Download Enzymatic Evidence for Differences in the

Enzymatic Evidence for Differences in the Placement of Rh Antigens Within the
Red Cell Membrane
By Kimita Suyama and Jack Goldstein
Intact erythrocytes of different Rh genotypes w e r e subjected to various enzyme treatments, t h e effects of which
w e r e monitored by separating t h e membrane proteins by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
and performing Western blotting using a n antibody preparation t h a t recognizes only Rh-related polypeptides. W e
found t h a t treatment of intact cells with either phospholipase A, or proteases such as papain did not alter t h e size of
Rh antigen-containing polypeptides. In contrast, phospholipase A, treatment followed by papain digestion cleaved a
fraction of t h e s e polypeptides. This cleavage appears, from
such digestions of Rh(D) positive and negative cells of
different genotypes, to occur solely at t h e extracellular
domain of Rh(D) polypeptide, while t h e extracellular domains of other Rh antigen-containing polypeptides a r e
unaffected. Digestion of red blood cell g h o s t s and insideo u t vesicles with trypsin showed t h a t Rh(D), (C/c), and
(E/e) antigen-containing polypeptides span t h e lipid bilayer
having cytoplasmic domains susceptible to t h e action of
proteases. T h e size of t h e cleavage products at t h e
cytoplasmic domain of -D-/-Dcells was found to
differ from t h a t of other Rh(D) positive genotypes, d u e
possibly to a difference in folding of Rh(D) polypeptide at its
cytoplasmic domain and within t h e cellular membrane of
t h e s e cells.
0 1990 by The American S o c i e t y of Hematology.
M
NY; and MPL + TDM + CWS adjuvant emulsion from RIB1
Immunochemical Research Inc, Hamilton, MT.
Enzymatic treatment of erythrocytes. RBCs were prepared by
washing three times in saline and three times in phosphate buffered
saline (PBS), pH 7.4, spinning at 2,500 rpm for 8 minutes and
aspirating the buffy coat. For phospholipase A, digestion, the
procedure of Verkleij et all' was followed with some modification.
Cells, at a concentration of 20% unless otherwise indicated, were
incubated with phospholipase A, (20 U/mL) in buffer containing 20
mmol/L Tris-HCI, 150 mmol/L NaCl, 25 mmol/L glucose, and 10
mmol/L CaCl,, pH 7.4, at 37°C for 2 hours. Under these conditions
phospholipase A, specifically converts 60% to 70% of the lecithin
present in the outer lipid bilayer of intact cells into lisolecithin
without hemolysing the cells." Treated cells were then immediately
washed twice in buffer containing 20 mmol/L Tris-HC1, 150
mmol/L NaCl, 25 mmol/L glucose, and 3 mmol/L EDTA, pH 7.4,
and once in PBS, pH 7.4. Aliquots of these cells were then further
digested by papain, again at cell concentrations of 20%,in PBS, pH
7.4. Incubation was for 2 hours at 37OC, 10 mmol/L dithiothreitol
was added to the buffer, and the enzyme level was either 0.1% papain
in 2.7 mmol/L L-cysteine or 0.25% papain in 6.7 mmol/L Lcysteine. Incubations were followed immediately with three washes
in PBS, pH 7.4. Membrane samples to be applied to 12% acrylamide
mini-gels were prepared from similar volumes of packed cells by
lysing the cells in 5 mmol/L sodium phosphate buffer, pH 7.4, and
washing the membranes at least three times with the same buffer to
remove adhering hemoglobin.
