Molecular evolution of dentin phosphoprotein among toothed and toothless animals

Abstract

Background: Dentin sialophosphoprotein (DSPP) is the largest member of the SIBLING family and is the most abundant noncollagenous protein in dentin. DSPP is also expressed in non-mineralized tissues including metabolically active ductal epithelia and some cancers. Its function, however, is poorly defined. The carboxy-terminal fragment, dentin phosphoprotein (DPP) is encoded predominantly by a large repetitive domain that requires separate cloning/sequencing reactions and is, therefore, often incomplete in genomic databases. Comparison of DPP sequences from at least one member of each major branch in the mammalian evolutionary tree (including some "toothless" mammals) as well as one reptile and bird may help delineate its possible functions in both dentin and ductal epithelia.

Results: The BMP1-cleavage and translation-termination domains were sufficiently conserved to permit amplification/cloning/sequencing of most species' DPP. While the integrin-binding domain, RGD, was present in about half of species, only vestigial remnants of this tripeptide were identified in the others. The number of tandem repeats of the nominal SerSerAsp phosphorylation motif in toothed mammals (including baleen whale and platypus which lack teeth as adults), ranged from approximately 75 (elephant) to >230 (human). These repeats were not perfect, however, and patterns of intervening sequences highlight the rapidity of changes among even closely related species. Two toothless anteater species have evolved different sets of nonsense mutations shortly after their BMP1 motifs suggesting that while cleavage may be important for DSPP processing in other tissues, the DPP domain itself may be required only in dentin. The lizard DSPP had an intact BMP1 site, a remnant RGD motif, as well as a distinctly different Ser/Asp-rich domain compared to mammals.

Conclusions: The DPP domain of DSPP was found to change dramatically within mammals and was lost in two truly toothless animals. The defining aspect of DPP, the long repeating phosphorylation domain, apparently undergoes frequent slip replication and recombination events that rapidly change specific patterns but not its overall biochemical character in toothed animals. Species may have to co-evolve protein processing mechanisms, however, to handle increased lengths of DSP repeats. While the RGD domain is lost in many species, some evolutionary pressure to maintain integrin binding can be observed.

Figure 1

Phylogeny and divergence timescales of mammalian species whose DPP sequences were compared. The phylogenetic and divergence time estimates are compilations of results reported in other molecular genetic studies and were based on genes other than DSPP [22-32].

Figure 2

Alignment of DPP's deduced amino acid sequences from the BMP1-cleavage domain through the RGD/vestigial integrin-binding motifs. (A) Note the conservation of the BMP1/tolloid-related protease cleavage domain (MQXDD). The underlined MQGDDP sequences were directly encoded by the 5' PCR primer during the production of an amplicon for that species. Due to direct sequencing of the original amplicon, only a portion of the bat sequence was available. The conserved RGD domains (red) are aligned with the vestigial tripeptides (bold). (B) In addition to the loss of their original RGD domains, the platypus was found to contain an RGD domain in a portion of DSP while the common shrew had two RGD motifs within DPP and 5' to the stop codon (*).

Figure 3

The DPP sequences of 26 mammalian species and green anole. Specific motifs are highlighted: SSD-like (highlighted in grey and includes a few simple variations on the tripeptide motif such as SSN and SSE); SKSD-like (blue); RGD (yellow); SSSSS (green); and dipeptides (pink). The extended carboxy-terminal regions for platypus and anole are highlighted in red. Within the anole sequence, serines encoded by TCN-type codons within the repeat domain are in black bold and arginines are highlighted in blue. All serines within the mammalian SSD repeat (except for SKSD) are AGC/T type codons. Stop codons are noted as *. A larger font file of these sequences is available as Additional File 3.

Figure 4

Order and transcription direction (arrows) of SIBLING genes plus adjacent PKD2 and SPARCL1 genes in mammals, green anole, and chicken. Note that the order of the DSPP and DMP1-like genes are reversed between mammals and the anole without changing their direction of transcription relative to the surrounding genes. Row labeled Chicken^A is our interpretation of the relative directions of all six genes' transcription as based on the version 2.1 chicken genome from the Genome Sequencing Center at the Washington University School of Medicine (St. Louis, MO) as compared to the interpretation by Sire et al. [40] (Chicken^B).