Topology and Organization of Human Rh (Rhesus) Blood Group-related Polypeptides*

Transcription

1 THE JOURNAL OF Bromrca CHEMISTRY by The Amercan Socety for Bochemstry and Molecular Bology, Inc. Val. 269, No. 9, Issue of March 4, pp , 1994 Prnted n U.S.A. Topology and Organzaton of Human Rh (Rhesus) Blood Group-related Polypeptdes* (Receved for publcaton, August 9, 1993, and n revsed form, October 21, 1993) Sophe A. C. Eyers$, Kay Rdgwell, Wllam J. Mawby$, and Mchael J. A. Tanned From the Department of Bochemstry, School of Medcal Scences, Unversty of Brstol, Unversty Walk, Brstol BS8 ITD, Unted Kngdom The Rh blood group antgens are assocated wth nonglycosylated human erythrocyte membrane protens of molecular mass 30 kda (the Rhso polypeptdes) and a glycoproten of kda (the Rh glycoproten). We have studed the topology of ths famly of protens n the erythrocyte membrane. We confrmed the predcted cytosolc localzaton of the C and N termn of the Rh proten famly. We located Lys-196 and Arg-323 of the Rh glycoproten to the cytosol, and Glu-34 to the extracellular sde of the plasma membrane n erythrocytes, by N-termnal sequencng of Rh glycoproten peptdes pro- regons. Studes wth Rh-specfc monoclonal antbodes have also duced by proteolyss at the cytoplasmc or extracellular dentfed Rh-related glycoprotens that co-precptate wth the sde of the membrane. We also show that a glycan chan s present on only one (Am-37) of the three potental Rh30 polypeptdes and mgrate as a dffuse band on SDS-PAGE N-glycan addton stes n the Rh glycoproten. Studes (Moore and Green, 1987; Avent et al., 1988b). The avalable of the Rh glycoproten fragments that co-mmunopre- evdence suggests that there s only one Rh glycoproten polycptated wth the Rhso polypeptdes suggest there s an nteracton between the Rhso polypeptdes and amno acds of the Rh glycoproten. A model for the organzaton of the components of the Rh complex n the red cell membrane s proposed. The antgens of the human Rh (Rhesus) blood group system are mportant n transfuson medcne and mmunohematology because of ther mmunogencty n man and nvolvement n hemolytc dsease of the newborn (revewed by Race and Sanger (1968) and Hadley and Kumpel (1993)). Some of the erythrocyte membrane protens that carry Rh antgens have been characterzed by mmunoprecptaton studes usng ant- Rh(C/c), ant-rh(d), or ant-rh(e/e) antsera and Rh-related monoclonal antbodes of the R6A type (revewed by Agre and Cartron (1991) and Anstee and Tanner (1993)). Two Rh-related polypeptdes of 30 and 32 kda, the Rh30 polypeptdes, have been dentfed and these are unusual n that they are not glycosylated (Moore et al., 1982; Gahmberg et al., 1982; Rdgwell et al., 1983; Moore and Green, 1987). The N-termnal proten sequences of both these polypeptdes have been determned (Avent et al., 1988b; Bloy et al., 1988) and cdna clones encodng members of ths group of polypeptdes have been solated. Two cdna clones, Rh30A (RhMb) and Rh13, predct hydrophobc membrane protens of 417 amno acds whch are very smlar n amno acd sequence (Avent et al., 1990; Cherf- Zahar et al., 1990; Le Van Km et al., 1992b; Kaj and Ikemoto, 1992; Kaj et al., 1993). Hydropathy analyss of the cdna clones suggests that the protens contan 12 or 13 membrane * Ths work was supported n part by a grant from the Medcal Research Councl. The costs of publcaton of ths artcle were defrayed n part by the payment of page charges. Ths artcle must therefore be hereby marked advertsement n accordance wth 18 U.S.C. Secton 1734 solely to ndcate ths fact. $ Recpent of a Medcal Research Councl Studentshp. 8 Supported by the Central Blood Laboratores Authorty. 1 To whom correspondence should be addressed spannng domans (Avent et al., 1990; Cherf-Zahar et al., 1990). It has been suggested that the C termn of the protens are located on the extracellular sde of the plasma membrane (Krahmer and Prohaska, 1987; Bloy et al., 1990; Cherf-Zahar et al., 1990) consstent wth the protens havng odd an number of up to 13 membrane spannng regons. However, other evdence (Avent et al., 1992) suggests that the C termn are on the cytoplasmc sde of the plasma membrane, and that the protens have an even number of up to 12 membrane spannng peptde (Rdgwell et al., 1994). Recently a cdnaclone (Rh5OA) was solated for the Rhso glycoproten (Rdgwell et al., 1992). The Rh5OA cdna clone predcts a polypeptde of 409 amno acds wth three potental N-glycosylaton stes, two of whch are predcted to be extracellular. The Rh5OA glycoproten amno acd sequence s clearly homologous to that of the Rh3OA polypeptde, and the Rh3OA and Rh50A protens probably both have the same topology, and belong to a famly of structurally related membrane protens (Rdgwell et al., 1992). Snce there s dsagreement about the topology of the Rhrelated polypeptdes, we have further nvestgated ths queston wth dfferent members of the Rh proten famly. We have studed the locaton of ther C and N termn usng polyclonal antbodes reactve wth the N- and C-termnal amno acd sequences of the Rh3OA proten and the C termnus of the Rh5OA proten. Although the