Trypsin treatment of unsealed ghosts and sealed inside-out
vesicles. Unsealed RBC ghosts were prepared as described by
Jennings et al,I9and sealed inside-out vesicles by the method of Steck
and Kant.*' A 5% suspension of either unsealed ghosts or sealed
inside-out vesicles in 10 mmol/L sodium phosphate buffer, pH 7.4,
OLECULAR PROPERTIES of antigens of the Rh
blood group system are currently the subject of
intensive investigation.' Rh(D) antigen, the most important
clinically, and the two commonly detected antigenic alleles,
(C/c) and (E/e), which are usually expressed along with it,
have been shown to be associated with polypeptides having
apparent molecular weights (mol wts) in the range of 28,000
to 35,000 daltons.*-*These appear to be nonglyc~sylated~
integral membrane proteins recently shown to contain thiollinked fatty acids." Thus far, only partial amino acid
sequences have been determined."-I3 Reports indicating
slight but reproducible electrophoretic differences between
Rh(D), (C), and (E) p01ypeptides,8"~as well as those
showing variation in their one- and two-dimensional peptide
"maps" after radioiodination and pr~teolysis,"~'~~'~
suggest
that these antigens are components of similar but not
structurally identical proteins.
What, if any, relationship these antigens have in maintaining viable erythrocyte membrane structures, or why Rh(D) is
more immunoreactive than (C/c) or (E/e) is not yet known.
As a first step toward answering these questions, we describe
results of enzymatic treatment of intact cells, ghosts, and
inside-out vesicles that provide further evidence for Rh
antigen-containing polypeptides spanning the membrane of
the red blood cell (RBC). Our results also suggest that
Rh(D) polypeptide differs from Rh (E/e) and (C/c) polypeptides in the manner in which its extracellular domain is
presented on the surface of the cell.
MATERIALS AND METHODS
Materials. Fresh RBCs (cDE/cDE, CDe/CDe, cde/cde) were
obtained from The New York Blood Center (New York, NY) and
rare RBCs (Rh-null regulator type, -D-/-D-,
cdE/cdE, Cde/
d e ) from Ortho Diagnostic Systems, Plainfield, NJ. Human monoclonal anti-Rh(D) was purchased from Bioproducts for Science Inc,
Indianapolis, IN, and goat anti-rabbit immunoglobulin G (1gG)HRP conjugate from Cooper Biomedical, Malvern, PA. Anti-human
IgG-agarose, phospholipase A, from Naja mocambique mocambique, trypsin from bovine pancreas, and N-#I-tosyl-L-lysinechloromethyl ketone (TLCK) were provided by Sigma, St Louis, MO.
Papain was ordered from Aldrich, Milwaukee, WI; L-cysteine from
Calbiochem, San Diego, CA; dextran T-1 10 from Pharmacia,
Piscataway, NJ; nitrocellulose paper from Schleicher & Schuell,
Keene, NH; 4-chlore 1-naphthol from Bio-Rad, Rockville Center,
Blood, Vol75, No 1 (January 1). 1990 pp 255-260
From the Lindsley F, KimbaN Research Institute of the New
York Blood Center, New York, NY.
Submitted June 26, 1989; accepted August 30, 1989.
Supported in part by the Ofice of Naval Research contract No.
NO001 4-84-C-0543.
Address reprint requests to Jack Goldstein, PhD, The New York
Blood Center, 310 E 67th St, New York. N Y 10021.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1990 by The American Society of Hematology.
0006-4971/90/7501-0023$3.00/0
255
SUYAMA AND GOLDSTEIN
256
containing 150 mmol/L KCI, was incubated with 50 pg/mL trypsin
at 37°C for 45 minutes. Then TLCK was added immediately to a
final concentration of 50 pg/mL. Five minutes later the ghosts or
vesicles were recovered by centrifugation at 22,000 x g for 30
minutes.
Wesrern blot immunosraining assay. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was performed
under the nonreducing conditions reported by Laemmli” using 12%
acrylamide gel concentrations and membrane samples diluted with
an equal volume of loading buffer. Transfer to nitrocellulose paper
and immunostaining of the separated membrane proteins was done
as reported by Burnette” and modified by Bio-Rad Laboratories as
described under catalog number 170-3930 entitled “Mini Trans-Blot
Electrophoretic Transfer Cell Instruction Manual” (1986, p I). The
rabbit antibody preparation recently reported by us to immunostain
Rh antigen-containing polypeptides’ was used at a 1:1,000 dilution
for studies described here.
