Connie M. Westhoff

Identification of the erythrocyte Rh blood group glycoprotein as a mammalian ammonium transporter

The Rh blood group proteins are well known as the erythrocyte targets of the potent antibody response that causes hemolytic disease of the newborn. These proteins have been described in molecular detail; however, little is known about their function. A transport function is suggested by their predicted structure and from phylogenetic analysis. To obtain evidence for a role in solute transport, we expressed Rh proteins in Xenopusoocytes and now demonstrate that the erythroid Rh-associated glycoprotein mediates uptake of ammonium across cell membranes. Rh-associated glycoprotein carrier-mediated uptake, characterized with the radioactive analog of ammonium [14C]methylamine (MA), had an apparent EC50 of 1.6 mm and a maximum uptake rate (V max) of 190 pmol/oocyte/min. Uptake was independent of the membrane potential and the Na+ gradient. MA transport was stimulated by raising extracellular pH or by lowering intracellular pH, suggesting that uptake was coupled to an outwardly directed H+ gradient. MA uptake was insensitive to additions of amiloride, amine-containing compounds tetramethyl- and tetraethylammonium chloride, glutamine, and urea. However, MA uptake was significantly antagonized by ammonium chloride with inhibition kinetics (IC50 = 1.14 mm) consistent with the hypothesis that the uptake of MA and ammonium involves a similar H+-coupled counter-transport mechanism.

Ammonia secretion from fish gill depends on a set of Rh glycoproteins

Ammonia excretion from the gill in teleost fish is essential for nitrogen elimination. Although numerous physiological studies have measured ammonia excretion, the mechanism of ammonia movement through the membranes of gill epithelial cells is still unknown. Mammalian Rh glycoproteins are members of a family of proteins that mediate ammonia transport in bacteria, yeast, and plants. We identified the Rh glycoprotein homologs, fRhag, fRhbg, fRhcg1, and fRhcg2, of the pufferfish, Takijugu rubripes. North‐ern blot, in situ hybridization, and immunohistochem‐istry revealed that the pufferfish erythroid Rh glycop‐rotein homologue fRhag was present in red blood cells and the hematological organs (spleen and kidney) in fish. All four pufferfish Rh glycoproteins are specifically localized in the gill and line the pillar cells, pavement cells, and the mitochondrion‐rich cells. Het‐erologous expression in Xenopus oocytes showed that they mediate methylammonium (an analog of ammonium) transport. These results suggest that pufferfish Rh glycoproteins are involved in ammonia excretion from the gill. These findings challenge the classic view that ammonia excretion in the fish gill occurs by passive diffusion.—Nakada, T., Westhoff, C. M., Kato, A., Hirose, S. Ammonia secretion from fish gill depends on a set of Rh glycoproteins. FASEB J. 21, 1067–1074 (2007)

METAL BINDING PROPERTIES OF A MONOCLONAL ANTIBODY DIRECTED TOWARD METAL-CHELATE COMPLEXES

A monoclonal antibody that recognizes cadmium-EDTA complexes has been produced by the injection of BALB/c mice with a metal-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to recognize 16 different metal-EDTA complexes was assessed by measuring equilibrium binding constants using a KinExA™ immunoassay instrument. The antibody bound to cadmium- and mercury-EDTA complexes with equilibrium dissociation constants of 21 and 26 nM, respectively. All other metal-EDTA complexes tested, including those of Mn(II), In(III), Ni(II), Zn(II), Co(II), Cu(II), Ag(I), Fe(III), Pb(II), Au(III), Tb(III), Ga(III), Mg(II), and Al(III) bound with affinities from 20- to 40,000-fold less than that determined for the cadmium-EDTA complex. With the exception of mercury and magnesium, the binding of divalent metal-chelate complexes was well-correlated with the size of the metal ion. The amino acid sequences of the heavy and light chain variable regions were deduced from polymerase chain reaction-amplified regions of the corresponding genes and subsequently used to construct molecular models of the antigen binding region. The key residue for cadmium binding in the model for 2A81G5 appeared to be histidine 96 in the heavy chain.

