From by guest on July 24, 2013. For personal use only.

1991 78: 551-563

Molecular biology of the Rh antigens
P Agre and JP Cartron

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org by guest on July 24. Recent advances in RBC membrane biochemistry have established that the Discovery. respectively. 725 N Wolfe St. and she experienced an immediate and severe transfusion reaction.From bloodjournal.” The antithetical antigen d is not immunologically detectable and is not expressed. suggesting that the Rh antigens are of physiologic importance (see below). THE RH POLYPEPTIDES T ‘251-labeledon the extracellular surface of intact RBCs. Baltimore. Emeritus Director of the Johns Hopkins Hematology Division and Prof Charles Salmon. Soon ~ after delivery. commonly referred to as “Rh positive.. membranes from Rh positive RBCs (obtained from individuals with presumed genotype DID or D / d ) were noted by autoradiography to have a diffuse but strongly labeled band of approximately 28 to 33 Kd. OOiO 551 . 0006-4971 I9117803-0035$3. the mother of a stillborn infant had received a blood transfusion donated by her husband. Two reports published in the winter of 1982 drastically changed the direction of Rh research. RBCs express multiple RBC membrane abnormalities. Address reprint requests to Peter Agre.. Apparent membrane skeleton linkage.”’ Ironically. virtually all normal RBCs bear the antithetical antigens C and/or c in addition to E andlor e. Moore et al’” Edinburgh and Gahmberg” in Helsinki independently observed a previously unrecognized protein that could be Blood. anti-c and anti-E. but is easier to communicate and is more compatible with current molecular studies (see below). and most biochemical methods therefore actually kill the antigenic reactivities that identify and define the Rh antigens. Likewise.” The isolated Rh polypeptides also failed to adsorb to lentil lectin affinity columns and were not degraded by glycosidases nor by alkaline hydrolysis. because the surface antigens detected by the human and rabbit antibodies were not identical. D is the major Rh antigen detected on the surface of RBCs obtained from individuals with the presumed genotypes DID or Did. Endogenous phosphorylation was not detected either. and the Caisse Nationale d’AssurancesMaladies des Travaillers Salaries.” Whether from Rh positive or negative individuals. MD. 1991.8 The latter system does not explain all of the complexities of Rh. despite the presence of an exofacial tyrosine that can be surface ‘251-labeled. For personal use only. hr’)’ or whether there exist multiple closely linked alleles each coding for an independent protein specific for a different Rh antigen (Cic. Emeritus Director of the Institut National de Transjiuion Sanguine. and autoimmune hemolytic The historic discovery of the Rh D antigen was made just over 50 years ago by Levine and Stet~on. accepted April 4. Rh antigenic reactivity is lost after membranes are solubilized or transferred onto immunoblot membranes. and unfortunately many early research efforts proved to be false leads. “Rh” is in fact a misnomer. the Rh polypeptides on intact RBC membranes resisted proteolytic degradation.rh’. 2013. Institut National de Transfusion Sanguine. Baltimore. National Institutes of Health ROI HL33991. A similar but much fainter band was seen in membranes from typical Rh negative RBCs (Fig 1). presumably due to increased binding of SDS. and the latter antigen was subsequently named “LW” in honor of Landsteiner and Wiener: Early investigators soon observed that Rh was very complex.hematologylibrary. 0 I991 by The American Society of Hematology. Johns Hopkins University School of Medicine. Lockard Conley. MD 21205. 1991: pp 551-563 From the Departments of Medicine and Cell Biology. and INSERM Unite U76.” These new bands together are now referred to as the “Rh polypeptides. the Rh polypeptides contained no detectable carbohydrate when examined with methods for labeling terminal sugars. with multiple antigenic variants. Anti-D antibodies were added to the membranes before detergent solubilization. Written in honor of Prof C. A controversy arose concerning whether Rh is a single protein containing multiple antigenic epitopes (referred to as Rho... The apparent lack of carbohydrate was indeed an extraordinary finding. Submitted Januaiy IO. or E/e).” The Rh polypeptides were shown to be highly unusual RBC membrane proteins. which normally removes 0-linked oligosaccharides. The investigators detected an antibody in her serum that agglutinated red blood cells (RBCs) from 80% of randomly selected type 0 donors. hence “Rh negative. REVIEW ARTICLE Molecular Biology of the Rh Antigens By Peter Agre and Jean-Pierre Cartron HISTORICAL BACKGROUND HE RH BLOOD group antigens are of large clinical importance because of their involvement in hemolytic disease of the newborn. and the ‘xI-labeled band was specifically immunoprecipitated with antibodies specific for D from Rh D positive RBCs. Johns Hopkins University School ofMedicine. transfusion medicine.. hence “anti-Rhesus” or “anti-Rh. Paris. Did.” Most surprisingly. P. Exceedingly rare individuals lack all of these antigens and are referred to as Rh.” The Rh polypeptides were found to have electrophoretic mobilities varying from 28 to 32 Kd relative to molecular weight standards in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE).'^ Also.’ Rh.. The antibody specificity was identical to antibodies raised in rabbits injected with RBCs from Rhesus monkeys. is an Established Investigator of the American Heart Association. MD. The molecular basis of the Rh antigens has proven to be a very difficult problem in membrane biology. immunoprecipitated similar but less heavily radiolabeled bands from Rh c and Rh E positive RBCs. No 3 (August 1). Supported in part by NATO Collaborative Research 0556188. Although the glycophorins and the band 3 anion transporter were also labeled.. which is known to occur to very hydrophobic protein^. Vol78. Institut National de la Sante et de la Recherche Medicale. 1991. There has been little agreement about the molecular identity of the Rh antigens until recently. because all known blood group antigens and virtually all mammalian transmembrane proteins are glycoproteins.A.

Fatiy acylation ofthe Rhplypeptides.'' conditions in which thc spcctrin-actin mcmbranc skclcton is insolublc. 2013. CDdcde (Dd). Note the absolute lack of Rh in the immunoprecipitatesfrom dd RBCs." This distancc is approximatcly the lcngth of an cxtcndcd spcctrin tctramcr. intermediate labeling on Dd RBCs. Rockford.2'Thc mcmhranc skclcton was also shown to prcscrvc Rh antigenic activity whcn mcmhrancs wcrc soluhilizcd in nonionic dctcrgcnts.'" Othcr studics dcmonstratcd a latticc-likc distribution on thc mcmhranc surfacc with fcncstrationsof approximatcly I O nm. hccausc initial rcports dcscribcd immunoprccipitation of Rh polypcptidc from Triton-solubilizcd mcmbrancs. IL)." The membrane skclcton is a complcx of spcctrin. Note the strong wtiace Iakling of Rh p o w p t i d e s on OD RBCs. actin. ccrtain glycoprotcins missing from Rh. Undcr ccrtain conditions thc Rh polypcptidcs wcrc found to prccipitatc with thc mcmbranc skcIcton.?' although partial dcficicncics of scvcral othcr mcmbranc componcnts in spherocytosis mcmbrancs suggcst that Rh dcficicncy is not a primary phcnomcnon in t h a t disordcr cithcr. and the small amount of band 3 which was nonspoclfiully immunoprecipitated from all preparations.?' Morcovcr. indicating that thc mcmbranc skclcton is not a major constraint.'* suhscqucnt data analysis indicatcd that thc D antigcns wcrc spaccd a mcan distancc of M nm and 92 nm from thc ncarcst ncighbors on thc RRC mcmbrancs of prcsumcd Rh D homozygotcs and hctcrozygotcs. Hughcs-Joncsat SI Mary's Hospital in hndon. Thc primary membrane dcfcct in the subtypc of hcrcditary elliptocytosis inhcritcd in linkagc with Rh phcnotypc was shown to rcsult from dcficicncics or dysfunctions of mcmbranc skclcton protcin 4." Paticnts with ccrtain congcnital ancmias arc known to have dcfcctivc RBC membrane skclctons. Reprintedwith permission. The anti-D immunoprecipitates are from 5 vol of the same RBCs. England. N." Whcn Triton X-IOOcxtractability of thc Rh polypcptidc from wholc RRC mcmhrancs was comparcd with cxtractahility from spcctrin-dcplctcd mcmbranc vcsiclcs.. a partial dcficicncy of Rh antigcns was found on the mcmbrancs of hcrcditary sphcrocytosis RBCs. For personal use only. and is thc intraccllular structure to which ccrtain transmcmhranc protcins arc attached." Thc incrcascd solubility of Rh polypcptidc whcn complcxcd with anti-D is most likcly confcrrcd by thc watcr-solublc Ig rathcr than a sccondary cffcct on the mcmbranc skclcton. and othcr investigators dcmonstratcd a rclationship hctwccn mcmhranc lipids and Rh antigcnic by guest on July 24. rcspcc- tivcly. whcrcas glycophorin A. Autoradiograph of radiolabehd Rh polypeptides on momh n a of intact RBCs [left panel) or immunoprecipitatedwlth anti-D (right panel). although mcmbranc skclcton linkagc has not bccn dircctly cstahlishcd. Rh D rcactivity was clutcd from RBC mcmbrancs with butanol3 and rcstorcd with cxogcnous phosphatidylcho- .1 gcnc coincidcntally resides ncar the Rh locus on the short arm of chromosomc no." mcmbranc skclcton is thc sourcc of cell shape. I. deformability.. whcn asscsscd quantitativcly. this hchavior may simply rcflcct thc rclativc insolubility of thc Rh polypcptidc in Triton X-100.hematologylibrary.."" Intcrcstingly." This opcrational dcfinition of thc mcmbranc skclcton will not distinguish protcins that arc insoluble duc to tight association with skclcton components from protcins that arc inhcrcntly insoluble in thcsc dctcrgcnts. RBCs also hchavc as if linkcd to thc mcmhranc skclcton (scc hclow). thc solubilities wcrc similar. Early investigations by Masouredis ct al at the University of California at San Dicgo providcd scvcral obscrvations a b u t thc distribution of Rh antigcns on thc surfacc of RBCs by immunoclcctron microscopy using fcrritin-lahclcd antihodics."' Although it was initially concludcd that thc Rh immunc complcxcs wcrc randomly distributcd on thc RBC surface.C."'. was largcly soluble. a nonskclcton-linkcd protcin. Fig 1.1. and scvcral associatcd protcins that arc insoluhlc in low conccntrations on nonionic dctcrgcnts such as 1% Triton X-IO. and weak labeling on dd RBCs. 552 AGRE AND CARTRON DD Dd dd DD Dd dd Glycophorins Rh membranes anti-D immppt. Early invcstigation by Floyd Grccn at the Statc Univcrsity of Ncw York in Buffalo. thc 4. A fraction of thc Rh polypcptidc is obviously soluhlc in 1% (volhrol) Triton X-IW.From bloodjournal. and cdelcde (dd lanes) were 'Waboied using lodogen (Pime. with lcss than 30% of thc Rh polypcptidc rcmaining with thc insoluhlc mcmbranc skclctons. bccausc subscqucnt invcstigators dcmonstratcd that physical shaking of intact RBC mcmbrancs in 5% (wthol) Triton X-IO() produccs an altogcthcr diffcrcnt rcsult." Linkagc of thc Rh polypcptidc to thc mcmhranc skclcton would hc a logical cxplanation for thcsc findings. Ncvcrthclcss.I O at 0°C for 15 minutcs. Intact RBCs from individuals with presumed Rh genotypes cD€/cD€ (DO).3'."'.:' Whilc a rclativcly weak interaction bctwccn thc Rh polypcptidcs and thc mcmhranc skclcton may cxist. 70% to 80% of thc Rh polypcptidcs wcrc still asstxiatcd with the insoluhlc mcmbranc skclcton after incubation in up to 5% (volhrol) Triton X .