RESULTS
Action of papain on Rh antigens of RBCs pretreated with
phospholipase A,. Treatment of intact Rh(D) positive
RBCs (cDE/cDE) with either phospholipase A2 or papain
did not affect the size of Rh-containing polypeptides. This
was determined by preparing membranes from such cells,
separating the membrane proteins by SDS-PAGE, and
subjecting them to immunostaining with an antibody preparation recently found to react with Rh-related polypeptides.’
Under these conditions only one immunostained band in
the 35-Kd region of the gel was observed (Fig I A , lanes 2
through 6). However, treatment of these cells in a sequential
manner, first with phospholipase A,, followed by papain,
produced a second antibody reactive band in approximately
the 30-Kd region of the gel (Fig I A , lane 7). Identical results
were obtained with CDe/CDe cells (data not shown). Increased cleavage of a 35-Kd protein band to 30 Kd was
neither obtained by raising the level of papain from 0.25% to
0.8% nor by increasing the concentration of RBCs from 20%
to 65%. Longer incubations with phospholipase A, and
A
papain also did not alter the relative amounts of the 35-Kd
and 30-Kd bands, or produce other detectable cleavage
products. Other proteases such as trypsin, chymotrypsin, and
the carboxypeptidases (A, B, and Y), when used in place of
papain, did not cleave any of the proteins in the 35-Kd
region, while pronase produced only small amounts of the
30-Kd cleavage product (data not presented). When - D-/
-D- cells were treated in the same manner as other Rh(D)
positive cells, significantly more cleavage of Rh antigen
(which in this case consists of only Rh(D) polypeptide and
thus exhibits an apparent mol wt under our SDS-PAGE
conditions of 33-Kd to 30-Kd product) was observed. (Fig
1 B, lanes 2 and 3).
In contrast, treatment of Rh(D) negative cells (cde/cde,
cdE/cdE) with phospholipase A2and papain did not result in
any detectable production of 30-Kd protein or any other
cleavage product from the intact Rh-containing polypeptides
in the 35-Kd region (Fig 2A, lanes 2 through 5; Fig 29, lanes
2 and 3). Identical results were obtained with Cde/cde cells
(data not shown). We had shown previously that our antibody preparation would not react with any membrane
proteins obtained from Rh-null regulator-type cells.’ Incubation of such cells with phospholipase A, followed by papain
did not produce any immunoreactive cleavage products (Fig
29, lanes 4 and 5), thus ruling out the possibility that such
treatment was producing a cross-reacting non-Rh related
digestion product.
Rh(D) polypeptide was isolated from both treated and
untreated Rh( D) positive cells (cDE/cDE) by immune
complex formation with a human polyclonal anti-D preparation as previously described.n Immunoblots of the isolated
protein (Fig 3, lanes 1 and 2) show that phospholipase A, and
papain digestion cleaved some, but not all, of the Rh(D)
polypeptide producing the 30-Kd product. That only polypeptide containing the Rh(D) antigen is being cleaved under
these conditions is strongly suggested by these results and
I 2 3 4 5 6 7
MW-
9655-
9655-‘
3629-
3629 -
18-
18-
B
2 3
Fig 1. Western blots of Rh polypeptidesbefore and after enzyme treatment of intact Rh-positive (cDE/cDE, - D- / -D- 1 RBCs. Total
membrane proteins prepared from similar volumes of pecked cells were separated by SDS-PAGE using e 12% polyacrylamide gel,
transferred to nitrocellulose paper. and reacted with rabbit antibody (1:1,OOO dilution), followed by goat anti-rabbit IgG conjugated with
horseradish peroxidase (1:2,OOO). (A) Lane 1, standard marker proteins; lanes 2 through 7. immunoreactive bands resulting from the
following treatments of cDE/cDE cells: lane 2, untreated: lane 3, buffer; lane 4, phospholipase A,; lane 5. papain (0.1%); lane 6.
phospholipase A, followed by buffer; lane 7, phospholipase A, followed by papain (0.1%).Identical results were obtained using CDe/CDe
cells. (E) Lane 1, standard marker proteins: lanes 2 and 3. immunoreactive bands resulting from the following treatments of - D- / - D cells: lane 2, phospholipase A, followed by buffer; lane 3, phospholipase A, followed by papain (0.25%).
