Molecular cloning, expression profiles and promoter analysis of insulin-like growth factor binding protein-4 (IGFBP-4) in Japanese flounder (Paralichthys olivaceus)

Abstract

We cloned and characterized cDNA sequence of insulin-like growth factor binding protein-4 (IGFBP-4) from Japanese flounder (Paralichthys olivaceus). The 1493 bp full-length cDNA sequence contained an open reading frame (ORF) of 780 bp, which encoded a protein of 259 amino acids. The deduced amino acid sequences included a putative signal peptide of 28 amino acid residues resulting in a mature protein of 231 amino acids. Twenty cysteine residues and two conserved IGFBPs motif (GCGCCXXC and CWCV) were found in the N- and C-terminal domain. In the over 13 kbp genomic sequence, four exons, three introns, and 5'-/3'-flanking sequences were identified. Sequence alignment and phylogenetic analysis showed that Japanese flounder IGFBP-4 was indeed the ortholog of the human IGFBP-4 gene and shared high identities with other teleost IGFBP-4 genes. The promoter region was also analyzed and several potential transcription factor (TF) binding sites were determined which may modulate the IGFBP-4 expression. Quantitative real-time PCR analysis revealed that IGFBP-4 mRNA was observed in various tissues, with intestine showing the highest expression. The maternal transcripts of IGFBP-4 gene existed in the early embryonic stages and then increased in the following stages until hatching, suggesting that IGFBP-4 may be involved in the fish early development. The expression level of IGFBP-4 mRNA was relatively higher at 3 days post hatching (dph) and 15 dph, and gradually decreased during the metamorphosis period. All these results indicated that IGFBP-4 plays a significant role in IGF regulating vertebrate growth and development.