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1991 78: 551-563

Molecular biology of the Rh antigens

P Agre and JP Cartron

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REVIEW ARTICLE Molecular Biology of the Rh Antigens

By Peter Agre and Jean-Pierre Cartron

HISTORICAL BACKGROUND ‘251-labeledon the extracellular surface of intact RBCs. HE RH BLOOD group antigens are of large clinical Although the glycophorins and the band 3 anion trans- T importance because of their involvement in hemolytic porter were also labeled, membranes from Rh positive disease of the newborn, , and autoim- RBCs (obtained from individuals with presumed genotype mune hemolytic The historic discovery of the Rh DID or D/d) were noted by autoradiography to have a D antigen was made just over 50 years ago by Levine and diffuse but strongly labeled band of approximately 28 to 33 Stet~on.~Soon after delivery, the mother of a stillborn Kd. A similar but much fainter band was seen in mem- infant had received a donated by her branes from typical Rh negative RBCs (Fig 1). Anti-D husband, and she experienced an immediate and severe antibodies were added to the membranes before detergent transfusion reaction. The investigators detected an anti- solubilization, and the ‘xI-labeled band was specifically body in her serum that agglutinated red blood cells (RBCs) immunoprecipitated with antibodies specific for D from Rh from 80% of randomly selected type 0 donors. The D positive RBCs. Likewise, anti-c and anti-E, respectively, antibody specificity was identical to antibodies raised in immunoprecipitated similar but less heavily radiolabeled rabbits injected with RBCs from Rhesus monkeys, hence bands from Rh c and Rh E positive RBCs.” These new “anti-Rhesus” or “anti-Rh.”’ Ironically, “Rh” is in fact a bands together are now referred to as the “Rh poly- misnomer, because the surface antigens detected by the peptides.” human and rabbit antibodies were not identical, and the The Rh polypeptides were shown to be highly unusual latter antigen was subsequently named “LW” in honor of RBC membrane proteins.” The Rh polypeptides were Landsteiner and Wiener: found to have electrophoretic mobilities varying from 28 to Early investigators soon observed that Rh was very 32 Kd relative to molecular weight standards in sodium complex, with multiple antigenic variants. A controversy dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- arose concerning whether Rh is a single protein containing PAGE), presumably due to increased binding of SDS, multiple antigenic epitopes (referred to as Rho,rh’, hr’)’ or which is known to occur to very hydrophobic protein^.'^ whether there exist multiple closely linked alleles each Also, despite the presence of an exofacial tyrosine that can coding for an independent protein specific for a different be surface ‘251-labeled,the Rh polypeptides on intact RBC Rh antigen (Cic, Did, or E/e).8 The latter system does not membranes resisted proteolytic degradation. Endogenous explain all of the complexities of Rh, but is easier to phosphorylation was not detected either.” communicate and is more compatible with current molecu- Most surprisingly, the Rh polypeptides contained no lar studies (see below). D is the major Rh antigen detected detectable carbohydrate when examined with methods for on the surface of RBCs obtained from individuals with the labeling terminal sugars.” The isolated Rh polypeptides presumed genotypes DID or Did, commonly referred to as also failed to adsorb to lentil lectin affinity columns and “Rh positive.” The antithetical antigen d is not immunolog- were not degraded by glycosidases nor by alkaline hydroly- ically detectable and is not expressed, hence “Rh negative.” sis, which normally removes 0-linked oligosaccharides. The Whether from Rh positive or negative individuals, virtually apparent lack of carbohydrate was indeed an extraordinary all normal RBCs bear the antithetical antigens C and/or c in finding, because all known blood group antigens and addition to E andlor e. Exceedingly rare individuals lack all virtually all mammalian transmembrane proteins are glyco- of these antigens and are referred to as Rh,,,,.’ Rh,,,, RBCs proteins. express multiple RBC membrane abnormalities, suggesting Apparent membrane skeleton linkage. Recent advances that the Rh antigens are of physiologic importance (see in RBC membrane biochemistry have established that the below). The molecular basis of the Rh antigens has proven to be a very difficult problem in membrane biology, and unfortu- From the Departments of Medicine and Cell Biology, Johns Hopkins nately many early research efforts proved to be false leads. University School of Medicine, Baltimore, MD, and INSERM Unite Rh antigenic reactivity is lost after membranes are solubi- U76, Institut National de Transfusion Sanguine, Paris. lized or transferred onto immunoblot membranes, and Submitted Januaiy IO, 1991; accepted April 4, 1991. Supported in part by NATO Collaborative Research 0556188, most biochemical methods therefore actually kill the anti- National Institutes of Health ROI HL33991, Institut National de la genic reactivities that identify and define the Rh antigens. Sante et de la Recherche Medicale, and the Caisse Nationale There has been little agreement about the molecular d’AssurancesMaladies des Travaillers Salaries. P.A. is an Established identity of the Rh antigens until recently. Investigator of the American Heart Association. Written in honor of Prof C. Lockard Conley, Emeritus Director of THE RH POLYPEPTIDES the Johns Hopkins Hematology Division and Prof Charles Salmon, Emeritus Director of the Institut National de Transjiuion Sanguine. Discovery. Two reports published in the winter of 1982 Address reprint requests to Peter Agre, MD, Johns Hopkins Univer- drastically changed the direction of Rh research. Moore et sity School ofMedicine, 725 N Wolfe St, Baltimore, MD 21205. al’” Edinburgh and Gahmberg” in Helsinki independently 0 I991 by The American Society of Hematology. observed a previously unrecognized protein that could be 0006-4971I91 17803-0035$3.OOiO

Blood, Vol78, No 3 (August 1). 1991: pp 551-563 551 From www.bloodjournal.org by guest on October 31, 2014. For personal use only.