" Phospholipasc A? digestion also produced partial loss of Rh D antigenic rcactivity." Digestion of intact RBCs with phospholipasc A? produced increased mcmbranc fluorcsccncc in normal but not Rh. Francc. addition of 'H-palmitatc to invcrted mcmbranc vesicles dcmonstratcd that the Rh polypcptidcs cxclusivcly wcrc acylatcd at multiple sitcs near the inncr Icaflct of the phospholipid bilayer.6. by guest on July 24. Approximately six mcmbranc proteins arc known to bccomc 'H-palmitylatcd when intact RBCs are incubatcd with 'H-palmitic acid.hematologylibrary. c. 57 Kd and 32 Kd wcrc the two most prominently labclcd (Fig 2). but could be rcstorcd by rcduction of disulfides and could be prcvcntcd by prior incubation with anti-D antibodics. Howcvcr. Thcsc different MoAbs permitted improved quantitation of numbcr of Rh antigen sites per RBC. 32-Kd 'H-palmitylatcd protein was labclcd comparably in Rh D negative and positive RBCs but could only be immunoprccipitatcd with anti-D from thc lattcr." Immunologic and nonimmtrnologk approaches. Thc fatty acylation was also found to bc rcvcrsiblc and was entirely rcmovcd during chase with unlabeled palmitate...' Investigators at the Univcrsity of Bristol and the South Wcstcrn Regional Blood Transfusion Centre in England also dcvclopcd pancls of murine MoAbs with Rh-related specificity by immunizing mice with human RBCs. two mcmbranc bands of M. thc M. CDelcDE (lanes 3. The cells were washed. phcnotypc and it was concluded that thesc proteins correspond to thc Rh polypcptidcs.." It remains to be established whether the palmitylation of Rh polypcptidcs is important in their primary antigenic rcactivity or whcthcr they are supporting structural modifications of possiblc physiologic importancc (scc bclow).* This numbcr is somcwhat higher than cstimates prcviously madc with polyclonal antisera. fluorograph of membranes (lanes 2 through 4)." Thc murine MoAbs are thought to be reactive with nonpolymorphic components of the Rh structural protcins. Invcstigators at the Institut National dc Transfusion Sanguine (INTS) in Paris.5. There appcars to be a total of approximately 10' Rh antigens per normal RBC with c (or C). and 7).w When human RBCs wcrc incubatcd with 'H-palmitate.. and the R h ." and all appear to retain the capacity to become fatty acylatcd. Using a radiolabeled impcrmeant malcimidc that apparently dcstroyed Rh antigenic rcactivity (N-malcoylmcthioninc ["SI sulphonc). IW7. Biochemical studies of thc Rh polypcptidcs wcrc impeded by the difficulty in isolating them in sufficicnt quantitics. A portion of the membranes were then incubated with anti-D antibody or nonimmune globulin and immunoprecipitates collected. Reprintedwith permission. produccd cnhanccment or rcduction of Rh antigcnic rcactivity. Pionccring work by Grcen also cstablishcd thc important obscrvation that Rh D and C antigcnic rcactivitics wcrc depcndcnt on a thiol group located at or near the outer surfacc of the RBC mcmbranc.JU Invcstigators in Paris. Palmitic acid appears to bc covalently attachcd to the Rh polypeptides by thioestcr linkages onto frcc sulfhydryls on certain cystcinc residues within the molecule. phenotype (lane 4).From bloodjournal. or nonimmune globulin (lanes 7 and 8 )from RBCs of presumed genotype cdelcde (lanes 1. and E (or e ) cach comprising about % of the total. oxidation of a single sulfhydryl was obscrvcd to lcad to loss of antigen sitcs. It was found that thc 'H-palmitatc can be clutcd from thc Rh polypcptides and othcr RBC acylprotcins with 1 mol/L hydroxylamine at neutral pH. and E antigens by virally transforming B cells from humans with circulating Rh antibodics. RBCs. RBCs. 32-Kd band failed to labcl in mcmbrancs of a variant of the Rh. In preliminary studies. For personal use only. Furthcrmore. respcctivcly. and membranes were prepared. Rh D immunoprecipitatedwith anti-D (lanes 5 and 6)." ISOLATION OF THE RH POLYPEPTIDES 1 2 3 4 5 6 7 8 Fig 2 Fluorograph of membranes from RBCs incubated with 'H-palmitic acid. the fatty acylation was not rcduccd by inhibitors of protcin synthcsis.4'." Alterations in the membrane cholcstcrol phospholipid ratio by cholcstcrol cnrichmcnt or dcplction.. Moreovcr. membrane proteins of M. 32 and 34 Kd wcrc labeled on normal RBCs...''-' Thesc findings all indirectly suggcstcd a strong association of the Rh antigens and membrane lipids. it must bc cmphasizcd that all studies of palmitylation to date have involvcd isotopic labclings. MOLECULAR BIOLOGY OF THE RH ANTIGENS 553 line." Thc 'H-palmitylation of Rh polypcptidcs within maturc RBCs is most likely the result of continuous exchange of frcc palmitate for palmitatc estcrificd onto cystcinc rcsiducs within Rh polypcptidcs. and elution of native fatty acids from the Rh polypcptidcs has not yct bccn reported. F~ofacial free siilflydtyl.'' " and investigators dcmonstratcd that the Rh polypcptidcs are major fatty acylatcd mcmbrane protcins.JO J? The Rh polypcptidcs wcrc subscqucntly found to contain an cxofacial-frec sulfhydryl. Using a variety of sulfhydryl-reactivc probcs. The Rh D polypeptide was immunoprccipi- . and thc attachcd 'H-palmitate was not metabolically dcgradcd to another molccular species.2'. D.'' which could be prcvcntcd by prior addition of anti-D antibody. Intact RBCs were incubated ovemight in tissue culture medium containing up to 1 mCi 'H-palmitic acid. and 8)... but the actual biochcmical explanation rcmaincd unclear. but not on membranes of Rh. variant of the R h . Coomassie-stained SDSPAGE of RBC membranes (lane 1). dcvclopcd cell lines secreting monoclonal antibodics (MoAbs) spccific for Rh D." Dcspitc thc apparcntly large evolutionary divcrsity. Bristol. and thc New York Blood Center cach immunopurificd thc Rh polypcptidcs by addition of large quantitics of the monoclonal or polyclonal anti-D to membranes from RBCs that had been surface "'I-labclcd.2. The M. nonhuman Rh homologs have bccn idcntificd.

