THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

VOl . 264, No. 23, Issue of August 15, PP. 13770-13774, 1989 Printed in U.S.A.

Purification and Characterization of an Erythrocyte Membrane Protein Complex Carrying Duffy Blood Group Antigenicity POSSIBLE RECEPTOR FOR PLASMODIUM VIVAX AND PLASMODIUM KNOWLESI MALARIA PARASITE*

(Received for publication, February 23, 1989)

Asok ChaudhuriS, Valerie ZbrzeznaS, Carol Johnson§, Margaret NicholsY, Pablo Rubinsteinq, W. Laurence Marsh§, and A. Oscar Pogo$(( From the Laboratories of ice11 Biolom. Blmmunohematology, and Wmmunogenetics, Lindsley F. Kimball Research Institute of the New York Blood Cehkr, New Yi;k,New York 10021

A murine monoclonal antibody, named anti-Fy6, which agglutinates all human red cells except those of Fy(a-b-) phenotype was used for purification and characterization of Duffy antigens. Duffy antigens are multimeric red cell membrane proteins composed of different subunits of which only one, designated pD protein, reacts in immunoblots with the murine mono- clonal antibody anti-Fy6. Affinity-purified detergent- soluble antigen-antibody complex obtained from red cells, surface-labeled with lzeI yielded a complex pat- tern of bands when separated by polyacrylamide gel electrophoresis. Proteins that react with anti-Fy6 in immunoblots are: pA and pB (>lo0 kDa) and pD (36- 46 kDa). Electroeluted pD protein aggregates and gen- erates bands of similar molecular mass to pA and pB proteins. Electroeluted pA and pB proteins disaggre- gate yielding pD protein. Oligomers and monomers of pD protein are present in red cells carrying Duffy antigens and absent in Fy(a-b-) cells. Six other pro- teins of molecular weight ranging from 68 to 21 kDa either associate or co-purify with pD protein. These proteins are only present in Duffy antigen positive cells. The pD protein is different in Fy(a+b-) and Fy(a-b+) cells by fingerprint analysis. Human antisera identify the same proteins in red cell carrying Duffy antigens as the murine monoclonal antibody anti-Fy6.

The Duffy blood group system now includes four major phenotypes, Fy(a+b-), Fy(a-b+), Fy(a+b+), and Fy(a-b-1, defined by the antisera anti-Fy” and anti-Fyb (1). Neither antiserum agglutinates Duffy Fy(a-b-) cells, the predomi- nant phenotype in blacks. Antisera that define the other phenotypes, Fy3, Fy4, and Fy5, are very rare. Very few ex- amples of anti-Fya or anti-Fyb antibodies have been found in black patients who have been transfused with red cells car- rying the Fy” or Fyb antigens (2). Blacks with Fy(a-b-) erythrocytes cannot be infected by the human malaria para- site Plasmodium vivax (3). These cells are also resistant to in vitro invasion by Plasmodium knowlesi (a simian parasite) that invades Fy(a+) or Fy(b+) human red cells (4). Receptors

* This work was supported by National Institutes of Health Grant HL 39021. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I( To whom correspondence should be addressed: Laboratory of Cell Biology, Lindsley F. Kimball Research Institute of the New York Blood Ctr., 310 E. 67th St., New York, NY 10021.

for the invasion by these parasites seem to be related to Duffy antigens.

There are limited data on the biochemical nature of Duffy antigens. Davies et al. (5) have obtained a soluble extract from Fy(a+b-), Fy(a-b+) and Fy(a+b+) erythrocytes which inhibits Duffy antigen activity. This extract seems to contain a glycoprotein fraction of 35-55 kDa. Moore et al. (6) have shown that Fy“ antigen can be immunoprecipitated from radioiodinated red cell membrane and is associated with pro- teins of 39.5, 64, and 88 kDa. Hadley et al. (7) have identified a protein with an apparent molecular mass of 35-43 kDa in Fy(a+) erythrocytes, but not in Fy(a-) red cells, by immu- noblotting with a potent anti-Fya serum. Nichols et al. (8) have obtained a murine monoclonal antibody, named anti- Fy6, which agglutinates all human red cells except those of Fy(a-b-) phenotype and identifies a “new” Duffy specificity (8). Anti-Fy6 reacts with a broad protein band of 36-46 kDa, in immunoblots of detergent soluble red cell membrane pro- teins (8).