Rh50A cdna sequence contans three potental N-glycan addton stes, we show that the Rh glycoproten contans a sngle N-glycan located n the frst extracellular loop of the proten. We have also determned the locaton of proteolytc cleavage stes n the amno acd sequence of the natve Rh glycoproten n the erythrocyte membrane. Ths data provdes expermental evdence for the topologcal locaton of several stes n the amno acd sequence of the Rh glycoproten, and by homology, the Rh30 polypeptde famly. MATERIALS AND METHODS Rh-specfc Polyclonal Antbodes and Monoclonal Antbodes-The polyclonal antbodes reactve wth the C and N termn of the Rh30A proten and the C termnus of the RhBOA glycoproten have been descrbed elsewhere (Rdgwell et al., 1994). Rh30A ant-c was rased to an l8-amn0 acd peptde correspondng to the C-termnal resdues of the predcted Rh30A polypeptde. Rh30A ant-n was rased to an 11-amno acd peptde correspondng to the N-termnal resdues 2-12 of the predcted Rh30A polypeptde. RhGP ant-c was rased to a 14- amno acd peptde that corresponds to the C-termnal resdues 39& 409 of the predcted RhBOA glycoproten sequence. Mouse monoclonal antbodes BRIC 69 and BRIC 207 are of the R6A type and react wth the Rh protens rrespectve of the Rh phenotype of the red cells. BRIC 69, BRIC 207, and the human monoclonal ant-rh(d) antbody (AB5) were provded by Dr. D. J. Anstee, Internatonal Blood Group Reference

2 6418 Topology of Human Rh (Rhesus) Polypeptdes Laboratory, Brstol, Unted Kngdom. separated on a 12% SDS-PAGE Trcne gel. Deglycosylaton of Erythrocytes-Intact washed erythrocytes were The Rh glycoproten tryptc peptdes were solated from 3 ml of resuspended n an equal volume of peptde N-glycosdase F (PNGase F)l trypsn-dgested erythrocyte membranes by mmunoprecptaton usng reacton buffer (112.5 mm NaCl, 25 I" sodum phosphate, 50 m~ EDTA, 7 ml of RhGP ant-c polyclonal rabbt antserum. To purfy the peptdes 5 mm glucose, 5 pd phenylmethylsulfonyl fluorde, ph 7.5) contanng for N-termnal amno acd sequencng, the mmunoprecptated pep- 200 untdml PNGase F (Oxford Glycosystems), and ncubated for 18 h tdes were separated by SDS-PAGE as descrbed above, except that no at 37 "C. Deglycosylated cells were washed n 0.1 M phosphate, ph 8.0, P-mercaptoethanol was added to the gel sample buffer. The peptdes n untl no lyss was observed. the gel were transferred onto Problott membrane (Appled Bosystems) Immunoprecptaton from Intact Erythrocytes and Erythrocyte and staned wth Serva Blue G (Serva) as descrbed by the membrane Mernbranes-Rh30 polypeptdes and Rh glycoprotens were mmunopre- supplers. The staned peptdes were sequenced usng a Blott cartrdge cptated from ntact erythrocytes as descrbed by Moore et al. (1982) and a modfed Fastblott cycle (Rdgwell et al., 1994). except that KI washng steps were ncluded as follows to reduce nonspecfc background (Bennett, 1983). Pror to overnght ncubaton wth antbody at 4 "C, ntact erythrocytes were washed frst wth phosphate- RESULTS buffered salne (PBS), then wth 10 volumes of ce-cold 1 M KI, 5 mm Immunoprecptaton of Rh Polypeptdes wth Rabbt Polysodum phosphate, ph 8.0 (KI/5P8), and fnally wth ce-cold PBS untl clonal Antbodes-Three Rh-specfc antpeptde rabbt polyno lyss was observed. Erythrocyte membranes were prepared as de- clonal antbodes were used n ths work. Rh30A ant-c s a scrbed by Dodge et al. (1963) and washed once wth ce-cold W5P8, polyclonal antbody that was rased aganst the C-termnal 18 then wth an excess of 5 I" sodum phosphate, ph 8.0 (5P8), and ncubated overnght at 4 "C wth the antbody. Membranes contanng amno acd resdues of the Rh3OA polypeptde; Rh30A ant-n bound antbody were then washed twce wth 5P8, twce wth W5P8, was rased aganst resdues 2-12 at the N termnus of Rh30A; and two more tmes wth 5P8 pror to the addton of proten A-Sepha- and RhGP ant-c was rased aganst the C-termnal 14 amno rose (Boprocessng Ltd.). When the Rh glycoproten was mmunopre- acds of the Rh5OA glycoproten. These antbodes detect the cptated from PNGase F deglycosylated ntact erythrocytes or erythro- Rh30 polypeptdes and Rh glycoprotens on mmunoblots of red cyte membranes from PNGase F-treated erythrocytes, the ntact erythrocytes and erythrocyte membranes were not washed KI. wth For cell membranes rrespectve of the Rh Cc, D, Ee phenotypes of large scale mmunoprecptatons from erythrocyte membranes usng the cells (Rdgwell et al., 1994). The antbodes were used to the Rh-specfc antpeptde polyclonal antbodes, 10 I" EDTA and study the cellular locaton of the N and C termn of the Rh30 0.1% (w/v) dgtonn were added to the antbody ncubaton mxture, polypeptdes and the C termn of the Rh glycoproten by comand all buffers contaned 1 m~ EDTA and 0.1% dgtonn to prevent parng ther ablty to mmunoprecptate the Rh polypeptdes resealng of the membranes. Immunoprecptates were analyzed usng from ntact erythrocytes and leaky erythrocyte membranes. the Trcne buffer SDS-PAGE system of Schagger & von Jagow (1987). Proteolytc Dgeston of Intact Erythrocytes and Erythrocyte Erythrocyte membranes were strpped of perpheral membrane Membrane-Erythrocytes