Abstract
The present study, for the first time, reported twelve A2M isoforms in Tenualosa ilisha, through SMRT sequencing. Hilsa shad, T. ilisha, an anadromous fish, faces environmental stresses and is thus prone to diseases. Here, expression profiles of different A2M isoforms in four tissues were studied in T. ilisha, for the tissue specific diversity of A2M. Large scale high quality full length transcripts (>0.99% accuracy) were obtained from liver, ovary, testes and gill transcriptomes, through Iso-sequencing on PacBio RSII. A total of 12 isoforms, with complete putatative proteins, were detected in three tissues (7 isoforms in liver, 4 in ovary and 1 in testes). Complete structure of A2M mRNA was predicted from these isoforms, containing 4680 bp sequence, 35 exons and 1508 amino acids. With Homo sapiens A2M as reference, six functional domains (A2M_N,A2M_N2, A2M, Thiol-ester_cl, Complement and Receptor domain), along with a bait region, were predicted in A2M consensus protein. A total of 35 splice sites were identified in T. ilisha A2M consensus transcript, with highest frequency (55.7%) of GT-AG splice sites, as compared to that of Homo sapiens. Liver showed longest isoform (X1) consisting of all domains, while smallest (X10) was found in ovary with one Receptor domain. Present study predicted five putative markers (I-212, I-269, A-472, S-567 and Y-906) for EUS disease resistance in A2M protein, which were present in MG2 domains (A2M_N and A2M_N2), by comparing with that of resistant and susceptible/unknown response species. These markers classified fishes into two groups, resistant and susceptible response. Potential markers, predicted in T. ilisha, placed it to be EUS susceptible category. Putative markers reported in A2M protein may serve as molecular markers in diagnosis of EUS disease resistance/susceptibility in fishes and may have a potential for inclusion in the marker panel for pilot studies. Further, challenging studies are required to confirm the role of particular A2M isoforms and markers identified in immune protection against EUS disease.
Highlights
The alpha-2-Macroglobulin (A2M) is a broad-spectrum protease-binding protein and evolutionarily conserved component of the innate immune system in vertebrates [1]
Alternative splicing (AS) of multiple exons is a major source of transcripts and leads to isoform and proteome diversity in eukaryotic organisms [10,11] and better understanding of the disease response requires the knowledge of full complement of mRNA isoforms [12,13]
The recent isoform sequencing (Iso-Seq) based on Single Molecule Real Time (SMRT) technology offers the rapid identification of AS events accurately [16], where entire RNA molecules can be sequenced without any fragmentation or post-sequencing assembly
Summary
The alpha-2-Macroglobulin (A2M) is a broad-spectrum protease-binding protein and evolutionarily conserved component of the innate immune system in vertebrates [1]. It provides protection against invading pathogens by trapping and inhibiting all classes of microbial and parasitic proteases [1]. The protective role of this non-specific protease inhibitor has been studied in various bacterial, viral and fungal diseases affecting aquaculture [2,3]. Previous studies have used conventional methods of cloning and sequencing for identification of A2M isoforms in aquatic animals i.e. common carp [14,6]and Chinese shrimp [15] against microbial infection. The recent isoform sequencing (Iso-Seq) based on Single Molecule Real Time (SMRT) technology offers the rapid identification of AS events accurately [16], where entire RNA molecules can be sequenced without any fragmentation or post-sequencing assembly
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