cells, indicating comparable "sensitivity" of FRET detection in both.
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• The authors study used a carefully characterized series of fungal glucans to determine that Dectin-1A activation is specifically influenced by the degree of β-glucan triple helical structure, not merely through its affinity or size.
• DSTORM failed to detect ligand inducible Dectin-1 nanodomains on a length scale of ≥15 nm, suggesting that Dectin-1 aggregation events are limited to small collections of ≤7 receptors.

Plasmids and Transfection of Dectin-1 Constructs

• PUNO1-hDectin-1A was stably transfected into HEK-293 cells for use in their calcium .
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• Stable transfection of mEmerald-Dectin-1A was used for Syk immunoblotting experiments.
• All other experiments involving exogenous protein expression used transient transfection.

Fungal Growth/Preparation

• Following a 3-minute centrifugation at 6000 rpm, the supernatant was removed, and the cells were resuspended in 4% paraformaldehyde and sterile phosphate-buffered saline (PBS) for 15 minutes.
• The cells were centrifuged and washed with sterile PBS three times.
• The cell concentration was then determined using a disposable hemocytometer (C-Chip; Bulldog Bio catalog no. DHC-N01).

Glucan Particles

• Glucan microparticles were prepared from lyophilized C. albicans SC5314 yeast derived from stationary phase culture in YPD.
• It is made available under and residue slurry was adjusted to neutral pH and washed thrice with ultrapure water.
• Pyrogen free reagent and glassware was used throughout preparation, and particles were stored at 4°C in sterile, pyrogen free water.

Soluble Glucan Chromatographic Analysis

• The molecular weight was assessed by gel permeation chromatography (GPC) and multi-angle light scattering (MALS).
• Samples (100 μg) were injected and eluted with a mobile phase of 0.15 M sodium chloride containing 0.02% sodium azide at a flow rate of 0.5 mL/min using two Waters Ultrahydrogel 500 columns and one Waters Ultrahydrogel 250 column connected serially.
• The samples were run with the column temperature at 18°C.
• The Mw was calculated using Wyatt Astra software using data resulting from measurements of the angular variation of scattered light using the MALS detector coupled with the concentration measured by the refractive index signal.

Soluble Glucan Linkage Analysis

• Desalted and lyophilized samples of the fractions were dissolved in dimethylsulfoxide (DMSO) and treated with NaOH and methyl iodide to methylate all free hydroxyl groups [75] .
• The purified material was then hydrolyzed with trifluoroacetic acid, the reducing ends of the resulting sugars were reduced with NaBD 4 , and then the resulting free hydroxyl groups were acetylated with acetic anhydride.
• The mixture of partially methylated alditol acetates was analyzed by gas chromatography.
• CC-BY 4.0 International license a 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 derivative corresponding to a particular linkage has been identified by a characteristic retention time and mass spectrum using a mass detector.

Biolayer interferometry

• Advanced Kinetics Biolayer interferometry experiments were conducted using the Personal Assay BLItz System.
• Biosensors tips were initially loaded with Dectin-1A:FC fusion protein (Invivogen, #fc-hdec1a) at 13 ug/ml.
• A global fitting was performed on the curves obtained using the BLItz software.

Congo Red Spectroscopic Assay

• A Analysis was conducted on the plasma membrane by masking out internal cellular compartments on the images.
• For their fungal contact site studies, analysis was conducted on the plasma membrane that was in contact with the fungus and a separate masking for plasma membrane that was not in contact with any yeast.
• A bi-exponential fit was performed to the decay curve.
• For donor only as well as donor and acceptor on opposite sides of the plasma membrane (negative control), the decay curve indicated a negative amplitude for one of the components, thus indicating a mono-exponential decay.
• Lifetime values of the second component (τDA) of the decay curve were used to calculate FRET efficiency using the equation: 𝐹𝑅𝐸𝑇𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = 1 − ./0 ./ ×100.

Raster Image Correlation Analysis/Number and Brightness

• Protocols on RICS and N&B analysis have been previously described in more depth and analysis of diffusion coefficient and receptor density were performed using SimFCS .
• HEK-293 cells expressing Emerald-Dectin1A-C-10 were plated at 40,000 cells in a 35 mm glass bottom MatTEK dishes 24h prior to imaging using equipment described in "Microscopy and Image Analysis" section above.
• The 473 nm diode laser operated at 0.1% power was used in these images.
• Data acquisition was on an Olympus IX-71 microscope equipped with an objective based TIRF illuminator using an oil-immersion objective (PlanApo N, 150×/1.45 NA; Olympus) in an oblique illumination configuration.
• The second H-SET pass determined clustering using the DBSCAN algorithm [80] which depends on the two parameters.

FRET Efficiency Calculation (Model)

• With the assumption that the concentration of excited donors is lower than the acceptor concentration, the authors can consider only one donor molecule.
• In addition, if the orientation factor for dipolar coupling between donor and acceptor is identical for all donor-acceptor pairs, the FRET efficiency equation is as follows, (the pairs are considered to be rotating freely [81] ):.
• The Förster distance (R 0 ) of the pair of donor and acceptor fluorophores used for glucan stimulation experiments also matches that used for donors and acceptors in their FLIM FRET experiments, namely R 0 = 5.24 nm [82] .
• The FRET efficiency in this simulation calculates the combination of FRET resulting from acceptors surrounding one excited donor, which are located with the specified radial distance.

Figures

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Full text

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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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
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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

3
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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4
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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5
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
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