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Detection of anticentromere antibodies using recombinant human CENP-A protein.

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Vol. 39, No. 5, May 1996, pp 863-867
0 1996, American College of Rheumatology
Objective. To evaluate CENP-A reactivity with
anticentromere antibodies (ACA) using recombinant
protein (rCENP-A).
Methods. Human CENP-A antigen was overexpressed in insect cells using the baculovirus system. We
tested for ACA activity against the full-length recombinant polypeptide by immunoblot and by enzyme-linked
immunosorbent assay (ELISA).
Results. Of the ACA+ sera studied (n = 38),
95% were positive when tested against the rCENP-A in
the ELISA system. Of the ACA- sera (n = 1001, only
2% gave false-positive results in the assay. There was
good correlation between the recombinant and bona fide
antigens in assaying for ACA reactivity.
Conclusion. CENP-A is a significant ACA target.
The availability of the rCENP-A assay is a valuable
adjunct to the previously described rCENP-B assay in
analyses of the clinical significance of ACA.
Autoantibodies are characteristic of the rheumatic disease systemic sclerosis (SSc; scleroderma)
(1,2). Anticentromere antibodies (ACA) are the primary specificity associated with the limited cutaneous
form of the disease, which includes the CREST syndrome (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias). The
molecular nature of a number of autoantigens has been
Supported in part by NIH research grants AR-40601 and
Dongxu Sun, PhD, Antigona Martinez, BS, Sallie 0. Hoch,
PhD: The Agouron Institute, La Jolla, California; Kevin F. Sullivan,
PhD: The Scripps Research Institute, La Jolla, California; Gordon
C. Sharp, MD: University of Missouri, Columbia.
Address reprint requests to Sallie 0. Hoch, PhD, The
Agouron Institute, 505 Coast Boulevard South, La Jolla, CA 920374696.
Submitted for publication May 9, 1995; accepted in revised
form December 8, 1995.
defined over the last decade, and a recurring observation is that many of the traditional antigen designations
actually encompass a number of protein species. The
recognition that ACA reactivity was directed against
multiple polypeptides was due in large part to the work
of Earnshaw and Rothfield (3). Those investigators
discriminated the major centromere protein targets as
CENP-A, CENP-B, and CENP-C, with estimated
molecular weights of 17 kd, 80 kd, and 140 kd,
CENP-A was the first of the polypeptides to be
recognized as a common target. Using the immunoblot
technique to survey a number of ACA+ scleroderma
sera, Guldner et a1 (4)showed that all of the sera tested
reacted with a 19.5-kd nuclear protein. The protein
blot proved to be an effective means of measuring sera
reactivity against the various CENP antigens in tandem, but such surveys tended to be somewhat ambiguous in that different studies emphasized different
CENP proteins. Yet the development of more sensitive assays targeted to each of the CENP antigens,
individually isolated from natural cell sources, was
deterred by the very limited concentration of these
polypeptides. An alternative is to use recombinant
protein antigens for such assays. A complementary
DNA (cDNA) encoding CENP-A, a 16-kd basic protein with a carboxy-terminal histone H3-like domain,
has recently been described (5). Transient expression
of this cDNA in HeLa and Indian muntjac cells
confirmed the centromere staining pattern.
We report here on the stable eukaryotic cell
expression of the CENP-A cDNA using baculovirusmediated infection of insect cells. The recombinant
human antigen (rHuCENP-A) that is produced is a
nonfusion protein, identical in length and sequence to
that encoded by the human CENP-A cDNA clone.
The protein is immunoreactive as assayed by both
protein blot and enzyme-linked immunosorbent assay
(ELISA). Screening of ACA+ scleroderma sera confirmed CENP-A as a significant target.
Isolation of rHuCENP-A. The 140-residue human
CENP-antigen was expressed using the BacPAK baculovirus expression system (Clontech, Palo Alto, CA) (Martinez
et al: manuscript in preparation). Suspension cultures of Sf9
insect cells were grown in Grace’s medium (Gibco BRL,
Gaithersburg, MD) containing 10% (volumeholume) fetal
calf serum. The cells were infected at a multiplicity of
infection of 2-5, and were harvested 3 days postinfection,
after visualization of a cell lysate by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and
Coomassie blue staining to estimate the rCENP-A expression. The harvested cells were washed with phosphate
buffered saline (PBS) and stored at -80°C.