552 AGRE AND CARTRON

DD Dd dd DD Dd dd tivcly." This distancc is approximatcly the lcngth of an cxtcndcd spcctrin tctramcr.'" Othcr studics dcmonstratcd a latticc-likc distribution on thc mcmhranc surfacc with fcncstrations of approximatcly IO nm." Linkagc of thc Rh polypcptidc to thc mcmhranc skclcton would hc a logical cxplanation for thcsc findings, although mcmbranc skclcton linkagc has not bccn dircctly cstah- lishcd. Undcr ccrtain conditions thc Rh polypcptidcs wcrc found to prccipitatc with thc mcmbranc skcIcton.3'.2'Thc mcmhranc skclcton was also shown to prcscrvc Rh anti- genic activity whcn mcmhrancs wcrc soluhilizcd in nonionic Glyco- dctcrgcnts.?' Morcovcr, ccrtain glycoprotcins missing from phorins Rh,,,, RBCs also hchavc as if linkcd to thc mcmhranc skclcton (scc hclow). A fraction of thc Rh polypcptidc is obviously soluhlc in 1% (volhrol) Triton X-IW. hccausc initial rcports dcscribcd immunoprccipitation of Rh poly- pcptidc from Triton-solubilizcd mcmbrancs."'.'' conditions in which thc spcctrin-actin mcmbranc skclcton is insolu- Rh blc." 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. Ncvcrthclcss, whcn asscsscd quantita- tivcly. 70% to 80% of thc Rh polypcptidcs wcrc still asstxiatcd with the insoluhlc mcmbranc skclcton after incubation in up to 5% (volhrol) Triton X-IO at 0°C for 15 minutcs. whcrcas glycophorin A. a nonskclcton-linkcd protcin, was largcly soluble."'.:' membranes anti-D immppt. Whilc a rclativcly weak interaction bctwccn thc Rh polypcptidcs and thc mcmhranc skclcton may cxist. this Fig 1. Autoradiograph of radiolabehd Rh polypeptides on mom- hchavior may simply rcflcct thc rclativc insolubility of thc hnaof intact RBCs [left panel) or immunoprecipitatedwlth anti-D Rh polypcptidc in Triton X-100. bccausc subscqucnt invcs- (right panel). Intact RBCs from individuals with presumed Rh geno- tigators dcmonstratcd that physical shaking of intact RBC types cD€/cD€ (DO),CDdcde (Dd), and cdelcde (dd lanes) were 'Waboied using lodogen (Pime, Rockford. IL). Note the strong mcmbrancs in 5% (wthol) Triton X-IO() produccs an wtiace Iakling of Rh powptides on OD RBCs, intermediate label- altogcthcr diffcrcnt rcsult, with lcss than 30% of thc Rh ing on Dd RBCs, and weak labeling on dd RBCs. The anti-D immunopre- polypcptidc rcmaining with thc insoluhlc mcmbranc skclc- cipitates are from 5 vol of the same RBCs. Note the absolute lack of Rh tons." Whcn Triton X-IOOcxtractability of thc Rh polypcp- in the immunoprecipitatesfrom dd RBCs. and the small amount of band 3 which was nonspoclfiully immunoprecipitatedfrom all prepa- tidc from wholc RRC mcmhrancs was comparcd with rations. Reprintedwith permission." cxtractahility from spcctrin-dcplctcd mcmbranc vcsiclcs, thc solubilities wcrc similar, indicating that thc mcmbranc mcmbranc skclcton is thc sourcc of cell shape, deformabil- skclcton is not a major constraint." ity, and is thc intraccllular structure to which ccrtain Paticnts with ccrtain congcnital ancmias arc known to transmcmhranc protcins arc attached." The membrane have dcfcctivc RBC membrane skclctons. Thc primary skclcton is a complcx of spcctrin. actin, and scvcral associ- membrane dcfcct in the subtypc of hcrcditary elliptocytosis atcd protcins that arc insoluhlc in low conccntrations on inhcritcd in linkagc with Rh phcnotypc was shown to rcsult nonionic dctcrgcnts such as 1% Triton X-IO." This opcra- from dcficicncics or dysfunctions of mcmbranc skclcton tional dcfinition of thc mcmbranc skclcton will not distin- protcin 4.1; thc 4.1 gcnc coincidcntally resides ncar the Rh guish protcins that arc insoluble duc to tight association locus on the short arm of chromosomc no. I."" Intcrcst- with skclcton components from protcins that arc inhcrcntly ingly, a partial dcficicncy of Rh antigcns was found on the insoluble in thcsc dctcrgcnts. mcmbrancs of hcrcditary sphcrocytosis RBCs,?' although Early investigations by Masouredis ct al at the University partial dcficicncics of scvcral othcr mcmbranc componcnts of California at San Dicgo providcd scvcral obscrvations in spherocytosis mcmbrancs suggcst that Rh dcficicncy is abut thc distribution of Rh antigcns on thc surfacc of not a primary phcnomcnon in that disordcr cithcr. RBCs by immunoclcctron microscopy using fcrritin-lahclcd Fatiy acylation of the Rhplypeptides. Early invcstigation antihodics."' Although it was initially concludcd that thc Rh by Floyd Grccn at the Statc Univcrsity of Ncw York in immunc complcxcs wcrc randomly distributcd on thc RBC Buffalo, N.C. Hughcs-Joncs at SI Mary's Hospital in hndon, surface,'* suhscqucnt data analysis indicatcd that thc D England, and othcr investigators dcmonstratcd a rclation- antigcns wcrc spaccd a mcan distancc of M nm and 92 nm ship hctwccn mcmhranc lipids and Rh antigcnic rcactivity. from thc ncarcst ncighbors on thc RRC mcmbrancs of Rh D rcactivity was clutcd from RBC mcmbrancs with prcsumcd Rh D homozygotcs and hctcrozygotcs. rcspcc- butanol3 and rcstorcd with cxogcnous phosphatidylcho- From www.bloodjournal.org by guest on October 31, 2014. For personal use only.

MOLECULAR BIOLOGY OF THE RH ANTIGENS 553

line." Digestion of intact RBCs with phospholipasc A? Moreovcr, the fatty acylation was not rcduccd by inhibitors produced increased mcmbranc fluorcsccncc in normal but of protcin synthcsis. Thc fatty acylation was also found to not Rh,,,, RBCs." Phospholipasc A? digestion also pro- bc rcvcrsiblc and was entirely rcmovcd during chase with duced partial loss of Rh D antigenic rcactivity,'' which unlabeled palmitate." could be prcvcntcd by prior addition of anti-D antibody." Thc 'H-palmitylation of Rh polypcptidcs within maturc Alterations in the membrane cholcstcrol phospholipid ratio RBCs is most likely the result of continuous exchange of by cholcstcrol cnrichmcnt or dcplction, respcctivcly, pro- frcc palmitate for palmitatc estcrificd onto cystcinc rcsi- duccd cnhanccment or rcduction of Rh antigcnic rcactiv- ducs within Rh polypcptidcs. In preliminary studies, addi- ity.''-' Thesc findings all indirectly suggcstcd a strong tion of 'H-palmitatc to invcrted mcmbranc vesicles dcmon- association of the Rh antigens and membrane lipids, but stratcd that the Rh polypcptidcs cxclusivcly wcrc acylatcd the actual biochcmical explanation rcmaincd unclear. at multiple sitcs near the inncr Icaflct of the phospholipid Approximately six mcmbranc proteins arc known to bilayer." Dcspitc thc apparcntly large evolutionary divcr- bccomc 'H-palmitylatcd when intact RBCs are incubatcd sity, nonhuman Rh homologs have bccn idcntificd," and all with 'H-palmitic acid,'' " and investigators dcmonstratcd appear to retain the capacity to become fatty acylatcd." It that the Rh polypcptidcs are major fatty acylatcd mcm- remains to be established whether the palmitylation of Rh brane protcins.w When human RBCs wcrc incubatcd with polypcptidcs is important in their primary antigenic rcactiv- 'H-palmitate, membrane proteins of M, 57 Kd and 32 Kd ity or whcthcr they are supporting structural modifications wcrc the two most prominently labclcd (Fig 2). The M, of possiblc physiologic importancc (scc bclow). 32-Kd 'H-palmitylatcd protein was labclcd comparably in F~ofacialfree siilflydtyl. Pionccring work by Grcen also Rh D negative and positive RBCs but could only be cstablishcd thc important obscrvation that Rh D and C immunoprccipitatcd with anti-D from thc lattcr. Furthcr- antigcnic rcactivitics wcrc depcndcnt on a thiol group more, thc M, 32-Kd band failed to labcl in mcmbrancs of a located at or near the outer surfacc of the RBC mcmbranc. variant of the Rh,,,, phcnotypc and it was concluded that Using a variety of sulfhydryl-reactivc probcs, oxidation of a thesc proteins correspond to thc Rh polypcptidcs. How- single sulfhydryl was obscrvcd to lcad to loss of antigen cvcr, it must bc cmphasizcd that all studies of palmitylation sitcs, but could be rcstorcd by rcduction of disulfides and to date have involvcd isotopic labclings, and elution of could be prcvcntcd by prior incubation with anti-D antibod-