PLACEMENT OF RH ANTIGENS IN THE RBC MEMBRANE
257
96-
96-
55-
55-
'
-
3 6-
36
29-
29-
18-
I8
.
-
Fig 2. Western blots of R h polypeptides before and after enzyme treatment of intact Rh-negative (cde/cde. cdE/cdEl and Rh-null
regulator-type cells. Separation end immunostaining of membrane proteins were the same as described in the legend to Fig 1. (AI Lane 1,
standard marker proteins; lanes 2 through 5, immunoreactive bands resulting from the following treatments of cde/cde cells: lane 2,
untreated; lane 3, phospholipase A,; lane 4, papain; lane 5, phospholipase A, followed by papain. (Bl Lane 1. standard marker proteins;
lanes 2 and 3,immunoreactive bands resulting from the following treatments of cdElcdE cells; lane 2, phospholipase A, followed by buffer;
lane 3, phospholipase A, followed by papain (0.25%) (identical results were obtained using Cde/cde cells); lane 4, standard marker
proteins; lanes 5 and 6, the lack of immunoreactive bands after the following treatments of Rh-null regulator-type cells: lane 5,
phospholipase A, followed by buffer; lane 6, phospholipase A, followed by papain (0.25%).
those described earlier; namely, that formation of the 30-Kd
band is detected only after digestion of Rh(D) positive
(including - D - / - D - )
cells but not after such treatment
of Rh(D) negative cells having C/c and E/e antigens.
-.
I
-
2
Fig 3. Western blots of Rh(Dl polypeptide
isolated from treated end untreated intact
cDE/cDE cells by immune complex formation
with human polyclonal anti-D. Separation of
the immune complexes (15 pg) and immunostaining with rabbit antibody were the same as
described in the legend t o Fig 1. Lane 1,
immune complex from untreated cells; lane 2,
immune complex from phospholipase A, followed by papain-treated cells.
A
MW
MW
'
18
Trypsin digestion of unsealed ghosts obtained from untreated or phospholipase A, and papain treated Rh(D)
positive (cDElcDE, - D-1- D - ) and negative (cdelcde)
cells. Immunoblots of tryptic digests of unsealed ghosts
prepared from either untreated or phospholipase A> and
papain-treated Rh( D) positive (cDE/cDE) cells showed the
presence of two new polypeptide bands having apparent mol
wts of approximately 20,000 and 17,000, respectively (Fig
4A, lanes 2 through 5). Identical results were obtained with
other Rh(D) positive (CDe/CDe, data not shown) and
Rh(D) negative (cde/cde) tryptic digests (Fig 4B, lanes 2
through 5). These results indicate that prior treatment of
intact cells with phospholipase A, and papain, with formation of the 30-Kd cleavage product, was not a prerequisite for
8
364
29
b
Fig 4. Western blots of R h polypeptides before and after trypsin digestion of ghosts prepared from either untreated or phospholipase
A, and papain-treated R h positive (cDE/cDEl and negative (cde/cdel intact cells. Total membrane proteins prepared from similar volumes
of packed ghosts were separated and immunostained as described in the legend to Fig 1. ( A ) Lane 1, standard marker proteins; lanes 2
through 5. immunoreactive bands obtained from cDE/cDE cells: lane 2. trypsin digestion of ghosts from cells treated with phospholipase A,
followed by papain (0.1%);lane 3,ghosts from cells treated with phospholipase A, followed by papain (0.1%);lane 4, trypsin digestion of
ghosts from nontreated cells; lane 5. ghosts from nontreated cells. Identical results were obtained using (CDe/CDel cells. ( 6 ) Lane 1,
standard marker proteins; lanes 2 through 5, immunoreactive bands obtained from cde/cde cells: lane 2. ghosts from nontreated cells; lane
3.trypsin digestion of ghosts from nontreated cells; lane 4. ghosts from cells treated with phospholipase A, followed by papain (0.1%I; lane
5, trypsin digestion of ghosts from cells treated with phospholipase A, followed by papain (0.1%I.