c.hematologylibrary.OOO. cach was found to bc a distinct protcin. which was found to cross-rcact with thc Rh c and c polypcptidcs on immunoblots. Variations in thc dcgrada- tion patterns indicated that Rh D is distinct from the C/c and E/c polypcptidcs.""'"' Mcanwhilc. including hydroxylapatite chromatography and prcparativc clcctrophorcsis of SDS-solubilizcd mcmbranc skclctons'l or mcmbranc vcsiclcs. Thc amino acid composition of Rh polypcptidcs obtaincd by immunopurifications and nonimmunc purifications wcrc vcry similar. composite Fig 3. Scvcral rcports indirectly confrmcd that thc M. A composite drawing (lower right panel) shows the iodopeptide corresponding to an exofacial domain of the protein (arrow)." Morcovcr. bcing compriscd of approximatcly 37% hydrophobic rcsiducs. Specific iodopeptidesare indicated: those shared by all Rh D.1 Kd.'" Nonhuman homolop of the Rh polypeptide. D.'' Thc c and E polypcptidcs wcrc nearly identical. Although thc Rh c. M. followcd by digestion with papain rcsultcd in partial dcgradation of thc Rh D polypcptide but not o f thc Rh C/c or E/c polypcptidcs. Two-dimensionaliodopeptide map of Rh D. c. Using this approach. 2013. For personal use only. digestion of intact RBCs with phospholipasc A. confirming an inherent diffcrcncc bctwccn thcsc Rh polypcptidcs. and E/c polypcptidcs was dcmonstratcd with an antiscrum to denatured purified Rh D polypcptide."' Using the hydroxylapatitc method . approximatcly 60. of 31." Thc isolatcd Rh polypcptidcs wcrc protcolytically digcstcd and analyzed by onc-dimcnsional SDS-PAGE autoradiography. 554 AGRE AND CARTRON tatcd from D positive RBCs and purificd to homogcncity by SDS-PAGE. and iodopeptides unique for c and E (vertical stripes). and E polypeptides were found to bc highly rclatcd. invcstigators at Johns Hopkins isolatcd Rh polypcptidcs from surfacc "'I-labclcd Rh D positive human RBCs by nonimmunc by guest on July 24. iodopeptide unique for E (open figure). arrow). Carcful analysis of onc-dimcnsional SDSPAGE gradicnt gcls of immunoprccipitatcd Rh polypcptidcs showcd a small but rcproduciblc diffcrcncc in thc mobilitics of thc diffcrcnt polypcptidcs.'b Thcsc valucs probably corrcspond to thc two mcmbrane protcins labclcd with thc impcrmcant sulfhydryl reagent. Immunologic studies of divcrsc mammalian spccics have shown that antigens which arc related but not identical to the Rh antigens cxist only on the RBCs of higher order primatcs but not other spccics. of 33. and finally spotted on thin-layer plates and analyzed in two dimensions: (1) horizontal.thin-layerchromatography. c.-". Additional cvidcncc undcrscoring thc similarity of thc C/c." Rh polypeptide. c. and E polypeptides show they are highly related but not identical. D." Rh polypcptidcs isolated by thc nonimmune method also showed distinct polymorphisms when Rh D negative and Rh D positive prcparations wcrc compared by twodimcnsional iodopcptidc maps" using Elder et al's method." In other studies.y arc polymovhic. 32-Kd Rh polypcptidcs arc compriscd of multiplc spccics (hcncc "Rh polypcptidcs") which contain thc polymorphisms underlying thc Rh antigcn system. Reprintedwith permission. or E polypeptides separately immunoprecipitated from the RBCs of a single individual (presumed Rh genotype cD€/cD€)with human MoAbs specific for D.9 Kd whcrcas c and E migratcd with an M. electrophoresis.From bloodjournal. (2) vertical. The SDS-denatured purified proteins were first radiolabeled with Inl by chloramine T oxidation. a singlc iodopcptidc conserved among c. 32-Kd bands wcrc cut from SDSPAGE gcls of mcmbrancs" or immunoprccipitatcd with Rh-specific MoAbs from surfacc "'I-labclcd RBCs of diffcrcnt Rh phcnotypcs. D. and E was idcntificd as that l"I-labclcd on the exofacial surfacc of intact RBCs (Fig 3. iodopeptides unique for Rh D (diagonal bars)." Thc nonimmunc mcthod Icd to a ncarly 200-fold purification and pcrmittcd calculation of thc total numbcr of Rh polypcptidcs pcr nativc RBC.% Morcovcr. and then were completely digested with crchymotypsin. and E polypeptides (solid spots). Rh polypcptidcs immunoprccipitatcd from a singlc unit of blood from an individual with prcsumcd Rh gcnotypc cDE/ cDE with MoAbs spccific for c. or E. and E wcrc also analyzcd by two-dimcnsional iodopeptidc maps (Fig 3)." . Autoradiograph of two-dimensional iodopeptide maps prepared from Rh D. D migratcd with an M.". respectively. whilc D was dcfinitcly rclatcd although lcss similar. D.

to 60-Kd glycoproteins had been discovered to coprecipitate along with the Rh polypeptides (discussed below)?’” The N-terminal amino acid sequence of these glycoproteins showed them to be ancestrally related to the Rh by guest on July 24. suggesting the existence of a family of Rh-related molecules. Despite minor differences in the amount of flanking untranslated DNA sequence. the iodopeptide corresponding to the surface ‘“I-labeled exofacial tyrosine was found only in the human preparations. or in the Jurkat lymphoblastic or HL60 promyelocytic cell lines. The Rh cDNA was used to search for poly(A)+ RNA by Northern analysis. . 32-Kd proteins were isolated from the membranes of monkey.s9 MEMBRANE ORGANIZATION OF THE RH POLYPEPTIDES Fig 4.7-kb Rh transcript was also found in RNA preparations from fetal liver. and both groups isolated clones of approximately 1. probably because of low abundance of the Rh cDNA and because of codon degeneracy for the amino acids identified in the N-terminal sequence.49@’ The amino acid sequences for the first 41 residues of the two Rh polypeptides were identical. Note also that the Rh-related glycoprotein bears a partial homology (bottom sequence).to 60-Kd glycoproteins are referred to here as “Rh-related glycoproteins. N-terminal amino acid sequences of Rh polypeptidesand an Rh-related glycoprotein.4 kb. and rat RBCs and analyzed by two-dimensional iodopeptide maps. A group of M. 35. confirming that the cloned Rh cDNA represents a new area of biologic research. The PCR products were radiolabeled and used to select plaques from A libraries constructed from human bone marrow. Overlapping amino acid sequences were independently reported by three research groups each having used different methods to isolate the Rh The recoveries from the sequencing were low. The ultimately useful probes for isolating an Rh cDNA were derived from N-terminal amino acid sequence determined by classical protein sequencing methods (Fig 4). In the end. both groups successfully used the polymerase chain reaction (PCR) with oligonucleotide primers derived from proximal and distal segments of the N-terminal amino acid sequence to amplify cDNA templates prepared from thalassemic spleen erythroblasts and peripheral reticulocytes. The isolated cDNA was shown to localize to chromosome lp34.7-kb and minor 3.60Both groups had previously attempted to use the traditional method of screening recombinant libraries with oligonucleotides corresponding to the N-terminal amino acid sequence previously determined from purified Rh polypeptide. with only five clones found after screening 1. HEL.From bloodjournal.” Isolation o f an Rh cDNA. similar M. For personal use only. Note that the amino acid sequence of the presumed Clc or €/e polypeptide diverges from the D polypeptide after the 41st residue (top sequences).6’ The Rh recombinants were found to be of low abundance. MOLECULAR BIOLOGY OF THE RH ANTIGENS 555 for isolating Rh polypeptides. Thus. Analysis of the primary sequence. the nonhuman homologs to the Rh polypeptides appear to have significantly different primary structures.” Amino acid sequences from the Rh D polypeptide and the Rh-related glycoprotein (glyRhl were entirely determined by sequencing the purified proteins. cat. search of existing databanks failed to identify any related sequences.“ Because of this. subsequent sequence was derived from cloned cDNA. MOLECULAR CLONING OF THE RH POLYPEPTIDES Determination of the N-terminal amino acid sequence. respectively. 35. and major 1. cow.39Of approximately one dozen iodopeptides in each preparation. Nevertheless. indicating that a partial block of the N-terminus may exist.1 by in situ hybridization with metaphase chromosomes. the M. The presumed Clc or Ele polypeptides (isolated by R6A immunoprecipitation)were sequenced to the 41st residue. Identical amino acid sequences were determined for the first 20 amino acid residues from Rh polypeptides isolated by hydroxylapatite chromatography from RBCs of multiple Rh D negative and positive phenotypes?’ This sequence contained the amino acid sequence determined for the first 16 residues from the Rh D polypeptide isolated by immunoprecipitation from Rh D positive RBCs with human monoclonal anti-D antibodiess8In addition. but not in adult human liver. While high-affinity antibodies to Rh D and other Rh antigens are widely available.5-kb signals were found in preparations from adult erythroblasts. several short segments of internal amino acid sequences were determined from proteolytic fragments of Rh polypeptides?’ C/c o r E/# 0 (I) S S K V P R S U R R C L P L U R L 7 L E R R L I L L F V ( 1 ) S S K V P R S U R R C L P L U R L T L E R R L l L L F V ( I ) n(c)F T F P L g l u Ah -n R I u L E I R . kidney.‘Om Identical residues are enclosed within boxes. these are specific for the Rh antigens within the native membrane and have not been found to react with denatured Rh polypeptides. 2013.n I u L F G - The Bristol investigators used the murine R6A MoAb (which most likely recognizes nonpolymorphic regions of the C/c and E/e polypeptides) and the human anti-D MoAb to immunopurify Rh polypeptides and successfully determined N-terminal amino acid sequences for extremely long stretches of the proteins (Fig 4).2 million phages. only two iodopeptides were found to be conserved by the human and all nonhuman preparations.61which corresponds to the Rh locus previously deduced by segregation analysis. Immunologic identification of Rh polypeptides expressed from cDNAs in recombinant libraries has therefore not proven feasible. A cDNA specific for one species of Rh polypeptide was isolated independently by the research groups in Pari?’ and Bristol. and K562 erythroid cell lines and the MEGOl megakaryocytic line. the open reading frames reported by the Paris and Bristol groups were identical?’” This result was not surprising because both groups had obtained commercially available cDNA libraries that had been prepared from the marrow of the same individual. The 1. This cloning strategy was not successful.3-p36.hematologylibrary. Interestingly.