native fatty acids from the Rh polypcptidcs has not yct bccn ics.JO J? The Rh polypcptidcs wcrc subscqucntly found to reported. contain an cxofacial-frec sulfhydryl. Using a radiolabeled Palmitic acid appears to bc covalently attachcd to the Rh impcrmeant malcimidc that apparently dcstroyed Rh anti- polypeptides by thioestcr linkages onto frcc sulfhydryls on genic rcactivity (N-malcoylmcthioninc ["SI sulphonc), two certain cystcinc residues within the molecule. It was found mcmbranc bands of M, 32 and 34 Kd wcrc labeled on that thc 'H-palmitatc can be clutcd from thc Rh polypcp- normal RBCs, but not on membranes of Rh,,,, RBCs." tides and othcr RBC acylprotcins with 1 mol/L hydrox- ylamine at neutral pH, and thc attachcd 'H-palmitate was ISOLATION OF THE RH POLYPEPTIDES not metabolically dcgradcd to another molccular species. Immunologic and nonimmtrnologk approaches. Biochem- ical studies of thc Rh polypcptidcs wcrc impeded by the 1234 5678 difficulty in isolating them in sufficicnt quantitics. Invcstiga- tors at the Institut National dc Transfusion Sanguine (INTS) in Paris, Francc, dcvclopcd cell lines secreting monoclonal antibodics (MoAbs) spccific for Rh D, c, and E antigens by virally transforming B cells from humans with circulating Rh antibodics.' Investigators at the Univcrsity of Bristol and the South Wcstcrn Regional Blood Transfu- sion Centre in England also dcvclopcd pancls of murine MoAbs with Rh-related specificity by immunizing mice with human RBCs." Thc murine MoAbs are thought to be reactive with nonpolymorphic components of the Rh struc- tural protcins. Thcsc different MoAbs permitted improved quantitation of numbcr of Rh antigen sites per RBC. There appcars to be a total of approximately 10' Rh antigens per Fig 2 Fluorograph of membranes from RBCs incubated with normal RBC with c (or C), D, and E (or e)cach comprising 'H-palmitic acid. Intact RBCs were incubated ovemight in tissue about % of the total.2'.4',* This numbcr is somcwhat higher culture medium containing up to 1 mCi 'H-palmitic acid. The cells were washed, and membranes were prepared. A portion of the than cstimates prcviously madc with polyclonal anti- sera. IW7.JU membranes were then incubated with anti-D antibody or nonimmune globulin and immunoprecipitatescollected. Coomassie-stained SDS- Invcstigators in Paris, Bristol, and thc New York Blood PAGE of RBC membranes (lane 1). fluorograph of membranes (lanes 2 Center cach immunopurificd thc Rh polypcptidcs by addi- through 4). Rh D immunoprecipitatedwith anti-D (lanes 5 and 6), or tion of large quantitics of the monoclonal or polyclonal nonimmune globulin (lanes 7 and 8)from RBCs of presumed genotype cdelcde (lanes 1,2,5, and 7). CDelcDE (lanes 3,6,and 8). and the Rh, anti-D to membranes from RBCs that had been surface variant of the Rh, phenotype (lane 4). Reprintedwith permission." "'I-labclcd. The Rh D polypeptide was immunoprccipi- From www.bloodjournal.org by guest on October 31, 2014. For personal use only.

554 AGRE AND CARTRON tatcd from D positive RBCs and purificd to homogcncity by tion patterns indicated that Rh D is distinct from the C/c SDS-PAGE.""'"' Mcanwhilc, invcstigators at Johns Hop- and E/c polypcptidcs."." kins isolatcd Rh polypcptidcs from surfacc "'I-labclcd Rh Rh polypcptidcs isolated by thc nonimmune method also D positive human RBCs by nonimmunc methods, including showed distinct polymorphisms when Rh D negative and hydroxylapatite chromatography and prcparativc clcctro- Rh D positive prcparations wcrc compared by two- phorcsis of SDS-solubilizcd mcmbranc skclctons'l or mcm- dimcnsional iodopcptidc maps" using Elder et al's method.% branc vcsiclcs." Thc nonimmunc mcthod Icd to a ncarly Morcovcr, Rh polypcptidcs immunoprccipitatcd from a 200-fold purification and pcrmittcd calculation of thc total singlc unit of blood from an individual with prcsumcd Rh numbcr of Rh polypcptidcs pcr nativc RBC, approximatcly gcnotypc cDE/ cDE with MoAbs spccific for c, D, and E 60,OOO. Thc amino acid composition of Rh polypcptidcs wcrc also analyzcd by two-dimcnsional iodopeptidc maps obtaincd by immunopurifications and nonimmunc purifica- (Fig 3). Although thc Rh c, D, and E polypeptides were tions wcrc vcry similar, bcing compriscd of approximatcly found to bc highly rclatcd, cach was found to bc a distinct 37% hydrophobic rcsiducs.-"." protcin.'' Thc c and E polypcptidcs wcrc nearly identical, Rh polypeptide.y arc polymovhic. Scvcral rcports indi- whilc D was dcfinitcly rclatcd although lcss similar. Using rectly confrmcd that thc M, 32-Kd Rh polypcptidcs arc this approach, a singlc iodopcptidc conserved among c, D, compriscd of multiplc spccics (hcncc "Rh polypcptidcs") and E was idcntificd as that l"I-labclcd on the exofacial which contain thc polymorphisms underlying thc Rh anti- surfacc of intact RBCs (Fig 3, arrow). gcn system. Carcful analysis of onc-dimcnsional SDS- Additional cvidcncc undcrscoring thc similarity of thc PAGE gradicnt gcls of immunoprccipitatcd Rh polypcp- C/c, D, and E/c polypcptidcs was dcmonstratcd with an tidcs showcd a small but rcproduciblc diffcrcncc in thc antiscrum to denatured purified Rh D polypcptide, which mobilitics of thc diffcrcnt polypcptidcs. D migratcd with an was found to cross-rcact with thc Rh c and c polypcptidcs M, of 31.9 Kd whcrcas c and E migratcd with an M, of 33.1 on immunoblots." Morcovcr, digestion of intact RBCs with Kd.'b Thcsc valucs probably corrcspond to thc two mcm- phospholipasc A, followcd by digestion with papain rc- brane protcins labclcd with thc impcrmcant sulfhydryl sultcd in partial dcgradation of thc Rh D polypcptide but reagent." not of thc Rh C/c or E/c polypcptidcs, confirming an In other studies, M, 32-Kd bands wcrc cut from SDS- inherent diffcrcncc bctwccn thcsc Rh polypcptidcs.'" PAGE gcls of mcmbrancs" or immunoprccipitatcd with Nonhuman homolop of the Rh polypeptide. Immuno- Rh-specific MoAbs from surfacc "'I-labclcd RBCs of dif- logic studies of divcrsc mammalian spccics have shown that fcrcnt Rh phcnotypcs." Thc isolatcd Rh polypcptidcs wcrc antigens which arc related but not identical to the Rh protcolytically digcstcd and analyzed by onc-dimcnsional antigens cxist only on the RBCs of higher order primatcs SDS-PAGE autoradiography. Variations in thc dcgrada- but not other spccics."' Using the hydroxylapatitc method