were washed n PBS and membranes were protens wth KI pror to ncubaton wth the antbodes. In prepared by lyss n 5P8 (Dodge et al., 1963). For trypsn dgeston ntact erythrocytes or erythrocyte membranes were washed and resuspended n 10 volumes of 10 mm Trs, ph 7.4, 150 I" KC1 contanng mg/ml tosylphenylalanyl chloromethyl ketone-treated tryp- sn (Sgma), and ncubated 37 "C for 45 mn. For SDS-PAGE analyss, sample buffer (Laemml, 1970) contanng protease nhbtors (5 pg/ml were added to KI washed ntact RhD+ve erythrocytes and the tosyllysyl chloromethyl ketone, 0.5 p~ phenylmethylsulfonyl fluorde, resultng mmunoprecptates were mmunoblotted wth the 10 pg/ml soybean trypsn nhbtor (Sgma)) was added the to dgest. For same antbodes. Nether Rh30A ant-n nor RhGP ant-c mmmunoprecptaton from trypsn-treated erythrocytes or trypsn- munoprecptated the Rh polypeptdes from the KI-washed ntreated erythrocyte membranes, the proteolyss reacton was stopped by tact erythrocytes (Fg. 1, b and e). However, mmunoblots of the the addton of the above protease nhbtors, and the dgested materal mmunoprecptates from KI-washed leaky erythrocyte memwas washed several tmes wth PBS (n the case of ntact erythrocytes) or 5P8 (n the case of erythrocyte membranes). For dgeston wth chybranes demonstrated the presence of the kDa Rh30 polymotrypsn, ntact erythrocytes or erythrocyte membranes were washed peptdes n the Rh30A ant-n mmunoprecptate and the dfwth 10 m~ Trs, ph 8.5,112.5 mm NaCI, resuspended n the same buffer fusely mgratng Rh glycoproten n the RhGP ant-c contanng 2.5 mg/ml chymotrypsn (Sgma), and ncubated for 45 mn mmunoprecptate (Fg. 1, a and f). In smlar experments at 37 "C. The reacton was stopped wth the protease nhbtors phenyl- (data not shown), Rh30A ant-c mmunoprecptated the Rh30 methylsulfonyl fluorde (0.5 p ~ and ) chymostatn (0.1 mg/ml). Staphy- polypeptdes from KI-washed leaky red cell membranes but not lococcus aureus V8 protease (0.5 mg/ml; Sgma) dgestons were camed from KI-washed ntact erythrocytes, consstent wth the results out under the same condtons as the chymotrypsn dgestons except that the reacton was stopped usng 125 pg/ml a-macroglobuln (Boehrnger Mannhem) and 0.5 p~ phenylmethylsulfonyl fluorde. Dgested ntact erythrocytes or erythrocyte membranes were mmunoprecptated wthout KI washng as descrbed above. Immunoblottng-Immunoblottng was performed as descrbed by Mallnson et al. (1986) except that samples were separated usng the SDS-PAGE Trcne buffer system of Schagger & von Jagow (19871, Immoblon (Mllpore) was used as the transfer membrane and 5% (w/v) bovne dred skmmed mlk powder n PBS, 0.2% (w/v) Tween 20 (Sgma) was used as blockng agent. Immunoprecptaton and N-termnal Sequencng of Rh Glycoproten Proteolytc Peptdes-2.5 ml of washed packed erythrocytes were deglycosylated usng PNGase F, dgested wth S. aureus V8 protease, and mmunoprecptated wthout KI washng, as descrbed above, usng 10 ml of culture supernatant of BRIC 69 (Avent et al., 1988a). The BRIC 69 mmunoprecptate was solublzed from the proten A-Sepharose usng 400 pl of gel sample buffer (Laemml, 1970) from whch glycerol and P-mercaptoethanol had been ommtted, and concentrated usng a 10- order to ensure that ths treatment dd not uncover any extracellular antbody eptopes, the ntact erythrocytes were also treated wth KI before ncubaton wth the antbodes. The polyclonal antbodes Rh30A ant-n and RhGP ant-c descrbed by Avent et al. (1992). No Rh glycoprotens were mmunoprecptated from PNGase F-treated ntact erythrocytes by.rhgp ant-c (Fg. In). However, a 30.5-kDa band was mmunoprecptated by RhGP ant-c from membranes prepared from ntact erythrocytes that had been deglycosylated wth PNGase F (Fg. lm). Ths band represents the deglycosylated Rh glycoproten (Fg. 11 ). For each of the above mmunoprecptaton experments a parallel control mmunoprecptaton was carred out wth the premmune rab- bt serum approprate to each antbody. None of the premmune sera mmunoprecptated Rh-specfc bands (Fg. 1). These mmunoprecptaton experments demonstrate that theptopes reactve wth Rh3OA ant-c, Rh30A ant-n, and RhGP ant-c are only accessble to the antbodes n leaky red cell membranes and not n ntact erythrocytes. We conclude that both the C and N termn of the Rh famly of polypeptdes are located kda cut-off Durapore concentrator (Mllpore). 5% glycerol was added to on the cytoplasmc sde of the red cell membrane. the concentrated solublzed mmunoprecptate, and protens were Protease Cleavage of Rh Glycoproten Intact Erythrocytes-We nvestgated the susceptblty of the Rh gly- The abbrevatons used are: PNGase F, peptde N-glycosdase F; PAGE, polyacrylamde gel electrophoress; PBS, phosphate-buffered sa- coproten to proteolyss n ntact erythrocytes n order to obtan lne; Trcne, N-[2-hydroxy-l,l-bs(hydroxymethyl~ethyllglycne. further nformaton on ts topology wthn the membrane. In-

