Identification, molecular characterization, and in silico structural analysis of larval salivary glands Netrin-A as a potent biomarker from Lucilia sericata (Diptera: Calliphoridae).

The greenbottle blowfly Lucilia sericata (L. sericata) is increasingly used in larval therapy of chronic wounds. Netrins as bifunctional proteins are in the superfamily of Laminins secreted from larval salivary glands. The Netrin protein has a significant instructive role in axon guidance, causing neuronal outgrowth, angiogenesis, and cell migration. It seems to be crucial in wound healing and acts as a potential biomarker in diagnosing some clinical diseases. This survey aimed to identify molecular features and analyze in silico structural configuration of Netrin-A in L. sericata larvae. The larvae were reared under standard maggotarium conditions. The nucleic acid sequence of L. sericata Netrin-A (LSN-A) was then identified using rapid amplification of circular DNA ends (RACE) and rapid amplification of genomic ends (RAGE). Parts of the Netrin-A gene, including the middle, 3'-, and 5'-ends, were identified, TA cloned in pTG19 plasmid, and transferred into DH5ɑ Escherichia coli. Each part was sequenced and assembled using SeqMan software. This gene structure was further subjected to in silico analysis. The DNA of LSN-A was identified to be 2407 bp, while its mRNA sequence was recognized as 2115 bp by Oligo0.7 software. It translated the Netrin-A protein with 704 amino acid residues. Its estimated molecular weight was 78.6 kDa. Sequencing of this fragment and its BLAST analysis revealed laminin-based high (95%) similarity with the mRNA sequence of Lucilia cuprina Netrin-A. The 3-D structure of Netrin-A drawn by SWISS-MODEL exhibited its partial resemblance to the reference molecule Netrin-1 of Homo sapiens. This study supports the molecular and structural analyses of LSN-A protein, which could lead to wound treatment. Ultimately, it can be an effective candidate to ameliorate injury. Our next attempt is to produce LSN-A recombinant protein for use in biomedical sciences.

Molecular characterization of the netrin-1 UNC-5 receptor in Lucilia sericata larvae.

Larval therapy with Lucilia sericata is a promising strategy in wound healing. Axon guidance molecules play vital roles during the development of the nervous system and also regulate the capacity of neuronal restoration in wound healing. Netrin-1, one of the proteins that larvae secrete, plays a useful role in cell migration and nerve tissue regeneration. The UNC-5 receptor combines with a netrin-1 signal and transmits the signal from one side of the membrane to the other side, initiating a change in cell activity. In the current study, we identified the full length of the UNC-5 receptor mRNA in L. sericata using different sets of primers, including exon junction and specific region primers. The coding sequence (CDS) of the UNC-5 receptor was sequenced and identified to include 633 base-pair nucleic acids, and BLAST analysis on its nucleotide sequence revealed 96% identity with the Lucilia cuprina netrin-1 UNC-5 receptor. The protein residue included 210 amino acids (aa) and coded for a protein with 24 kD weight. This gene lacked the signal peptide. Furthermore, the UPA domain is conserved in UNC-5. It lied at the interval of 26-131 aa. We identified the CDS of netrin-1 UNC-5 receptor in L. sericata. It could be applied to research activities implementing a new essential component design in wound healing.