Pharyngeal Polysaccharide Deacetylases Affect Development in the Nematode C. elegans and Deacetylate Chitin In Vitro

Abstract

Chitin (β-1,4-linked-N-acetylglucosamine) provides structural integrity to the nematode eggshell and pharyngeal lining. Chitin is synthesized in nematodes, but not in plants and vertebrates, which are often hosts to parasitic roundworms; hence, the chitin metabolism pathway is considered a potential target for selective interventions. Polysaccharide deacetylases (PDAs), including those that convert chitin to chitosan, have been previously demonstrated in protists, fungi and insects. We show that genes encoding PDAs are distributed throughout the phylum Nematoda, with the two paralogs F48E3.8 and C54G7.3 found in C. elegans. We confirm that the genes are somatically expressed and show that RNAi knockdown of these genes retards C. elegans development. Additionally, we show that proteins from the nematode deacetylate chitin in vitro, we quantify the substrate available in vivo as targets of these enzymes, and we show that Eosin Y (which specifically stains chitosan in fungal cells walls) stains the C. elegans pharynx. Our results suggest that one function of PDAs in nematodes may be deacetylation of the chitinous pharyngeal lining.

Tracking developmentally regulated post-synthetic processing of homogalacturonan and chitin using reciprocal oligosaccharide probes

“…Analogous to methyl esterification of HG, the degree of acetylation has important functional implications. In many cases, the biological significance of chitin acetylation state is poorly understood, but the quantity and ratio of chitin to chitosan is highly dynamic depending on the developmental phase and cellular interactions (Christodoulidou et al, 1999;Baker et al, 2011;Heustis et al, 2012;Luschnig et al, 2006;Wang et al, 2006). For example, tracheal tube elongation in Drosophila melanogaster is regulated by luminal CDA proteins (Luschnig et al, 2006;Wang et al, 2006).…”

Group I chitin deacetylases are essential for higher order organization of chitin fibers in beetle cuticle

“…arthropods (2)(3)(4)(5)(6)(7). In insects, genes encoding CDA-like proteins have been identified and classified into five groups (8 -11).…”

“…Group I CDAs (CDA1s and CDA2s) have a peritrophin-A type chitin-binding domain (CBM14) and a low-density lipoprotein receptor A-type domain in addition to the CE-4 domain. They influence development and molting by acting on extracellular matrix-associated chitin in cuticle, trachea, and pharyngeal lining (5,(12)(13)(14)(15)(16). In Drosophila melanogaster embryos, disruption of DmCDA1 (serpentine) and DmCDA2 (vermiform) genes resulted in an abnormal chitin core in the tracheal lumen and elongated tracheal tubes (12,13).…”

Developmental genetics of the Caenorhabditis elegans pharynx

“… 113 Specific aspects remain to be understood in detail, including cell fusions 124 and the secretion and deposition of many extracellular components, including glycans, 125 specialized cuticle 126 and chitin. 12 One of the most pressing issues toward a better understanding of axon guidance in the C. elegans pharynx concerns the source and distribution of the guidance cues. In vivo reporters suggest that some neurons may act as morphogen sources within the pharynx.…”

“…The pharyngeal lumen is lined with a cuticle that connects with that of the epidermis and includes specialized structures that serve as sieves and, in the posterior bulb, as a grinder; chitosan, a deacetylated form of chitin, may be a component of the pharyngeal cuticle. 12 The basal surfaces of the marginal and muscle cells face a basal lamina continuous with that covering the hypodermis and intestine. Components of this basal lamina are likely produced by body-wall muscles.…”