The apolipoprotein L family of programmed cell death and immunity genes rapidly evolved in primates at discrete sites of host-pathogen interactions

Abstract

Apolipoprotein L1 (APOL1) is a human protein that confers immunity to Trypanosoma brucei infections but can be countered by a trypanosome-encoded antagonist SRA. APOL1 belongs to a family of programmed cell death genes whose proteins can initiate host apoptosis or autophagic death. We report here that all six members of the APOL gene family (APOL1-6) present in humans have rapidly evolved in simian primates. APOL6, furthermore, shows evidence of an adaptive sweep during recent human evolution. In each APOL gene tested, we found rapidly evolving codons in or adjacent to the SRA-interacting protein domain (SID), which is the domain of APOL1 that interacts with SRA. In APOL6, we also found a rapidly changing 13-amino-acid cluster in the membrane-addressing domain (MAD), which putatively functions as a pH sensor and regulator of cell death. We predict that APOL genes are antagonized by pathogens by at least two distinct mechanisms: SID antagonists, which include SRA, that interact with the SID of various APOL proteins, and MAD antagonists that interact with the MAD hinge base of APOL6. These antagonists either block or prematurely cause APOL-mediated programmed cell death of host cells to benefit the infecting pathogen. These putative interactions must occur inside host cells, in contrast to secreted APOL1 that trafficks to the trypanosome lysosome. Hence, the dynamic APOL gene family appears to be an important link between programmed cell death of host cells and immunity to pathogens.

Figure 1.

Genome organization and coding exons of the human APOL gene family. (A) The genome organization of the six human APOL genes is shown. (B) Coding exons for human APOL genes and isoforms are shown. Differences between isoforms in noncoding regions are not shown.

Figure 2.

Phylogenetic relationships in the APOL gene family in primates. A phylogenetic tree was created from the large-exon sequences of each APOL gene, which includes ∼70%–85% of each coding sequence. Pseudogenes are shown in parentheses, and probabilities of individual clades are shown at central topological junctions. Within individual APOL gene clades, sequences cluster according to the established primate phylogeny (Purvis 1995). The tree shows numerous duplications of APOL genes: APOL2.1 is the result of a duplication that occurred after the divergence of APOL1 and APOL2; APOL7 is the result of a duplication that occurred after the divergence of APOL1/L2 and APOL3/L4; and, depending on the location of the tree root, which is unknown, the APOL1/L2 and APOL3/L4 clades may themselves be products of a duplication that occurred early in primate evolution.

Figure 3.

Lineage-specific evidence for positive selection in primate APOL6. A cladogram is shown with maximum-likelihood estimates of lineage-specific d_N/d_S during primate evolution. Numbers in parentheses are the estimated number of nonsynonymous and synonymous changes, respectively, for each branch. Values of d_N/d_S are also colored according to the class of d_N/d_S to which they are predicted to belong (by GABranch analysis): Bolded values belong to class d_N/d_S = 1.49; bolded, italicized values belong to class d_N/d_S = 5.92; and nonbolded values belong to class d_N/d_S = 0.63. Branch lengths in the figure are not proportional to time.

Figure 4.

Codons under positive selection in primate APOL genes, and a cluster of rapidly evolving codons on an APOL6 MAD alpha helix. (A) We used maximum-likelihood-based tests to estimate positive selection in APOL1, APOL2, and APOL2.1 (combined); APOL3 and APOL4 (combined); and APOL6. We did not analyze APOL5 due to lack of available sequences. The genes are shown to scale and are aligned with one another. The dotted lines show two codons that are under positive selection in both APOL1/L2/L2.1 and APOL6. Gene coordinates and amino acids are shown for human genes, and the isoforms shown include APOL1 isoform a and APOL3 isoform1. APOL2.1 is not shown in the figure. Asterisks indicate stop codons. (B) A group of rapidly evolving codons in the APOL6 MAD tightly cluster on one face of an alpha helix, the predicted structure for the region. Rapidly evolving codons are shown in bold. Sites are also shaded according to hydrophobicity, and the helix has 3.6 amino acids per turn. The rapidly evolving sites of the MAD are located at the base of a pH-sensitive hinge structure. The helix appears to be amphipathic, and rapidly evolving sites appear to be at the hydrophobic/hydrophilic interface of the helix. MAD: membrane-addressing domain; SID: SRA-interacting domain.

Figure 5.

Cartoon model for how MAD and SID antagonists use distinct mechanisms to cause or prevent APOL6-mediated PCD of host cells. (A, i) In the absence of pathogen antagonists, we predict that the APOL6 MAD functions like the APOL1 MAD during trypanosome infections. That is, at pH 7 in an endosome, the MAD hinge is in closed conformation with salt bridges between adjacent alpha helices. At pH 5 in an endolysosome, the hinge opens and allows APOL1 to dissociate from HDL particles to form a pore in the lysosomal membrane, killing the trypanosome. An important difference between APOL1 and APOL6 is that secreted APOL1 kills trypanosomes, whereas APOL6 is not secreted and induces apoptosis of host cells. (ii) We predict that pathogen antagonists manipulate APOL6 function by interacting at the base of the MAD hinge—a region under strong positive selection (shown in dark gray). By recognizing this critical region, antagonists may be able to manipulate APOL6 in different ways. One possibility is that antagonists promote infections by preventing APOL6-mediated apoptosis. In this case, antagonists interact with the MAD hinge to keep it from opening (shown in figure), prolonging infection of the cell. Alternately, antagonists may further infections by prematurely causing apoptosis. In this case, antagonists could interact with the MAD base to open it, initiating apoptosis prematurely (not shown in figure). (B) The trypanosome antagonist SRA binds APOL1 to prevent APOL1-mediated trypanosome lysis during infections. The same region of APOL1 that SRA binds, the SID, is under positive selection in APOL6 (shown in dark gray) and other APOL genes. Hence, we predict that additional SID antagonists, like SRA but distinct in mechanism from MAD antagonists, interact with the SID to block APOL-mediated cell death of host cells during infections.