Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5'-flanking region. BLAST and phylogenetic analyses reveal that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (<26%) with other apoLp-IIIs. Homology modeling of Tm apoLp-III shows a bundle of five amphipathic alpha helices, including a short helix 3'. The 'helix-short helix-helix' motif was predicted to be implicated in lipid binding interactions, through reversible conformational changes and accommodating the hydrophobic residues to the exterior for stability. Highest level of TmapoLp-III mRNA was detected at late pupal stages, albeit it is expressed in the larval and adult stages at lower levels. The tissue specific expression of the transcripts showed significantly higher numbers in larval fat body and adult integument. In addition, TmapoLp-III mRNA was found to be highly upregulated in late stages of L. monocytogenes or E. coli challenge. These results indicate that TmapoLp-III may play an important role in innate immune responses against bacterial pathogens in T. molitor.
Keywords: ATCC; American Type Culture Collection; Apolipophorin-III; BHI; Brain–Heart infusion; ENA-EBI; EST; European Nucleotide Archive-European Bioinformatics Institute; Expressed sequence tag; Expression analysis; GRAVY; Grand average of hydropathy; Innate immunity; LPS; LTA; Lipopolyssacharide; Lipoteichoic acid; Listeria monocytogenes; ML; MSA; Maximum likelihood; Multiple sequence alignment; NCBI; NJ; NMR; National Centre for Biotechnology Information; Neighbor-joining; Nuclear magnetic resonance; PAMP; PDB; PPO; Pathogen associated molecular patterns; Prophenoloxidase; Protein data bank; Quantitative real-time PCR; RC; Reliability class; SE; SMART; Simple modular architecture research tool; Standard error; TB; Tenebrio molitor; Terrific broth; qRTPCR.
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