Using the anti-Fy6 antibody we have developed a procedure for purification and characterization of Duffy antigens in human red cells. Duffy antigens appear to be multimeric red cell membrane proteins composed of different subunits. A protein of 36-46 kDa is the only subunit which interacts with anti-Fy6 antibody by immunoblotting. This subunit is the major component of the Duffy antigen and has the property of forming discrete oligomers of molecular mass higher than 100 kDa. A preliminary communication of these findings has been presented elsewhere (22).

EXPERIMENTAL PROCEDURES

Materials-Human blood was obtained from our institution and hemagglutination tests were performed according to standard proce- dures. Goat anti-mouse IgG horseradish peroxidase conjugate was obtained from Bio-Rad. Anti-mouse IgG-Sepharose beads were ob- tained from Cappel. Protein A-Sepharose CL-4B beads were obtained from Pharmacia LKB Biotechnology Inc. The murine monoclonal anti-Fy6 antibody was obtained and purified as described elsewhere (8). A murine monoclonal antibody which reacts specifically with type M glycophorin A was produced as described (9). A murine monoclonal antibody which reacts specifically with type N glyco- Fhorin A and B and rabbit antibody which reacts with types MN glycophorin A were supplied by Dr. 0. Blumenfeld (Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY) (10). Monoclonal antibody R1.3 which reacts with both glycophorin A and B was supplied by Dr. D. J. Anstee (South Western Regional Trans- fusion Center, Bristol, United Kingdom). Rabbit polyclonal antibody to human Band 3 protein was supplied by Dr. V. Marchesi (Depart- ment of Pathology, Yale University School of Medicine). Rabbit antibody to actin was supplied by Dr. D. Fischman (Department of Cell Biology/Anatomy Cornel1 University Medical College). Ultra- pure urea was obtained from Schwarz/Mann. Chymotrypsin was

13770

A Multimeric Erythrocyte Membrane Protein and Duffy Antigen 13771

obtained from Boehringer Mannheim. SDS' (sequencing grade) was obtained from Calbiochem. Triton X-100, Tween 20, and Hepes were from Sigma.

Monoclonal Antibody Binding to Red Cells-Red cells were washed three times in cold PBS (pH 7.4), resuspended in the same solution, and mixed continuously overnight a t 4 "C with anti-Fy6 antibody a t a concentration of 10 pg/ml of packed red cells. This concentration, determined with radioiodinated antibody, exceeds the concentration required to saturate Duffy antigen sites. Unbound antibody was removed by washing the red cells with cold PBS. Red cell ghosts were prepared by hypotonic lysis with 20 volumes of cold 5 mM sodium phosphate buffer (pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride and 100 kallikrein-inactivating units/ml Trasylol (aprotinin). Then the ghosts were washed exhaustively until they were light pink in color. Ghosts were centrifuged for 30 min at 43,000 X g; supernatant was decanted, and the pellet was made to 50 mM Hepes-NaOH, pH 8.0, 1 mM phenylmethylsulfonyl fluoride, 100 kallikrein-inactivating units/ml Trasylol, and frozen a t -20 "C. Frozen ghosts prepared in this way can be stored for months without loss of Duffy antigens.

Preparation of a Detergent-soluble Erythrocyte Membrane Frac- tion-Frozen ghosts were thawed and centrifuged for 30 min a t 43,000 X g. The pellet was resuspended in 50 mM Hepes-NaOH, pH 8.0, 1 mM phenylmethylsulfonyl fluoride, 100 kallikrein-inactivating units/ ml Trasylol to three times the initial volume of packed red cells. Triton X-100 (peroxide-free) was added to a final concentration of 1%, and the solution was mixed gently for 1 h a t room temperature. Shells were removed by centrifugation for 30 min at 43,000 X g. The supernatant was concentrated 4-fold in an Amicon concentrator using a PM Y10 filter (Amicon Corp.) under nitrogen pressure.