3 Topology of Human Rh (Rhesus) Polypeptdes a b c d e f g h k j ~ 6419 m on p :-:--&.# FIG.1. Immunoprecptaton of Rh polypeptdes wth rabbt polyclonal antsera. Immunoblots of erythrocyte membrane protens separated by Trcne SDS-PAGE. u-e, Rh30A ant-n mmunoblots of (a)control erythrocyte membranes; fb-c) materal mmunoprecptated by Rh30Aant-N from ( b )leaky erythrocyte membranes;( c ) ntact erythrocytes;(d and e ) materal mmunoprecptated by the premmune serumfor Rh30A ant-n from ( d l leaky erythrocyte membranes; and ( e ) ntact erythrocytes. f-j, RhGP ant-c mmunoblots of C f, control erythrocyte membranes; ( g and h ) materal mmunoprecptated by RhGP ant-c from (g)ntact erythrocytes; ( h ) leaky erythrocyte membranes; ( a n d j ), materal mmunoprecptatedby the premmune serumfor RhGP ant-cfrom () ntact erythrocytes;( j )leaky erythrocyte membranes.k-p, RhGP a n t 4 mmunoblots of (k)control erythrocyte membranes; ( I ) control erythrocyte membranes from PNGase F deglycosylated ntact erythrocytes; ( m and n), materal mmunoprecptated by RhGP ant-cfrom ( m )erythrocyte membranes preparedfrom PNGase F-treated ntact erythrocytes; (n)ntact PNGase F-treated erythrocytes;( 0 and p ), materal mmunoprecptatedby the premmune serumfor RhGP ant-cfrom (0)erythrocyte membranes prepared from PNGase F-treated ntact erythrocvtes: -. (4). ntact PNGase F-treated erythrocytes. Immunoblotsu-e, f-g, and k-p are from dfferent gels. Molecular mass s nkda. tact erythrocytes were deglycosylated wth PNGase F, treated three dfferent experments. Theseresults suggest that there s wth S. aureus V8 protease, and thenmmunoprecptated wth a dfference n theaccessblty of the cytoplasmc porton of the the ant-rh(d)monoclonal antbody A B 5 (Avent et al., 1988a). Rh glycoproten to trypsn, whch s dependent on Rh phenothe mmunoprecptated proteolytc fragments were separated type. by SDS-PAGE and mmunoblotted wth RhGP ant-c. When The 28.5-kDa S. aureus V8 protease fragment (RhGP.V8) of ntact glycosylated erythrocytes were dgested wth S. aureus the Rh glycoproten was precptated by the ant-rh(d) monov8 protease (Fg. 2), the M, of the dffuse band correspondng to clonal antbody AB5 (Fg. 3, a d ), and by the Rh-specfc monothe Rh glycoproten showed a slght shft toa lower M, (Fg. 2, clonal antbodes of the R6A type, BRIC 69 andbric 207 (data c and dl. When ntact erythrocytes were deglycosylated wth not shown; Avent et al., 1988a). Ths suggests that the RhGP.V8 PNGase F pror to treatment wth S. aureus V8 protease, the peptde s ether tself part of the antbody eptope or t nterapparent molecular mass of the band detectedwthrhgp acts wth the protens carryng the Rh antbody eptopes. To ant-c clearly shfted from 30.5 kda (ntact deglycosylated pro- further nvestgate ths queston, we used BRIC 69 and BRIC ten) to 28.5 kda (Fg. 2, a and b). 207 to mmunoprecptate protens from trypsn-treated eryththese resultsshow that therh glycoproten n redblood cells rocyte membranes,andthen used Rh30A ant-n, Rh30A contans a n S. aureus V8 protease cleavage ste whch s extra- ant-c, and RhGP ant-c to nvestgate the presence of Rh30 cellular. The s. aureus V8 protease fragment (RhGP.V8) ex- polypeptde and Rh glycoproten fragments n mmunoblots of tends from the proteolytc cleavage ste to the C termnus of the the mmunoprecptates. No trypsn cleavage products reactve Rh glycoproten and can be mmunoprecptated wth the ant- wth RhGP ant-c were detected n mmunoblots of the A B 5, Rh(D)-specfc monoclonal antbody AB5 and s also N-glyco- BRIC 69, or BRIC 207 mmunoprecptates (Fg. 3, a<), alsylated. When ntact erythrocytes were treated wth trypsn though the expected C-termnal tryptc fragments of the Rh (Fg. 2, e and f ), the Rh glycoproten dd not show a detectable glycoproten were clearly present when the mmunoprecptashft nm,, suggestng that t contans no extracellular trypsn ton from the membranes was done usng the RhGP ant-c cleavage stes. polyclonal antbody (Fg. 3d 1. However, trypsn cleavage prodprotease Deatment of the Rh Glycoproten n h a k y Erythro- ucts reactve wth Rh30A ant-n and ant-c were detected n cyte Membranes-The susceptblty of the Rh glycoproten to mmunoblots of the BRIC 69 and BRIC 207 monoclonal anttrypsn cleavage n leaky erythrocyte membranes was nvest- body mmunoprecptates. Ths shows that these monoclonal gated. Untreated membranes or membranes from PNGase F- antbodes precptate the C-termnal and N-termnal tryptc treated erythrocytes were dgested wth trypsn. The resultng peptdes derved from the Rh3o polypeptdes but not the Cmembrane proten fragments were separated bysds-page termnal fragments of the Rh glycoproten. Therefore, unlke and mmunoblotted wth RhGP ant-c. Trypsn dgeston of the RhGP.V8 fragment, thec-termnal tryptc fragmentsof the leaky erythrocyte membranes gave rse to a t least 9 tryptc Rh glycoproten do not reman assocated wththe protens that fragments thatreacted wth RhGPant-C (Fg. 2, g and ), and carry the Rh(D), BRIC 69, and BRIC 207 antbody eptopes n whch therefore all extend to the C termnus of the Rh glyco- the red cell membrane. N-termnal Amno Acd Sequencng of Rh Glycoproten Proproten. PNGase F treatment of the erythrocytes pror to trypsn-treatment of the membranes dd not affect the pattern of teolytc Peptdes-In order to dentfy the locaton of the extrarhgp ant-c reactve