SUYAMA AND GOLDmIN
258
the production of 20-Kd and 17-Kd polypeptide bands.
Furthermore, unsealed ghosts prepared from both Rh(D)
positive or negative cells could be digested with trypsin to
yield these immunoreactive bands. This suggests that the
20-Kd and 17-Kd polypeptides are cleavage products produced by the action of trypsin at the cytoplasmic domains of
Rh(D), (C/c), and (E/e) antigens.
In contrast, trypsin digestion of unsealed ghosts obtained
from untreated -D-/-Dcells cleaved the Rh(D) polypeptide to produce two barely separable bands in about the
31-Kd to 32-Kd region of the gel (Fig 5, lanes 2 and 3).
Phospholipase A, and papain treatment of intact -D-/
-D- cells produced the expected incomplete conversion of
the 33-Kd Rh(D) polypeptide to the 30-Kd cleavage product
(Fig 5, lane 4). Digestion of unsealed ghosts prepared from
these cells with trypsin further cleaved these polypeptides to
produce a second band in the 26-Kd region of the gel (Fig 5,
lane 5). The size of the trypsin cleavage products obtained in
these studies suggests that the arrangement of the cytoplasmic domain of Rh(D) polypeptide in -D-/-Dcells is
different from that found in other Rh(D) positive cells.
Trypsin digestion of inside-out vesicles obtained from
Rh(D)positive (CDeICDe, - D-1- D - ) and Rh(D)negative
(cdelcde) cells. To confirm that the results obtained with
unsealed ghosts represented cleavage at the cytoplasmic
domains of Rh antigens, we prepared inside-out vesicles from
Rh(D) positive (CDe/CDe) and Rh(D) negative (cde/cde)
cells that had not been pretreated with phospholipase A, plus
papain, and digested these vesicles with trypsin. Such treatment produced two immunoreactive cleavage products of
identical size (Fig 6A, lanes 2 and 3; Fig 6B, lanes 2 and 3) to
those found as a result of trypsin digestion of unsealed ghosts
A
MW
2 3
XIO-3
9655-
I ! 3
u
9 4
36-
2918-
Fig 6. Western blots of R h polypeptides before and after
trypsin digestion of inside-out vesicles prepared from R h positive
(CDe/CDe) and negative (cdelcde) cells. Total membrane proteins
prepared from approximately 200 pg of starting inside-out vesicles
were separated and immunostained as described in the legend t o
Fig 1. ( A ) Lane 1, standard marker proteins; lane 2, insideout
vesicles from CDe/CDe cells; lane 3,trypsin digestion of insideout
vesicles from CDelCDe cells. Identical results were obtained using
cDE/cDE cells. (6)Lane 1, standard marker proteins; lane 2.
inside-out vesicles from cdelcde cells; lane 3. trypsin digestion of
inside-out vesicles from cde/cde cells.
(Fig 4A, lanes 2 through 5; Fig 4B, lanes 2 through 5 ) .
Identical results were obtained using cDE/cDE cells (data
not shown). Similarly, trypsin cleaved the Rh(D) polypeg
tides of inside-out vesicles prepared from - D - / - D- cells,
producing two partially separable cleavage products (Fig 7,
lanes 2 and 3) identical in size (31 to 32 Kd) to those found
MW
XIO-3-
96i
55i
291
36
I8i
Fig 5. Western blots of Rh polypeptides before and after
trypsin digestion of ghosts prepared from either untreated or
phospholipase A, and papain-treated - D- / - D - intact cells.
