Genomic organization, sequence characterization and expression analysis of Tenebrio molitor apolipophorin-III in response to an intracellular pathogen, Listeria monocytogenes.
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.
['Noh|Ju Young|JY|', 'Patnaik|Bharat Bhusan|BB|', 'Tindwa|Hamisi|H|', 'Seo|Gi Won|GW|', 'Kim|Dong Hyun|DH|', 'Patnaik|Hongray Howrelia|HH|', 'Jo|Yong Hun|YH|', 'Lee|Yong Seok|YS|', 'Lee|Bok Luel|BL|', 'Kim|Nam Jung|NJ|', 'Han|Yeon Soo|YS|']
D000595:Amino Acid Sequence D000818:Animals D001053:Apolipoproteins D001483:Base Sequence D001665:Binding Sites D018076:DNA, Complementary D023281:Genomics D007814:Larva D008089:Listeria monocytogenes D008969:Molecular Sequence Data D010802:Phylogeny D017433:Protein Structure, Secondary D016415:Sequence Alignment D017421:Sequence Analysis D013712:Tenebrio
Expression analysis Tenebrio molitor Apolipophorin-III Listeria monocytogenes Innate immunity ATCC American Type Culture Collection BHI Brain–Heart infusion ENA-EBI EST European Nucleotide Archive-European Bioinformatics Institute Expressed sequence tag GRAVY Grand average of hydropathy LPS LTA Lipopolyssacharide Lipoteichoic acid 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 Terrific broth qRTPCR