Fig 3. Two-dimensionaliodopeptide map of Rh D, c, and E polypeptides show they are highly related but not identical. Autoradiograph of two-dimen- sional iodopeptide maps prepared from Rh D, c, or E polypeptides separately immunoprecipitated from the RBCs of a single individual (presumed Rh geno- type cD€/cD€)with human MoAbs specific for D, c, or E, respectively. The SDS-denatured purified proteins were first radiolabeled with Inl by chloramine T oxidation, and then were completely digested with crchymotypsin, and finally spotted on thin-layer plates and analyzed in two dimensions: (1) horizon- tal, electrophoresis; (2) vertical, thin-layer chromatog- raphy. A composite drawing (lower right panel) shows the iodopeptide corresponding to an exofacial do- main of the protein (arrow). Specific iodopeptidesare indicated: those shared by all Rh D, c, and E polypep- tides (solid spots), iodopeptides unique for Rh D (diagonal bars), iodopeptide unique for E (open fig- . composite ure), and iodopeptides unique for c and E (vertical stripes). Reprinted with permission." From www.bloodjournal.org by guest on October 31, 2014. For personal use only.

MOLECULAR BIOLOGY OF THE RH ANTIGENS 555 for isolating Rh polypeptides, similar M, 32-Kd proteins The Bristol investigators used the murine R6A MoAb were isolated from the membranes of monkey, cow, cat, and (which most likely recognizes nonpolymorphic regions of rat RBCs and analyzed by two-dimensional iodopeptide the C/c and E/e polypeptides) and the human anti-D MoAb maps.39Of approximately one dozen iodopeptides in each to immunopurify Rh polypeptides and successfully deter- preparation, only two iodopeptides were found to be mined N-terminal amino acid sequences for extremely long conserved by the human and all nonhuman preparations. stretches of the proteins (Fig 4).49@’The amino acid se- Thus, the nonhuman homologs to the Rh polypeptides quences for the first 41 residues of the two Rh polypeptides appear to have significantly different primary structures. were identical. A group of M, 35- to 60-Kd glycoproteins Interestingly, the iodopeptide corresponding to the surface had been discovered to coprecipitate along with the Rh ‘“I-labeled exofacial tyrosine was found only in the human polypeptides (discussed below)?’” The N-terminal amino preparations. acid sequence of these glycoproteins showed them to be ancestrally related to the Rh polypeptides, suggesting the MOLECULAR CLONING OF THE RH POLYPEPTIDES existence of a family of Rh-related molecules.“ Because of Determination of the N-terminal amino acid sequence. this, the M, 35- to 60-Kd glycoproteins are referred to here While high-affinity antibodies to Rh D and other Rh as “Rh-related glycoproteins.” antigens are widely available, these are specific for the Rh Isolation of an Rh cDNA. A cDNA specific for one antigens within the native membrane and have not been species of Rh polypeptide was isolated independently by found to react with denatured Rh polypeptides. Immuno- the research groups in Pari?’ and Bristol.60Both groups had logic identification of Rh polypeptides expressed from previously attempted to use the traditional method of cDNAs in recombinant libraries has therefore not proven screening recombinant libraries with oligonucleotides corre- feasible. The ultimately useful probes for isolating an Rh sponding to the N-terminal amino acid sequence previously cDNA were derived from N-terminal amino acid sequence determined from purified Rh polypeptide. This cloning determined by classical protein sequencing methods (Fig strategy was not successful, probably because of low abun- 4). dance of the Rh cDNA and because of codon degeneracy Overlapping amino acid sequences were independently for the amino acids identified in the N-terminal sequence. reported by three research groups each having used dif- In the end, both groups successfully used the polymerase ferent methods to isolate the Rh The chain reaction (PCR) with oligonucleotide primers derived recoveries from the sequencing were low, indicating that a from proximal and distal segments of the N-terminal amino partial block of the N-terminus may exist. Identical amino acid sequence to amplify cDNA templates prepared from acid sequences were determined for the first 20 amino acid thalassemic spleen erythroblasts and peripheral reticulo- residues from Rh polypeptides isolated by hydroxylapatite cytes, respectively. The PCR products were radiolabeled chromatography from RBCs of multiple Rh D negative and and used to select plaques from A libraries constructed from positive phenotypes?’ This sequence contained the amino human bone marrow, and both groups isolated clones of acid sequence determined for the first 16 residues from the approximately 1.4 kb. The isolated cDNA was shown to Rh D polypeptide isolated by immunoprecipitation from localize to chromosome lp34.3-p36.1 by in situ hybridiza- Rh D positive RBCs with human monoclonal anti-D tion with metaphase chromosomes,61 which corresponds to antibodiess8In addition, several short segments of internal the Rh locus previously deduced by segregation analysis.6’ amino acid sequences were determined from proteolytic The Rh recombinants were found to be of low abun- fragments of Rh polypeptides?’ dance, with only five clones found after screening 1.2 million phages. The Rh cDNA was used to search for poly(A)+ RNA by Northern analysis, and major 1.7-kb and C/c or E/# (I) S S K V P R S U R R C L P L U R L 7 LE R R L I L L F V minor 3.5-kb signals were found in preparations from adult 0 (1)SSKVPRSURRCLPLURLTLERRLlLLFV-- -- erythroblasts. The 1.7-kb Rh transcript was also found in glu Ah (I) n(c)F T F P L n R I u L E I R n I u -L F G RNA preparations from fetal liver, HEL, and K562 eryth- roid cell lines and the MEGOl megakaryocytic line, but not in adult human liver, kidney, or in the Jurkat lymphoblastic or HL60 promyelocytic cell lines.s9

MEMBRANE ORGANIZATION OF THE RH POLYPEPTIDES Analysis of the primary sequence. Despite minor differ- Fig 4. N-terminal amino acid sequences of Rh polypeptidesand an ences in the amount of flanking untranslated DNA se- Rh-related glycoprotein. The presumed Clc or Ele polypeptides quence, the open reading frames reported by the Paris and (isolated by R6A immunoprecipitation)were sequenced to the 41st residue; subsequent sequence was derived from cloned cDNA.” Bristol groups were identical?’” This result was not surpris- Amino acid sequences from the Rh D polypeptide and the Rh-related ing because both groups had obtained commercially avail- glycoprotein (glyRhl were entirely determined by sequencing the able cDNA libraries that had been prepared from the purified proteins.‘Om Identical residues are enclosed within boxes. marrow of the same individual. Nevertheless, search of Note that the amino acid sequence of the presumed Clc or €/e polypeptide diverges from the D polypeptide after the 41st residue existing databanks failed to identify any related sequences, (top sequences). Note also that the Rh-related glycoprotein bears a confirming that the cloned Rh cDNA represents a new area partial homology (bottom sequence). of biologic research. From www.bloodjournal.org by guest on October 31, 2014. For personal use only.