Purification of Antigen-Antibody Complex-A 0.1 volume of PBS solution, 10 times the normal concentration, was added to the deter- gent extract. The detergent extract was then incubated with Sepha- rose 4B beads coupled to anti-mouse IgG for 1 h a t room temperature. The ratio of beads to detergent extract was 1:lOO (v/v). The anti- mouse I@-Sepharose beads were removed by centrifugation, and washed in a solution containing PBS and 0.5% Triton X-100 a t a 1:20 (v/v) ratio of beads to washing solution. The washings were done at room temperature for 15 min and repeated three times. Elution was done by incubating the beads in a solution containing 62.5 mM Tris-HC1 (pH 6.8), 0.5% SDS at a 1:2 (v/v) ratio beads to eluant. The incubation was a t 65 "C for 10 min and repeated three times. The eluted material was concentrated in an Amicon concentrator with PM Y10 filter (Amicon Corp.) under nitrogen pressure.

Preparative Polyacrylamide Gel Electrophoresis-PAGE in the presence of 0.1% SDS was performed according to Laemmli (11) with the following modifications: the acrylamide concentration was lo%, polymerization was done overnight to destroy oxidizing reagents, and 0.1 mM thioglycolate was added in the upper chamber. To the con- centrated solution of affinity purified material, the following chemi- cals were added urea to 4 M, SDS to 2%, and 0-mercaptoethanol to 5%. Before electrophoresis, the solution was heated at 65 "C for 15 min. After electrophoresis the gels were fixed for 30 min in 10% isoamyl alcohol and 5% acetic acid and stained with 0.002% Coo- massie Blue R-250 until marker protein bands were seen. Regions that corresponded between the 36-46-kDa region and above the 96- kDa region were excised, destained with several changes of 5% acetic acid, and washed with distilled water. Gel pieces were stored a t -20 'C or used immediately.

Electroelution-Gel pieces, cut into 4 X 4 mm cubes were delivered into the elution chamber of an Elutrap apparatus (Schleicher and Schuell) and eluted overnight in 50 mM ammonium bicarbonate, 0.1% SDS solution a t 100 volts (constant). Fresh 50 mM ammonium bicarbonate, 0.1% SDS solution was added, and electroelution was continued for an additional 6-8 h. Eluted material was concentrated by Centricon microconcentrator (Amicon Corp).

Analytical Polyacrylamide Gel Electrophoresis-Bio-Rad minigels (Bio-Rad) were used for analytical purposes. 10% polyacrylamide gels were prepared in the presence of 0.1% SDS and were polymerized overnight. In the upper chamber 0.1 mM thioglycolate was added. T o the concentrated solution of affinity purified material, the following chemicals were added: urea to 4 M, SDS to 2%, and 8-mercaptoethanol to 5%. Before electrophoresis, the solution was heated at 65 "C for 15 min. Samples treated differently are explained in the respective figure legends. After electrophoresis, the gels were silver-stained according

' The abbreviations used are: SDS, sodium dodecyl sulfate; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; PAGE, poly- acrylamide gel electrophoresis; PBS, phosphate-buffered saline.

to the procedure of Morrissey (12) with the following modifications: treatment periods were reduced to 15 min and washing with distilled water was done for 1-2 h. Immunoblots of minigels were done ac- cording to the procedure of Towbin et ai. (13) with 20% fetal calf serum as blocking agent. Incubation with primary (anti-Fy6; 2 pg/ ml) and secondary antibodies (goat anti-mouse IgG horseradish per- oxidase conjugate) were done for 1 h. Color was developed according to manufacturer's protocol. For determinations of molecular mass the best fit plot of the mobilities of pre-stained standards (Rainbow, Amersham Corp.) was used.

