Characterization of Human RhCG and Mouse Rhcg as Novel Nonerythroid Rh Glycoprotein Homologues Predominantly Expressed in Kidney and Testis

Abstract

In mammals, the Rh family includes the variable Rh polypeptides and invariant RhAG glycoprotein. These polytopic proteins are confined to the erythroid lineage and are assembled into a multisubunit complex essential for Rh antigen expression and plasma membrane integrity. Here, we report the characterization of RhCG and Rhcg, a pair of novel Rh homologues present in human and mouse nonerythroid tissues. Despite sharing a notable similarity to the erythroid forms, including the 12-transmembrane topological fold, the RHCG/ Rhcg pair is distinct in chromosome location, genomic organization, promoter structure, and tissue-specific expression. RHCG and Rhcg map at 15q25 of human chromosome 15 and the long arm of mouse chromosome 7, respectively, each having 11 exons and a CpG-rich promoter. Northern blots detected kidney and testis as the major organs of RHCG or Rhcg expression. In situ hybridization revealed strong expression of Rhcg in the kidney collecting tubules and testis seminiferous tubules. Confocal imaging of transiently expressed green fluorescence protein fusion proteins localized RhCG exclusively to the plasma membrane, a distribution confirmed by cellular fractionation and Western blot analysis. In vitro translation and ex vivo expression showed that RhCG carries a complex N-glycan, probably at the 48 NLS 50 sequon of exoloop 1. These results pinpoint RhCG and Rhcg as novel polytopic membrane glycoproteins that may function as epithelial transporters maintaining normal homeostatic conditions in kidney and testis.The Rh antigens, originally identified in human red blood cells (RBCs), 1 are potent immunogens and, when incompatible, cause hemolytic disease of the newborn and blood transfusion reaction (1). They are defined by two erythroid-specific transmembrane (TM) proteins, the Rh polypeptides and the Rhassociated glycoprotein (RhAG), which form a multisubunit complex and display exodomains as D or CcEe antigens (2). The Rh polypeptides are highly polymorphic and are distinguished from RhAG by two biochemical features, i.e. palmitoylation but no glycosylation. In contrast, RhAG is largely invariant at the population level and is thought to modulate the assembly of the Rh complex and its surface expression. Human Rh polypeptides and RhAG are encoded by RHCED and RHAG loci that reside on chromosomes 1 and 6, respectively (3, 4). Despite this difference, these RH genes have originated from a common ancestor during evolution in that they are homologous in coding sequence and are grossly similar in exon/intron organization (5, 6). Although their exact functions remain to be identified, Rh proteins (and their complex) are necessary for the maintenance of RBC morphology and plasma membrane integrity. The Rh deficiency syndrome, a rare inherited form of hemolytic anemia, is caused by mutations at the RHAG or RHCED locus (2). In this disorder, RBCs deficient in all Rh antigens exhibit spherostomatocytosis and multiple membrane abnormalities (7), implying that Rh proteins have some functional roles in membrane ph...

Rhesus glycoprotein gene expression in the mangrove killifishKryptolebias marmoratusexposed to elevated environmental ammonia levels and air

“…They share similar topology that resembles bacterial Amt (Blakey et al, 2002) and mammalian Rh membrane channels (Liu et al, 2000;Liu et al, 2001), with cytoplasmic N-and Cterminals. K. marmoratus RhBG is predicted to have ten transmembrane domains, while both RhCG1 and RhCG2 have 12 predicted transmembrane domains (Fig.·2).…”

“…In mammals, three Rh genes are identified so far: RhAG, RhBG and RhCG. RhAG expression is restricted to the erythrocyte membrane, whereas RhBG and RhCG are expressed in various tissues in the mammalian systems, including liver, kidney and gastrointestinal tract (Handlogten et al, 2005;Liu et al, 2000;Liu et al, 2001;. Studies in mammalian and plant systems reveal that at least some of those Rh genes encode proteins that mediate NH 3 /NH 4 + movement (Khademi et al, 2004;Zheng et al, 2004;Mayer et al, 2006), but the form of ammonia (NH 3 gas or NH 4 + ion) being transported, and whether the transport is active or passive, are still under much debate (Bakouh et al, 2004;Khademi et al, 2004;Nakhoul et al, 2005).…”

Human Rhesus B and Rhesus C glycoproteins: properties of facilitated ammonium transport in recombinant kidney cells

“…Full-length cDNAs encoding RhBG and RhCG were amplified by PCR from human kidney Marathon-Ready cDNA (Clontech) with the use of published data [7,8], both strands were sequenced using an ABI PRISM 310 Genetic Analyser (Applied Biosystems, Warrington, U.K.) and subcloned in to the pCEP4 expression vector. The mutated cDNAs were constructed by in vitro mutagenesis from pCEP4-RhBG and pCEP4-RhCG double-stranded recombinant DNA according to the supplier's instructions (Stratagene).…”

“…Since RhAG expression is erythroid-restricted, this glycoprotein could not account for ammonium transport in the two organs which play a key role in ammonium metabolism and excretion: liver and kidney. However, two non-erythroid RhAG homologues, RhBG and RhCG, have been characterized and found to be expressed in various tissues including the kidney and the liver [5,7,8].…”

Human Rhesus-associated glycoprotein mediates facilitated transport of NH 3 into red blood cells

“…This discovery was followed by the characterization of two nonerythroid orthologs, RhBG and RhCG (9,10), and the identification of paralogs in a variety of living species, thereby suggesting the conservation of some critical function (reviewed in ref. 11).…”