A moonlighting function of a chitin polysaccharide monooxygenase, CWR-1, in Neurospora crassa allorecognition
- Department of Chemistry, University of California, Berkeley, United States
- Department of Plant and Microbial Biology, University of California, Berkeley, United States
- California Institute for Quantitative Biosciences, University of California, Berkeley, United States
- Department of Molecular and Cell Biology, University of California, Berkeley, United States
Figures
Figure 1 with 6 supplements
Characterization of the polysaccharide monooxygenase (PMO) domain from CWR-1 from a haplogroup 1 strain (FGSC2489) and functional dissection of CWR-1 domains in vivo.
(A) A schematic diagram depicting the series of truncated cwr-1 constructs studied. SP indicates signal peptide; GS linker indicates the glycine/serine-rich region that connects the PMO catalytic …
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Figure 1—source data 1
HPAEC-PAD source data.
Blank subtracted HPAEC-PAD traces. The samples were prepared as described in ‘Materials and methods'. The time and nC response are listed in the table.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig1-data1-v2.xlsx
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Figure 1—source data 2
ICP source data.
The amount of bound copper that was calculated for each polysaccharide monooxygenase (PMO) construct. The amount of copper was determined using ICP-MS and then divided by the protein concentration to generate a ratio. The protein concentration was determined using the absorbance at 280 nm with a NanoDrop UV-Vis with extinction coefficients predicted by Benchling (all Cys oxidized). The results from the technical triplicates are listed.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig1-data2-v2.xlsx
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Figure 1—source data 3
Horseradish peroxidase (HRP)-oxygen reduction assay source data.
The slopes from monitoring polysaccharide monooxygenase (PMO)-catalyzed oxygen reduction using an HRP-coupled assay. A PMO sample (2 µM) was incubated at room temperature with 100 µM Amplex red, 1.3 µM HRP (Sigma), 2 mM ascorbate in 50 mM MOPS pH 7.0. The change in absorbance at 540 nm vs. time (min) is listed for each biological replicate in this table.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig1-data3-v2.xlsx
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Figure 1—source data 4
p-Values.
Fusion test of the truncated versions of CWR-1 paired with cwr-2HG3; ΔΔΔ.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig1-data4-v2.xlsx
Figure 1—figure supplement 1
Alignment of characterized AA11s.
All of the AA11s besides AfAA11A have a very similar architecture to CWR-1, including the GS linker and X278 domain.
Figure 1—figure supplement 2
AA11 sequence similarity network (SSN) annotated with characterized proteins.
This SSN was generated using the EFI-EST BLAST feature with the CWR-1HG1 sequence as the search peptide. This search yielded approximately 1700 sequences (Supplementary file 1g), which were …
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Figure 1—figure supplement 2—source data 1
Sequence similarity network (SSN) data from the EFI-EST tool as described in ‘Materials and methods’ used to construct the figure.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig1-figsupp2-data1-v2.zip
Figure 1—figure supplement 3
Germling fusion phenotype and percentages among engineered stains.
(A) Pairings between a cwr-1HG1 strain and strains carrying cwr-2 alleles from the six different haplogroups. A strain harboring a cwr-1HG1 allele and expressing cytoplasmic GFP (his-3::Pcwr-1-cwr-1H…
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Figure 1—figure supplement 3—source data 1
p-Values.
Comparison of the fusion test of the cwr-1HG1 paired with strains harboring cwr-2 alleles from six different haplogroups.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig1-figsupp3-data1-v2.xlsx
Figure 1—figure supplement 4
Purification and size of polysaccharide monooxygenase (PMO) domains of CWR-1 haplogroup (HG) proteins.
(A) An SDS-PAGE gel showing purity of CWR-1 PMO domains encoded by cwr-1 alleles from the six different haplogroups. The haplogroup is denoted above each lane. Original data provided in Figure …
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Figure 1—figure supplement 4—source data 1
Purified polysaccharide monooxygenase (PMO) domain from the six haplogroups (HGs).
An SDS-PAGE stain-free gel of the purified CWR-1 PMO domains from each haplogroup. The order of the lanes from left to right is: ladder (Precision Plus Protein Standards unstained Bio-Rad) HG 6, HG4, HG3, HG5, HG2, HG1.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig1-figsupp4-data1-v2.zip
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Figure 1—figure supplement 4—source data 2
Whole-protein MS data.
Deconvoluted mass spectra of the purified polysaccharide monooxygenase (PMO) domains from each haplogroup and selected mutants. The whole-protein mass spectrometry is described in ‘Materials and methods'.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig1-figsupp4-data2-v2.xlsx
Figure 1—figure supplement 5
Oligosaccharide standard chromatograms and elution times.
(A) Chitooligosaccharide elution times. Chitooligosaccharides elute early on in the HPAEC-PAD trace. The chitotetraose and chitotriose elute at very similar times. An arrow denotes where the buffer, …
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Figure 1—figure supplement 5—source data 1
HPAEC-PAD source data.