Residue 284 is a cysteine that is predicted to lie very near the outer leaflet of the lipid bilayer in the sequence CHLIP . Individuals with Rh D positive RBCs appeared to have two Rh polypeptide genes. D. In addition. and E polypeptides are indeed closely related but distinct proteins.t e r m i n u ~ consistent ~ ~ ~ ~ ~with the model in (WH2 J 61)CLP (185)CLP (310)CLPVCC Fig 5. Consistent with the studies of Gahmberg. the isolated clone corresponded to the latter sequence. c. The gene organization of the Rh polypeptides also remains to be determined. Fig 5. the point where the sequence of D diverges from C/c or E/e (42). and 5th bilayer spanning domains are noted (4(see text). While most of the bilayer-spanning domains are exclusivelyhydrophobic. The N-terminus is thought to extend into the cytoplasm. an unblocked exofacial cysteine (284) and an exofacial tyrosine (400). The deduced amino acid sequence of the Rh polypeptide includes six cysteine residues?M' and the locations and the identity of surrounding amino acids have proven to be of significant interest. but the Rh polypeptide has a mobility of approximately 32 Kd by SDS-PAGE.N. and the other was a related sequence in which only 8 of 11 amino acids were identical (see Fig 4). two related 7-amino acid sequence repeats were found beginning at residues 392 and 407 and are also predicted to reside at exofacial sites close to the C-terminus. Certain bilayerspanning domains may turn while still within bilayer." Moreover. 185. Modeling suggests 13 bilayer-spanning domains with only very short connecting regions extending outside of the cell or protruding into the cytoplasm.& Although speculative. 2nd. For personal use only. only 5 of the last 13 residues correspond to those deduced from the Rh cDNA (Fig 4). use of different transcriptional initiation sites. and E polypeptides might all be products of a single Rh gene with the divergence arising from alternate splicing of mRNA.~ Most of the polypeptide is predicted to reside between the leaflets of the phospholipid bilayer (Fig 5 ) . the Paris group used the PCR with oligonucleotide primers flanking amino acids 8 through 26 and isolated a single 90-bp product containing a sequence corresponding exactly to that determined from the purified Rh p~lypeptide.@ Topology of cysteine residues. probably due to increased binding of SDS. When the PCR products were used together to screen the library. It was thought that the Rh c." The possibility that this represents a posttranslational proteolytic modification is unlikely. 3rd. Model of Rh polypeptide topology within the membrane lipid bilayer based on hydropathy analysis of cDNA sequence.60 Identification of an Rh isoform. and E iodopeptides (described above and in Fig 3). Digestion of Rh polypeptides from surface 'xI-labeled RBCs with carboxypeptidase Y established that an exofacial tyrosine exists at a location very near the C .6~ Hydropathy analysis of the deduced amino acid sequence for the Rh polypeptide demonstrated a striking degree of hydrophobi~ity?~. because a peptide isolated from the tryptic digestion of the Rh D polypeptide contains a 7-amino acid sequence corresponding to residues 400 through 406 of the predicted Rh protein ~equence. and 12th and 13th bilayer-spanning domains).) of the mature protein is located inside the cell. 9th and loth. Negatively charged residues located within the 1st. D. and the third and fifth are amphipathic helices. whereas the Rh D negative individuals have only one gene. 556 AGRE AND CARTRON The open reading frame of the Rh cDNA encodes 416 amino acids plus an initiating methionine that is removed from the mature protein.67The Rh D and the C.'~ The Bristol group used oligonucleotide primers flanking amino acids 29 through 46 from the amino acid sequence determined for the first 54 residues of Rh polypeptide immunoprecipitated with an anti-D MoAb (Fig 4 ) .From by guest on July 24.'* the primary amino acid sequence is notable for the lack of potential surface N-glycosylation sites.5 Kd. The calculated molecular weight of the deduced protein is 45. or differences in mRNA stability. ~ The ~ location of the C-terminus cannot be determined from the primary amino acid sequence. the cloned Rh cDNA sequence reported by both the Paris and Bristol groups may correspond to the Rh C/c or E/e sequences. Locations of selected deduced amino acid sequencesare indicated:intracellular cysteinesare thought to be palmitylation sites (residues 11. thereby crossing the bilayer twice with no extracellular or intracellular connecting loops (Fig 5. four of the first five domains each contain acidic residues that may be functionally important.M The N-terminus (NH. the tyrosine at the 4th residue was not found to contain '*'I after surface labeling intact R B C S . 2013. but probably does not correspond to the Rh D sequence. The PCR amplification gave two 88-bp products. Moreover.hematologylibrary. and the C-terminus(COOH) faces outward. The existence of tyrosines in the flanking sequence suggests that this region of the Rh polypeptides may correspond to the variant iodopeptides specific for the Rh c. the eight variant residues represent nonconservative substitutions. Recent cloning advances permitted restriction fragment length polymorphism analysis of DNA from individuals with Rh D positive and Rh D negative RBCs. and e polypeptides may therefore be products of a duplicated ancestral gene. Interestingly. based on lack of a cleavable leader peptide and flanking charge differences. It remains uncertain which of the Rh polypeptides corresponds to the deduced cDNA sequence. but was deduced by biochemical studies. D. Divergence between the different Rh polypeptides appears after the 41st amino acid residue. E. theoretical analysis of Rh genetics indicates that there are likely to be two separate but closely linked loci corresponding to D and CcEe. Of the 54 residues determined from sequencing the Rh D polypeptide. The extent of the divergence in more distal sequence remains to be established. The sequence of one product corresponded exactly to the Rh D sequence.w Thus. and 310). 4th and 5th. The Rh c.

After 47-Kd glycoprotein was identified on normal RBCs but not on Rh. cells. the Rh-related glycoproteins were still immunoprecipitated with Rh antibodies after the glycosidase digestion. RBCs.^' Terminal galactose residues on glycoproteins can be labeled on the surface of RBCs labeled with NaB3H4and galactose Rh-related glycoproteins immunoprecipitated from such 3H-labeled RBCs were analyzed by fluorography.72Interestingly. RBCs were shown to partially or totally lack several other distinct glycoproteins. The other five cysteines are predicted to lie near the inner leaflet of the bilayer and exist in the interesting sequence motif CLP (cys-leu-pro).. and BRIC-207). an M. Also. The biology of the LW antigen is complex. phenotype..75Using an MoAb with LW specificity. MOLECULAR BIOLOGY OF THE RH ANTIGENS 557 (cys-his-leu-ile-pro). it has been speculated that Rh might be the precursor of LW. 2013.. . indicating that the Rh antigenic reactivity is not simply the result of the attached glycan. although RBCs from Rh.52 These Rh-related glycoproteins were susceptible to endo-betagalactosidase digestions. Although the M.5-Kd band. 47.. Glycophorin B was shown to be reduced to approximately 30% of the normal and certain other blood group antigens including the DufQ (Fy5). the BRIC 125 protein was found in kidney and liver tissue as well as myeloid. 35 to 60 Kd were coprecipitated with anti-c and anti-E. The CLP motifs are the palmitylation sites on the Rh polypeptide.~~ Selective carboxypeptidase digestions indicate that the C-terminus of the LW protein is exofacial and involved in the antigenic reactivity.~ Immunohistochemical studies with the BRIC 69 and R6A antibodies indicated that the corresponding proteins are restricted to erythroid tissues. Each cysteine is adjacent to a leucine that may provide a hydrophobic side chain within a structural kink in the tertiary structure resulting from the following proline. and the LW antigenic reactivity was destroyed by endoglycosidase F (removes all asparagine-linked oligosaccharides) but not by endo-betagalactosidase (degrades only polylact~saminoglycans).. the investigators showed that the immunoprecipitations of the Rh-related glycoproteins were specifi~... RBCS. and megakaryocytic cell lines. no consensus sequence has been identified. lymphoid.. except the last which is CLPVCC (Fig 5). and the existence of an Rh complex remains to be established (see below). 32-Kd Rh polypeptides were found to lack carbohydrate. The LW antigen.’’ The BRIC 125 MoAb reacts with an M.. while similar bands of M. suggesting a functional While it had been speculated that LW is a glycosylated form of the Rh polypeptide. which is greatly reduced in Rh. by guest on July 24. heterogeneous band of M. and all three appear to be ~almitylated. a characteristic of the polylactosaminoglycans. individuals have reduced expression of the BRIC 125 glycoprotein. and U and Duclos antigens were also missing from Rh. 28.~’ A murine MoAb (2D10) was recently developed in Amsterdam by von dem Borne et and was found to react with the Rh-related glycoprotein^?^ Moreover it is likely that these glycoproteins are coprecipitated with Rh polypeptides during immunoprecipitations with human Rh antibodies as well with several murine MoAbs (R6A. immunohistochemical staining of their lymphocytes was normal. because chelation of Mg’+ by EDTA has recently been shown to block LW antigenic reactivity.. the topology of the cysteines in the established palmitylation sites of these other acylproteins resembles the cysteines within the Rh polypeptide.... For personal use 52-Kd glycoprotein. Nevertheless. two-dimensional iodopeptide maps of purified Rh polypeptide and purified LW glycoprotein conclusively demonstrated that Rh and LW are distinctly different cells.~’. but whether the cysteine and histidine residues are components of the Rh antigenic epitope or an essential supporting structure remains to be established.^^. histidine-reactive compounds had previously been shown to partially reduce all Rh antigenic reactivities.hematologylibrary. These CLP motifs are also flanked by multiple basic amino acids (not shown). On the basis of these phenotypic relationships. 37. The studies did not include quantitative immunoprecipitations.. 45 to 100 Kd was immunoprecipitated along with the Rh D polypeptide by anti-D. In addition to erythroid cells.79 Murine MoAbs developed by the Bristol investigators showed that at least two other membrane glycoproteins may be associated with the Rh polypeptides that are distinct from the LW and D u e antigens.From bloodjournal. however. and a large.. the family of complex asparagine-linked carbohydrates containing numerous galactose-N-acetylglucosamine repeating units and terminating in AB0 blood group structures. While palmitylation of certain other mammalian and viral glycoproteins has been established..” subsequent investigation showed that glycosylated higher molecular weight proteins with ancestrally related amino acid sequences (the “Rh-related glycoproteins”) were coprecipitated by Rh antibodies along with the Rh polypeptide^. with the greatest reductions being of the c and C antigens6’ These observations are entirely consistent with the location of this domain in the model in Fig 5. An exofacial sulfhydryl is known to be involved in the immunoreactivity of Rh D and C antigens (discussed above).. The protein recognized by the BRIC 69 MoAb was found to resemble the Rh-related R6A Both antibodies precipitated a broad complex of M. which may be the same as an Rh-related glycoprotein and which was shown by immunoblot to be missing from Rh. because the level of LW antigen expression is greater in Rh D positive than negative RBCs. being located within a few residues of the inner leaflet of the bilayer with adjacent basic amino acids and glycine or proline helix breakers7’ GLYCOPROTEINS ASSOCIATED WITH RH Rh-related glycoproteins with A B 0 spec@city.. In all cases these CLP motifs precede or follow a region of presumed a helix emerging from or entering the inner leaflet of the phospholipid bilayer. the BRIC 125 protein migrated as an M. Nevertheless.. The initial confusion of Rh and LW antigens was not unlikely. or LW negative membranes. 52-Kd glycoprotein on normal RBCs. BRIC-69. and LW antigens are altogether lacking in the Rh.’~ The motif would be predicted to be a suitable site for the addition of fatty acids from preformed fatty acyl-Coenzyme A complex.. The pathologOther glycoproteins deficient in Rhnus ically affected Rh.