Radioiodination-Labeling of membrane surface proteins was done as explained elsewhere (14). Protein radioiodination was done with Iodobeads (Pierce Chemical Co.) following manufacturer's protocol. Free '"1 was removed by Sephadex G-50 column chromatography and, if necessary, further removal was done by SDS-PAGE and electroelution.

Protein Determination-Pierce BCA method was used for protein determination and bovine serum albumin was used as standard (15).

RESULTS

Anti-Fy6 murine monoclonal antibody agglutinates all red cells containing the Duffy Fy" or Fyh blood group antigens (8). Interaction with proteins of apparent molecular mass of 40-46 kDa has been observed when Fy(a+) or Fy(b+) red cell ghosts were treated with detergents, and the detergent-soluble fraction was immunoblotted with anti-Fy6 (8). This antibody is, therefore, useful for purification and characterization of red cell membrane proteins having Duffy blood group anti- genicity.

Preliminary experiments indicated that solubilized Duffy antigens and anti-Fy6 monoclonal antibody reacted ineffi- ciently. A very stable antigen-antibody interaction was ob- tained, however, when Duffy antigens were bound in situ.

Affinity-purified Triton X-100-soluble antigen-antibody complex obtained from red cells, surface-labeled with '*'I, yielded a complex pattern of bands when separated by SDS- PAGE (Fig. 1). The bands were assigned alphabetical labels starting at the top of the gel: pA and pB, two heavily radio- labeled broad bands with mobilities higher than 200 kDa and about 100 kDa, respectively; PC, a band of approximately 68 kDa; pD, a broad band of 36-46 kDa; pE, a band of approxi- mately 35 kDa; pF, a band of approximately 26 kDa; and pG,

A B k D a 1 2 3 4 5 6 4 5 6

200 '

92. 69.

45.

30. 21-

FIG. 1. Protein composition of purified Duffy complex. Hu- man red cells were: washed in cold PBS, '"I surface-labeled, incubated in cold PBS with and without anti-Fy6 monoclonal antibody, hemo- lyzed, and membrane proteins solubilized by Triton X-100. The antibody-antigen complex was purified by affinity chromatography using anti-mouse IgG Sepharose beads and eluted as explained under "Experimental Procedures." Equivalent amounts of each cell per Duffy phenotype (100 pI of packed red cells) were run on SDS-PAGE. Autoradiography was done with vacuum-dried gels and exposed for 24 h (A) and for 36 h ( B ) . Lanes 1 and 2, are Fy(a-b-) cells incubated without and with anti-Fy6 respectively. Lanes 3 and 4 are Fy(a+b-) cells incubated without and with anti-Fy6 respectively. Lane 5 is Fy(a-b+) cells, and lane 6 is Fy(a+b+) cells incubated with anti-Fy6. Asterisks indicate bands unique to Fy(a+b-) and Fy(a+b+) pheno- types.

13772 A Multimeric Erythrocyte Membrane Protein and Duffy Antigen

I 2 3 4 kDa pA. 100

PB. 92

w 45

L- 30

FIG. 2. Immunoblots of affinity-purified Duffy complex. Equal amounts of cells of each phenotype (250 pl of packed red cells) were analyzed on SDS-PAGE and immunoblotted with anti-Fy6 monoclonal antibody as explained under “Experimental Procedures.” Lanes 1-4 are: Fy(a+b-), Fy(a-b+), Fy(a+b+), and Fy(a-b-) phe- notypes respectively. Notice that heavy (H) and light ( L ) IgG chains of the monoclonal antibody reacted with Goat Anti-Mouse IgG horse- radish peroxidase conjugate.

a band of approximately 21-18 kDa. Bands pD and pG seem to be duplex proteins. In some gels the two components of pG run well apart (Fig. 3E) .

