A moonlighting function of a chitin polysaccharide monooxygenase, CWR-1, in Neurospora crassa allorecognition
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
This study identifies an important role for a lytic polysaccharide monooxygenase in allorecognition in the filamentous fungus Neurospora crassa, which is independent of the catalytic activity of this remarkable class of proteins. The study's findings are strongly supported through an interdisciplinary approach, combining microscopy with genetics and biochemistry. The study will be of great interest to fungal biologists and microbiologists, as well as biochemists studying carbohydrate-active enzymes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Genetics and Genomics (eLife)
Abstract
Organisms require the ability to differentiate themselves from organisms of different or even the same species. Allorecognition processes in filamentous fungi are essential to ensure identity of an interconnected syncytial colony to protect it from exploitation and disease. Neurospora crassa has three cell fusion checkpoints controlling formation of an interconnected mycelial network. The locus that controls the second checkpoint, which allows for cell wall dissolution and subsequent fusion between cells/hyphae, cwr (cell wall remodeling) , encodes two linked genes, cwr-1 and cwr-2 . Previously, it was shown that cwr-1 and cwr-2 show severe linkage disequilibrium with six different haplogroups present in N. crassa populations. Isolates from an identical cwr haplogroup show robust fusion, while somatic cell fusion between isolates of different haplogroups is significantly blocked in cell wall dissolution. The cwr-1 gene encodes a putative polysaccharide monooxygenase (PMO). Herein we confirm that CWR-1 is a C1-oxidizing chitin PMO. We show that the catalytic (PMO) domain of CWR-1 was sufficient for checkpoint function and cell fusion blockage; however, through analysis of active-site, histidine-brace mutants, the catalytic activity of CWR-1 was ruled out as a major factor for allorecognition. Swapping a portion of the PMO domain (V86 to T130) did not switch cwr haplogroup specificity, but rather cells containing this chimera exhibited a novel haplogroup specificity. Allorecognition to mediate cell fusion blockage is likely occurring through a protein–protein interaction between CWR-1 with CWR-2. These data highlight a moonlighting role in allorecognition of the CWR-1 PMO domain.
Article activity feed
-
-
Author Response
Reviewer #2 (Public Review):
Detomasi et al investigated the mechanism behind allorecognition in the filamentous fungus Neuraspora crassa. Previous work had identified two proteins cwr-1 and cwr-2 that control recognition of haplotype and the following cell wall dissolution and subsequent fusion of hyphae. This work is a systematic study of the role of cwr-1 in the allorecognition of six haplogroups. Cwr-1 is predicted to be a chitin-active lytic polysaccharide monooxygenase belonging to the AA11 enzyme family. The activity of the isolated cwr-1 enzyme on chitin is confirmed and it is shown that the catalytic domain is sufficient to confer an allorecognition checkpoint. Surprisingly, and in contrast to previously published data, enzyme activity of cwr-1 is not required. This is shown by the introduction of mutant …
Author Response
Reviewer #2 (Public Review):
Detomasi et al investigated the mechanism behind allorecognition in the filamentous fungus Neuraspora crassa. Previous work had identified two proteins cwr-1 and cwr-2 that control recognition of haplotype and the following cell wall dissolution and subsequent fusion of hyphae. This work is a systematic study of the role of cwr-1 in the allorecognition of six haplogroups. Cwr-1 is predicted to be a chitin-active lytic polysaccharide monooxygenase belonging to the AA11 enzyme family. The activity of the isolated cwr-1 enzyme on chitin is confirmed and it is shown that the catalytic domain is sufficient to confer an allorecognition checkpoint. Surprisingly, and in contrast to previously published data, enzyme activity of cwr-1 is not required. This is shown by the introduction of mutant cwr-1 lacking key residues for activity in a cwr-1 deletion strain followed by screening for fusion events with an incompatible cwr-2 allele.
The strength of this study is the rigor by which all experiments have been designed and carried out. The data sets from the biological assays are complete and treated appropriately with statistical tools. The enzymology is for the most part very comprehensive with eg. full-length mass spectrometry to verify the mutant enzymes. The enzyme activity assays using chitin as substrate are carried out at a high standard using HPAEC detection of soluble products.