556 AGRE AND CARTRON

The open reading frame of the Rh cDNA encodes 416 Fig 5. In addition, two related 7-amino acid sequence amino acids plus an initiating methionine that is removed repeats were found beginning at residues 392 and 407 and from the mature protein. Consistent with the studies of are also predicted to reside at exofacial sites close to the Gahmberg,'* the primary amino acid sequence is notable C-terminus.60 for the lack of potential surface N-glycosylation sites. The Identification of an Rh isoform. It remains uncertain calculated molecular weight of the deduced protein is 45.5 which of the Rh polypeptides corresponds to the deduced Kd, but the Rh polypeptide has a mobility of approximately cDNA sequence. Interestingly, the Paris group used the 32 Kd by SDS-PAGE, probably due to increased binding of PCR with oligonucleotide primers flanking amino acids 8 SDS." The possibility that this represents a posttransla- through 26 and isolated a single 90-bp product containing a tional proteolytic modification is unlikely, because a pep- sequence corresponding exactly to that determined from tide isolated from the tryptic digestion of the Rh D the purified Rh p~lypeptide.'~The Bristol group used polypeptide contains a 7-amino acid sequence correspond- oligonucleotide primers flanking amino acids 29 through 46 ing to residues 400 through 406 of the predicted Rh protein from the amino acid sequence determined for the first 54 ~equence.6~ residues of Rh polypeptide immunoprecipitated with an Hydropathy analysis of the deduced amino acid sequence anti-D MoAb (Fig 4). The PCR amplification gave two for the Rh polypeptide demonstrated a striking degree of 88-bp products. The sequence of one product corresponded hydrophobi~ity?~,~Most of the polypeptide is predicted to exactly to the Rh D sequence, and the other was a related reside between the leaflets of the phospholipid bilayer (Fig sequence in which only 8 of 11 amino acids were identical 5). Modeling suggests 13 bilayer-spanning domains with (see Fig 4). When the PCR products were used together to only very short connecting regions extending outside of the screen the library, the isolated clone corresponded to the cell or protruding into the cytoplasm. Certain bilayer- latter sequence.w spanning domains may turn while still within bilayer, Thus, the cloned Rh cDNA sequence reported by both thereby crossing the bilayer twice with no extracellular or the Paris and Bristol groups may correspond to the Rh C/c intracellular connecting loops (Fig 5, 4th and 5th, 9th and or E/e sequences, but probably does not correspond to the loth, and 12th and 13th bilayer-spanning domains). While Rh D sequence. Divergence between the different Rh most of the bilayer-spanning domains are exclusively hydro- polypeptides appears after the 41st amino acid residue. Of phobic, four of the first five domains each contain acidic the 54 residues determined from sequencing the Rh D residues that may be functionally important, and the third polypeptide, only 5 of the last 13 residues correspond to and fifth are amphipathic helices. those deduced from the Rh cDNA (Fig 4). Moreover, the The N-terminus is thought to extend into the cytoplasm, eight variant residues represent nonconservative substitu- based on lack of a cleavable leader peptide and flanking tions. The existence of tyrosines in the flanking sequence charge differences." Moreover, the tyrosine at the 4th suggests that this region of the Rh polypeptides may residue was not found to contain '*'I after surface labeling correspond to the variant iodopeptides specific for the Rh c, intact RBCS.~~The location of the C-terminus cannot be D, and E iodopeptides (described above and in Fig 3). The determined from the primary amino acid sequence, but was extent of the divergence in more distal sequence remains to deduced by biochemical studies. Digestion of Rh polypep- be established. tides from surface 'xI-labeled RBCs with carboxypeptidase The gene organization of the Rh polypeptides also Y established that an exofacial tyrosine exists at a location remains to be determined. The Rh c, D, and E polypeptides very near the C-terminu~~~~~~consistent with the model in are indeed closely related but distinct proteins. It was thought that the Rh c, D, and E polypeptides might all be products of a single Rh gene with the divergence arising from alternate splicing of mRNA, use of different transcrip- tional initiation sites, or differences in mRNA stability.& Although speculative, theoretical analysis of Rh genetics indicates that there are likely to be two separate but closely linked loci corresponding to D and CcEe.67The Rh D and the C, c, E, and e polypeptides may therefore be products of a duplicated ancestral gene. Recent cloning advances permitted restriction fragment length polymorphism analy- J 61)CLP (185)CLP (310)CLPVCC (WH2 sis of DNA from individuals with Rh D positive and Rh D negative RBCs. Individuals with Rh D positive RBCs Fig 5. Model of Rh polypeptide topology within the membrane appeared to have two Rh polypeptide genes, whereas the lipid bilayer based on hydropathy analysis of cDNA sequence.N,MThe N-terminus (NH,) of the mature protein is located inside the cell, and Rh D negative individuals have only one gene.@ the C-terminus(COOH) faces outward. Locations of selected deduced Topology of cysteine residues. The deduced amino acid amino acid sequences are indicated:intracellular cysteines are thought sequence of the Rh polypeptide includes six cysteine to be palmitylation sites (residues 11, 185, and 310); an unblocked residues?M' and the locations and the identity of surround- exofacial cysteine (284) and an exofacial tyrosine (400); the point ing amino acids have proven to be of significant interest. where the sequence of D diverges from C/c or E/e (42). Negatively charged residues located within the 1st. 2nd. 3rd. and 5th bilayer Residue 284 is a cysteine that is predicted to lie very near spanning domains are noted (4(see text). the outer leaflet of the lipid bilayer in the sequence CHLIP From www.bloodjournal.org by guest on October 31, 2014. For personal use only.