tryptc fragments on mmunoblots (Fg. cellular aureus V8 protease and cytoplasmc trypsn cleav2k). In partcular, there was no dfference n themoblty of the age stes n the Rh glycoproten amno acd sequence, the 21.5-kDa tryptc fragment (RhGP.Tl) and the kDa tryp- proteolytc fragments RhGP.V8, RhGP.Tl, and RhGP.T4 were tc fragment (RhGP.T4), whether the erythrocytes had been solated by mmunoprecptaton, separated by SDS-PAGE, and deglycosylated wth PNGase F or not, showngthat theydo not ther N-termnal amno acd sequences determned. The Ncarry N-glycan chans. Although the same tryptc peptdes were termnal amno acd sequence obtaned from the 28.5-kDa RhGP.V8 fragment, solated from PNGase F-deglycosylated observed from membranes of erythrocytes of Rh phenotype CCDee, ccdee, or ccdee, there were dfferences n the amounts CCDee erythrocytes usng the monoclonal antbody BRIC 69, of each of the tryptc fragments (notably RhGP.Tl)that reacted corresponded to amno acds predcted by the Rh5OA wth RhGP ant-cn tryptc dgests of membranes from cells of cdna clone (Rdgwell et al., 1992), except that aspartc acd dfferent Rh phenotypes (Fg. 2, m-o), when low concentratons was observed n the RhGP.V8 peptde sequence at theresdue of the protease were used. These dfferences were reproducedn where the Rh50A cdna predcts asparagne (amnoacd 37). s.

4 lbpology of Human Rh (Rhesus) Polypeptdes 6420 h * " r-", 4 k A I m n FIG.2. Proteolyss of Rh glycoproten n erythrocyte membranes.immunoprecptaton and mmunoblottngof Rh-related polypeptdes from protease-treated erythrocytes and erythrocyte membranes. a d, RhGP ant-c mmunoblota of materal mmunoprecptated by the RhD specfc monoclonal antbody A B 5 from ( a ) PNGase F-deglycosylated, S.aureus V8 protease-treated ntact erythrocytes; ( 6 ) PNGase F-deglycosylated ntact erythrocytes; (c) S. aureus V8 protease treated ntact erythrocytes; ( d )untreated ntact erythrocytes. e - k, RhGP ant-c mmunoblots of membranes from (e, h, and 1 ) undgested control erythrocytes; ( f, trypsn (100 pg/ml) treated ntact erythrocytes; (g and ) trypsn (100 pp/ml)-treated erythrocyte membranes; ( j ) PNGase F-deglycosylated erythrocytes; ( k ) trypsn-treated erythrocyte membranes from PNGase F-deglycosylated erythrocytes. m-o, trypsn (10 pp/ml) treated leaky erythrocyte membranes from cells of Rh phenotype ( m )ccddee; (n)ccddee; (0)CCDDee. Molecular mass s n kda. a b c d e f g F as, 46 2% t a L m am 46- * h htf* 2%- j k 1 Cherf-Zahar et al., 1990). The cytosolc locaton of the N ter- of the Rh famly of polypeptdes s predcted by the ab$9.we" mn sence of cleaved leader sequences and the chargedstrbuton - A"&". Frc. 3. Immunoprecptaton of proteolytc fragments of Rhrelated polypeptdesfrom erythrocyte membranes.immunoprecptaton and mmunoblottng of Rh-related polypeptdes from protease-treatederythrocytesanderythrocytemembranes. a d, RhGP ant-cmmunoblots of materalmmunoprecptated from trypsntreated leaky erythrocyte membranesby ( a )monoclonal antbody A B 5 ; ( 6 ) monoclonal antbody BRIC 69; (c) monoclonal antbody BRIC 207; ( d ) polyclonal antbody RhGP ant-c. e-f, RhGP ant-c mmunoblotof materal mmunoprecptated from ntact erythrocytes by (e) monoclonal antbody BRIC 69 and ( f, monoclonal antbody BRIC 207. g, Rh30A ant-cmmunblots of (g)trypsn-treatedleakyerythrocyte membranes; ( h ) materal mmunoprecptated from trypsn-treated leaky erythrocyte membranesby ( h )monoclonal antbody BRIC 69; () monoclonal antbody BRIC 207. j-l, Rh3OA ant-n mmunoblots of ( j ) trypsn-treated leaky erythrocyte membranes.k and 1, materal mmunoprecptated from trypsn-treated leaky erytrocyte membranes by ( k ) monoclonal antbody BRIC 69 and ( I ) monoclonal antbody BRIC 207. Molecular mass s nkda. The 21.5-kDa RhGP.Tl and kDa RhGP.T4 fragments solated by mmunoprecptaton from trypsn-treated erythrocyte membranes (ccdee phenotype) usng therhgp ant-c polyclonal antbody were also sequenced. The tryptc peptde RhGP.T1 corresponded to amno acds of the predcted Rh5OA amno acd sequence, and the N-termnal sequence of RhGP.T4 corresponded to amno acds of the Rh50A cdna sequence. These resultslocate the S. aureus V8 protease cleavage ste at Glu-34 of the Rh glycoproten to the extracellular face of the erythrocyte membrane, and the two trypsn cleavage stes at Lys-196 and Arg-323 to the cytoplasmc surface of the membrane. DISCUSSION The N a n d C Termn of the Rh Proten Famly Are Located n the Red Cell Cytoplasm-Hydropathy analyss andamno acd sequence algnment of Rh30A and Rh5OA suggests that the Rh30 polypeptdes and Rh glycoprotens have a smlar topology n theerythrocyte membrane (Avent et al., 1990; Rdgwell et al., 1992). Two dfferent membrane orentatons have been proposed for the Rh30 polypeptdes. Both predct a cytosolc N termnus, but one report suggests that the C termnus of the Rh30 polypeptde s located n the redcell cytoplasm (Avent et al., 19921, whle other workers have suggested t s at the extracellular face of the red cell membrane (Bloy et al., 1990; flankng the frst membrane spannng segment (Avent et al., 1990; Cherf-Zahar et al., 1990; Rdgwell