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Gene
2008 Nov 15;4241-2:11-7. doi: 10.1016/j.gene.2008.07.035.
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Identification and characterization of integrin-binding sialoprotein (IBSP) genes in reptile and amphibian.
Shintani S
,
Kamakura N
,
Kobata M
,
Toyosawa S
,
Onishi T
,
Sato A
,
Kawasaki K
,
Weiss KM
,
Ooshima T
.
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Integrin-binding sialoprotein (IBSP) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family; and the whole SIBLING family is further included in a larger secretory calcium-binding phosphoprotein (SCPP) family. SIBLING proteins are known to construct a part of the non-collagenous extracellular matrices of calcified tissues, and considered to have arisen by duplication and subsequent divergent evolution of a single ancient gene. To understand the alterations of SIBLING molecules associated with the evolution of calcified tissues in vertebrates, we initiated a search for lower vertebrate orthologs of SIBLING genes. In the present study, an IBSP ortholog from a reptile (caiman) and two distinct orthologs from an amphibian (African clawed toad) were identified and characterized. As expected, the toad IBSP genes were transcribed only in calcified tissue (jaw and tibia), as also seen in mammals. The caiman, toad, avian, and mammalian IBSPs share several unique features specific for IBSP and apparently have similar properties. Furthermore, analysis of the sequences suggested that the IBSP molecule might have gradually intensified its functions related to calcification during its evolutionary process through tetrapods.
Fig. 1.
Nucleotide and translated amino acid sequences of caiman IBSP cDNA clone. Amino acid residues are shown with the IUPAC-IUB single letter code. The signal peptide is underlined with a dotted line. The stop codon is indicated by an asterisk (â) and polyadenylation signals are underlined with a solid line. Primer positions and orientations are indicated by double lines with arrowheads above their boxed locations in the sequence. The sequences can be accessed in the GenBank database under accession No. EU007686.
Fig. 2.
Southern blot analysis of the IBSP genes. Genomic DNA was digested with BamHI (Bam), BglII (Bgl), and EcoRV (Eco) restriction enzymes, then hybridized with the IBSP cDNA probe. (A) Caiman IBSP gene; genomic DNA derived from a spectacled caiman (Caiman crocodilus apaporiensis), which is a near relation of Paleosuchus palpebrosus, was used. (B) Toad IBSP genes.
Fig. 3.
RT-PCR analysis of toad tissues. (A) cDNA was amplified from jaw, tibia, heart, and liver specimens using the IBSP cDNA-specific primer sets IBSPX17 and IBSPX14 for toad-A (Lane a), and IBSPX22 and IBSPX28 for toad-B (lane b). (B) cDNA was amplified with primers GAPDH1 and GAPDH2, and used as a positive control.
Fig. 4.
Neighbor-joining tree of amino acid sequences of IBSP proteins. The scale bar represents the distance based on the proportion of amino acid differences. Gapped sites were removed from all sequences before distance estimates were made. Numbers on the nodes indicate the percentage recovery of that node in 1000 bootstrap replications.
Fig. 5.
Structural organization of human, avian, and two toad IBSP genes. Exons are numbered according to the human IBSP gene. Coding lengths of exons with the number of amino acids are shown beneath the boxes. Open and shaded boxes indicate untranslated and translated exon regions, respectively. Exon 1 of the avian IBSP is illustrated by a box with dotted lines, because it was determined to be absent. The amino acid numbers of exon 1 of the toad genes are expressed as question marks, because it is unclear whether they are present. The letters âGluâ and âThrâ enclosed in the open boxes with arrows show the locations of the poly-Es and Thr-rich regions, respectively.
Fig. 6.
Amino acid sequence alignment of IBSP genes from human, bovine, rat, mouse, chicken, caiman, and toad specimens. Amino acid residues are shown with the IUPAC-IUB single letter code. Identity with the simple majority consensus sequence at the top is indicated by a dash (-) and deletions introduced for optimal alignment by asterisks (â). Vertical lines indicate exon borders of the human, chicken, and toad IBSP genes, and numbers on both sides of the line denote exon numbers. Dotted vertical line shows the cleavage site for eliminating signal peptides. The poly-Es are surrounded by grizzled boxes. The conserved signature for the phosphoserine motifs and the Thr-rich regions are indicated by open boxes with solid lines and dotted lines, respectively. The white letters on shaded backgrounds represent the conserved RGD sequences.
Supplementary Figure S1.
Nucleotide and translated amino acid sequences of toad-A cDNA clone. Amino acid residues are shown with the IUPAC-IUB single letter code. The signal peptide is underlined with a dotted line. The stop codon is indicated by an asterisk (â) and the polyadenylation signals are underlined with a solid line. Primer positions and orientations are indicated by double lines with arrowheads above their boxed locations in the sequence. Vertical lines show the positions of the exon boundaries. The sequences can be accessed in the GenBank database under accession no. EU007687.
Supplementary Figure S2.
Nucleotide and translated amino acid sequences of toad-B cDNA clone. The indicators are the same as used in Fig. S1. The sequences can be accessed in the GenBank database under accession no. EU007688.