The Rh blood group system in review: A new face for the next decade

h is the most well-recognized blood group system after ABO, probably because of the dramatic presentation of a fetus suffering hemolytic disease of the newborn (HDN) following maternal alloimmunization to the D antigen. Even individuals not associated with medicine have heard of the “Rh factor” and are aware that it has some importance in pregnancy. The earliest recorded description of the syndrome dates to the 1600s from a French midwife who attended the delivery of a set of twins, one of which was hydropic and the other was jaundiced and died of kernicterus. The agent responsible for the wide range of fetal symptoms, from mild jaundice to fetal demise, remained obscure until 1941. Levine and colleagues observed that the delivery of a stillborn fetus and the adverse reaction in the mother to a blood transfusion from the father were related and were the result of an immune reaction to a paternal antigen. Serologists’ relationship with the offending blood group system began when it was confused with a Rhesus monkey red blood cell (RBC) protein, now termed LW, and much argument and debate ensued over who should receive credit for its discovery. Becoming aware of the antigen, however, was only the beginning of the story. This blood group system would become notorious for its complexity, with numerous antigens and multiple nomenclatures defining it. Several seminal events characterized the history of the Rh system. One of the most important was the observation that ABO mismatch between a mother and the fetus had a partial protective effect against immunization R to D. This suggested the rationale for the development of Rh immune globulin (RhIG). Although immunoglobulin M (IgM) antibodies did not provide protection, immunoglobulin G (IgG) anti-D was effective. By the early 1960s, a mere 20 years after the discovery of Rh incompatibility, an effective treatment was available. Despite their clinical importance, the extremely hydrophobic nature of the Rh proteins made biochemical studies difficult, and the proteins were not successfully isolated until the late 1980s. This led to the cloning of the genes in the 1990s and to major advances in our understanding of the Rh system. The molecular bases of most Rh antigens have been determined, and the RH gene structure explains why this system is so polymorphic. Specifically, the conventional Rh antigens are encoded by two genes, RHD and RHCE, but numerous gene conversion events between them create hybrid genes. The resulting novel hybrid proteins containing regions of RhD joined to RhCE, or the converse, generate the myriad of different Rh antigens. The goal of this review is to highlight the insights gained since the cloning of the genes, describe applications for RH molecular testing to clinical practice, introduce other members of the Rh family of proteins that are present in other tissues, and focus on the next piece of the Rh puzzle, that is, efforts to determine the structure and function of the Rh family of proteins.

International Society of Blood Transfusion Committee on Terminology for Red Cell Surface Antigens: Cape Town report.

The Committee met in Cape Town during the 2006 Inter-national Society of Blood Transfusion (ISBT) Congress (seeAppendix 1 for Committee members). Some changes to theclassification documented in Blood Group Terminology 2004[1] were agreed and are described below. The full updatedclassification can be found on the Blood Group Terminologywebsite at http://www.blood.co.uk/ibgrl. New antigens wereadded to the MNS, Kell, Scianna, Cromer, Indian, Knops,and JMH systems (Table 1). In line with convention, aminoacid positions are numbered with the translation-initiatingmethionine as 1, although the more traditional numberingfor glycophorin A, with number 1 representing the first aminoacid of the mature protein, is also provided.

International Society of Blood Transfusion Committee on terminology for red blood cell surface antigens: Macao report

The committee met in Macao Special Administrative Region,China, during the 2008 International Society of Blood Trans-fusion (ISBT) Congress. Some changes to the classificationdocumented in Blood Group Terminology 2004 [1] and updatedin 2007 [2] were agreed and are described below. The fullupdated classification can be found on the blood groupterminology website at http://www.blood.co.uk/ibgrl. A newblood group system, the RHAG system, was established andnew antigens were added to the Rh, Kell, and Dombrocksystems (Table 1). A total of 308 antigens are now recognized,270 of which are clustered in 30 blood group systems.

