Mineralized tissue and vertebrate evolution: the secretory calcium-binding phosphoprotein gene cluster

Abstract

Gene duplication creates evolutionary novelties by using older tools in new ways. We have identified evidence that the genes for enamel matrix proteins (EMPs), milk caseins, and salivary proteins comprise a family descended from a common ancestor by tandem gene duplication. These genes remain linked, except for one EMP gene, amelogenin. These genes show common structural features and are expressed in ontogenetically similar tissues. Many of these genes encode secretory Ca-binding phosphoproteins, which regulate the Ca-phosphate concentration of the extracellular environment. By exploiting this fundamental property, these genes have subsequently diversified to serve specialized adaptive functions. Casein makes milk supersaturated with Ca-phosphate, which was critical to the successive mammalian divergence. The innovation of enamel led to mineralized feeding apparatus, which enabled active predation of early vertebrates. The EMP genes comprise a subfamily not identified previously. A set of genes for dentine and bone extracellular matrix proteins constitutes an additional cluster distal to the EMP gene cluster, with similar structural features to EMP genes. The duplication and diversification of the primordial genes for enameldentinebone extracellular matrix may have been important in core vertebrate feeding adaptations, the mineralized skeleton, the evolution of saliva, and, eventually, lactation. The order of duplication events may help delineate early events in mineralized skeletal formation, which is a major characteristic of vertebrates.

Figure 1

Casein, salivary, and EMP gene cluster on human 4q13. Locations of the gene products (milk, saliva, and enamel) are shown on the top. Two salivary protein gene complexes, proximal and distal, are shown. Gene symbols, locations, and transcriptional polarities are indicated at the bottom. Filled and open boxes represent the locations of functional genes and pseudogenes, respectively. The scale represents sequence contigs. The 3′ half of AMBN is not included in the human draft genome sequence (gap). The length of AMBN was inferred from the mouse gene.

Figure 2

Structures and intron phases. White, gray, and black regions represent UTR, SP, and the mature protein, respectively. (A) Exon–intron structures of EMP, casein, and salivary protein genes on 4q13 and AMEL on Xp22 are shown. The length of each exon (bp) is shown in the boxes. The phases of introns are indicated at the bottom of exon boundaries. CSN1S2L2 is a pseudogene, but the exon–intron boundaries were determined unambiguously by comparing bovine and porcine CSN1S2 sequences with the human genome sequence. (B) Exon–intron structures of SPARCL1 and dentine/bone ECM protein genes on 4q21 are shown. The structure of SPARC is the same as SPARCL1 except that exon 4 is missing in the 5′ region of SPARC. (C) The structure of the first four exons of the primordial EMP gene is shown. A protein kinase phosphorylates the Ser residue (P) in the SXE motif coded by the 3′ end of exon 3. The introns are exclusively phase-0.

Figure 3

Phylogenetic trees for EMPs and casein-salivary proteins. MEGA II was used to construct gene trees based on the neighbor-joining method and to calculate substitution rates and bootstrap values. Both trees were drawn based on the timing of the inferred first gene duplication. (A) A phylogenetic tree for the three EMPs was constructed based on the amino acid sequences coded by exons 2–4, which are conserved among all EMP genes. The topology was the same when using sequences coded by exons 2 and 3. (B) A phylogenetic tree for Ca-sensitive CSN and three salivary protein genes was constructed based on the nucleotide sequences of the last exons (3′ UTR): exon 16 of CSN1S1, exon 18 of bovine CSN1S2 (human has two pseudogenes), exon 9 of CSN2, and exon 6 of STATH/HTNs. The same topology was obtained when using human CSN1S2L2. No ubiquitous repeat sequences were identified in these regions.

Figure 4

Divergence of the primordial EMP gene. The phylogeny of Ca-sensitive CSNs (milk), STATH/HTN (saliva), and EMP genes (enamel) was based on the two phylogenetic trees in Fig. 3. The primordial EMP gene appeared early in vertebrate evolution, perhaps in conodont (parenthesized). Immunohistochemical studies suggested the possibility that AMEL and ENAM emerged before the divergence of shark (parenthesized). AMBN diverged before caiman (reptiles). The primordial Ca-sensitive CSN diverged from one of the EMP genes, probably either from ENAM or AMBN before the emergence of monotremes. STATH then diverged from CSN1S2 before the divergence of rodents. Except for AMEL, genes are arranged in this order on chromosome 4.