et al., 1992). Our mmunoprecptaton studes wth a rabbt polyclonal antbody rased aganst the N-termnal 12 amno acds of the Rh30A polypeptde (Rh3OA ant-n) provde expermental evdence for the cytosolc locaton of the N termnus of ths famly of protens. TheRh30polypeptde was mmunoprecptated by Rh3OA ant-n from leaky erythrocyte membranes butnot from ntact erythrocytes. Both the Rh30 polypeptdes and Rh glycoprotens could be mmunoprecptated from leaky erythrocyte membranes, but not from ntact erythrocytes, by antbodes reactveaganst the C termn of the Rh30 polypeptde and the Rh glycoproten (Rh30A ant-c andrhgp ant-c, respectvely). The removal of bulky glycans from the erythrocyte surface by PNGase F treatment dd not uncover the RhGP ant-c eptope n ntact erythrocytes: the Rh glycoprotens could only be mmunoprecptated from leaky erythrocyte membranes, whether thered cells were deglycosylated or not. These resultssuggest that thec termn of both the Rhs0 polypeptde and the Rh glycoproten are located n the cytoplasm. Ths s further demonstrated by the susceptblty of the Rh30A ant-c antbody eptope to carboxypeptdase Y dgeston. Ths eptope was not destroyed by carboxypeptdase Y dgeston of ntact erythrocytes, but was lost after exopeptdase dgeston of leaky membrane preparatons (Avent et al., 1992). A SngleSteCarres the N-Glycan Chan on the Rh Glycoproten-The susceptblty of the Rh glycoproten to the endoglycosdases PNGase F and endo-&galactosdase has been descrbed elsewhere (Moore and Green, 1987; Mallnson et al., 1990; Avent et al., 1988a; Rdgwell et al., 1994). The Rh5OA cdna sequence contans three consensus N-glycan addton stes, but the topologcal model for the proten derved from hydropathy analyss (Rdgwell et al., 1992) suggests thatonly two of these are extracellular, and therefore lkely to carry N-glycan chans (Asn-37 and Asn-355). Expresson of the Rh50A cdna clone n the rabbt retculocyte lysate cell-free translaton system has suggested that only one of these stes may be glycosylated (Rdgwell et al., 1994). The molecular mass of the largest C-termnal fragment obtaned by trypsn treatment of the Rh glycoproten n erythrocyte membranes (RhGPTl,21.5 kda) dd not changewhen the membranes were derved from erythrocytes pretreated wth PNGase F, whch suggests that tdoes not carry an N-glycan chan. However, the PNGase F deglycosylated Rh glycoproten showed a change n molecular mass from 30.5 to 28.5 kda after S. aureus V8 protease cleavage n ntacterythrocytes, and ths

5 Topology Polypeptdes of Human Rh (Rhesus) 6421 extracellular membrane szeofgtermnal ' fragment on SDWAGE FIG. 4. Schematc representaton of the membrane topology of the RhSOA polypeptde. The structure s based on hydropathy analyss of the Rh50A polypeptde (Rdgwell et al., 1990). The predcted glycosylaton stes (Asn-37, Am-355, and Asn-274) are shown, but only Am-37 actually carres an N-glycan chan. The S. aureus V8 protease (Glu-34) and trypsn (Lys-196, Arg-323, and Lys-384) cleavage stes are also shown. The C and N termn of the Rhso polypeptdes and the C termnus of the Rh glycoproten are located n the cytoplasm, and hydropathy analyss and amno acd sequence algnment of the Rh3o polypeptdes and Rhso glycoproten suggest that they have the same topology n the membrane. Ths topologcal model s therefore lkely to be applcable to the famly of Rh polypeptdes. fragment (RhGP.V8) extends to the C termnus of the proten snce t reacts wth RhGP ant-c. The presence of the glycan chan on Asn-37 of the Rh glycoproten was confrmed by N-termnal sequencng of the kda RhGP.V8 fragment. The N-termnal amno acd sequence obtaned from the deglycosylated RhGPV8 peptde sequence corresponded to amno acds of the proten sequence of the Rh5OAcDNA, except that Asp was obtaned at cycle 3 nstead of Asn. Ths resdue corresponds to Asn-37 of the sequence of the ntact proten, whch s a potental N-glycan addton ste. Ths demonstrates that Asn-37 s normally glycosylated, snce PNGase F removes N-glycans from glycoprotens by cleavage of the asparagnyl-glucosamne bond wth consequent hydrolyss of the Asn to an Asp (Alexander and Elder, 1982). Glycosylaton ofksn-37 may also explan the msassgnment of resdues 37 and 38 n the orgnal N-termnal amno acd sequence determnaton of the Rh glycoproten (Rdgwell et al., 1992). These results show that the 28.5-kDa S. aureus V8 protease fragment (Rbp,V8) of the Rh glycoproten carres an N-glycan chan, but that the C-termnal 21.5-kDa tryptc peptde (Rh- GET11 does not. Thus only the most N-termnal of the consensus N-glycan addton ste (at Asn-37) s actually glycosylated (Fg. 4). Gavel and von Hejne (1990) examned the frequency of glycosylated and nonglycosylated potental glycan acceptor stes n a large number of glycoprotens. They found that the closer a predcted N-glycan addton ste s to the C termnus, the less lkely t s to be N-glycosylated. They also noted that the ncdence of glycosylaton was lower for Asn-X-Ser N-glycan acceptor stes than Asn-X-Thr stes. The consensus Rh5OA N- glycan acceptor stes at Asn-274 and Asn-355 (Fg. 4) are closer to the C termnus than to the