Total membraneproteins prepared from similar volumes of packed
ghosts were separated and immunostained as described in the
legend t o Fig 1. Lane 1, standard marker proteins; lanes 2 through
5. immunoreactive bends obtained from -D- / -D- cells: lane 2.
ghosts from nontreated cells; lane 3, trypsin digestion of ghosts
from nontreated cells; lane 4. ghosts from cells treated with
phospholipase A, followed by papain (0.1%); lane 5. trypsin
digestion of ghosts from cells treated with phospholipase A,
followed by papain (0.1%).
I
I
2
3
96
:i
55i
18
Fig 7. Western blots of R h polypeptides before and after
trypsin digestion of inside-out vesicles prepared from -D- / -Dcells. Total membrane proteins prepared from approximately 200
pg of starting inside-out vesicles were separated and immunostained as described in the legend t o Fig 1. Lane 1, standard
marker proteins; lane 2. inside-out vesicles from -D- / -Dcells; lane 3, trypsin digestion of inside-out vesicles from -D-/
- D - cells.
259
PLACEMENT OF RH ANTIGENS IN THE RBC MEMBRANE
with trypsin-treated unsealed ghosts from the same genotype
(Fig 5, lanes 3 and 4).
DISCUSSION
Using a rabbit polyclonal antibody preparation recently
shown to specifically immunostain Rh antigen-related
polypeptides: we have been able to monitor the effects of
various enzyme treatments on the structural integrity of
Rh(D), (C/c), and (E/e) antigen-containing polypeptides in
situ. Phospholipase A, treatment of intact erythrocytes has
been reported to significantly reduce the antigenic activity of
Rh(D)
Similar incubations with proteases, on
the other hand, apparently have no effect on the ability of
Rh(D) antigen to interact with antibody, and no degradation
of the polypeptide is observed."
Treatment of intact cells with phospholipase A, under
conditions where no hemolysis occurs, followed by digestion
with papain again without producing hemolysis, resulted in
cleavage at the extracellular domain of Rh(D) polypeptide.
In contrast, such treatment of cells having various Rh(D)
positive and negative phenotypes showed that the (C/c) and
(E/e) antigen-containing polypeptides were not susceptible
to cleavage. This suggests that the structure of the extracellular domain of Rh(D) polypeptide is unique among Rh
antigens and could possibly be a factor contributing to its
superior immunogenicity. It also appears that the 30-Kd
cleavage product of the Rh(D) polypeptide either still
contains one or more intact D antigenic epitopes or is
associated in some way with undigested polypeptide, since
both are recovered by immune complex formation using
human polyclonal anti-D. The former explanation seems
more likely because Gorick et alZ5reported the presence of at
least three epitopes on the Rh(D) polypeptide and believe
these epitopes to be present at varying depths within the lipid
bilayer.
Digestion of unsealed ghosts and inside-out vesicles with
trypsin resulted in the cleavage of Rh(D), (C/c), and (E/e)
polypeptides, thus confirming that they span the lipid bilayer.
The size of the digestion products obtained from ghosts and
vesicles of Rh(D) positive and negative cells was the same,
indicating similar cytoplasmic domains for the Rh(D),
(C/c), and (E/e) polypeptides. However, the size of the
products of Rh(D) polypeptide from ghosts and vesicles of
-D - / - D - cells was significantly higher. These bands
were not discernible in the digests obtained from other
Rh(D) positive cells. Although our evidence is not conclusive,
it could reflect a difference in folding of the Rh(D) polypeptide within the lipid bilayer and at the cytoplasmic domain in
-D-/-Dcells where other common Rh antigens are
lacking.
ACKNOWLEDGMENT
We thank E. Rotter and S. Colt for their expert technical
assistance; Dr L. Lenny for her critical reading of the manuscript,
and the Philip Levine Laboratory, Ortho Diagnostic Systems, Inc,
for providing Rh-null, - D - / - D- ,cdE/cdE, and Cde/cde erythrocytes.
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