hemolytic disease of the newborn affects Rh D positive infants of Rh negative mothers. and E antigens” and the failure to detect several unrelated glycoproteins in Rh.. Rh-related glycoproteins.From bloodjournal. mutation. because the entire complex is greatly reduced or altogether missing presumably due to a single Rh."^^ and the acetylcholine receptor. Therefore.. It is unlikely that several genes are each altered in Rh. D. The actual demonstration of the components needed for Rh antigenicity may ultimately require expression of specific genetic material by transfection. One hypothesis is that Rh polypeptides are required for the correct transport or cell surface expression of certain glycoproteins..92 Using radiation inactivation as a measure of molecular size.. certain of these other glycoproteins are clearly not immunoprecipated by anti-D along with the Rh polypeptides.^^ The abundance of the different possible components of the Rh membrane complex is quite variable. the size of the Rh membrane complex has not been accurately mea- sured. the Rh D antigen is the most immunogenic of all the RBC antigens. 2013... and the 1D8 antigens are each located on a different 32-Kd Rh polypeptides are apparently not glycosylated also suggests that they may be translated and processed in association with a specific glycoprotein(s). It has also been shown that the Rh polypeptide is not a precursor for the various glycoproteins (see above). because the structural genes for LW. glycophorin B.’~ Several lines of investigation support the Rh membrane complex hypothesis. RBCs both suggest that the Rh polypeptides and these glycoproteins may exist together within a large membrane complex (see above).hematologylibrary. the Rh antigen was estimated to be M. ‘Z51-labeled Rh polypeptides in detergent-solubilized membranes behaved as if complexed with a membrane glycoprotein(s). Nevertheless. patients.. The genetic polymorphism underlying the Rh antigens appears to lie within the gene(s) that encodes the Rh polypeptides.000 copies of the Rh polypeptides per cell. so the Rh membrane complexes might be heterogeneous containing various combinations of the different components. However.. the ability to coprecipitate Rh-related glycoproteins with antibodies specific for Rh c.. Except for instances of spontaneous or drug-induced autoimmunity..” Moreover. All existing information suggests that the Rh D antigenic epitope is not a simple peptide sequence but is likely to be a precise surface conformation that results from the cooperative interactions of at least one of the Rh polypeptides with surrounding glycoproteins and also requires the presence of a specific exofacial free sulfhydryl and also possibly requires certain adjacent lipid structures. Very recent investigation using [3H]palmitate-labeled Rh polypeptides demonstrates that they migrate as a large complex of M. Because of instability in the solubilized state. This explanation suggests that the Rh antigen is a large membrane complex containing the Rh polypeptides. Rh D antigenic reactivity is lost after RBC membranes are solubilized in detergents. and possibly also other unrelated glycoprotein~. and the nucleotide and deduced amino acid sequences should soon be identified..” Obviously. the converse is not true. Because of its lability. an occurrence that would theoretically . more direct evidence is required to bring further support to the Rh membrane complex hypothesis and to demonstrate a direct association of the different glycoproteins. and DufQ blood groups are phenotypically normal and are not deficient in Rh antigens. 60 Kd and 174 Kd by two different investigator^.. Rh D is only immunogenic for individuals who are Rh D negative. Early investigation showed that a small amount of Rh D antigenic reactivity transiently survived solubilization in deoxycholate and was roughly estimated by ultrafiltration to have an M. Cells lacking glycophorin B (S-s-U).. Rh-related glycoprotein cDNAs..~~ WHY I S RH D SO ANTIGENIC? After ABH. 170 Kd when analyzed by velocity sedimentation and gel filtration. phenotype raises the question of how multiple abnormalities might simultaneously arise.. by guest on July 24. there is no obvious physiologic advantage associated with being Rh D positive. For personal use only. simultaneous transfection and expression of Rh polypeptide cDNAs. The fact that the M. ranging from 5. RBCs. because most of the Rh polypeptide is predicted to reside between the leaflets of the lipid bilayer with minimal projection into the extracellular space (see Fig 5). The lack of a single polypeptide chain may prevent the cell surface expression of multichain complexes by altering the assembly or intracellular transport.^^... although the basis of this extreme antigenic reactivity remains very poorly understood.. Isolation of the Rh polypeptide cDNA provided little additional insight. the Rh polypeptides very likely play a fundamental role in the Rh complex structure. RBCS. it is conceivable that continuous and reversible exchange occurs between the components of multisubunit Rh membrane complexes (see above).’~. Likewise. because the amorph and regulator type Rh. attempts to biochemically reconstitute the Rh antigens are likely to be difficult.’~ ASSEMBLY OF RH WITHIN A MEMBRANE COMPLEX The pleitropic defects in several antigens and polypeptides in the Rh. and the members of the hypothetical Rh membrane complex do not appear to be tightly associated.”. regulation of the expression of the other genetic loci by mutations in the Rh locus is also unlikely. and possibly cDNAs for other structural components may be needed. This appears to be a general mechanism that has been well documented for the assembly of T-cell receptors: cell adhesion integrins?= histocompatibility antigen^. whereas the isolated Rh polypeptides did not. yet exhibit the same phenotypic defects. 558 AGRE AND CARTRON Other membrane components recognized by murine MoAbs 1D8 and BS58 but still uncharacterized at molecular levels have also been found to be missing from Rh. mutations arise from different genetic backgrounds. Because the latter are phenotypically normal.% The stoichiometric composition of this complex is yet to be defined but may be a tetramer composed of two Rh polypeptides and two Rh-related gly~oproteins. of less than 300 Kd. 30.000 copies of LW to approximately 100. Because multiple associated structural components may also be required. LW. While these glycoproteins are all deficient in Rh....

