Purification of an active, oligomeric chitin synthase complex from the midgut of the tobacco hornworm

Maue, Lars; Meissner, Derek; Merzendorfer, Hans

doi:10.1016/j.ibmb.2009.06.005

Cited by 36 publications

(21 citation statements)

References 45 publications

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“…Previous studies performed in insect and fungal systems provided in vitro evidence that chitin synthases form di- or oligomeric complexes [29,30,31,32]. Based on these studies, we examined Chs3 oligomerization in vivo using BiFC analysis [36].…”

Section: Resultsmentioning

confidence: 99%

“…First evidence for oligomerization of chitin synthases in insects has been provided by Maue et al [32] by purifying an active, oligomeric chitin synthase complex from the midgut of Manduca sexta . Y2H and CoIP experiments performed in yeast also suggested that Chs3 is a self-interacting protein [29,30,31].…”

Section: Discussionmentioning

confidence: 99%

“…In another Y2H study, the dimerization domain was narrowed down to the regions between amino acids 226 and 452 [30] and the N-terminal region comprising the first 171 amino acids [31]. Additional evidence that chitin synthases form oligomeric complexes were derived from biochemical studies in insects, that reported successful purification of active, oligomeric complexes of the midgut-specific chitin synthase 2 from Manduca sexta larvae (MsChs2) [32]. …”

Section: Introductionmentioning

confidence: 99%

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In Vitro and In Vivo Studies on the Structural Organization of Chs3 from Saccharomyces cerevisiae

Gohlke

Muthukrishnan

Merzendorfer

2017

IJMS

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Chitin biosynthesis in yeast is accomplished by three chitin synthases (Chs) termed Chs1, Chs2 and Chs3, of which the latter accounts for most of the chitin deposited within the cell wall. While the overall structures of Chs1 and Chs2 are similar to those of other chitin synthases from fungi and arthropods, Chs3 lacks some of the C-terminal transmembrane helices raising questions regarding its structure and topology. To fill this gap of knowledge, we performed bioinformatic analyses and protease protection assays that revealed significant information about the catalytic domain, the chitin-translocating channel and the interfacial helices in between. In particular, we identified an amphipathic, crescent-shaped α-helix attached to the inner side of the membrane that presumably controls the channel entrance and a finger helix pushing the polymer into the channel. Evidence has accumulated in the past years that chitin synthases form oligomeric complexes, which may be necessary for the formation of chitin nanofibrils. However, the functional significance for living yeast cells has remained elusive. To test Chs3 oligomerization in vivo, we used bimolecular fluorescence complementation. We detected oligomeric complexes at the bud neck, the lateral plasma membrane, and in membranes of Golgi vesicles, and analyzed their transport route using various trafficking mutants.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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In Vitro and In Vivo Studies on the Structural Organization of Chs3 from Saccharomyces cerevisiae

Gohlke

Muthukrishnan

Merzendorfer

2017

IJMS

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“…Therefore, binding of etoxazole to the 5TMS cluster could block chitin translocation across the membrane; alternatively, it could impair oligomerization of CHS units, a crucial step in the formation of a functional CHS complex (25) similar to cellulose synthesizing rosette complexes (2,25,26), or the proper function of the adjacent coiled-coil motif. The replacement of the isoleucine residue by the aromatic phenylalanine in the 5TMS helix of the CHS1 might prevent etoxazole from interacting with the transmembrane helices to affect chitin synthesis.…”

Section: Discussionmentioning

confidence: 99%

Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods

Leeuwen

Demaeght

Osborne

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Because of its importance to the arthropod exoskeleton, chitin biogenesis is an attractive target for pest control. This point is demonstrated by the economically important benzoylurea compounds that are in wide use as highly specific agents to control insect populations. Nevertheless, the target sites of compounds that inhibit chitin biogenesis have remained elusive, likely preventing the full exploitation of the underlying mode of action in pest management. Here, we show that the acaricide etoxazole inhibits chitin biogenesis in Tetranychus urticae (the two-spotted spider mite), an economically important pest. We then developed a populationlevel bulk segregant mapping method, based on high-throughput genome sequencing, to identify a locus for monogenic, recessive resistance to etoxazole in a field-collected population. As supported by additional genetic studies, including sequencing across multiple resistant strains and genetic complementation tests, we associated a nonsynonymous mutation in the major T. urticae chitin synthase (CHS1) with resistance. The change is in a C-terminal transmembrane domain of CHS1 in a highly conserved region that may serve a noncatalytic but essential function. Our finding of a target-site resistance mutation in CHS1 shows that at least one highly specific chitin biosynthesis inhibitor acts directly to inhibit chitin synthase. Our work also raises the possibility that other chitin biogenesis inhibitors, such as the benzoylurea compounds, may also act by inhibition of chitin synthases. More generally, our genetic mapping approach should be powerful for high-resolution mapping of simple traits (resistance or otherwise) in arthropods.

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“…The oligomerization of chitin synthase is crucial for the generation of insoluble chitin fibrils, as pre-aligned catalytic units could facilitate the proper alignment of nascent sugar chains before coalescence (Sacristan et al, 2013;Gohlke et al, 2017). Evidence of trimeric ChsB complexes from the larval midgut of Manduca sexta (tobacco hornworm) have been reported (Maue et al, 2009), which are presumed to be further oligomerized to form higher-order complexes. Thus, we hypothesized that OfChtIII is localized between newly synthesized chitin chains produced by an oligomerized chitin synthase complex and a newly formed insoluble chitin fibril.…”

Section: A Deduced Role For Ofchtiiimentioning

confidence: 99%

The deduced role of a chitinase containing two nonsynergistic catalytic domains

Liu

Zhu

Wang

et al. 2018

Acta Cryst Sect D Struct Biol

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The glycoside hydrolase family 18 chitinases degrade or alter chitin. Multiple catalytic domains in a glycoside hydrolase family 18 chitinase function synergistically during chitin degradation. Here, an insect group III chitinase from the agricultural pest Ostrinia furnacalis (OfChtIII) is revealed to be an arthropod-conserved chitinase that contains two nonsynergistic GH18 domains according to its catalytic properties. Both GH18 domains are active towards single-chained chitin substrates, but are inactive towards insoluble chitin substrates. The crystal structures of each unbound GH18 domain, as well as of GH18 domains complexed with hexa-N-acetyl-chitohexaose or penta-N-acetylchitopentaose, suggest that the two GH18 domains possess endo-specific activities. Physiological data indicated that the developmental stage-dependent gene-expression pattern of OfChtIII was the same as that of the chitin synthase OfChsA but significantly different from that of the chitinase OfChtI, which is indispensable for cuticular chitin degradation. Additionally, immunological staining indicated that OfChtIII was co-localized with OfChsA. Thus, OfChtIII is most likely to be involved in the chitin-synthesis pathway.

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Purification of an active, oligomeric chitin synthase complex from the midgut of the tobacco hornworm

Cited by 36 publications

References 45 publications

In Vitro and In Vivo Studies on the Structural Organization of Chs3 from Saccharomyces cerevisiae

In Vitro and In Vivo Studies on the Structural Organization of Chs3 from Saccharomyces cerevisiae

Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods

The deduced role of a chitinase containing two nonsynergistic catalytic domains

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