MOLECULAR BIOLOGY OF THE RH ANTIGENS 557

(cys-his-leu-ile-pro). An exofacial sulfhydryl is known to be be established (see below). Nevertheless, the investigators involved in the immunoreactivity of Rh D and C antigens showed that the immunoprecipitations of the Rh-related (discussed above). Also, histidine-reactive compounds had glycoproteins were specifi~.~’ previously been shown to partially reduce all Rh antigenic A murine MoAb (2D10) was recently developed in reactivities, with the greatest reductions being of the c and Amsterdam by von dem Borne et and was found to react C antigens6’ These observations are entirely consistent with the Rh-related glycoprotein^?^ Moreover it is likely with the location of this domain in the model in Fig 5, but that these glycoproteins are coprecipitated with Rh polypep- whether the cysteine and histidine residues are components tides during immunoprecipitations with human Rh antibod- of the Rh antigenic epitope or an essential supporting ies as well with several murine MoAbs (R6A, BRIC-69, and structure remains to be established. BRIC-207). The other five cysteines are predicted to lie near the The LW antigen. The initial confusion of Rh and LW inner leaflet of the bilayer and exist in the interesting antigens was not unlikely, because the level of LW antigen sequence motif CLP (cys-leu-pro), except the last which is expression is greater in Rh D positive than negative RBCs, CLPVCC (Fig 5). These CLP motifs are also flanked by and LW antigens are altogether lacking in the Rh,,,, multiple basic amino acids (not shown). In all cases these phenotype. On the basis of these phenotypic relationships, CLP motifs precede or follow a region of presumed a helix it has been speculated that Rh might be the precursor of emerging from or entering the inner leaflet of the phospho- LW.75 Using an MoAb with LW specificity, an M, 37- to lipid bilayer. Each cysteine is adjacent to a leucine that may 47-Kd glycoprotein was identified on normal RBCs but not provide a hydrophobic side chain within a structural kink in on Rh,,,, or LW negative membranes, and the LW antigenic the tertiary structure resulting from the following proline. reactivity was destroyed by endoglycosidase F (removes all The CLP motifs are the palmitylation sites on the Rh asparagine-linked oligosaccharides) but not by endo-beta- polypeptide, and all three appear to be ~almitylated.’~The galactosidase (degrades only polylact~saminoglycans).~~Se- motif would be predicted to be a suitable site for the lective carboxypeptidase digestions indicate that the C-ter- addition of fatty acids from preformed fatty acyl-Coenzyme minus of the LW protein is exofacial and involved in the A complex. While palmitylation of certain other mamma- antigenic reactivity. The biology of the LW antigen is lian and viral glycoproteins has been established, no consen- complex, however, because chelation of Mg’+ by EDTA has sus sequence has been identified. Nevertheless, the topol- recently been shown to block LW antigenic reactivity, ogy of the cysteines in the established palmitylation sites of suggesting a functional While it had been speculated these other acylproteins resembles the cysteines within the that LW is a glycosylated form of the Rh polypeptide, Rh polypeptide, being located within a few residues of the two-dimensional iodopeptide maps of purified Rh polypep- inner leaflet of the bilayer with adjacent basic amino acids tide and purified LW glycoprotein conclusively demon- and glycine or proline helix breakers7’ strated that Rh and LW are distinctly different Other glycoproteins deficient in Rhnuscells. The patholog- GLYCOPROTEINS ASSOCIATED WITH RH ically affected Rh,,,, RBCs were shown to partially or totally Rh-related glycoproteins with AB0 spec@city. Although lack several other distinct glycoproteins. Glycophorin B was the M, 32-Kd Rh polypeptides were found to lack carbohy- shown to be reduced to approximately 30% of the normal drate,” subsequent investigation showed that glycosylated and certain other blood group antigens including the higher molecular weight proteins with ancestrally related DufQ (Fy5), and U and Duclos antigens were also missing amino acid sequences (the “Rh-related glycoproteins”) from Rh,,,, cells.79Murine MoAbs developed by the Bristol were coprecipitated by Rh antibodies along with the Rh investigators showed that at least two other membrane polypeptide^.^^.^' Terminal galactose residues on glycopro- glycoproteins may be associated with the Rh polypeptides teins can be labeled on the surface of RBCs labeled with that are distinct from the LW and Due antigens.’’ The NaB3H4and galactose Rh-related glycoproteins BRIC 125 MoAb reacts with an M, 47- to 52-Kd glycopro- immunoprecipitated from such 3H-labeled RBCs were ana- tein on normal RBCs, which is greatly reduced in Rh,,,, lyzed by fluorography, and a large, heterogeneous band of RBCs. After deglycosylation, the BRIC 125 protein mi- M, 45 to 100 Kd was immunoprecipitated along with the Rh grated as an M, 28.5-Kd band. In addition to erythroid cells, D polypeptide by anti-D, while similar bands of M, 35 to 60 the BRIC 125 protein was found in kidney and liver tissue Kd were coprecipitated with anti-c and anti-E.52 These as well as myeloid, lymphoid, and megakaryocytic cell lines. Rh-related glycoproteins were susceptible to endo-beta- Interestingly, although RBCs from Rh,,,, individuals have galactosidase digestions, a characteristic of the polylac- reduced expression of the BRIC 125 glycoprotein, immuno- tosaminoglycans, the family of complex asparagine-linked histochemical staining of their lymphocytes was normal. carbohydrates containing numerous galactose-N-acetylglu- The protein recognized by the BRIC 69 MoAb was found to cosamine repeating units and terminating in AB0 blood resemble the Rh-related R6A Both antibod- group structures.72Interestingly, the Rh-related glycopro- ies precipitated a broad complex of M, 35- to 52-Kd teins were still immunoprecipitated with Rh antibodies glycoprotein, which may be the same as an Rh-related after the glycosidase digestion, indicating that the Rh glycoprotein and which was shown by immunoblot to be antigenic reactivity is not simply the result of the attached missing from Rh,,,, RBCS.~’,~Immunohistochemical studies glycan. The studies did not include quantitative immunopre- with the BRIC 69 and R6A antibodies indicated that the cipitations, and the existence of an Rh complex remains to corresponding proteins are restricted to erythroid tissues. From www.bloodjournal.org by guest on October 31, 2014. For personal use only.

558 AGRE AND CARTRON

Other membrane components recognized by murine Mo- sured. Early investigation showed that a small amount of Abs 1D8 and BS58 but still uncharacterized at molecular Rh D antigenic reactivity transiently survived solubilization levels have also been found to be missing from Rh,,,, in deoxycholate and was roughly estimated by ultrafiltration RBCS.’~,’~ to have an M, of less than 300 Kd.92 Using radiation inactivation as a measure of molecular size, the Rh antigen ASSEMBLY OF RH WITHIN A MEMBRANE COMPLEX was estimated to be M, 60 Kd and 174 Kd by two different The pleitropic defects in several antigens and polypep- investigator^.^^,^^ The abundance of the different possible tides in the Rh,,,, phenotype raises the question of how components of the Rh membrane complex is quite variable, multiple abnormalities might simultaneously arise. It is ranging from 5,000 copies of LW to approximately 100,000 unlikely that several genes are each altered in Rh,,,, copies of the Rh polypeptides per cell, so the Rh membrane patients, because the structural genes for LW, glycophorin complexes might be heterogeneous containing various com- B, and the 1D8 antigens are each located on a different binations of the different components. Rh D antigenic chromosome. Likewise, regulation of the expression of the reactivity is lost after RBC membranes are solubilized in other genetic loci by mutations in the Rh locus is also detergents, and the members of the hypothetical Rh mem- unlikely, because the amorph and regulator type Rh,,,, brane complex do not appear to be tightly associated. mutations arise from different genetic backgrounds, yet Therefore, it is conceivable that continuous and reversible exhibit the same phenotypic defects. It has also been shown exchange occurs between the components of multisubunit that the Rh polypeptide is not a precursor for the various Rh membrane complexes (see above). Very recent investi- glycoproteins (see above). One hypothesis is that Rh gation using [3H]palmitate-labeled Rh polypeptides demon- polypeptides are required for the correct transport or cell strates that they migrate as a large complex of M, 170 Kd surface expression of certain glycoproteins. This explana- when analyzed by velocity sedimentation and gel filtration.% tion suggests that the Rh antigen is a large membrane The stoichiometric composition of this complex is yet to be complex containing the Rh polypeptides, Rh-related glyco- defined but may be a tetramer composed of two Rh proteins, and possibly also other unrelated glycopro- polypeptides and two Rh-related gly~oproteins.~~ tein~.”.’~ Several lines of investigation support the Rh membrane WHY IS RH D SO ANTIGENIC? complex hypothesis. ‘Z51-labeledRh polypeptides in deter- After ABH, the Rh D antigen is the most immunogenic gent-solubilized membranes behaved as if complexed with a of all the RBC antigens, although the basis of this extreme membrane glycoprotein(s), whereas the isolated Rh poly- antigenic reactivity remains very poorly understood. Isola- peptides did not.” Moreover, the ability to coprecipitate tion of the Rh polypeptide cDNA provided little additional Rh-related glycoproteins with antibodies specific for Rh c, insight, because most of the Rh polypeptide is predicted to D, and E antigens” and the failure to detect several reside between the leaflets of the lipid bilayer with minimal unrelated glycoproteins in Rh,,,, RBCs both suggest that the projection into the extracellular space (see Fig 5). Rh polypeptides and these glycoproteins may exist together All existing information suggests that the Rh D antigenic within a large membrane complex (see above). epitope is not a simple peptide sequence but is likely to be a The fact that the M, 30- to 32-Kd Rh polypeptides are precise surface conformation that results from the coopera- apparently not glycosylated also suggests that they may be tive interactions of at least one of the Rh polypeptides with translated and processed in association with a specific surrounding glycoproteins and also requires the presence of glycoprotein(s). Nevertheless, the Rh polypeptides very a specific exofacial free sulfhydryl and also possibly requires likely play a fundamental role in the Rh complex structure, certain adjacent lipid structures. Because of its lability, because the entire complex is greatly reduced or altogether attempts to biochemically reconstitute the Rh antigens are missing presumably due to a single Rh,,,, mutation. The lack likely to be difficult. The actual demonstration of the of a single polypeptide chain may prevent the cell surface components needed for Rh antigenicity may ultimately expression of multichain complexes by altering the assem- require expression of specific genetic material by transfec- bly or intracellular transport. This appears to be a general tion. The genetic polymorphism underlying the Rh antigens mechanism that has been well documented for the assembly appears to lie within the gene(s) that encodes the Rh of T-cell receptors: cell adhesion integrins?= histocompat- polypeptides, and the nucleotide and deduced amino acid ibility antigen^,"^^ and the acetylcholine receptor.” Obvi- sequences should soon be identified. Because multiple ously, more direct evidence is required to bring further associated structural components may also be required, support to the Rh membrane complex hypothesis and to simultaneous transfection and expression of Rh polypep- demonstrate a direct association of the different glycopro- tide cDNAs, Rh-related glycoprotein cDNAs, and possibly teins. While these glycoproteins are all deficient in Rh,,,, cDNAs for other structural components may be needed. RBCs, the converse is not true. Cells lacking glycophorin B Except for instances of spontaneous or drug-induced (S-s-U), LW, and DufQ blood groups are phenotypically autoimmunity, Rh D is only immunogenic for individuals normal and are not deficient in Rh antigens. Moreover, who are Rh D negative. Because the latter are phenotypi- certain of these other glycoproteins are clearly not immuno- cally normal, there is no obvious physiologic advantage precipated by anti-D along with the Rh polypeptides. associated with being Rh D positive. However, hemolytic Because of instability in the solubilized state, the size of disease of the newborn affects Rh D positive infants of Rh the Rh membrane complex has not been accurately mea- negative mothers, an occurrence that would theoretically From www.bloodjournal.org by guest on October 31, 2014. For personal use only.