'" The deduced amino acid sequence of the cloned Rh polypeptide does not contain the consensus sequence characteristic of and the likely existence of large Rh membrane complexes suggests the possibility that the Rh polypeptides may be associated with other proteins with catalytic activities.. and multiple palmitic acids covalently linked to sites on the Rh polypeptide near the inner leaflet may function to directly influence the organization of surrounding phospholipids.. with phosphatidylcholine (PC) being enriched in the outer leaflet. Interestingly.. Nevertheless.From bloodjournal. RBCs may contain mutant Rh polypeptides that are devoid of Rh antigenic reactivity but may still contribute to PS transbilayer movements. patients and found that the PS analogs were cross-linked to a similar M. For personal use only. the Rh D positive phenotype would be expected to eventually predominate over the Rh D negative by guest on July 24. These data were interpreted as showing that Rh. so the Rh polypeptides within the membranes of RBCs of the common Rh phenotypes must therefore function similarly. many of the Rh D positive fetuses would have developed hydrops fetalis and would have died in utero. 115-Kd Mg2'-ATPase I1 present in chromaffin granules.. In subsequent pregnancies. RBCs are pleomorphic but always have some degree of stomatocytosis and spherocytosis.' The lack of severe clinical manifestations suggests that Rh may be a fine-tuning mechanism in RBC membrane physiology. The possibility that the Rh polypeptides may be noncatalytic subunits of the PS flippase has been raised.lW The same investigators studied RBCs from several Rh. 2013. the incidence of the Rh D positive phenotype is 99%...' POTENTIAL PHYSIOLOGIC FUNCTION(S) OF RH Several lines of evidence indicate that the Rh polypeptides play a fundamental role in the physiology of RBC membranes that is unrelated to their antigenic reactivities. but specific details remain lacking. RBCS. Radiolabeled PS analogs with photoactivatable cross-linking groups become nearly exclusively associated with an M.'''~~'~~~'~' The PSlabeled protein was specifically immunoprecipitated by Rh MoAbs. The explanation for evolutionary selection of the D gene may have resulted from a reproductive advantage by Rh D positive mothers.6' Therefore... the Rh polypeptides may provide a passive restraint to PS in the inner leaflet of the phospholipid bilayer.% Physiologic roles in membrane stability and volume regulation have been suggested for Rh. Indeed.phenofype. ce. Such a notion is consistent with observations of other blood group antigens that are often functionally important structures.. presumably D and CcEe.lo5 and a similar protein has been identified in RBCS. The generation of echinocytes or stomatocytes was monitored after addition of PC or PS which located in the outer or inner leaflet of the bilayer respectively. in ancient times. Alternatively. patients suffer from a chronic hemolytic anemia. and control cells did not behave differently... in certain genetically isolated populations such as Japan. suggesting a common functional ~ignificance. phenotype bear several membrane defects.. and a relative deficiency of membrane cholesterol. while phosphatidylethanolamine (PE) is enriched in the inner leaflet and phosphatidylserine (PS) is located entirely in the inner An ATP-dependent phosphatidylserine translocase ("PS flippase"). which is usually of mild to moderate clinical severity.'~ Pathology ofthe Rh. RBCs from individuals with the rare Rh. This finding is presently difficult to reconcile with the observations that Rh.. The role of Rh in PS flippase and overall membrane phospholipid organization is currently being debated. TX.. and Rh antibodies did not interfere with flip- . The multiple bilayer spanning domains of the membrane model of an Rh polypeptide (Fig 5) is reminiscent of known membrane transporters.~' PhosphatidylserineB@pase? Phospholipids are known to be asymmetrically distributed between the leaflets of the normal RBC lipid bilayer.95 reduced RBC cation and water contents.. theoretically because of the extreme antigenic reactivity associated with Rh D. Once introduced. PS flippase activity was studied in intact RBCs by Daleke et a1 at Stanford and Indiana Universities.."-'" It has recently been proposed by Zachowski et al and Morrot et a1 at the Curie Institute in Paris that PS flippase is similar to the M. which is inactivated by sulfhydryl oxidation.. 32-Kd transmembrane protein with several properties similar to the Rh p~lypeptides. RBCs have severely reduced or absent Rh polypeptides. whereas Rh D negative individuals only have one gene... and PS flippase activity was found to be normal in these Rh.. although use of isotopic amounts of photoactivatable PS analog may not distinguish reduced from normal level of Rh polypeptides. Some Rh D negative mothers will become sensitized by their Rh D positive fetuses. the distantly related proteins isolated from RBCs of several nonhuman species should function equivalently to their human counterpart^.'^ The fatty acylation characteristic of the human Rh polypeptides appears to be conserved amongst all nonhuman Rh homologs.. The RBCs from humans of all of the frequent Rh phenotypes are obviously normal..... and have increased sensitivity to osmotic lysis.. thereby canceling the selection against Rh D positive infants. the function of the polypeptide is not apparent. stillborn hydropic infants and their mothers would probably both have perished due to the lack of obstetrical care.. RBCs... and provide certain clues to the functional importance of Rh. MOLECULAR BIOLOGY OF THE RH ANTIGENS 559 affect one in seven pregnancies among multiparous women in the United States. Rh. 32-Kd band.'" Another candidate for the PS flippase has been proposed by Schroit et al at MD Anderson Cancer Center in Houston. Rh. membrane phospholipid distribution has been reported to be abnormal in Rh. Likewise. However. because they are immune from Rh D sensitization and their offspring will not suffer hemolytic disease.hematologylibrary. into a population. membranes have characteristically hyperactive membrane ATPases. it is likely that the D gene represents an ancient genetic accident during which an ancestral Rh gene was imperfectly duplicated... has been identified as the enzyme responsible for this. Rh. Rh. Very recent work by the group in Paris has demonstrated that Rh D positive individuals have two Rh polypeptides genes..

Churchill Livingstone. NY. observations that led these investigators to conclude that flippase is not related to the Rh polypeptide. Shotton DM. Cartron JP: Human monoclonal antibody against .(D) antigen. including certain Rh-related glycoproteins. Bloy C. Singher H: A 'D-like' antigen in rhesus monkey. recent research has significantly advanced the molecular understanding of these important blood group antigens. Branton DM: The molecular structure of human erythrocyte spectrin.1979 19.1940 6. James V: Nearest neighbour analyses on the distribution of Rh antigens on erythrocyte membranes. and Northern analysis indicated that Rh is erythroid specific. David Anstee. The Rh polypeptides contain an exofacial free sulfhydryl that is important for Rh antigenic reactivity and several intracellular sulfhydryls that appear to be palmitylated. Gahmberg CG: Molecular identification of the human Rh. Alan Schroit. but until recently have been poorly understood at a molecular level. 1980 4. Nature 295529.(D) antigenic sites on human erythrocyte membranes. McClelland D B L Isolation of membrane components associated with human red cell antigens Rh. Pollack W. Gahmberg CG: Molecular characterization of the human red cell Rho (D) antigen.1944 9. ACKNOWLEDGMENT The authors thank Drs Floyd Green.. The cDNA coding for a 416-amino acid Rh polypeptide was recently isolated but was not found to share sequence homology with any known protein. Race RR: An incomplete antibody in human serum.1973 16. J Immunol137:240. Biophysical and electron microscopic studies. Woodrow CF.1987 10. Engelfriet CP.1986 23.(D). New York. Nash R. Moore S. Proc Natl Acad Sci USA 68:1416.hematologylibrary. The Rh polypeptides are a family of nonglycosylated M. committed RBC progenitors. Race RR. Rouger P. Br J Haematol40657. Victoria EJ: Antigen site densities and ultrastructural distribution patterns of red cell Rh antigens. Karhi KK: Association of Rh. Michael Tanner. Simons K Solubilization of membranes by detergents. but most of the molecule appears to reside between the leaflets of the phospholipid bilayer.1975 14. Shojania AM: Hematological aspect of Rh deficiency syndrome: A case report and review of the literature. J Immunol133:334. some of which may exist within the hypothetical Rh membrane complex (described previously). While the physiologic role of Rh is yet to be defined. 2013. RBC membranes but may not be directly associated with the Rh polypeptides in the mature RBC plasma membrane. New York.1983 13. Contreras M: Blood Transfusion in Clinical Medicine (ed 8).1971 17. individuals that express several membrane defects. Singer SJ: Quantitative twodimensional ultrastructural distribution of Rh.. Proc SOCExp Biol Med 43:223.(D)-positive human red cells. Science 100:595. Lemiew R: Protective effect of the membrane skeleton on the immunologic reactivity of the human red cell Rh.1982 11. although the more primitive BFU-e cells were devoid of Rh.. Transfusion 1694. The Rh antigens within the native membranes are thought to exist as a complex of Rh polypeptides and multiple other membrane components. J Supramol Structure 1:233. FEBS Lett 174:7. 30. Stetson RE: An unusual case of intragroup agglutination. and David Daleke for information and valuable discussions. Bazin R. Goossens D.. Salmon C. Landsteiner K. Levine P.1984 22. Bennett V: The spectrin-actin junction of the erythrocyte membrane-skeleton. Sanger R: Blood Groups in Man (ed 6). 1987 3.. Nature 153:771. Celano M. the structural basis of this remains to be established. Fischman DA. several clues indicate that it may play a role in the organization of membrane phospholipids or synthesis or membrane expression of various glycoproteins. AGRE AND CARTRON pase activity in normal RBCs.Blackwell. the Rh polypeptides may confer stability to certain other membrane components that are missing from Rh. Helenius A. FEBS Lett 140:93.1982 12. Wiener AS: The Rh series of allelic genes. While our knowledge of Rh is still very incomplete. NY. 1975 2. Rh polypeptides are present in RBCs from normal humans and other mammalian species and are probably required for normal membrane integrity. Mollison PL.1989 15. Therefore. the actual physiologic role of Rh remains to be defined and is being investigated. NY. Gahmberg CG. Blanchard D. For personal use only.. Am J Hematol24:267. Biochim Biophys Acta 988:107. Peter Issitt. because they appear to be missing from the RBCs of the rare Rh.. Steck T L Selective solubilization of proteins and phospholipids from red blood cell membranes by nonionic detergents. Lambin P.1976 20. 1944 8. J Mol Biol131:303.1961 7. REFERENCES 1. Biochim Biophys Acta 415:29. (E). Tanner MJA.(D) polypeptides with the membrane skeleton in Rh.'" Otherpossible roles. James NT. Despite these lines of evidence. J Immunol87:6."* This analysis suggests a physiologic need for Rh rather early in the differentiating red cell. Masouredis SP. human Rh positive and human Rh negative red blood cells. SUMMARY The RBC Rh antigens are of large clinical importance. Nicolson GL.1984 21. EMBO J 32-Kd transmembrane proteins that are core structural components of the Rh antigens and have been purified and partially characterized biochemically. (c). Sudora EJ.1939 5. Garratty G: Acquired Immune Hemolytic Anemia. and Fy". Masouredis SP. JAMA 113:126. Ridgwell K. While it is thought that this assembly may be important for the Rh antigenic by guest on July 24. Paradis G.From bloodjournal. Yu J. Burke BE. Petz LD. Mahan L. Analysis of Rh expression made with fluorescence-activated cell sorting demonstrated that some Rh antigens are already located in the plasma membrane in CFU-e. Levine P. Fenichel R. Anstee DJ: The Rhesus (D) polypeptide is linked to the human erythrocyte cytoskeleton. New York.Blackwell.(D) antigen. The Rh polypeptides appear necessary for the membrane expression of several glycoproteins.1978 18. Wiener AS: An agglutinable factor in human blood recognized by immune sera for rhesus blood.