These proteins are present in Fy(a+b-), Fy(a-b+), and Fy(a+b+) phenotypes and absent in the Fy(a-b-) phenotype. There are, however, quantitative and qualitative differences in the individuals shown in Fig. 1. In Fy(a-b+) phenotype all proteins are more abundant. In Fy(a+b+) red cells there are much less pE and pF proteins (Fig. 1B). A band of about 56 kDa is present only in Fy(a+b+) red cells, and another band of about 62 kDa is present only in Fy(a+b-) red cells (see bands with dsterisks in Fig. 1B). The same pattern of radio- labeled proteins as that shown in Fig. 1 was obtained when the antigen-antibody complex was formed with human anti- Fya serum and surface radioiodinated Fy(a+b-) red cells (not shown).

We have extended this study to several individuals within each phenotype. The differences in the amount of PA, pB, and pD proteins among individuals of the same phenotype were prominent (not shown). The quantities of the minor bands, like PC, pE, pF, and pG, also changed within each phenotype (not shown). We are conducting population and family studies to determine the pattern of variability in each phenotype and whether the pattern is heritable.’

Not all the isolated proteins reacted with the anti-Fy6 antibody by Western immunoblotting (Fig. 2). Proteins that reacted were in the PA, pB, and pD regions and were classified as having Duffy blood group activity. In addition, a smear of Duffy antigen activity was observed between these regions. Although the PC band and the bands unique to Fy(a+b-) and Fy(a+b+) phenotypes are present in the smear of Duffy antigen activity located between the pB and pD bands, they did not react with anti-Fy6 antigen. Proteins migrating more rapidly than the pD protein did not react with anti-Fy6 antibody. Thus, proteins pE, pF, and pG were nonreactive. Duffy antigen activity was not detected in an equivalent amount of Fy(a-b-) cells, nor in samples concentrated ap- proximately ten times (not shown). Moreover, when Fy(a+b-) erythrocytes were challenged with anti-Fya serum and the affinity-purified antigen-antibody complex, was sep- arated by SDS-PAGE and immunoblotted with anti-Fy6 an- tibody, a similar pattern to that shown in Fig. 2 was observed (not shown).

The most rapidly migrating form of proteins having Duffy antigen activity is pD protein. It appears to be a single protein species, since electroelution and re-run on SDS-PAGE shows

A. Chaudhuri, V. Zbrzezna, and A. 0. Pogo, manuscript in prep- aration.

one silver-stained band (Fig. 3A). Moreover, a single protein peak was obtained when the electroeluted pD protein was analyzed by high performance liquid chromatography (not shown). Upon storage either at -20 “C or at 4 “C (in the refrigerator), this homogeneous protein generated discrete bands with mobilities similar to pA and pB bands. An iden- tical pattern was obtained by immunoblotting with anti-Fy6 or silver staining (Fig. 3, B and C). Conversely, electroeluted pA and pB bands, when re-run on SDS-PAGE and immuno- blotted, yielded pA and pB bands, the typical Duffy reactive pD band, and a smear of Duffy antigen activity between these bands (Fig. 3 0 ) . A complex pattern of radiolabeled protein bands having the same electrophoretic mobilities as PA, pB, pD, and pF was observed (Fig. 3E) . It appears that after the first run most of the PC and pE bands as well as the pG duplex band dissociated.

Specific antibodies were used by Western immunoblotting to determine whether Band 3 co-migrates with pA and pB proteins, whether actin co-migrates with pD proteins, whether pE protein is the monomeric form of glycophorin A and its

A B C

D

“ 9 2 ‘.

30 - 21 .

