The Rh protein family: gene evolution, membrane biology, and disease association

Abstract

The Rh (Rhesus) genes encode a family of conserved proteins that share a structural fold of 12 transmembrane helices with members of the major facilitator superfamily. Interest in this family has arisen from the discovery of Rh factor's involvement in hemolytic disease in the fetus and newborn, and of its homologs widely expressed in epithelial tissues. The Rh factor and Rh-associated glycoprotein (RhAG), with epithelial cousins RhBG and RhCG, form four subgroups conferring upon vertebrates a genealogical commonality. The past decade has heralded significant advances in understanding the phylogenetics, allelic diversity, crystal structure, and biological function of Rh proteins. This review describes recent progress on this family and the molecular insights gleaned from its gene evolution, membrane biology, and disease association. The focus is on its long evolutionary history and surprising structural conservation from prokaryotes to humans, pointing to the importance of its functional role, related to but distinct from ammonium transport proteins.

Fig. 1

Distribution of Rh genes and their coexistence with Amt genes in eukaryotes. Plus and minus indicate presence and absence, respectively, of Rh (left) or Amt genes (right). The results were obtained from tblastn/blastp search using Rh or Amt protein queries against genome databases. Taxonomic divisions are modified from the eukaryotic tree (www.ncbi.nlm.nih.gov/sutils/genom_tree.cgi). Certain taxa lack both Rh and Amt

Fig. 2

Gene gain and gene loss in the genesis and evolution of the Rh protein family. Left Gene gain and loss are denoted by copy numbers (circled) on the tree trunk (not to scale). Bending arrows indicate gene duplications (green) or gene contractions (black). The increase from three to six genes may arise by a genome-wide duplication. Vertical arrows to the truck point to evolutionary events with which Rh gene duplications coincide: a origin of an ancient Rh gene and its branch off Amt genes in Bacteria and Archaea below the arrow; b origin of epithelia; c origin of erythrocyte. Right Exon remodeling in Rh family genes from representative taxa is shown. E6 (exon 6), the most conserved exon encoding 46 amino acids in metazoans, is used as a reference

Fig. 3

Rh and Amt are distantly related families but have gone through divergent and independent evolution. a The maximum likelihood (ML) joint tree of 111 Rh proteins (red) and 260 Amt (blue) proteins. Bacterial NeRh and archaeal FaAmt and TvAmt are at the base of the respective families. b The ML joint protein tree of 18 Rh and 30 Amt in species that harbor both types of genes. The values at nodes are the bootstrap proportion from ML. This expanded analysis covers species from bacteria to invertebrates

Fig. 4

Membrane topology of N. europaea Rh protein as a model for the Rh family. TMH0 is absent in the crystals and is based on hydropathy plot, whereas TMH 1-11 are from the 3D structure. Closely packed TMH are colored the same except TMH0. The residues positioned at the bilayer leaflets are numbered. Twin-His H170/H324 and twin-Phe F110/F218 are bold (red). Positive K/R charges are colored pink and negative D/E charges green. The α-helical bundle-forming sequence in the C-tail is boxed

Fig. 5

3D structure of the N. europaea Rh protein. Each drawing is oriented such that the periplasm is above and cytoplasm is below. a Ribbon diagram of a monomer. b Ribbon diagram of a homotrimer. c Diagram of the putative central channel in a monomer. The constriction of extracellular vestibule (3.5 Å) and membrane-crossing (28 Å) are denoted. Twin-Phe barrier, twin-His site, and CO₂-binding site are illustrated. d Atomic details of CO₂-binding residues in the pocket. e Interactions between Tyr41 and TMH1. Potential hydrogen bonds between Tyr41 and Ser217 are shown as black dotted lines

Fig. 6

Chromosomal location and disease association of human Rh family genes. a RH, RHBG, RHAG, and RHCG reside in chromosomes 1, 6, and 15. The exon–intron structure and orientation of each gene as well as Rh-negative and Rh-positive haplotypes are shown. c Centromere, p short arm, q long arm, HDFN hemolytic disease of the fetus and newborn, OHSt over-hydrated hereditary stomatocytosis, MDD-RE recurrent early-onset major depressive disorders, dRTA distal renal tubular acidosis. Question mark denotes unknown. b Diagram of mutations of RH and RHAG genes. Amorph type Rh_null (upper); regulator Rh_null, Rh_mod, and OHSt (lower). The 12-TMH of Rh and RhAG is based on NeRh (Fig. 4). Duclos, DSLK, and Ol^a located on the ECLs are point changes of RhAG likely to be neutral antigenic polymorphisms