Reconstruction of the Carbohydrate 6-O Sulfotransferase Gene Family Evolution in Vertebrates Reveals Novel Member, CHST16, Lost in Amniotes

Abstract

Glycosaminoglycans are sulfated polysaccharide molecules, essential for many biological processes. The 6-O sulfation of glycosaminoglycans is carried out by carbohydrate 6-O sulfotransferases (C6OSTs), previously named Gal/GalNAc/GlcNAc 6-O sulfotransferases. Here, for the first time, we present a detailed phylogenetic reconstruction, analysis of gene synteny conservation and propose an evolutionary scenario for the C6OST family in major vertebrate groups, including mammals, birds, nonavian reptiles, amphibians, lobe-finned fishes, ray-finned fishes, cartilaginous fishes, and jawless vertebrates. The C6OST gene expansion likely started early in the chordate lineage, giving rise to four ancestral genes after the divergence of tunicates and before the emergence of extant vertebrates. The two rounds of whole-genome duplication in early vertebrate evolution (1R/2R) only contributed two additional C6OST subtype genes, increasing the vertebrate repertoire from four genes to six, divided into two branches. The first branch includes CHST1 and CHST3 as well as a previously unrecognized subtype, CHST16 that was lost in amniotes. The second branch includes CHST2, CHST7, and CHST5. Subsequently, local duplications of CHST5 gave rise to CHST4 in the ancestor of tetrapods, and to CHST6 in the ancestor of primates. The teleost-specific gene duplicates were identified for CHST1, CHST2, and CHST3 and are result of whole-genome duplication (3R) in the teleost lineage. We could also detect multiple, more recent lineage-specific duplicates. Thus, the vertebrate repertoire of C6OST genes has been shaped by gene duplications and gene losses at several stages of vertebrate evolution, with implications for the evolution of skeleton, nervous system, and cell-cell interactions.

Keywords: Gal/GalNAc/GlcNAc 6-O sulfotransferases; carbohydrate 6-O sulfotransferases; vertebrate; whole-genome duplication.

Fig. 1.

—Maximum likelihood phylogeny of chordate C6OST sequences. The phylogeny is supported by aLRT and UFBoot analyses. UFBoot supports for deep nodes are shown. Red filled arrowheads indicate unreliable nodes (≤75%) in both UFBoot and aLRT, yellow filled arrowheads indicate nodes with low aLRT support only, and red open arrowheads indicate nodes with low UFBoot support only. The phylogeny is rooted with the Drosophila melanogaster C6OST sequences CG9550 and CG31637.

Fig. 2.

—Phylogeny of CHST1 and CHST16, and conserved synteny between CHST1- and CHST16-bearing chromosome regions, including CHST1a- and CHST1b-bearing regions in teleost fishes. The placement of the CHST1 and CHST16 branches within the full C6OST phylogeny is indicated in the bottom left. Sequence names include species names followed by chromosome/linkage group designations (if available) and gene symbols. Asterisks indicate incomplete sequences. For node support details, see figure 1 caption. Some node support values for shallow nodes have been omitted for visual clarity. Neighboring genes identified in the vicinity of CHST1a and CHST1b genes are indicated in blue. For medaka chromosome 6, CHST16-neighboring genes are to the left and CHST1-neighboring genes are to the right.

Fig. 3.

—Phylogeny of CHST3 branch of C6OST sequences, and conserved synteny across CHST3-bearing chromosome regions, including CHST3a- and CHST3b-bearing regions in teleost fishes. See figure 2 caption for phylogeny details.

Fig. 4.

—Phylogeny of CHST2 and CHST7, and conserved synteny between CHST2- and CHST7-bearing chromosome regions, including CHST2a- and CHST2b-bearing regions in teleost fishes. Genes with uncertain synteny relationships are indicated in gray. See figure 2 caption for phylogeny details.

Fig. 5.

—Phylogeny of CHST4, CHST5, and related genes, including CHST6 and “CHST4/5-like” genes. See figure 2 caption for phylogeny details.

Fig. 6.

—Proposed evolution of C6OST genes through the vertebrate whole-genome duplications (1R, 2R and 3R) (left) and C6OST gene repertoires in representative vertebrate species (right). A and B indicate alternative duplication scenarios through 1R/2R. The uncertain divergence of jawless vertebrates relative to 1R and 2R is indicated by dashed lines. Crossed-over boxes indicate gene losses. Open boxes indicate genes with unresolved phylogenetic positions. Some species-specific or lineage-specific duplicates are indicated by “X2” etc. within boxes. Asterisks indicate species included in the larger phylogenies shown in supplementary figures S1–S5 and S7, Supplementary Material online.

Fig. 7.

—Proposed scenario of C6OST gene evolution after 1R and 2R. Crossed-over boxes indicate gene losses. This figure shows turtles as the sister clade to archosaurs, however, this position is still contested (Gilbert and Corfe 2013). The cyprinid-specific whole-genome duplication and the allotetraploidization in Xenopus laevis are not shown.