MOLECULAR BIOLOGY OF THE RH ANTIGENS 559 affect one in seven pregnancies among multiparous women deficiency of membrane cholesterol.% Physiologic roles in in the United States. membrane stability and volume regulation have been sug- Very recent work by the group in Paris has demonstrated gested for Rh, but specific details remain lacking. Interest- that Rh D positive individuals have two Rh polypeptides ingly, membrane phospholipid distribution has been re- genes, presumably D and CcEe, whereas Rh D negative ported to be abnormal in Rh,,,, RBCS.~' individuals only have one gene, ce.6' Therefore, it is likely PhosphatidylserineB@pase? Phospholipids are known to that the D gene represents an ancient genetic accident be asymmetrically distributed between the leaflets of the during which an ancestral Rh gene was imperfectly dupli- normal RBC lipid bilayer, with phosphatidylcholine (PC) cated. The explanation for evolutionary selection of the D being enriched in the outer leaflet, while phosphatidyletha- gene may have resulted from a reproductive advantage by nolamine (PE) is enriched in the inner leaflet and phosphati- Rh D positive mothers, because they are immune from Rh dylserine (PS) is located entirely in the inner An D sensitization and their offspring will not suffer hemolytic ATP-dependent phosphatidylserine translocase ("PS disease. Some Rh D negative mothers will become sensi- flippase"), which is inactivated by sulfhydryl oxidation, has tized by their Rh D positive fetuses. In subsequent pregnan- been identified as the enzyme responsible for this."-'" It cies, many of the Rh D positive fetuses would have has recently been proposed by Zachowski et al and Morrot developed hydrops fetalis and would have died in utero. et a1 at the Curie Institute in Paris that PS flippase is similar However, in ancient times, stillborn hydropic infants and to the M, 115-Kd Mg2'-ATPase I1 present in chromaffin their mothers would probably both have perished due to the granules,lo5 and a similar protein has been identified in lack of obstetrical care, thereby canceling the selection RBCS.'" against Rh D positive infants. Once introduced, into a Another candidate for the PS flippase has been proposed population, the Rh D positive phenotype would be ex- by Schroit et al at MD Anderson Cancer Center in pected to eventually predominate over the Rh D negative Houston, TX. Radiolabeled PS analogs with photoactivat- phenotype, theoretically because of the extreme antigenic able cross-linking groups become nearly exclusively associ- reactivity associated with Rh D. Indeed, in certain geneti- ated with an M, 32-Kd transmembrane protein with several cally isolated populations such as Japan, the incidence of properties similar to the Rh p~lypeptides.'''~~'~~~'~'The PS- the Rh D positive phenotype is 99%.' labeled protein was specifically immunoprecipitated by Rh MoAbs.lW The same investigators studied RBCs from POTENTIAL PHYSIOLOGIC FUNCTION(S) OF RH several Rh,,,, patients and found that the PS analogs were Several lines of evidence indicate that the Rh polypep- cross-linked to a similar M, 32-Kd band, and PS flippase tides play a fundamental role in the physiology of RBC activity was found to be normal in these Rh,,,, RBCs. These membranes that is unrelated to their antigenic reactivities. data were interpreted as showing that Rh,,,, RBCs may Such a notion is consistent with observations of other blood contain mutant Rh polypeptides that are devoid of Rh group antigens that are often functionally important struc- antigenic reactivity but may still contribute to PS transbi- tures. The multiple bilayer spanning domains of the mem- layer movements. This finding is presently difficult to brane model of an Rh polypeptide (Fig 5) is reminiscent of reconcile with the observations that Rh,,,, RBCs have known membrane transporters. Nevertheless, the function severely reduced or absent Rh polypeptides, although use of the polypeptide is not apparent. of isotopic amounts of photoactivatable PS analog may not The RBCs from humans of all of the frequent Rh distinguish reduced from normal level of Rh polypeptides. phenotypes are obviously normal, so the Rh polypeptides The possibility that the Rh polypeptides may be noncata- within the membranes of RBCs of the common Rh pheno- lytic subunits of the PS flippase has been raised.'" The types must therefore function similarly. Likewise, the dis- deduced amino acid sequence of the cloned Rh polypeptide tantly related proteins isolated from RBCs of several does not contain the consensus sequence characteristic of nonhuman species should function equivalently to their and the likely existence of large Rh membrane human counterpart^.'^ The fatty acylation characteristic of complexes suggests the possibility that the Rh polypeptides the human Rh polypeptides appears to be conserved may be associated with other proteins with catalytic activi- amongst all nonhuman Rh homologs, suggesting a common ties. Alternatively, the Rh polypeptides may provide a functional ~ignificance.'~ passive restraint to PS in the inner leaflet of the phospho- Pathology ofthe Rh,,,phenofype. RBCs from individuals lipid bilayer, and multiple palmitic acids covalently linked with the rare Rh,,,, phenotype bear several membrane to sites on the Rh polypeptide near the inner leaflet may defects, and provide certain clues to the functional impor- function to directly influence the organization of surround- tance of Rh. Rh,,,, patients suffer from a chronic hemolytic ing phospholipids. anemia, which is usually of mild to moderate clinical The role of Rh in PS flippase and overall membrane severity.' The lack of severe clinical manifestations suggests phospholipid organization is currently being debated. PS that Rh may be a fine-tuning mechanism in RBC membrane flippase activity was studied in intact RBCs by Daleke et a1 physiology. Rh,,,, RBCs are pleomorphic but always have at Stanford and Indiana Universities. The generation of some degree of stomatocytosis and spherocytosis, and have echinocytes or stomatocytes was monitored after addition increased sensitivity to osmotic lysis. Rh,,,, membranes have of PC or PS which located in the outer or inner leaflet of the characteristically hyperactive membrane ATPases,95 re- bilayer respectively. Rh,,,, and control cells did not behave duced RBC cation and water contents, and a relative differently, and Rh antibodies did not interfere with flip- From www.bloodjournal.org by guest on October 31, 2014. For personal use only.

