Stanniocalcin has deep evolutionary roots in eukaryotes

Abstract

Vertebrates have a large glycoprotein hormone, stanniocalcin, which originally was shown to inhibit calcium uptake from the environment in teleost fish gills. Later, humans, other mammals, and teleost fish were shown to have two forms of stanniocalcin (STC1 and STC2) that were widely distributed in many tissues. STC1 is associated with calcium and phosphate homeostasis and STC2 with phosphate, but their receptors and signaling pathways have not been elucidated. We undertook a phylogenetic investigation of stanniocalcin beyond the vertebrates using a combination of BLAST and HMMER homology searches in protein, genomic, and expressed sequence tag databases. We identified novel STC homologs in a diverse array of multicellular and unicellular organisms. Within the eukaryotes, almost all major taxonomic groups except plants and algae have STC homologs, although some groups like echinoderms and arthropods lack STC genes. The critical structural feature for recognition of stanniocalcins was the conserved pattern of ten cysteines, even though the amino acid sequence identity was low. Signal peptides in STC sequences suggest they are secreted from the cell of synthesis. The role of glycosylation signals and additional cysteines is not yet clear, although the 11th cysteine, if present, has been shown to form homodimers in some vertebrates. We predict that large secreted stanniocalcin homologs appeared in evolution as early as single-celled eukaryotes. Stanniocalcin's tertiary structure with five disulfide bonds and its primary structure with modest amino acid conservation currently lack an established receptor-signaling system, although we suggest possible alternatives.

FIG. 1.—

Alignment of stanniocalcin homologs from a variety of eukaryotes. Putative full-length protein sequences from representative species were chosen and aligned by MUSCLE v3.7, with additional manual alignment at the N-terminus and C-terminus outside of the conserved cysteine core region (indicated by a line). Salient features were highlighted as follows: black—conserved cysteine residues, green—predicted signal peptides, blue—predicted N-glycosylation sites, and yellow—unique hydrophobic region. Cysteine residues in addition to the ten conserved cysteines are highlighted in orange, and the 11^th cysteine residues in vertebrates, which are dimerization sites, are highlighted in red. The accession numbers of the displayed sequences are as follows: Human STC1 (NCBI NP_003146), Human STC2 (NCBI NP_003705), Zebra fish STC1 (NCBI NP_956833), Zebra fish STC2 (NP_001014827), Amphioxus STCa (NCBI XP_002603782), Amphioxus STCc (NCBI FE592411), Annelid STC (JGI 227571), Mollusc STC (JGI 165566), Nematode STC (NCBI NM_001083148), Cnidarian STC (NCBI XP_002157469), Sponge STC (NCBI GO093006), Fungus STC (NCBI XP_001891263), and Ciliate STC (NCBI XP_001015058).

FIG. 2.—

Amino acid identity matrix of stanniocalcin homologs. Putative full-length protein sequences from representative species were aligned by MUSCLE v3.7 and an amino acid identity matrix was then produced in BioEdit 7.0.5. Values correspond to the percentage of identical sites between two sequences using only the core region between conserved cysteines 1 and 10. The accession numbers of the displayed sequences are listed in the legend of figure 1.

FIG. 3.—

Phylogenetic tree of stanniocalcin homologs from multicellular and unicellular eukaryotes. Stanniocalcin homologs listed in table 1 were aligned by MUSCLE v3.7, trimmed to the region only including the ten conserved cysteine residues, and degapped (for the alignment file, see Supplementary material, Supplementary material online). Maximum likelihood trees were then constructed using PhyML 3.0 with modifications outlined in Methods. The unrooted tree is shown with the vertebrate STC1 and STC2 clades compressed and expanded 4× adjacent to the main tree in order to provide viewable branch lengths. The scale bars represent the substitutions per site, and the numbers at each node are approximate likelihood ratio test for branches (aLRT, SH-like) support values. Support values under 50 were removed.

FIG. 4.—

Diagram of the evolution of several proteins related to calcium control in eukaryotes. Stanniocalcin (STC) has a remarkable conservation across eukaryotes whereas calcitonin (Calc) and parathyroid hormone (PTH) are restricted, to date, to the chordates or vertebrates, respectively. Only proteins with known gene models and/or ESTs are included in the figure. Organisms with VDCCs are included only if they have four-domain calcium channels, which includes a variety of unicellular organisms. Organisms such as amoeboflagellates and ciliates do not have four-domain VDCCs but do have one- or two-domain calcium channels. Two important calcium sensors, calmodulin (CaM) and the calcium-sensing receptor (CaSR) are included in the figure. CaM is ubiquitous in eukaryotes but CaSR has been verified only in vertebrates, although distantly related receptors that are also in family C of the GPCRs are present throughout metazoans.