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

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, theRHCG/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 Rhcgexpression. 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 vitrotranslation and ex vivo expression showed that RhCG carries a complex N-glycan, probably at the48NLS50 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.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  C. Huang,et al.  Molecular biology and genetics of the Rh blood group system. , 2000, Seminars in hematology.

[3]  M. Reid,et al.  Molecular basis for Rhnull syndrome: Identification of three new missense mutations in the Rh50 glycoprotein gene , 1999, American journal of hematology.

[4]  C. Huang,et al.  Rh50 glycoprotein gene and rhnull disease: a silent splice donor is trans to a Gly279-->Glu missense mutation in the conserved transmembrane segment. , 1998, Blood.

[5]  S. Tyerman,et al.  Characterization of an ammonium transport protein from the peribacteroid membrane of soybean nodules. , 1998, Science.

[6]  N. Saitou,et al.  Conserved evolution of the Rh50 gene compared to its homologous Rh blood group gene. , 1998, Biochemical and biophysical research communications.

[7]  T. Omi,et al.  Identification of 5' flanking sequence of RH50 gene and the core region for erythroid-specific expression. , 1998, Biochemical and biophysical research communications.

[8]  C. Huang,et al.  The Human Rh50 Glycoprotein Gene , 1998, The Journal of Biological Chemistry.

[9]  G. Matassi,et al.  Organization of the human RH50A gene (RHAG) and evolution of base composition of the RH gene family. , 1998, Genomics.

[10]  B. André,et al.  The Rh (rhesus) blood group polypeptides are related to NH4+ transporters. , 1997, Trends in biochemical sciences.

[11]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[12]  O. Blumenfeld,et al.  Alternative splicing of a novel glycophorin allele GPHe(GL) generates two protein isoforms in the human erythrocyte membrane. , 1997, Blood.

[13]  B. Carritt,et al.  Evolution of the human RH (rhesus) blood group genes: a 50 year old prediction (partially) fulfilled. , 1997, Human molecular genetics.

[14]  Cheng-Han Huang.  Alteration of RH gene structure and expression in human dCCee and DCW- red blood cells: phenotypic homozygosity versus genotypic heterozygosity , 1996 .

[15]  P. Gane,et al.  Candidate gene acting as a suppressor of the RH locus in most cases of Rh-deficiency , 1996, Nature Genetics.

[16]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[17]  M. Tanner,et al.  Studies on the glycoprotein associated with Rh (rhesus) blood group antigen expression in the human red blood cell membrane. , 1994, The Journal of biological chemistry.

[18]  R. Durbin,et al.  2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans , 1994, Nature.

[19]  P. Henderson,et al.  The 12-transmembrane helix transporters. , 1993, Current opinion in cell biology.

[20]  A. Joyner,et al.  A mouse model of Greig cephalo–polysyndactyly syndrome: the extra–toesJ mutation contains an intragenic deletion of the Gli3 gene , 1993, Nature Genetics.

[21]  H. Heng,et al.  High-resolution mapping of mammalian genes by in situ hybridization to free chromatin. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[22]  C. MacGeoch,et al.  Isolation of cDNA clones for a 50 kDa glycoprotein of the human erythrocyte membrane associated with Rh (rhesus) blood-group antigen expression. , 1992, The Biochemical journal.

[23]  P. Agre,et al.  Molecular biology of the Rh antigens. , 1991, Blood.

[24]  A. Schroit,et al.  Involvement of Rh blood group polypeptides in the maintenance of aminophospholipid asymmetry. , 1990, Biochemistry.

[25]  M. Kilberg,et al.  Substrate-dependent adaptive regulation and trans-inhibition of System A-mediated amino acid transport. Studies using rat hepatoma plasma membrane vesicles. , 1990, Biochimica et biophysica acta.

[26]  M. Frohman,et al.  Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[27]  A. Schroit,et al.  Transbilayer movement of phosphatidylserine in erythrocytes: inhibition of transport and preferential labeling of a 31,000-dalton protein by sulfhydryl reactive reagents. , 1988, Biochemistry.

[28]  M. Kozak.  An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. , 1987, Nucleic acids research.

[29]  Marvin B. Shapiro,et al.  RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. , 1987, Nucleic acids research.

[30]  M. Frommer,et al.  CpG islands in vertebrate genomes. , 1987, Journal of molecular biology.

[31]  R. Doolittle,et al.  A simple method for displaying the hydropathic character of a protein. , 1982, Journal of molecular biology.

[32]  D. Mcclelland,et al.  Isolation of membrane components associated with human red cell antigens Rh(D), (\[cmacr]), (E) and Fya , 1982, Nature.

[33]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[34]  P. Mollison,et al.  Progress in Hematology , 1957 .

[35]  J. Klein,et al.  Molecular Biology and Evolution of Blood Group and MHC Antigens in Primates , 1997, Springer Berlin Heidelberg.

[36]  B. André,et al.  Copyright © 1997, American Society for Microbiology A Family of Ammonium Transporters in , 1997 .

[37]  W. Müller,et al.  Molecular cloning and primary structure of a Rhesus (Rh)-like protein from the marine sponge Geodia cydonium , 1997, Immunogenetics.

[38]  J. Cartron,et al.  Organization of the gene (RHCE) encoding the human blood group RhCcEe antigens and characterization of the promoter region. , 1994, Genomics.

[39]  R. Smith,et al.  Phosphatidylserine transport in Rhnull erythrocytes. , 1990, Blood.

[40]  E. Harlow,et al.  Antibodies: A Laboratory Manual , 1988 .