The acquisition of novel N-glycosylation sites in conserved proteins during human evolution

Abstract

Background: N-linked protein glycosylation plays an important role in various biological processes, including protein folding and trafficking, and cell adhesion and signaling. The acquisition of a novel N-glycosylation site may have significant effect on protein structure and function, and therefore, on the phenotype.

Results: We analyzed the human glycoproteome data set (2,534 N-glycosylation sites in 1,027 proteins) and identified 112 novel N-glycosylation sites in 91 proteins that arose in the human lineage since the last common ancestor of Euarchonta (primates and treeshrews). Three of them, Asn-196 in adipocyte plasma membrane-associated protein (APMAP), Asn-91 in cluster of differentiation 166 (CD166/ALCAM), and Asn-76 in thyroglobulin, are human-specific. Molecular evolutionary analysis suggested that these sites were under positive selection during human evolution. Notably, the Asn-76 of thyroglobulin might be involved in the increased production of thyroid hormones in humans, especially thyroxine (T4), because the removal of the glycan moiety from this site was reported to result in a significant decrease in T4 production.

Conclusions: We propose that the novel N-glycosylation sites described in this study may be useful candidates for functional analyses to identify innovative genetic modifications for beneficial phenotypes acquired in the human lineage.

Figure 1

Overall procedure for identifying gains of novel N-glycosylation sites during human evolution. Computational screening and manual inspection were employed to identify the acquisition of novel N-glycosylation sites in human proteins during human evolution.

Figure 2

Timing of acquisition and numbers of novel N-glycosylation sites in the human lineage. Numbers of novel N-glycosylation sites acquired in the human lineage of the mammalian phylogenetic tree are shown. The number of sites acquired is shown on each branch where the N-glycosylation site consensus motif emerged in the ancestor of the corresponding clade.

Figure 3

Multiple sequence alignments of human-specific N-glycosylation sites. The human-specific N-glycosylation modification sites and the surrounding regions for APMAP (A), CD166 (B), and thyroglobulin (C) proteins are presented. The N-glycosylation consensus sequences are highlighted in cyan. An adjacent N-glycosylation site (Asn-95) that is found in CD166 and is well conserved among mammals is indicated by plus signs (+++). The residues that are identical to those in the human sequence are indicated by dots (.). Dashes (−) denote alignment gaps. In some species, sequences were not determined. hum, humans; hac, humans and chimpanzees; aga, African great apes; gra, great apes; ape, apes; cat, catarrhines; sim, simians; pri, primates; eua, Euarchonta; gli, Glires; lau, Laurasiatheria; afr, Afrotheria; xen, Xenarthra; mar, Marsupialia; and mon, Monotremata.

Figure 4

Multiple sequence alignments of N-glycosylation sites that arose during human evolution. The N-glycosylation sites and the surrounding regions for pappalysin-1 (A), TSHR (B), and UD19 (C) proteins are presented. See Figure 3 for further details.

Figure 5

Schematic domain organizations of CD166 (A) and thyroglobulin (B). The N-glycosylation sites are indicated with lollipops, and human-specific sites are indicated in red. The domain organizations are derived from the UniProt database; the accession numbers are [Swiss-Prot:Q13740] (CD166) and [Swiss-Prot:P01216] (thyroglobulin).