- ..___

E 1 2 3 4

FIG. 3. In vitro polymerization of purified pD protein and depolymerization of pA and pB proteins. pD, PA, and pB proteins were purified in a preparative SDS-PAGE and electroeluted as ex- plained under “Experimental Procedures.” A, pD proteins were ob- tained from Fy(a+b-) (lane 1 ) and Fy(a-b+) (lane 2 ) cells, re-run immediately after electroelution, and silver-stained as explained un- der “Experimental Procedures.” B and C, pD proteins were obtained from Fy(a+b-) (lane I ) , Fy(a-b+) (lane 2), and Fy(a+b+) (lane 3 ) cells, re-run after 24 h at -20 “C, on two SDS-PAGE simultaneously. One gel was immunoblotted ( B ) and the other was silver stained (C). D, pA and pB proteins were purified from Fy(a+b-) (lane 1 ) and Fy(a-b+) (lane 2) cells, re-run after 24 h a t -20 “C and immuno- blotted. E, surface-labeled red cells were treated as explained in F’ig. 1 and under “Experimental Procedures.” The immunopurified samples h-xe divided into two aliquots. One aliquot was used to isolate pA and pB proteins; it was treated and re-run as in D. The other aliquot was used as molecular markers. Autoradiography was done with vacuum-dried gels. Lanes I and 3 are total immunopurified proteins from Fy(a+b-) and Fy(a-b+) erythrocytes, respectively. Lanes 2 and 4 are the re-run of pA and pB proteins obtained from Fy(a+b-) and Fy(a-b+) red cells, respectively. Note in lanes 1 and 3 that the two components of pG band ran well separated. The asterisk indicates the band usually seen in Fy(a+b-) red cells (see Fig. 1B). In Fy(a-b+) erythrocytes PC band is hardly seen due to the high intensity of the background.

A Multimeric Erythrocyte Membrane Protein and Duffy Antigen 13773

dimeric form is PC protein (they have similar electrophoretic mobilities (16)), and whether pF or pG is glycophorin B. None of these antibodies reacted with the proteins affinity-purified from equivalent amounts of red cells, as analyzed in Fig. 1, or even from 10 times more red cells (not shown).

The most prominent radioiodinated proteins, the most in- tense in Duffy antigen activity, were present in pA and pB bands. These bands must be oligomers of pD protein which may interact with other proteins. As indicated in Fig. 3, B and C , in vitro oligomerization of pD protein occurs in the absences of the other proteins.

To determine whether the red cell cytoskeleton contains proteins having Duffy antigen activity, an equivalent amount of detergent-soluble and -insoluble fractions and the affinity- purified fraction were analyzed by SDS-PAGE and immuno- blotted with anti-Fy6 antibody. Fig. 4 shows that most of the proteins reacting with the antibody were soluble in Triton X- 100. Although the amount of protein having Duffy antigen activity in lanes 1 and 3 was the same, the reaction was significantly less in the detergent-soluble fraction than in the purified fraction. I t was noted that in the presence of other cell membrane proteins, immunoblots of Duffy antigen do not give a strong signal. I t is not known whether this is due to inefficient electroblotting or interference with the anti-Fy6 probe.

In a separate experiment (not shown) purified and concen- trated pD protein reacted very weakly with two reagents usually used for carbohydrate detection; periodic acid-Schiff and concanavalin A (17, 18).

Amino acid composition and high performance liquid chro- matography of purified pD protein indicated that like brain proteolipid apoprotein (lipophilin) (19), it is a protein con- taining an abundant amount of hydrophobic amino acids (not shown).

It is informative to compare the peptide maps, by high resolution two-dimensional separation, of enzymatically di- gested pD protein obtained from Fy(a+b-), Fy(a-b+), and Fy(a+b+) red cells. Comparisons were made by using equal amounts of pD proteins iodinated with '''1 and analyzed at the same time. The patterns, presented in Fig. 5, showed a high degree of homology between pD protein isolated from

H- PD

L-

-4 5

FIG. 4. Duffy activities in Triton X-100-insoluble (cyto- skeleton) and -soluble fractions. Fy(a+b-) cells were incubated with anti-Fy6 as explained in Fig. 1 and under "Experimental Pro- cedures.'' After hemolysis, the ghost suspension was fractionated into Triton X-100-soluble and -insoluble fractions as explained under "Experimental Procedures." The detergent-soluble fraction was di- vided into two aliquots, and Duffy antigen-antibody complex was purified from one aliquot and immunoblotted. The aliquot of the detergent-soluble fraction that contained Duffy antigen-antibody complex and the insoluble (cytoskeleton) fraction were also immu- noblotted. Equal amounts of material (750 pl of packed red cells) were loaded in each lane. Lane 2 is the affinity-purified Duffy antigen- antibody complex and lanes 2 and 3 are detergent-insoluble (cyto- skeleton) and detergent-soluble fractions, respectively. The arrow- head indicates heavy I@ chain dimers of the monoclonal antibody reacting with goat anti-mouse horseradish conjugate.

t Fy (o+b-)

!