N termnus, and Asn-355 s part of a Asn-X-Thr consensus sequence. Locaton of an Extracellular Loop of the Rh Glycoproten- The RhGP.V8 peptde was produced by dgeston of ntact erythrocytes wth S. aureus V8 protease. The protease cleavage ste, between resdues Glu-34 and Gln-35 of the Rh glycoproten, must therefore extracellular. be Ths s consstent wth the predcted topology of Rh50A (Fg. 4), whch suggests resdues are located n the most N-termnal extracellular loop. Identfcaton of 7uo Cytosolc Loops of the Rh Glycoproten-The Rh glycoproten does not appear to be susceptble to trypsn cleavage n ntact erythrocytes, but trypsn treatment of erythrocyte membranes generated at least 9 tryptc peptdes contanng the C termnus. The stes of trypsn cleavage whch generate these fragments are located n the cytosol. The cleavage stes gvng rse to two of these peptdes, RhGP.Tl (21.5 kda) and RhGP.T4 (7-7.5 kda) were at Lys-196 and Arg-323 of the Rh glycoproten, respectvely. A 3-kDa C-termnal Rh glycoproten tryptc peptde was also observed n the total reacton mxture obtaned arer trypsn treatment of erythrocyte membranes (Fg. 2g). The 3-kDa fragment was not obtaned n RhGP ant-c mmunoprecptates derved from trypsn-treated erythrocyte membranes that were washed pror to mmunoprecptaton (Fg. 3d 1, and s therefore not membrane bound. Ths 3-kDa peptde arses from trypsn cleavage at Lys-384, snce there are no other potental trypsn cleavage stes n the Rh5OA cdna sequence that would generate a C-termnal fragment of ths sze. Lys-384 s therefore lkely to be located on the cytoplasmc sde of the membrane. Ths s further evdence for the cytosolc locaton of the C termnus of the Rh polypeptdes, as amno acd resdues are hydrophlc (Engelman et al., 1986) and ths fragment s clearly not membrane bound. Topology of the Rh Famly of Polypeptdes-The proten sequences predcted by the Rh30A polypeptde and Rh5OA glycoproten cdna clones are very hydrophobc and show extensve homology, partcularly n the membrane spannng regons. Hydropathy analyss predcts that the protens have a very smlar topology n the membrane. On ths bass, expermental evdence for elements of topology n ether of the polypeptdes s lkely to be applcable to both. Our studes suggest that a model for the topology of the Rh famly of protens contanng an even number up to 12 membrane spannng domans, as proposed by Avent et al. (19901, s correct. Fg. 4 llustrates ths model and shows the locaton of amno acd resdues establshed n the present study. Interacton between the Rh3* Polypeptdes and Rh Glycoprotens-Moore and Green (1987) suggested that the Rh30 polypeptdes and Rh glycoprotens nteract to form a complex n the erythrocyte membrane. The dfferent Rh antgens expressed on red cells depend on sequence dfferences n the Rh30 polypeptdes and not the Rh glycoprotens (Rdgwell et al., 1992). The changes n the trypsn dgeston patterns of the Rh glycoproten n cells of dfferent Rh phenotype (Fg. 2, 1-01 suggest that the structure of the Rh glycoproten may be slghtly dfferent when t s assocated wth dfferent Rh30 polypeptdes. Ths s consstent wth the observaton that the Rh glycoproten assocated wth dfferent members of the Rh30 polypeptde famly shows dfferent surface labelng patterns (Moore and Green, 1987; Avent et al., 1988a, 1988b; Barker et al., 1992), although the Rh glycoproten molecule nvolved appears to be the same n each case (Rdgwell et al., 1994). Our proteolyss studes have located one regon of nteracton between the Rh30 polypeptde and Rh glycoproten n the Rh complex. The 28.5-kDa RhGP.V8 peptde was co-precptated wth the Rh30 polypeptdes from S. aureus V8 protease-treated erythrocytes usng Rh-specfc monoclonal antbodes (Fg. 2a). However, tryptc peptdes contanng the Rh glycoproten C termnus, ncludng RhGP.Tl (whch results from tryptc cleavage at Lys-196 of the Rh glycoproten), were only mmunoprecptated by the RhGP ant-c polyclonal antbody (Fg. 3, ad) and not by the Rh-specfc monoclonal antbodes. The absence of Rh glycoproten tryptc fragments from the monoclonal antbody mmunoprecptates was not due to the destructon of the antbody eptopes by trypsn treatment of the membranes,

6 I trypsn 6422 Topology of Human Rh (Rhesus) Polypeptdes as the antbodes stll mmunoprecptated the Rh30 tryptc polypeptdes under these condtons (Fg. 3, h,, k, and I). These results suggesthat the Rh30 polypeptde nteracts wth the Rh glycoproten between the extracellular S. aureus V8 protease cleavage ste (Glu-34) and the cytoplasmc trypsn cleavage ste (Lys-196) of the Rh glycoproten. "hs regon encompasses fve predcted transmembrane helces (helces 2-6) n the N-termnal half of the Rh glycoproten. We do not know whether the regon between the N termnus and Glu-34 of the Rh glycoproten, whch contans the frst transmembrane helx, s also mmunoprecptated by the Rh-specfc monoclonal antbodes from S. aureus V8 protease-treated red cells. Although t s known that genetc dfferences n the Rh3o polypeptdes rather the Rh glycoproten specfy the dfferent Rh blood group antgens (Rdgwell et al., 1992), the stes on the Rh protens reactve wth Rh-specfc