The stability of extracellular matrices is in general ensured by cross-linking of its components. Previously, we had shown that the integrity of the layered
Drosophila
cuticle relies on the presence of a covalent cuticular dityrosine network. Production and composition of this structure remained unstudied. In this work, we present our analyses of the
schlaff
(
slf
) gene coding for a putative C-type lectin that is needed for the adhesion between the horizontal cuticle layers. The Slf protein mainly localizes between the two layers called epicuticle and procuticle that separate from each other when the function of Slf is reduced or eliminated paralleling the phenotype of a cuticle with reduced extracellular dityrosine. Localisation of the dityrosinylated protein Resilin to the epicuticle-procuticle interface suggests that the dityrosine network mediates the adhesion of the epicuticle to the procuticle. Ultimately, compromised Slf function is associated with massive water loss. In summary, we propose that Slf is implied in the stabilisation of a dityrosine layer especially between the epicuticle and the procuticle that in turn constitutes an outward barrier against uncontrolled water flow.
The formation of an extracellular matrix (ECM) presupposes an ordered delivery of its components to ensure its stereotypic architecture. The Drosophila cuticle is an ECM produced by the epidermis at its apical site and is characterized by a layered organization. To understand the mechanisms of cuticle assembly during development, we have investigated early aspects of protein N-glycosylation, i.e. the attachment of a dolichol-linked oligosaccharide to distinct Asn sites of a protein known to be essential for sorting in the secretory pathway. Mutations in the Drosophila alg5 gene wollknäuel (wol) that codes for an enzyme initiating the glucosylation of the dolichol-linked oligosaccharide decrease, as expected, glucosylation and the amounts of N-glycosylated proteins such as the cuticle-organizing factor Knickkopf, without affecting their correct localization. At the same time, the polarity determinants Crumbs and atypical protein kinase C accumulate at the apical plasma membrane in wol deficient embryos. In part, these perturbations may also be caused by the unfolded protein response, which is commonly triggered by ER stress and downsizes transcription and translation in general. In any case, they are associated with the loss of cuticle layering and aberrant apical plasma membrane organization suggesting that glucosylation, either directly or indirectly through controlling protein degradation, is important for the efficient and balanced deployment of the biochemical functions of secreted and membrane-associated proteins during epidermal differentiation.
The dopamine monoxygenase N-terminal (DOMON) domain is found in extracellular proteins across several eukaryotic and prokaryotic taxa. It has been proposed that this domain binds to heme or sugar moieties. Here, we have analyzed the role of four highly conserved amino acids in the DOMON domain of the Drosophila melanogaster Knickkopf protein that is inserted into the apical plasma membrane and assists extracellular chitin organization. In principal, we generated Knickkopf versions with exchanged residues tryptophan(299), methionine(333), arginine(401), or histidine(437), and scored for the ability of the respective engineered protein to normalize the knickkopf mutant phenotype. Our results confirm the absolute necessity of tryptophan(299), methionine(333), and histidine(437) for Knickkopf function and stability, the latter two being predicted to be critical for heme binding. In contrast, arginine(401) is required for full efficiency of Knickkopf activity. Taken together, our genetic data support the prediction of these residues to mediate the function of Knickkopf during cuticle differentiation in insects. Hence, the DOMON domain is apparently an essential factor contributing to the construction of polysaccharide-based extracellular matrices.
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