Human-Specific Mutations and Positively Selected Sites in MARCO Confer Functional Changes

Abstract

Macrophage Receptor with COllagenous structure (MARCO) is a class A scavenger receptor that binds, phagocytoses, and modifies inflammatory responses to bacterial pathogens. Multiple candidate gene approach studies have shown that polymorphisms in MARCO are associated with susceptibility or resistance to Mycobacterium tuberculosis infection, but how these variants alter function is not known. To complement candidate gene approach studies, we previously used phylogenetic analyses to identify a residue, glutamine 452 (Q452), within the ligand-binding Scavenger Receptor Cysteine Rich domain as undergoing positive selection in humans. Herein, we show that Q452 is found in Denisovans, Neanderthals, and extant humans, but all other nonprimate, terrestrial, and aquatic mammals possess an aspartic acid (D452) residue. Further analysis of hominoid sequences of MARCO identified an additional human-specific mutation, phenylalanine 282 (F282), within the collagenous domain. We show that residue 282 is polymorphic in humans, but only 17% of individuals (rs6761637) possess the ancestral serine residue at position 282. We show that rs6761637 is in linkage disequilibrium with MARCO polymorphisms that have been previously linked to susceptibility to pulmonary tuberculosis. To assess the functional importance of sites Q452 and F282 in humans, we cloned the ancestral residues and loss-of-function mutations and investigated the role of these residues in binding and internalizing polystyrene microspheres and Escherichia coli. Herein, we show that the residues at sites 452 and 282 enhance receptor function.

Keywords: MARCO; host–pathogen interactions; positive selection; scavenger receptor; single nucleotide polymorphism.

Fig. 1.

(A) Partial alignment of the collagenous domain of MARCO highlighting residue 282. Humans possess a unique phenylalanine residue at residue 282, except those with the rs6761637 polymorphism, who possess the ancestral serine residue. For a list of species included in each group, see table 2. (B) The global minor allele frequency of rs6761637 (F282S) is 16.8% and varies in frequency between 3.6% in European to 34.5% in African populations. The F282S polymorphism is found at higher frequencies in East Asian (12.3%, n = 629), South Asian (18.6%, n = 657), and African (34.5%, n = 902) populations, relative to European (3.6%, n = 535) and Mixed American (4.8%, n = 468) populations. Each population is composed of multiple subpopulations (i.e., the Mixed American population is composed of Mexican Ancestry from Los Angeles, Puerto Ricans from Puerto Rico, Colombians from Medellin, Colombia, and Peruvians from Lima, Peru). Complete descriptions of each population are available in the 1000 Genomes project. (C) The rs6761637 SNP is in linkage disequilibrium with other polymorphisms associated with infection. Haplotype mapping illustrates that rs6761637 is in LD with four SNPs associated with susceptibility to pulmonary tuberculosis in the Chinese Han population (rs2278589, rs67517405, rs12998782, and rs17009726, n = 163) and one in the Gambian population (rs13389814, n = 179). No LD was observed between rs6761637 and the rs41279766 SNP from the European population (n = 535). All analyses were considered significant if P < 0.05. P values are available in supplementary table S1, Supplementary Material online.

Fig. 2.

Partial alignment of the SRCR domain of MARCO highlighting a residue undergoing positive selection at position 452 (Yap et al. 2015). Aquatic (n = 2) and terrestrial (n = 15) mammals possess a conserved aspartic acid residue at position 452, nonhuman primates (n = 5) vary. Neanderthals (n = 1), Denisovans (n = 1), and humans (n = 1) possess a glutamine residue. For a list of species included in each group, see table 2.

Fig. 3.

Structural comparisons of wildtype human SRCR, Q452A, and Q452D SRCR domain models. (A) Ribbon diagrams of the respective SRCR variants with residue 452 highlighted in red. (B) Molecular surface model of the respective SRCR variants with residue 452 highlighted in red. (C) Molecular surface model highlighting the RGRAEVYY motif in black and residue 452 in red. (D) Coulombic surface charge modeling applied. Red = −10, white = 0, blue = +10 in kcal/(mol·e) at 298 K. (E) Solvent Accessible Surface area (sas) modeling applied. Red = 160 Å, white = 80 Å, blue = 0 Å. Raw sas values of arginine 13, of the SRCR domain RGRAEVYY, circled in black, are shown below each model in Ų.

Fig. 4.

Mutation of sites Q452 and F282 does not affect expression of MARCO. HEK 293 T cells transfected with empty vector, MARCO, MARCOII, Q452A, Q452D, or F282S were examined for total protein expression by western blot (A) and surface expression by flow cytometry (B). V, vector; M, MARCO; II, MARCOII; A, Q452A; D, Q452D; S, F282S.

Fig. 5.

Residues at positions 452 and 282 of MARCO influence ligand association and bacterial internalization. (A) Mutation of glutamine residue 452 to alanine (Q452A) reduces ligand-coated microsphere association in HEK 293 T cells, whereas mutation to the ancestral aspartic acid (Q452D) does not. Mutation of residue phenylalanine 282 to the ancestral serine (F282S) also reduces microsphere association. Statistical comparisons relative to MARCO were made using one-way ANOVA with Tukey’s post hoc test. Error bars indicate mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Experiments were performed a minimum of three times with a minimum of three technical replicates. (B) Immunofluorescence microscopy of microsphere association in HEK 293 T transfected with empty vector, MARCO, MARCOII, or variants Q452A, Q452D, and F282S. Scale bars represent 25 µm. (C) Mutation of residue 452 to alanine (Q452A) or residue 282 to serine (F282S) greatly reduces the phagocytic index of RAW 264.7 macrophages. Phagocytic index was calculated as number of internalized bacteria divided by the number of RAW 264.7 macrophages counted. Statistical comparisons relative to MARCO were made using one-way ANOVA with Tukey’s post hoc test. Error bars indicate mean ± SEM. **P < 0.01.