Molecular testing for transfusion medicine

Purpose of reviewMolecular testing methods were introduced to the blood bank and transfusion medicine community more than a decade ago after cloning of the genes made genetic testing for blood groups, that is genotyping, possible. This review summarizes the progress made in the last decade in applying genotyping to prenatal practice and clinical transfusion medicine. Recent findingsAssays that target allelic polymorphisms prevalent in all populations are reproducible and highly correlated with red blood cell phenotype. For some blood groups, assays that detect silencing mutations are also required for accurate typing, and for ABO and Rh, multiple regions of the genes must be sampled. Genotyping is a powerful adjunct to serologic testing and is superior for typing transfused patients, for D-zygosity determination, for noninvasive fetal typing, and for antigen-matching in sickle cell patients. SummaryImplementation of molecular testing for transfusion medicine has been a conservative process and limited primarily to reference laboratory environments. With the development of high-throughput platforms, genotyping is poised to move into the mainstream, revolutionizing the provision of antigen-negative donor units. This will enable electronic selection of units antigen matched to recipients at multiple blood group loci, potentially eliminating alloimmunization and significantly improving transfusion outcomes.

DIIIa and DIII Type 5 are encoded by the same allele and are associated with altered RHCE*ce alleles: clinical implications

BACKGROUND: The partial D phenotype DIIIa was originally reported to be associated with 455A>C in Exon 3, 602C>G in Exon 4, and 667T>G in Exon 5. Other alleles with these changes were subsequently identified and designated DIII Types 5, 6, and 7, as they had additional alterations. The observation that DNA samples associated with the DIIIa phenotype had more changes than those originally reported motivated us to reanalyze the DIIIa probands (BP and DJ) from the original study. We also studied additional DIIIa samples to clarify the RHD background and establish the associated RHCE.

Severe anaphylactic reactions following transfusions of platelets to a patient with anti-Ch

The high-frequency Chido (Ch) antigen, found predominantly in plasma, is a determinant of the C4d fragment of the C4 molecule and is acquired by red cells during in vivo complement activation. Antibodies are made by Ch- people who lack C4S. It has often been reported that anti-Ch (and anti-Rg) do not cause hemolytic transfusion reactions. Reported here is a case of a transfusion reaction caused by anti-Ch. The antibody did not cause red cell destruction, but did cause a life-threatening anaphylactic reaction during transfusion of plasma proteins in pooled platelets. The antibody was of the IgG4 subclass and might have caused a short-term, sensitizing anaphylactic response. This case, and one previously reported in which a patient with anti-Rg experienced a severe reaction to fresh-frozen plasma and a plasma derivative, illustrates that these antibodies can cause severe, life-threatening reactions in patients who receive plasma-containing components.

Rh complexities: serology and DNA genotyping.

O ur ability to investigate the RH genes has provided enormous insight into the complexity and origin of the numerous antigens in the Rh system. RH gene diversity has exceeded all estimates predicted by serology, as more than 120 RHD and more than 50 different RHCE alleles have been documented, and new alleles are still being discovered. Although the genetic information finally explains longstanding questions about the Rh system, most importantly, it has direct relevance for improved decision making in the selection of red blood cells (RBCs) for transfusion, for RhIG administration, and for prenatal care. The goal of this article is to demonstrate, through examples from our laboratory, the power of molecular testing for the Rh blood group system. As such, some of the shortcomings of Rh serologic testing and serologic reagents will be discussed; however, it is to be emphasized that serologic methods and reagents have served us well, and should continue to do so in the future. The power lies in the combination of the two approaches. As we begin to consider what blood bank testing in the future might involve, potential possibilities to examine include the practice of repeat testing, or determining the Rh type on two occasions “to get it right.” An improved future approach might include both a serologic phenotype and an RH genotype. One would envision that the RH genotype would become part of the permanent health record and, unlike serologic testing, would not be repeated on each sample. RH genotyping will become economically feasible with the availability of highthroughput platforms. Currently, RH molecular testing is used in our laboratory to resolve serologic Rh typing discrepancies, to aid complex antibody identification, and to discriminate alloversus autoantibodies. Samples are referred primarily from laboratories using reagents licensed by the Food and Drug Administration (FDA), and thus, our experience may differ somewhat from Europe, where many more monoclonal anti-D are available. Most European laboratories do not use the monoclonal immunoglobulin M (IgM) and immunoglobulin G blended clones that are in routine use in the United States and do not perform the indirect antiglobulin test for weak D.