Molecular characterization of matrix metalloproteinase-1 (MMP-1) in Lucilia sericata larvae for potential therapeutic applications

Background: The salivary glands of Lucilia sericata are the first organs to express specific endopeptidase enzymes. These enzymes play a central role in wound healing, and they have potential to be used therapeutically. Methods: Rapid amplification of cDNA ends and rapid amplification of genomic ends were used to identify the coding sequence of MMP-1 from L. sericata. Different segments of MMP1 gene, namely the middle part, 3' end, and 5' end, were cloned, sequenced, and analyzed using bioinformatics tools to determine the distinct features of MMP-1 protein. Results: Assembling the different segments revealed that the complete mRNA sequence of MMP-1 is 1932 bp long. CDS is 1212 bp long and is responsible for the production of MMP-1 of 404 amino acid residues with a predicted molecular weight of 45.1 kDa. The middle part, 3' end, and 5' end sequences were 933, 503, and 496 bp. In addition, it was revealed that the MMP-1 genomic sequence includes three exons and two introns. Furthermore, the three-dimensional structure of L. sericata MMP-1 protein was evaluated, and its alignment defined that it has high similarity to chain A of human MMP-2 with 100% confidence, 72% coverage, and 38% identity according to the SWISS-MODEL modeling analysis. Conclusions: MMP-1 of L. sericata has a close relationship with its homologs in invertebrates and other insects. The present study significantly contributes to understanding the function, classification, and evolution of the characterized MMP-1 from L. sericata and provides basic required information for the development of an effective medical bioproduct.

The myostatin gene of Mytilus chilensis evidences a high level of polymorphism and ubiquitous transcript expression.

Myostatin (MSTN) is a protein of the Transforming Growth Factor-ß (TGF-ß) superfamily and plays a crucial role in muscular development for higher vertebrates. However, its biological function in marine invertebrates remains undiscovered. This study characterizes the full-length sequence of the Mytilus chilensis myostatin gene (Mc-MSTN). Furthermore, tissue transcription patterns and putative single nucleotide polymorphisms (SNPs) were also identified. The Mc-MSTN cDNA sequence showed 3528 base pairs (bp), consisting of 161 bp of 5' UTR, 2,110 bp of 3' UTR, and an open reading frame of 1,257 bp encoding for 418 amino acids and with an RXXR proteolytic site and nine cysteine-conserved residues. Gene transcription analysis revealed that the Mc-MSTN has ubiquitous expression among several tissues, with higher expression in the gonads and mantle than in the digestive gland, gills, and hemolymph. Furthermore, high levels of polymorphisms were detected (28 SNPs in 3'-UTR and 9 SNPs in the coding region). Two SNPs were non-synonymous and involved amino acid changes between Glu/Asp and Thr/Ile. Until now, the MSTN gene has been mainly related to muscle growth in marine bivalves. However, the present study suggests a putative biological function not entirely associated to muscle tissue and contributes molecular evidence to the current debate about the function of the MSTN gene in marine invertebrates.

Functional characterization of a full length pregnane X receptor, expression in vivo, and identification of PXR alleles, in zebrafish (Danio rerio).

The pregnane X receptor (PXR) (nuclear receptor NR1I2) is a ligand activated transcription factor, mediating responses to diverse xenobiotic and endogenous chemicals. The properties of PXR in fish are not fully understood. Here we report on cloning and characterization of full-length PXR of zebrafish, Danio rerio, and pxr expression in vivo. Initial efforts gave a cDNA encoding a 430 amino acid protein identified as zebrafish pxr by phylogenetic and synteny analysis. The sequence of the cloned Pxr DNA binding domain (DBD) was highly conserved, with 74% identity to human PXR-DBD, while the ligand-binding domain (LBD) of the cloned sequence was only 44% identical to human PXR-LBD. Sequence variation among clones in the initial effort prompted sequencing of multiple clones from a single fish. There were two prominent variants, one sequence with S183, Y218 and H383 and the other with I183, C218 and N383, which we designate as alleles pxr*1 (nr1i2*1) and pxr*2 (nr1i2*2), respectively. In COS-7 cells co-transfected with a PXR-responsive reporter gene, the full-length Pxr*1 (the more common variant) was activated by known PXR agonists clotrimazole and pregnenolone 16α-carbonitrile but to a lesser extent than the full-length human PXR. Activation of full-length Pxr*1 was only 10% of that with the Pxr*1 LBD. Quantitative real time PCR analysis showed prominent expression of pxr in liver and eye, as well as brain and intestine of adult zebrafish. The pxr was expressed in heart and kidney at levels similar to that in intestine. The expression of pxr in liver was weakly induced by ligands for mammalian PXR or constitutive androstane receptor (NR1I3). The results establish a foundation for PXR studies in this vertebrate model. PXR allelic variation and the differences between the full-length PXR and the LBD in reporter assays have implications for assessing the action of PXR ligands in zebrafish.