Hughes-Jones NC. Staufenbiel M. J Biol Chem 252:6510. Staufenbiel M: Fatty acids covalently bound to erythrocyte proteins undergo a differential turnover in vivo. Bertles JF. Dahr W. Vox Sang 1032. Goldstein J. FEBS Lett 226:105.1988 52a. Rouger P. Lincoln PJ: Observations of the number of available c. Bloy C. Immunochemistry4:247.5'-dithiobis-(2-nitrobenzoic acid) and protection against loss of activity by bound anti-Rh.1986 36. Biochem J 256:1043. Kumpel B: Protein-sequence studies of Rh-related polypeptides suggest the presence of at least two groups of proteins which associated in the human red-cell membrane. Cartron J-P: Characterization of the C. McGuire M. Cherif-Zahar B. Colin Y: Molecular cloning and protein structure of a human blood group Rh polypeptide. Tanner MJA. Ridgwell K. Biochem Biophys Res Commun 54:1015. Denker BM. Vox Sang 21:210. Shinitzky M. Hartel-Schenk S. Mol Immunol21:433. Pickett RA. Elder JH.From bloodjournal. J Biol Chem 263: 18193. Pudlak W: The phospholipid requirement for Rh. J Immunol Methods 01:193.1988 46. Merry AH. Kratochvil C H Individual differences in chimpanzee blood.1979 34. Blood 72:1424. Agre P: Two-dimensional iodopeptide mapping demonstrates erythrocyte Rh D. Lazarides E: Ankyrin is fatty acid acylated in erythrocytes. Mol Immunol25:925.1988 38. Blanchard D. and E antigen sites on red cells. Wiener AS. Goossens D. Biochem J 213:267. Tanner MJA. Kent S: Regarding the size of Rh proteins. Bailly P.Martin AP. Smith B L Purification and partial characterization of the M. Goossens D. Agre P: Isolation of proteins related to the Rh polypeptides from non-human elythrocytes. Suyama K. J Cell Biol 111:322a. Agre P. Hughes-Jones NC. Anstee DJ. Blood 72:661. and E polypeptides are structurally homologous but nonidentical. 32. Suyama K.1971 48. Smith BL. Roberts SJ. Thomson EE. Basu MK. Schacter D. Blood 75:255. Goldstein J: Antibody produced against isolated Rh(D) polypeptide reacts with other Rh-related antigens.(D) antigen activity by 5. Biochem J 2715321. Rouger P. Souroujon M: Passive modulation of blood group antigens. Moor-JankowskiJ.1987 37. Anstee DJ: Absence of two membrane proteins containing extracellular thiol groups in Rh.1 deficiency. Salmon C. Hunt V A Loss of Rh antigen activity following the action of phospholipaseA2on red cell stroma. Maniatis T: Effects of modulating erythrocyte membrane cholesterol. Bloy C. c. human erythrocytes. Sherman S L Report of the committee on genetic constitution of chromosome 1. Mawby WJ. Blood 72:287. Masouredis SP.(D) antibody. Ridgwell K. Mol Immunol20769. J Biol Chem 247881. Beyreuther K. 1991 . Le Van Kim C. Lerner RA: Radioiodination of proteins in single polyacrylamidegel slices. Agre P: Distinct variants of erythrocyte protein 4. Hampton H. Smith BL. Biochem Biophys Res Commun 95:887. Smith BL. 1967 42. Blood 77:411. Prohaska R: Characterization of hyman red cell Rhesus specific polypeptides by limited proteolysis. Ridgwell K.1990 60. Green FA: Erythrocyte membrane sulfhydryl groups and Rh antigen activity.1987 53. Stratton F: The quantification of erythrocyteantigen sites with monoclonal antibodies.3-lp36. 1966 58.1987 24. For personal use only. Vox Sang 29:184. E. Suyama K. Agre P: Polymorphism in the M. Goldstein J: Enzymatic evidence for differences in the placement of Rh antigens within the red cell membrane. Blanchard D.1984 33. Green F A Erythrocyte membrane lipids and Rh antigen activity.(D) antigen activity. Saboori AM. Vorveck M L Lipid-protein interactions of erythrocyte membranes.1968 29. Immunology 51:793.1987 52.000 integral membrane protein associated with the erythrocyte Rh(D) antigen. 30. Snyder LM: Decreased amount of the Rh antigen D in hereditary spherocytosis. D. Blood 721622.1986 26.1988 56.1983 44. Green LAD. Blood 69:1491.1975 32. 1972 30. J Clin Invest 39:1450. Green F A Studies on the Rh(D) antigen.1988 27.1980 35. Salmon C. J Biol Chem 263:13615. Hermand P. Hum Genet 86:398. Green C: The identification of specific Rhesus polypeptide blood group ABH active glycoprotein complexes in the human red cell membrane. Cytogenet Cell Genet 51:67.1983 43.1991 62. MOLECULAR BIOLOGY OF THE RH ANTIGENS 561 Rh(D) antigen: Partial characterization of rh Rh(D) polypeptide from human erythrocytes. Bloy C. Saboori AM. Tchernia G. J Biol Chem 243:5519. Asimos A. Salmon C. Hermand P.hematologylibrary. 1965 41. 1989 28.1988 39. Mol Cell Biol7:2981. Krahmer M. Hui HL. Cherif-Zahar B. Blanchard D. Biochem J 244735.1 region by in situ hybridization. Odgren P. (c) and (E) antigens are carried by distinct polypeptide chains. Mohandas N..1977 55. Anstee DJ. 1990 (abstr) 25. C. SabooriA.. Cartron J-P: Determination of the N-terminal sequence of human red cell Rh(D) polypeptide and demonstration that the Rh(D). Senhauser DA. Cartron J-P.1960 49. Gordon EB. Heubusch P. Green F A The mode of attenuation of erythrocyte membrane Rh. demonstrable with absorbed human anti-Rho sera. Saboori A.1989 40. Agre P: Fatty aid acylation of human red cell membrane proteins and phospholipids. 1984 47. Conboy J. deVetten MP. Masouredis SP: Relationship between Rho (D) genotype and quantity of "'I anti-Rho (D) bound to red cells. 1989 63. Green F A Phospholipid requirement for Rh antigen activity. Salmon C: Human monoclonal antibodies against blood group antigens. Flamm M. and G antigens of the Rh blood group system with human monoclonal antibodies. 1988 59. Green FA. Cartron J-P. Avent ND. J Clin Invest 83:187. Colin Y: Localization of the human Rh blood group gene structure to chromosome lp34. Proc Natl Acad Sci USA 83:318. Smith JA. Bloy C. Aebersold R. Kan YW: Molecular basis of hereditary elliptocytosis due to protein 4. Avent ND. Br J Haematol73:537.1973 31. N Engl J Med 315:680. Le Van Kim C..1987 45. Blanchard D. Lucas FV. Szymanski IO. Moore S. Tanner by guest on July 24.1 inherited in linkage with elliptocytosisand Rh type in three Caucasian families. Lambin P. 1990 57. 1990 61. Agre P: The Rh polypeptide is a major fatty acid acylated erythrocyte membrane protein. Proc Natl Acad Sci USA 876243. Proc Natl Acad Sci USA 56:458. Proc Natl Acad Sci USA 85:4042. Bruns GAP. Araszkiewicz P. Champomier G. Staufenbiel M: Ankyrin-bound fatty acid turns over rapidly at the erythrocyte plasma membrane.1987 54. Proc Natl Acad Sci USA 76:4438. Cartron J-P.000 Rh protein purified from Rh(D) positive and negative erythrocytes. J Biol Chem 262:17497. 1988 50. Anstee DJ: cDNA cloning of a 30 kDA erythrocytemembrane protein associated with Rh (Rhesus)-blood-group-antigenexpression. Mattei MG. Green EJ. 2013.1988 51. Bailly P. Gardner B.