Blank subtracted HPAEC-PAD traces. The samples were prepared as described in ‘Materials and methods'. The time and nC response are listed in the table.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig1-figsupp5-data1-v2.xlsx
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Figure 1—figure supplement 5—source data 2
HPAEC-PAD source data.
Blank subtracted HPAEC-PAD traces. The samples were prepared as described in ‘Materials and methods'. The time and nC response are listed in the table.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig1-figsupp5-data2-v2.xlsx
Figure 1—figure supplement 6
MS/MS fragment is consistent with C1 oxidation.
A MS/MS spectrum from the product of PMOHG1-treated α-chitin. This protein was used as a representative sample for the others as the products do not appear different by HPAEC-PAD between the …
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Figure 1—figure supplement 6—source data 1
Tandem MS source data.
Raw MS data from tandem MS experiment as described in ‘Materials and methods'.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig1-figsupp6-data1-v2.zip
Figure 2
Comparison of CWR-1 polysaccharide monooxygenase (PMO) domains.
In all panels, blue indicates cwr-1 predicted proteins from HG1 isolates, red indicates HG2, light blue indicates HG3, orange indicates HG4, purple indicates HG5, and green indicates HG6 isolates. (A…
Figure 3 with 1 supplement
The polysaccharide monooxygenase (PMO) domain in CWR-1 functions to confer the allorecognition fusion checkpoint.
(A) Micrographs of the dominant fusion events between germlings expressing cwr-1 alleles from each haplogroup in a Δcwr-1Δ81Δcwr-2 GFP background when paired with cwr-1HG1 cwr-2HG1 germlings …
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Figure 3—source data 1
p-Values. Figure 3D.
Fusion test of the different CWR-1 full-length and CWR-1 chimeras paired with cwr-1HG1cwr-2HG1.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Germling fusion phenotype and fusion percentages in strains bearing cwr-1 from the different haplogroups and cwr-1 chimeras.
(A) Representative micrographs of fusion phenotypes of cells expressing cwr-1 alleles (from HG1-6) and paired the permissive Δcwr-1Δ81Δcwr-2 mutant stained with FM4-64. (B) Representative …
Figure 4 with 1 supplement
Catalytic activity of CWR-1 haplogroups.
(A, B) A comparison of the reaction products from CWR-1 from each of the six haplogroups on α-chitin and β-chitin. There were minor differences, but each haplogroup generates the same C1-oxidized …
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Figure 4—source data 1
HPAEC-PAD source data.
Blank subtracted HPAEC-PAD traces. The samples were prepared as described in ‘Materials and methods.’ The time and nC response are listed in the table.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig4-data1-v2.xlsx
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Figure 4—source data 2
HPAEC-PAD source data.
Blank subtracted HPAEC-PAD traces. The samples were prepared as described in ‘Materials and methods.’ The time and nC response are listed in the table.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig4-data2-v2.xlsx
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Figure 4—source data 3
ICP source data.
The amount of bound copper that was calculated for each construct. The amount of copper was determined using ICP-MS and then divided by the protein concentration to generate a ratio. The protein concentration was determined using the absorbance at 280 nm with a NanoDrop UV-Vis with extinction coefficients predicted by Benchling (all Cys oxidized). The results from the technical triplicates are listed.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig4-data3-v2.xlsx
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Figure 4—source data 4
Horseradish peroxidase (HRP)-oxygen reduction assay source data.
The slopes from monitoring polysaccharide monooxygenase (PMO)-catalyzed oxygen reduction using an HRP-coupled assay. A PMO sample (2 µM) was incubated at room temperature with 100 µM Amplex red, 1.3 µM HRP (Sigma), 2 mM ascorbate in 50 mM MOPS pH 7.0. The change in absorbance at 540 nm vs. time (min) is listed for each biological replicate in this table.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig4-data4-v2.xlsx
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Figure 4—source data 5
HPAEC-PAD source data.
Blank subtracted HPAEC-PAD traces. The samples were prepared as described in ‘Materials and methods.’ The time and nC response are listed in the table.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig4-data5-v2.xlsx
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Figure 4—source data 6
HPAEC-PAD source data.
Blank subtracted HPAEC-PAD traces. The samples were prepared as described in ‘Materials and methods.’ The time and nC response are listed in the table.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig4-data6-v2.xlsx
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Figure 4—source data 7
HPAEC-PAD source data.
Blank subtracted HPAEC-PAD traces. The samples were prepared as described in ‘Materials and methods.’ The time and nC response are listed in the table.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig4-data7-v2.xlsx
Figure 4—figure supplement 1
HPAEC-PAD traces of reaction before fusion experiment and cell fusion percentages.
(A) Purified CWR-1 polysaccharide monooxygenase (PMO) (from HG1 or HG4) was mixed with conidial cell wall sample, with or without ascorbate. All solutions used reagents at the same concentration as …
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Figure 4—figure supplement 1—source data 1
HPAEC-PAD source data.