Because of the highly surprising conclusion that the active domain but not the active site is required, the weakness of the manuscript lies in the inability to explain this finding.
The term "moonlighting" to describe the phenomenon, is not a very good one. I would recommend changing the title of the paper accordingly. There are already published studies that describe proteins (termed X325) that are highly similar to lytic polysaccharide monooxygenases (both in the overall fold and in the coordination of a single copper atom) that have a clear biological function but no detectable catalytic activity.
Moonlighting proteins comprise a subset of multifunctional proteins in which one polypeptide chain exhibits more than one physiologically relevant biochemical or biophysical function (Jeffery CJ. 1999 Moonlighting Proteins. Trends Biochem. Sci. 24, 8–11 doi:10.1016/S0968-0004(98)01335-8). CWR-1 fits this description. It is predicted to be a PMO and indeed utilizes chitin as a substrate and yields expected PMO-derived oxidative products, thus is clearly definable as a PMO. CWR-1 also is directly involved in allorecognition and as our paper shows, the chitin catalytic activity has nothing to do with allorecognition. So as defined, CWR-1 is a “moonlighting” protein. There does not appear to be another PMO that falls under this definition. The closest one would be GbpA, however activity on polysaccharides present on mucin has not been ruled out, so we decided to remove this comment from the manuscript. The “X325” family PMO-type proteins have an alternate activity, but not two separate activities within the same polypeptide. Although we do not yet know what physiological role is played by the chitin activity in N. crassa, it is not required to know this to conform to the definition. Thus, CWR-1 conforms to the moonlighting definition. We slightly changed the title of the manuscript to be less cumbersome.
-
Evaluation Summary:
This study identifies an important role for a lytic polysaccharide monooxygenase in allorecognition in the filamentous fungus Neurospora crassa, which is independent of the catalytic activity of this remarkable class of proteins. The study's findings are strongly supported through an interdisciplinary approach, combining microscopy with genetics and biochemistry. The study will be of great interest to fungal biologists and microbiologists, as well as biochemists studying carbohydrate-active enzymes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
Detomasi et al. investigated the role of a protein encoded by the cwr-1 gene that belongs to the cell wall remodeling locus that controls cell fusion checkpoints in Neurospora crassa. This protein corresponds to a putative polysaccharide monooxygenase (called PMO or LPMO) from family AA11 (according to the CAZy family). This class of enzymes is known for oxidative cleavage of recalcitrant polysaccharides but recently diverging functions have emerged. In this work, the authors clearly demonstrated LPMO activity towards chitin for several CWR-1 from different haplogroups. Mutagenesis and construction of chimeras allowed the authors to reveal that enzymatic activity was not required for cell fusion blockage. Beyond this very interesting observation, they identified a polymorphic region in the main catalytic …
Reviewer #1 (Public Review):
Detomasi et al. investigated the role of a protein encoded by the cwr-1 gene that belongs to the cell wall remodeling locus that controls cell fusion checkpoints in Neurospora crassa. This protein corresponds to a putative polysaccharide monooxygenase (called PMO or LPMO) from family AA11 (according to the CAZy family). This class of enzymes is known for oxidative cleavage of recalcitrant polysaccharides but recently diverging functions have emerged. In this work, the authors clearly demonstrated LPMO activity towards chitin for several CWR-1 from different haplogroups. Mutagenesis and construction of chimeras allowed the authors to reveal that enzymatic activity was not required for cell fusion blockage. Beyond this very interesting observation, they identified a polymorphic region in the main catalytic domain (corresponding to several loops) that was essential to trigger allorecognition. The authors suspect that this region is involved in the recognition of CWR-2, a transmembrane protein with two domains of unknown function. The authors propose a model highlighting the role of CWR-1 in allorecognition at the cell fusion checkpoint. These results open new prospects for the biological function of fungal PMOs/LPMOs not directly related to their enzymatic activity.