' 4 -* u

Fy (o-b+) b

c .E ._ E a

-

I L

I 00%

It t

1st Dimension

FIG. 5. Comparative two-dimensional chymotryptic pep- tide maps of pD proteins obtained from Fy(a+b-), Fy(a-b+), and Fy(a+b+) cells. pD protein was purified, iodinated with '''1 as explained under "Experimental Procedures," and digested with chy- motrypsin a t enzyme to substrate ratio of 1: lO (w/w) for 48 h at 37 "C in 0.05 M ammonium bicarbonate solution. Peptides were separated by thin-layer high-voltage electrophoresis in pH 4.8 buffer (pyridine, acetic acid, and water (3:3:394, by volume)) at 0.5 kV for 30 min (first dimension). After drying the thin-layer plate, the peptides were separated (second dimension) by ascending chromatography in a mixture of butanol, acetic acid, and water (41:5, by volume). The peptide positions recorded on tracing paper are shown on the r$ht side. In I is the summation of the maps of pD proteins obtained from both homozygotes, with peptides unique to Fy(a+b-) cells repre- sented by stippled spots and those unique to Fy(a-b+) cells by solid spots, whereas those common to both by blank spots. In I I is the drawing of peptides found in Fy(a+b+) cells. Peptides unique to Fy(a+b-) cells shown by stippledspots and those unique to Fy(a-b+) by black spots, whereas those common to both homozygotes are shown by blank spots.

Fy(a+b-) and Fy(a-b+) cells. However, there are subtle differences, and these differences are reinforced when the two peptide maps are compared with that of Fy(a+b+) cells. With the exception of five or six peptides from Fy(a+b-) and six or seven peptides from Fy(a-b+), all of the spots in the two maps are common, indicating that the proteins are closely related.

I t is interesting that the sample of Fy(a+b+) cells used in this study resembled Fy(a-b+) cells more than Fy(a+b-) cells. Whether the other proteins of the Duffy complex have different amino acid sequences in Fy(a+b-) and Fy(a-b+) cells remains to be demonstrated.

DISCUSSION

A murine monoclonal antibody was used to isolate and characterize a multimeric red cell membrane protein complex

13774 A Multimeric Erythrocyte Membrane Protein and Duffy Antigen

having Duffy blood group antigenicity. The major subunit of this complex, named pD protein, is the only protein that reacts with the murine monoclonal antibody in Western im- munoblots. It is an integral membrane protein of 36-46 kDa, not firmly associated with the cytoskeleton, and having the property of forming discrete oligomers of 100 kDa and higher molecular mass. Six other surface membrane proteins either associate or co-purify with pD protein. Four of them, named PC, pE, pF, and pG, are present in the three Duffy antigen positive red cell types. Two minor proteins, one of 62 kDa is present only in Fy(a+b-) cells, and the other of 56 kDa is present only in Fy(a+b+) cells. The human anti-Fya antibody identifies the same pD protein in Fy(a+b-) cells, and the other noncovalently associated polypeptides, as does the mu- rine monoclonal antibody. Within each Duffy phenotype there are individual variations in the amount of all Duffy proteins. None of these proteins were isolated from either equivalent amounts of Fy(a-b-) red cells or from 10 times more red cells of the same phenotype.