antbodes, such as ant-d or the R6A type of monoclonal antbodes have not been defned. It s lkely that they nteract only wth the Rh3o polypeptdes, n whch case the Rh glycoproten would be co-mmunoprecptated wth the Rh30 polypeptde because of a drect assocaton between the N-termnal half of the Rh glycoproten wth the Rh30 polypeptde. However, t s also possble that some part of the Rh glycoproten s nvolved together wth the Rh30 polypeptde n the bndng ste of the Rh-specfc antbodes. The co-mmunoprecptaton of the Rh30 polypeptdes and the Rh glycoproten could result from the formaton of an antbody brdge between the Rh30 polypeptdes and the N-termnal half of the Rh glycoproten. In ether case our results lead us to conclude that the N-termnal porton of the Rh glycoproten s located physcally close to the Rh30 polypeptdes. the RhSOAproten, and also dvdes ths proten nto two halves each contanng sx membrane spannng segments. We suggest A Model for the Organzaton of the Rh Complex-There s that the N-termnal half of the Rh glycoproten nteracts wth consderable evdence that the Rhao protens and Rhso glycothe homologous N-termnal half of the Rh30 polypeptde, so that protens are present a as complex n the erythrocyte membrane these portons of the protens form the center of the complex. It (Moore and Green, 1987; Avent et al., 1988a, 1988b). The Rh30 s nterestng that both the N- and C-termnal halves of the polypeptdes and the Rh glycoproten appear to form the core of Rh30 polypeptde are mmunoprecptated from trypsn-treated the complex, wth the addtonal nvolvement of glycophorn B, membranes by BRIC 69 and BRIC 207, the monoclonal antand possbly the CD47 glycoproten, blood group LW antgens, bodes of the R6A type (Fg. 3, g-). It may be that the N- and and Fy glycoproten (revewed by Agre and Cartron (1991) and C-termnal halves of the Rh30 polypeptde assocate together Anstee and Tanner (1993)). Hartel-Schenk and Agre (1992) more strongly than the correspondng portons of the Rh glydetermned the sze of the complexes contanng the Rh-asso- coproten. However, another possblty s that the bndng stes cated protens n nononc detergent-solublzed membranes, of the R6A type of mononoclonal antbodes comprse surface and estmated that they contan 170 kda of proten after correcton for bound detergent. Ths sze s consstent wth the complex beng based on an azpz-type tetramer made up of two Rh30 polypeptde molecules and two Rh glycoproten molecules. The addtonal protens such as glycophorn B whch are probably also present wll not be consdered n the followng dscusson. A model for the organzaton of the Rh30 polypeptdes and Rh glycoproten n the Rh complex s shown n Fg. 5. The N- and C-termnal halves of the Rh glycoproten are shown as comprsng two domans wthn the lpd blayer, separated by the trypsn cleavage ste at Lys-196, each contanng sx transmem- brane segments. Recent evdence for the presence of subdoman structures located n the lpd blayer n multspannng membrane protens has come from structural studes on two-dmensonal crystals of the sarcoplasmc retculum calcum pump (Toyoshma et al., 19931, and of human red cell band 3 (Wang et al., 1993). We assume that the sequence and topologcal homology between the Rh30 polypeptde and Rh glycoproten extends to ther tertary structure and that both protens have smlar doman structures. Consstent wth ths, the Rh30 polypeptde (n ths case the molecule(s) carryng CcEe antgens reactve wth the R6A-type antbodes) s cleaved by trypsn nto two smlar szed fragments (Fg. 3, g-), as has been prevously observed by Suyama and Goldsten, (1992). Ths cleavage s cleavage trypsn cleavage Rh glycoproten Rh gopolypeptdes 0 antbodes FIG. 5. A model for the organzaton of the Rhso polypeptdes and the Rh glycoproten n the Rh complex. Suggested organzaton of the Rh polypeptdes n the erythrocyte membrane as vewed from the external face of the membrane. The model proposes that the Rh polypeptdes are comprsed of two domans, each of whch contans sx transmembrane helces, whch can be separated by trypsn cleavage. It s suggested that the Rh,, polypeptdes and the Rh glycoproten nteract through ther N-termnal domans (30N and GPN), and that Rhspecfc monoclonal antbodes BRIC 69 and BRIC 207 may act as a brdge whch stablzes the assocaton between the N- and C-termnal halves of the Rh30 Polypeptdes. probably n the cytosolc loop at Lys-189, Lys-193, or Arg-201 of loops on the Rh,, polypeptde derved from both the N- and C-termnal halves of the molecule, and the monoclonal antbody acts as a brdge whch stablzes the assocaton between the two halves of the Rh30 polypeptde. Resoluton of ths queston and valdaton of the model n Fg. 5 wll clearly requre further nvestgaton. Acknowledgment-We clonal antbodes. are grateful to Dr. Davd Anstee for mono- REFERENCES Agre, P., and Cartron, J-P. (1991) Blood 78, Alexander, S., and Elder, J. H. (1982) Methods Enzymol. 79, Anstee, D. J., and Tanner, M. J. A. (1993) Ballere's Cln. Haematol. 6, Avent, N. D., Judson, P. A,, Parsons, S. F., Mallnson, G., Anstee, D. 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