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. 2023 Mar 18;13(1):4470.
doi: 10.1038/s41598-023-31271-x.

Molecular cloning, characterization and 3D modelling of spotted snakehead fbn1 C-terminal region encoding asprosin and expression analysis of fbn1

Priyanka Sathoria  1 , Bhawna Chuphal  2 , Umesh Rai  3 , Brototi Roy  4
Affiliations

Molecular cloning, characterization and 3D modelling of spotted snakehead fbn1 C-terminal region encoding asprosin and expression analysis of fbn1

Priyanka Sathoria et al. Sci Rep. .

Abstract

The FBN1 gene encodes profibrillin protein that is cleaved by the enzyme furin to release fibrillin-1 and a glucogenic hormone, asprosin. Asprosin is implicated in diverse metabolic functions as well as pathological conditions in mammals. However, till date, there are no studies on asprosin in any non-mammalian vertebrate. In this study, we have retrieved the spotted snakehead Channa punctata fbn1 gene (ss fbn1) from the testicular transcriptome data and validated it. The transcript is predicted to encode 2817 amino acid long putative profibrillin protein. Amino acid sequence alignment of deduced ss profibrillin with human profibrillin revealed that the furin cleavage site in profibrillin is well conserved in C. punctata. Further, differential expression of ss fbn1 was observed in various tissues with the highest expression in gonads. Prominent expression of furin was also observed in the gonads suggesting the possibility of proteolytic cleavage of profibrillin protein and secretion of asprosin in C. punctata. In addition, the C-terminal of the fbn1 gene of C. punctata that codes for asprosin protein has been cloned. Using in silico approach, physicochemical properties of the putative ss asprosin were characterized and post-translational changes were predicted. The putative ss asprosin protein sequence is predicted to consist of 142 amino acid residues, with conserved glycosylation sites. Further, the 3D model of ss asprosin was predicted followed by MD (molecular dynamics) simulation for energy minimization. Thus, the current study, for the first time in non-mammalian vertebrates, predicts and characterizes the novel protein asprosin using in silico approach.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(a) Tissue distribution of fbn1 gene in different tissues of C. punctata. Real-time quantitative PCR was used to quantify the gene expression in each tissue. Each data point represents the mean ± SEM of tissues collected from 6 fish (N = 6). Two technical replicates for each sample were used. (b) Tissue distribution of furin using semi quantitative PCR. The gene expression of furin was studied in tissues which exhibited prominent fbn1 expression (testis, ovary, heart, forebrain midbrain and hindbrain) using semi-quantitative PCR followed by resolving into the 1% agarose gel.
Figure 2
Figure 2
Amino acid alignment of C-terminal region of profibrillin of human and snakehead. The furin cleavage site has been highlighted in green colour and the predicted mature ss asprosin has been highlighted in yellow colour. The ‘*’ represents amino acids that are conserved, ‘:’ indicates amino acids with strongly similar properties, ‘.’ represents amino acids with less similar properties whereas the gaps represent mismatch residues.
Figure 3
Figure 3
Expression analysis of recombinant ss asprosin. (a) 15% SDS-PAGE showing the protein expression in the uninduced pellet, induced pellet, uninduced supernatant and induced supernatant samples observed using coomassie staining. (b) Western blotting using anti-His antibody for validation of recombinant asprosin in supernatant of induced and uninduced samples.
Figure 4
Figure 4
Sequence alignment of putative ss asprosin and human asprosin showing post-translational modifications. The glycosylation sites are highlighted in green and phosphorylation sites are highlighted in yellow.
Figure 5
Figure 5
Structural representation and stability parameters of ss asprosin after stimulation. (a) 3D model of ss asprosin in which arrow represent the β-sheets. (b) Ramachandran plot of amino acid residues of ss asprosin. (c) RMSD (Root mean square deviation) of the backbone Cɑ atoms of ss asprosin vs time. (d) RMSF (Root mean square fluctuation) of each residue of ss asprosin vs time. (e) Rg (Radius of gyration) vs time.
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