AGRE AND CARTRON pase activity in normal RBCs, observations that led these normal membrane integrity, because they appear to be investigators to conclude that flippase is not related to the missing from the RBCs of the rare Rh,,,, individuals that Rh polypeptide.'" express several membrane defects. The Rh polypeptides Otherpossible roles. The Rh polypeptides appear neces- contain an exofacial free sulfhydryl that is important for Rh sary for the membrane expression of several glycoproteins, antigenic reactivity and several intracellular sulfhydryls that some of which may exist within the hypothetical Rh appear to be palmitylated, but most of the molecule membrane complex (described previously). Therefore, the appears to reside between the leaflets of the phospholipid Rh polypeptides may confer stability to certain other bilayer. The cDNA coding for a 416-amino acid Rh polypep- membrane components that are missing from Rh,,,, RBC tide was recently isolated but was not found to share membranes but may not be directly associated with the Rh sequence homology with any known protein, and Northern polypeptides in the mature RBC plasma membrane. Analy- analysis indicated that Rh is erythroid specific. sis of Rh expression made with fluorescence-activated cell The Rh antigens within the native membranes are sorting demonstrated that some Rh antigens are already thought to exist as a complex of Rh polypeptides and located in the plasma membrane in CFU-e, committed multiple other membrane components, including certain RBC progenitors, although the more primitive BFU-e cells Rh-related glycoproteins. While it is thought that this were devoid of Rh."* This analysis suggests a physiologic assembly may be important for the Rh antigenic reactivity, need for Rh rather early in the differentiating red cell. the structural basis of this remains to be established. While Despite these lines of evidence, the actual physiologic role the physiologic role of Rh is yet to be defined, several clues of Rh remains to be defined and is being investigated. indicate that it may play a role in the organization of membrane phospholipids or synthesis or membrane expres- SUMMARY sion of various glycoproteins. While our knowledge of Rh is The RBC Rh antigens are of large clinical importance, still very incomplete, recent research has significantly ad- but until recently have been poorly understood at a molecu- vanced the molecular understanding of these important lar level. The Rh polypeptides are a family of nonglycosy- blood group antigens. lated M, 30- to 32-Kd transmembrane proteins that are core structural components of the Rh antigens and have been ACKNOWLEDGMENT purified and partially characterized biochemically. Rh poly- The authors thank Drs Floyd Green, David Anstee, Michael peptides are present in RBCs from normal humans and Tanner, Peter Issitt, Alan Schroit, and David Daleke for informa- other mammalian species and are probably required for tion and valuable discussions.

REFERENCES 1. Race RR, Sanger R: Blood Groups in Man (ed 6). New York, 14. Bennett V: The spectrin-actin junction of the erythrocyte NY,Blackwell, 1975 membrane-skeleton. Biochim Biophys Acta 988:107,1989 2. Mollison PL, Engelfriet CP, Contreras M: Blood Transfusion 15. Yu J, Fischman DA, Steck TL Selective solubilization of in Clinical Medicine (ed 8). New York, NY,Blackwell, 1987 proteins and phospholipids from membranes by 3. Petz LD, Garratty G: Acquired Immune Hemolytic Anemia. nonionic detergents. J Supramol Structure 1:233,1973 New York, NY,Churchill Livingstone, 1980 16. Nicolson GL, Masouredis SP, Singer SJ: Quantitative two- 4. Levine P, Stetson RE: An unusual case of intragroup aggluti- dimensional ultrastructural distribution of Rh,(D) antigenic sites nation. JAMA 113:126,1939 on human erythrocyte membranes. Proc Natl Acad Sci USA 5. Landsteiner K, Wiener AS: An agglutinable factor in human 68:1416,1971 blood recognized by immune sera for rhesus blood. Proc SOCExp 17. James NT, James V: Nearest neighbour analyses on the Biol Med 43:223,1940 distribution of Rh antigens on erythrocyte membranes. Br J 6. Levine P, Celano M, Fenichel R, Pollack W, Singher H: A Haematol40657,1978 'D-like' antigen in rhesus monkey, human Rh positive and human 18. Shotton DM, Burke BE, Branton DM: The molecular Rh negative red blood cells. J Immunol87:6,1961 structure of human erythrocyte spectrin. Biophysical and electron 7. Wiener AS: The Rh series of allelic genes. Science 100:595, microscopic studies. J Mol Biol131:303,1979 1944 19. Masouredis SP, Sudora EJ, Mahan L, Victoria EJ: Antigen 8. Race RR: An incomplete antibody in human serum. Nature site densities and ultrastructural distribution patterns of red cell 153:771,1944 Rh antigens. Transfusion 1694,1976 9. Nash R, Shojania AM: Hematological aspect of Rh deficiency syndrome: A case report and review of the literature. Am J 20. Gahmberg CG, Karhi KK: Association of Rh,,(D) polypep- Hematol24:267,1987 tides with the membrane skeleton in Rh,(D)-positive human red 10. Moore S, Woodrow CF, McClelland DBL Isolation of cells. J Immunol133:334,1984 membrane components associated with human red cell antigens 21. Ridgwell K, Tanner MJA, Anstee DJ: The Rhesus (D) Rh,(D), (c), (E), and Fy". Nature 295529,1982 polypeptide is linked to the human erythrocyte cytoskeleton. FEBS 11. Gahmberg CG: Molecular identification of the human Lett 174:7,1984 Rh,(D) antigen. FEBS Lett 140:93,1982 22. Paradis G, Bazin R, Lemiew R: Protective effect of the 12. Gahmberg CG: Molecular characterization of the human membrane skeleton on the immunologic reactivity of the human red cell Rho (D) antigen. EMBO J 2:223,1983 red cell Rh,(D) antigen. J Immunol137:240,1986 13. Helenius A, Simons K Solubilization of membranes by 23. Bloy C, Blanchard D, Lambin P, Goossens D, Rouger P, detergents. Biochim Biophys Acta 415:29,1975 Salmon C, Cartron JP: Human monoclonal antibody against From www.bloodjournal.org by guest on October 31, 2014. For personal use only.

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