Biochem J 234:649. Hermand P.1989 89. Annu Rev Biochem 55:1091. Tanner MJA.1973 101.. Cherif-Zahar B. Perkins HA.1979 73. Blanchard D. Daniels GL. France. von dem Borne AEG. Plow EF. Evans PR.1965 76. Mol Immunol4:361. Hermand P. Judson PA. Zwaal RFA. Anstee DJ. Montgomery Scientific.1984 98. Tillis D. 1986 70. Cell 5285. FL. Ann NY Acad Sci 127:884. Uzan G. Berthier R. Blood 75:2245. Lodish HF: Predicting the orientation of eukaryotic membrane-spanning proteins. Marguerie G: Biosynthesis and assembly of platelet GPIIb-IIIa in human megakaryocytes. Lomas CG. Kuypers F. Schmidt MFG: Fatty acylation of proteins. Joiner CH: Increased potassium transport and ouabain binding in human Rh null red blood cells. cells.. Blood 1991 (in press) 69. erythrocytes. Cuppoletti J: Molecular size of the Rh. J Biol Chem 254: 5458.1975 102. Transfusion 30222. Fukuda MN. Ashwell JD: Failure to synthesize the T cell CD3-zeta chain. Gahmberg C G External labeling of erythrocyte glycoproteins. Op den Kamp JAF: Rh. Seigneuret M.. Lebeck L. Admiraal LG. Biochim Biophys Acta 988:411.1989 71. Klausner RD..1984 94. Proc Natl Acad Sci USA 81:3751. Harper JR: Arg-Gly-Asp recognition by a cell adhesion receptor requires its 130-kDa alpha subunit. Anstee DJ. Biochem Biophys Res Commun 50:1027. Lippincott-Schwartz J. Green F A Reconstitution of Rh(D) antigen activity from human erythrocyte membranes solubilized by deoxycholate.1985 91. Comfurious P. Masouredis SP: Rh antigen immunoreactivity after histidine modification. Am J Hum Genet 41:1061. Tills D. 1990 84. Marinetti GV: The asymmetric arrangement of phospholipids in the human erythrocyte membrane. Glass AA. Lomas C. Cartron J-P: Recent advances in the biochemistry of blood group Rh antigens. Roelofsen B.1990 78. Roelofsen B.. Ellory JC. J Biol Chem 251:510. Mallinson G.. Blut 5413. Cell 36573. van de Graaf J. Mohandas N. Hakomori S: Developmental change and genetic defect in the carbohydrate structure of band 3 glycoprotein of human erythrocyte membrane. van Deenen LLM: The asymmetric distribution of phospholipids in the human red blood cell membrane. Cartron J-P: Genetic basis of the Rh-D positive and Rh-D negative polymorphism. 1976 96. J Biol Chem 262:1434. Sussman JJ. Harper JR.. Branks MJ. human erythrocytes have an abnormal membrane phospholipid organization. Cheresh DA. Blood 74:14. Tanner MJA.1984 103. Owens NA. Paris. Maciver AG. Lauf PK.(D) antigen of the human erythrocyte in situ by radiation inactivation. Eur J Immunol12:228. Sonnebom HH: Identification and partial characterization of the human erythrocyte membrane component(s) which express the antigens of the LW blood group system. 1987. Bos MJE.From bloodjournal.. Daleke DL. Mol Immunol 23:1039. Colfer HF. Evans R M Complex transcriptional units: Diversity in gene expression by alternative RNA processing. Le Van Kim C. Tippett P.1984 97.1985 92. For personal use only.hematologylibrary. a multisubunit integral membrane protein. Issitt PD: Applied Blood Group Serology (ed 3). Sonneborn H. Duperray A. Caswell MS.1986 68. 1985 80. Tippett P. Tanner MJA. J Biol Chem 265:21482. Leff SE. Vox Sang 58:219.1990 66.. Gorick BD. Cherif-Zahar B.1985 . Gielen W. Anstee DJ. Rapoport TA. Clark MR. Cartron JP: Comparative analysis by two-dimensional iopeptide mapping of the RhD protein and LW glycoprotein. Weissman A.. Goossens D. Race R R Modern concepts of the Blood group systems. Owen MJ.1990 74. Cherif-Zahar B. Von dem Borne AEG: Murine monoclonal antibody MB-2D10 recognizes Rh-related glycoproteins in the human red cell membrane. Moulds J. 562 AGRE AND CARTRON 64. Hughes-Jones N C Comparison of the reactions of the Rh-related murine monoclonal antibodies BS58 and R6A. Cartron J-P. Anstee DJ. Ridgwell K. Shohet SB: Red cell membranes and cation deficiency in Rh null syndrome. Blood by guest on July 24. Colin Y.1976 72. Bonifacino JS. Huestis WH: Incorporation and translocation of aminophospholipids in human erythrocytes. Hung CY.1982 82. Troesch A. Avent ND. Science 188:66. Fukuda M. Devaw P F ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane.1973 100. in Rouger P. Amette. Cartron J-P: Surface orientation and antigen properties of Rh and LW polypeptides of the human erythrocyte membrane. Victoria EJ. Raynal V.1986 67. Mallinson G. Miami. Sonneborn HH.1975 93. Martin PG. Green FA. Rosenfeld MG. Biochem J 221:931.. Lorusso DJ. Hughes-Jones NC: A molecular size determination of Rh(D) antigen by radiation inactivation. Verkleij AJ. Bergen MO. Ginsberg MH: Efficient surface expression of platelet GPIIb-IIIa requires both subunits. Biochim Biophys Acta 323:187.1987 79. Roelofsen B. 2013. Dahr W. Br J Haematol 75:254. p 69 85. Folkerd E. Zwaal RFA. Chagnon E.1986 77. Ballas S. Biochim Biophys Acta 406:83. Immunochemistry 14529. Ersat M. OToole TE. Tippett P: A speculative model for the Rh blood groups. Kordowicz M.1988 86. Blood 63:1046. Van Huffel V. Frachet P. Tanner MJA.1987 83.1987 88. Avent ND. Biochemistry 24: 5406. Kastelijn D. J Biol Chem 255:9678.1972 99. Merlie JP: Biogenesis of the acetylcholine receptor.1980 90. Krueger J: Characterization of the Ss sialoglycoprotein and its antigens in Rh. Kissonherghis A-M. Mallinson G. Germain RN. Bretscher M: Asymmetric lipid bilayer structure for biological membranes. Anstee DJ. Blood 74:1603.1988 81. Holmes C Monoclonal antibodies that recognize different membrane proteins that are deficient in Rh. Quill H Influence of allelic polymorphism on the assembly and surface expression of class I1 MHC (Ia) molecules. Edwards PAW: Monoclonal antibodies to human erythrocytes.1989 87. Bloy C. Loftus JC. Miller YE. van Deenen LLM: Organization of phospholipids in red blood cell membranes as detected by various purified phospholipases.1990 65. Bloy C. Cell 43:233. Hodges EE. Hartmann E. Mery AH. Comfurius P. human erythrocytes. Gordesky SE. Hermand P.1990 75. Nature 236:11.1977 95. Hui HL. Bloy C. Parsons SF. van Linde-Sibenius-Trip M. Jones C. Proc Natl Acad Sci USA 865786. Bentley CM. Daniels GL. Salmon C (eds): Monoclonal Antibodies Against Human Red Blood Cell and Related Antigens. Biochem J 251:499.. Overbeeke MAM: Murine monoclonal antibodies against a unique determinant of erythrocytes related to Rh and U antigens. Ann Hum Genet 50:241. Lodish HF: Biosynthesis of HLA-A and HLA-B antigens in vivo. Saito T. Palmer D K Identification of a cell-surface antigen produced by a gene on human chromosome 3 (cen-q22) and not expressed by Rh.

1989 106. Devaw P: Outside-inside translocation of aminophospholipids in the human erythrocyte membrane is mediated by a specific enzyme. Blood 66:660. photoactivatable phosphatidylcholine and phosphatidylserine: Transfer properties and differential photoreactive interaction with human erythrocyte membrane proteins. Nature 34075. kinases and other ATP-requiring enzymes and a common nucleotide binding fold. Connor J.hematologylibrary.1990 107. Madsen J: Radioiodinated. MOLECULAR BIOLOGY OF THE RH ANTIGENS 563 104.Biochemistry 29:10303. Nichols ME.1988 109. Devaw PF: Partial purification and characterization of the human erythrocyte Mg+ +ATPase. 2013.1987 108. Daleke D L Phosphatidylserine transport in Rh.000-Dalton protein by sulfhydrylreactive reagents.. Zachowski A. Devaw P F Control of transmembrane lipid asymmetry in chromaffin granules by an ATPdependent protein. Zachowski A. Favre E. Heme P.1986 105. Cartron JP: Involvement of Rh blood group polypeptidesin the maintenance of aminophospholipid asymmetry. Cribier S. erythrocytes.From bloodjournal.. FEBS Lett by guest on July 24. Henry JP.. A candidate aminophospholipid translocase. Biochemistry26:1812. For personal use only. EMBO J 8:945.1985 . Morrot G. Walker JE. Jansen J: Human erythroid progenitor cells express rhesus antigens. Biochemistry 25:2585. Falkenburg JHF. Fibbe WE. Saraste M. Rubinstein P.1982 111. Schroit AJ. Connor J. van der Vaart-Duinkerken N. Blood 76:1021. Bloy C.1990 112. Schroit AJ.1990 110. Runswick J. Smith RE. Schroit N:Transbilayer movement of phosphati- dylserine in erythrocytes: Inhibition of transport and preferential labeling of a 31. myosin. Zachowski A. Gay NJ: Distantly related sequences in the alpha and beta subunits of ATP synthase. Biochemistry272348.

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