The seminal work of Miller et al. (3,4) indicates that Duffy antigen is involved in the invasion of red cells by merozoites of P. viuax or P. knowlesi malaria parasite. Merozoites attach to the membrane of Duffy-positive and Duffy-negative red cells, but fail to penetrate the invaginated membrane of red cells lacking the Duffy determinant (20). Penetration, how- ever, occurs in Duffy-negative cells treated with trypsin or neuraminidase (20). These conflicting data are difficult to explain, and as was postulated by Miller and collaborators (20), it may be that structures associated with the Duffy determinant and not the Duffy antigen itself may be the requirement for invasion. With the possibility of obtaining the Duffy antigen and its associated proteins in pure form, it should be possible to test their role in parasite-red cell inter- actions. Moreover, these interactions may be determined for each protein in blots of electrophoretically separated proteins of the Duffy complex. Thus, Haynes et al. (21) have demon- strated that a 135-kDa protein synthesized by P. knowksi binds to an erythrocyte membrane protein having the same electrophoretic mobility as the monomeric form of pD protein. It will be extremely informative to determine whether this, or other parasite protein, binds to the proteins that co-purify with pD protein. This should facilitate the understanding of the mechanism of parasite penetration and suggest methods for blocking and interfering with the asexual malaria parasite life cycle.

If the Duffy antigen is the receptor of P. knowksi and P. uiuax, the parasite invasion may require multivalent associa- tion with the receptor. According to this idea, the receptor would be a complex of surface proteins composed of several subunits with one set contributing to attachment and the other to penetration. Attachment and penetration might re- quire the presence of pre-existing complexes on the cell sur- face or the ability to form such complexes by recruiting each component. Either mechanism could be controlled by factors which regulate subunit interaction within the membrane.

The relationship of anti-Fy3, anti-Fy4, and anti-Fy5 anti- bodies to PC, pD, pE, and pG proteins as their putative antigens is not known. What seems to be clear is that the murine monoclonal antibody, which defines the Fy6 specific- ity, recognizes a different pD protein having a common epi- tope in Fy(a+b-) and Fy(a-b+) phenotypes.

Is pD protein a glycoprotein? Hadley et al. (7) reported that neuraminidase treatment of erythrocyte ghosts increased the electrophoretic mobility of a protein band similar to pD protein. In our hands digestion with glycosidases produced no significant change in electrophoretic mobility of purified pD

protein (not shown). Moreover, purified and concentrated pD protein reacted very weakly with periodic acid-Schiff reagent (17) and bound very poorly to concanavalin A (18). Since these reagents can react with components other than carbo- hydrates, and since the reaction of pD protein is extremely weak, the presence of carbohydrates in pD protein remains to be determined by chemical analysis.

The monoclonal antibody only reacts with monomeric and polymeric forms of pD protein in Western immunoblots. This appears to be true with human anti-Fya serum (7). The other proteins, therefore, must interact with pD protein either in vivo, or immediately upon detergent dissolution of the eryth- roid membrane. Preliminary experiments indicate that the multimeric form of pD protein, as well as its interaction with the other proteins pre-exist detergent dissolution of the eryth- rocyte membrane.3 Studies of the conditions of polymerization and depolymerization of Duffy proteins are essential to obtain greater insight into the structural organization of this complex blood group antigen and its role in the molecular mechanism of parasite attachment and penetration.

Finally, peptide maps of pD protein obtained from Fy(a+b-) cells and Fy(a-b+) cells indicated a high degree of homology with differences in some peptides. It is interesting that pD protein from the sample of Fy(a+b+) cells studied here showed more homology to Fy(a-b+) cells than Fy(a+b-) cells. It could be that there is a different copy number of the two pD proteins in the heterozygote. This issue, as well as the structural differences of pD proteins, will be resolved with the cloning of the pD gene.

Acknowledgments-We thank Dr. 0. Blumenfeld and Dr. D. J. Anstee for providing anti-glycophorin antibodies, Dr. V. Marchesi for providing anti-Band 3 antibody, Dr. D. Fischman for supplying anti- actin antibody, H. Hlawaty for her technical assistance, and I. Yeager for reviewing and typing the manuscript.

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