dc.description.abstract
Chitin has a recognised importance in physiology but also as a biomaterial. In insects, it is the principal component of the apical extracellular matrix of epidermis, tracheae, foregut and hindgut. In these compartments, chitin is synthesised by the family A of Chitin Synthases (ChS), which in Drosophila is encoded by the gene kkv. Chitin is also the main constituent of the peritrophic matrix that lines the inner surface of the midgut. In this case, the polymer is synthesized by the family B of Chitin Synthases, encoded by ChS2 in Drosophila. Kkv alone is not sufficient to deposit chitin, and two interchangeable MH2-containing proteins, namely Expansion (Exp) and Rebuf (Reb), are equally required for chitin deposition. Kkv and Exp/Reb constitute the minimal genetic program necessary and sufficient for chitin deposition.
The aim of this work is to gain further insights into chitin deposition in apical extracellular matrices of Drosophila melanogaster. In particular, through a structure-function analysis approach, we aimed to investigate the molecular mechanisms of activity of Exp, Reb and Kkv in the tracheal system, focusing our attention on domains that we speculated could be involved in putative protein-protein interactions between these factors. Furthermore, we aimed to produce extracellular chitin in cell culture and to investigate how the differences between Kkv and ChS2, the two Chitin Synthases in Drosophila, correlate with chitin deposition and with the functional requirement of Exp/Reb.
We found that the Nα-MH2 domain of Exp and Reb, the only domain recognisable in these proteins, is necessary for the extracellular chitin deposition, but it is not involved in the subcellular localisation of the chitin machinery. By the comparison of amino acid sequences of homolog proteins to Exp, we identified a new highly conserved region. This conserved motif does not seem to be involved in chitin deposition but it may be relevant for Exp/Reb localisation. On the other hand, our results suggest that Exp/Reb proteins appear to regulate the organised distribution of Kkv at the apical membrane.
We found Kkv, or Kkv complex, assembles at the level of Golgi, then it is translocated to the membrane where the synthesis of chitin starts. Later, Kkv is endocytosed and recycled. From the structure-function analysis of Kkv, we found out that the conserved motif WGTRE is involved in Kkv subcellular localisation and that mis-localised Kkv is not able to deposit chitin, indicating that the localisation of the protein is important for its function. Instead, the coiled-coil domain of Kkv is dispensable for the chitin deposition activity of Kkv, but more experiments are necessary to determine a role for this domain.
It is possible to synthesise chitin in vitro in cell culture of S2 cells transfected with Kkv and Exp/Reb; however, the chitin machinery is not localised at the membrane and the chitin produced is present in intracellular particles. The analysis of chitin synthesis in vitro revealed that the cells need to be polarised to properly localise the chitin machinery and that polarised cells with Kkv and Exp/Reb activity produce less intracellular chitin particles than non-polarised cells with the same activity. We speculate that most of the chitin particles are extruded to the extracellular compartment.
Finally, we confirmed that ChS2 cannot exert the function of Kkv and that, in the embryo, ChS2 does not localise at the membrane. Furthermore, we found that ChS2 is not able to act in concert of Exp and Reb to deposit chitin.
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