-
Reviewer #2 (Public Review):
Detomasi et al investigated the mechanism behind allorecognition in the filamentous fungus Neuraspora crassa. Previous work had identified two proteins cwr-1 and cwr-2 that control recognition of haplotype and the following cell wall dissolution and subsequent fusion of hyphae. This work is a systematic study of the role of cwr-1 in the allorecognition of six haplogroups. Cwr-1 is predicted to be a chitin-active lytic polysaccharide monooxygenase belonging to the AA11 enzyme family. The activity of the isolated cwr-1 enzyme on chitin is confirmed and it is shown that the catalytic domain is sufficient to confer an allorecognition checkpoint. Surprisingly, and in contrast to previously published data, enzyme activity of cwr-1 is not required. This is shown by the introduction of mutant cwr-1 lacking key …
Reviewer #2 (Public Review):
Detomasi et al investigated the mechanism behind allorecognition in the filamentous fungus Neuraspora crassa. Previous work had identified two proteins cwr-1 and cwr-2 that control recognition of haplotype and the following cell wall dissolution and subsequent fusion of hyphae. This work is a systematic study of the role of cwr-1 in the allorecognition of six haplogroups. Cwr-1 is predicted to be a chitin-active lytic polysaccharide monooxygenase belonging to the AA11 enzyme family. The activity of the isolated cwr-1 enzyme on chitin is confirmed and it is shown that the catalytic domain is sufficient to confer an allorecognition checkpoint. Surprisingly, and in contrast to previously published data, enzyme activity of cwr-1 is not required. This is shown by the introduction of mutant cwr-1 lacking key residues for activity in a cwr-1 deletion strain followed by screening for fusion events with an incompatible cwr-2 allele.
The strength of this study is the rigor by which all experiments have been designed and carried out. The data sets from the biological assays are complete and treated appropriately with statistical tools. The enzymology is for the most part very comprehensive with eg. full-length mass spectrometry to verify the mutant enzymes. The enzyme activity assays using chitin as substrate are carried out at a high standard using HPAEC detection of soluble products.
Because of the highly surprising conclusion that the active domain but not the active site is required, the weakness of the manuscript lies in the inability to explain this finding.
The term "moonlighting" to describe the phenomenon, is not a very good one. I would recommend changing the title of the paper accordingly. There are already published studies that describe proteins (termed X325) that are highly similar to lytic polysaccharide monooxygenases (both in the overall fold and in the coordination of a single copper atom) that have a clear biological function but no detectable catalytic activity. -
Reviewer #3 (Public Review):
The authors here describe that the PMO domain of CWR-1 is active on chitin, which is demonstrated with beautiful and solid biochemistry data. Furthermore, they show that the catalytic activity of the PMO domain is dispensable for allorecognition in N. crassa. More specifically, they showed that the side loops of the PMO domain of CWR-1 are important for allorecognition and cell fusion. The chitin catalytic activity of the PMO domain of CWR-1 is not surprising, as other LPMOs from the same family (AA11) had already been characterized. This paper highlights the discovery that LPMOs are involved in cell wall remodeling of filamentous fungi and cell fusion. These findings certainly strengthen the emerging biological roles that LPMOs play in microorganisms, which are still limited.
The strengths of the paper are …
Reviewer #3 (Public Review):
The authors here describe that the PMO domain of CWR-1 is active on chitin, which is demonstrated with beautiful and solid biochemistry data. Furthermore, they show that the catalytic activity of the PMO domain is dispensable for allorecognition in N. crassa. More specifically, they showed that the side loops of the PMO domain of CWR-1 are important for allorecognition and cell fusion. The chitin catalytic activity of the PMO domain of CWR-1 is not surprising, as other LPMOs from the same family (AA11) had already been characterized. This paper highlights the discovery that LPMOs are involved in cell wall remodeling of filamentous fungi and cell fusion. These findings certainly strengthen the emerging biological roles that LPMOs play in microorganisms, which are still limited.
The strengths of the paper are the interdisciplinary approach, whereby microscopy is combined with genetics and biochemistry.
There are no major weaknesses in the paper.
-
