Evidence for positive selection in the C-terminal domain of the cholesterol metabolism gene PCSK9 based on phylogenetic analysis in 14 primate species

Abstract

Background: Cholesterol homeostasis is maintained through finely tuned mechanisms regulating intestinal absorption, hepatic biosynthesis and secretion as well as plasma clearance. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted enzyme of the serine protease family that reduces cellular uptake of plasma low-density lipoprotein (LDL) cholesterol by promoting LDL receptor (LDL-R) degradation. Species-specific positive selection has been noted in the LDLR promoter, leading to differential expression of LDLR among primates. Whether PCSK9 experienced significant selective pressure to maintain a functional relationship with its target protein, LDL-R, is unknown.

Methodology/principal findings: We compiled the sequences of the coding regions of PCSK9 from 14 primate species in the clade of Hominoids, Old World monkeys and New World monkeys. To detect selective pressure at the protein level, the ratios of nonsynonymous/synonymous substitution rate (d(N)/d(S)) under different evolutionary models were calculated across the phylogeny of PCSK9. Maximum likelihood analyses of d(N)/d(S) ratios for the aligned coding region sequences among 14 primate species indicated that PCSK9 was subject to a strong functional constraint (i.e., purifying selection). However, positive selection was noted in the functional carboxyl-terminal (C-terminal) domain in many branches across the phylogeny, especially in the lineage leading to the orangutan. Furthermore, at least five positively selected amino acids were detected in this lineage using the branch-site model A. In a sliding-window analysis, several d(N)/d(S) peaks in the C-terminal domain in both the human and the orangutan branches were noted.

Conclusions: These results suggest that among primates, differential selective pressure has shaped evolutionary patterns in the functional domains of PCSK9, an important regulator of cholesterol homeostasis.

Figure 1. Distribution of non-synonymous variations along PCSK9.

There are five functional domains in the PCSK9 protein –: 1) a signal peptide (SP) (1∼30 aa), 2) a prodomain (31∼147 aa), 3) a catalytic domain (148∼425 aa), 4) a putative P domain (426∼525 aa), and 5) a C-terminal domain (526∼691 aa). Gain-of-function mutations are only identified in families with hypercholesterolemia or subjects with high LDL cholesterol levels , and loss-of-function mutations in subjects with low levels of LDL cholesterol –. Some non-synonymous mutations have been identified in subjects with either high or low plasma LDL cholesterol , , and are labeled ‘both’ in the figure. Rare mutations found in families with autosomal dominant hypercholesterolemia are labeled with an asterisk. The gain-of-function mutations are: S127R, F216L, R237W, D374Y, H417Q, R469W, E482G, F515L and H553R. The loss-of-function mutations are: 14insL, E57K, Y142X, L253F, H391N, Q554E, and C679X. Mutations associated with either high- or low- plasma levels of LDL cholesterol subjects are: R46L, A53V, N425S, A443T, I474V, Q619P, and E670G.

Figure 2. Protein sequence alignment of PCSK9 in 14 primates.

“.” Indicates identity to the first sequence (i.e., human) in each alignment. “-” indicates an alignment gap, and “X” indicates a stop codon. The coordinates after 84 should be minus one to be consistent with that in Human reference sequence (NP_777596), since an insertion at position 84 was present in the dusky titi. The signal peptide (SP) domain (1–90) shows the evolution of Leucine (Leu) repeats (15–23) in PCSK9, and the C-terminal domain shows the premature stop codon (X) in the tamarin (686), and dusky titi (689).

Figure 3. Ratios of d _N/d _S estimated for the C-terminal domain of PCSK9 in indicated branches of the primate phylogeny.

Values of d _N/d _S along each branch were calculated by using the free-ratio model using the CODEML program in ‘PAML’ . Branch lengths were estimated by maximum likelihood under this model. A d _N/d _S value of >1 suggests that positive selection has acted along that lineage. ‘Inf’ indicates cases where d _S = 0. The phylogenetic tree was deduced from the entire coding sequence of PCSK9.

Figure 4. Positive selection or relaxed selective constraint of the C-terminal domain of PCSK9.

d _N is plotted versus d _S for all pairwise combinations of primate sequences. The pairwise ratios of d _N/d _S were calculated using the Nei-Gojobori method implemented in the package ‘PAML’ . Pairwise combinations of Hominoids (HOM), Old World monkeys (OWM), and New World monkeys (NWM) are plotted; for example, ‘Human’ represents the points that are making comparisons between human and another primate. We plotted the entire sequence, non C-terminal domain, and C-terminal domain separately. The higher pairwise d _N/d _S ratio in the C-terminal domain suggests that this domain is evolving in a non-neutral model, which maybe due to positive selection or relaxed selective constraint in some lineages. The entire sequence and non C-terminal domain of PCSK9 showed a net signature of purifying selection.

Figure 5. Sliding-window analysis of the cumulative d _N/d _S across primates (black), the lineage leading to human (green), and orangutan (blue).

The gene average d _S across primates is 0.5680, in the lineage leading to human is 0.0030, and orangutan is 0.0204.