Blank subtracted HPAEC-PAD traces. The samples were prepared as described in ‘Materials and methods.’ The time and nC response are listed in the table.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig4-figsupp1-data1-v2.xlsx
Figure 5 with 2 supplements
Characterization of CWR-1 polysaccharide monooxygenase (PMO) active-site variants.
(A, B) Comparison of the ability of active-site variants of the PMOHG1 and PMOHG6 domains and their ability to degrade β-chitin. Mutation of one or both of the histidine brace residues abolished …
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Figure 5—source data 1
HPAEC-PAD source data.
Blank subtracted HPAEC-PAD traces. The samples were prepared as described in ‘Materials and methods.’ The time and nC response are listed in the table.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig5-data1-v2.xlsx
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Figure 5—source data 2
HPAEC-PAD source data.
Blank subtracted HPAEC-PAD traces. The samples were prepared as described in ‘Materials and methods.’ The time and nC response are listed in the table.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig5-data2-v2.xlsx
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Figure 5—source data 3
ICP source data.
The amount of bound copper that was calculated for each construct. The amount of copper was determined using ICP-MS and then divided by the protein concentration to generate a ratio. The protein concentration was determined using the absorbance at 280 nm with a NanoDrop UV-Vis with extinction coefficients predicted by Benchling (all Cys oxidized). The results from the technical triplicates are listed.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig5-data3-v2.xlsx
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Figure 5—source data 4
Horseradish peroxidase (HRP)-oxygen reduction assay source data.
The slopes from monitoring polysaccharide monooxygenase (PMO)-catalyzed oxygen reduction using an HRP-coupled assay. A PMO sample (2 µM) was incubated at room temperature with 100 µM Amplex red, 1.3 µM HRP (Sigma), 2 mM ascorbate in 50 mM MOPS pH 7.0. The change in absorbance at 540 nm vs. time (min) is listed for each biological replicate in this table.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig5-data4-v2.xlsx
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Figure 5—source data 5
p-Values.
Fusion test of the different cwr-1HG1 histidine variants and tyrosine variant paired with a strain expressing cwr-2HG3; ΔΔΔ.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig5-data5-v2.xlsx
Figure 5—figure supplement 1
Deconvoluted mass spectrum of polysaccharide monooxygenase (PMO) histidine variants.
These PMOHG1 variants have a histidine to alanine substitution of the first histidine (His20; the first residue after the signal peptide), a second variant containing a His→Ala substitution at H78 …
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Figure 5—figure supplement 1—source data 1
Whole-protein MS data.
Deconvoluted mass spectra of the purified polysaccharide monooxygenase (PMO) domains from each haplogroup and selected mutants. The whole-protein mass spectrometry is described in ‘Materials and methods’.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig5-figsupp1-data1-v2.xlsx
Figure 5—figure supplement 2
X-band EPR and simulated spectra of PMOHG1 and PMOHG1 H78A variant.
The top black trace is the X-band continuous-wave (CW) spectrum from Cu(II) from PMOHG1. The top red trace is the simulated fit for WT PMOHG1 with the following parameters: g1 = 2.245, g2 = 2.065, …
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Figure 5—figure supplement 2—source data 1
EPR source data.
Measured raw data in xy format from the EPR experiment as described in ‘Materials and methods’.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig5-figsupp2-data1-v2.xlsx
Figure 6
CWR-1 chimeras to define the polysaccharide monooxygenase (PMO) haplogroup specificity region.
(A) A schematic depicting where the loops from PMOHG1 domain that were replaced with loops from PMOHG6 (green). SP means signal peptide; GS linker means the glycine/serine-rich region that connects …
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Figure 6—source data 1
p-Values.
Fusion test of the cwr-1HG1LSHG6 chimera paired with a strain expressing cwr-1HG1cwr-2HG1, cwr-2HG1; ΔΔΔ or cwr-2HG6; ΔΔΔ.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig6-data1-v2.xlsx
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Figure 6—source data 2
p-Values.
Comparison of the fusion test of the cwr-1HG1 or cwr-1HG1LS-L8-LCHG6 chimera paired with strains harboring cwr-2 alleles from six different haplogroups.
- https://cdn.elifesciences.org/articles/80459/elife-80459-fig6-data2-v2.xlsx
Figure 7
A schematic model that summarizes the role of CWR-1 and CWR-2 in allorecognition at the cell fusion checkpoint.
(A) The polysaccharide monooxygenase (PMO) domain, a glycine and serine-rich region, and a putative chitin-binding module, X278, of CWR-1 are shown. The PMO domain degrades chitin, and the region …
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/80459/elife-80459-mdarchecklist1-v2.docx
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Supplementary file 1
Description of plasmids, strains and materials used in this study.
(a) Plasmids to transform N. crassa. (b) Plasmids to transform E. coli. (c) Primers used in this study. (d) N. crassa strains used in this work. (e) Intra-haplogroup sequence ID. (f) Substrates used to test CWR-1 activity. (g) Sequence similarity network (SSN) data set.
- https://cdn.elifesciences.org/articles/80459/elife-80459-supp1-v2.xlsx