Moderately high salt efficiently solubilizes much of
the total Sf9 protein, but not the rCENP-A. The cell pellet
was extracted twice in buffer A (50 mM Tris HCl, pH 7.5, 10
mM MgCl,, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride [PMSF]) containing 0.5M NaC1. The suspension was sonicated 4 times for 40 seconds each at 4°C and
then centrifuged for 20 minutes at 25,OOOg. The supernatant
was discarded. The pellet containing the rCENP-A was
resuspended in buffer A containing 6M urea, sonicated twice
for 40 seconds each time, and centrifuged for 20 minutes at
65,OOOg. The residual pellet was reextracted in a minimal
volume of buffer A plus 6M guanidine HCl, and then slowly
diluted in a 30-fold volume of buffer A plus 6M urea. The
suspension was centrifuged a final time for 20 minutes at
65,OOOg. The 6M urea extract was loaded directly onto a
cation exchange column (Hi-Load S-Sepharose HP; Pharmacia, Piscataway, NJ) at room temperature. The adsorbed
protein was eluted with a linear NaCl gradient (0-1M) in
buffer B (50 mM Tris HCl, pH 7.5, 0.2 mM PMSF) containing 6M urea. Fractions containing rCENP-A were determined by SDS-PAGE.
Sera. Most patient sera were obtained from the
Antinuclear Antibody Laboratory at the University of
Missouri-Columbia School of Medicine. Sera designated
ACA+ were assayed by immunofluorescence staining of
HEp-2 cells. Additional sera were obtained from the serum
bank at the Agouron Institute and included ACA- rheumatic disease controls. Normal sera were obtained from
laboratory personnel and from Immunovision (Rogers, AR).
Immunoblot assays. Protein samples were fractionated on 12.5% polyacrylamide gels. The separated proteins
were transferred to polyvinylidene difluoride (PVDF) membranes overnight at 4°C at 60V in 0 . 5 ~
Laemmli buffer with
10% methanol and 0.01% SDS (6) (Westran; Schleicher and
Schuell, Keene, NH). A second antibody (rabbit anti-human
IgG, IgM, and IgA serum) was added prior to the addition of
iodinated protein A because, in our experience, a secondary
antibody enhances blots of the bona fide CENP-A.
Extracts were prepared from HeLa cell nuclei in 1M
MgCl, as described (6). Block gels were run at a protein
concentration of -50 pg/5 mm. Alternatively, acid extracts
of HeLa nuclei were prepared as described (6) and these
extracts were used as control samples containing both
CENP-A and the core histones.
Extracts were also prepared from baculovirusinfected insect cells producing rHuCENP-A. SDS-lysed
whole cell extracts were run on block gels at an equivalency
of the original culture volume of 0.15 mu5 mm.
ELISA for detection of anti-CENP-A antibodies. Recombinant CENP-A antigen was purified by fast protein
liquid chromatography (FPLC). This antigen was used to
coat 96-well polystyrene microtiter plates (Immulon-2;
Dynatech, Chantilly, VA) at a concentration of -20 ng/well
by incubation for 1.5 hours at room temperature. All dilutions were made in blocking reagent (0.5% bovine serum
albumin in PBS, pH 7.4, 0.05% Tween 20). Plates were
washed 4 times with water after each step. The remaining
free binding sites were coated for 1 hour in the blocking
reagent. Human serum (1:2,000 dilution) was added and
incubation continued for 1 hour at room temperature. Immune
complexes were detected with horseradish peroxidase coupled
to goat anti-human IgG using 0.2% o-phenylenediamine in
citratephosphate buffer, pH 5.0, containing 0.02% H,O,.
The reaction was stopped with 4.5M H,SO,. The plates were
read at 490 nm on an ELISA reader (Molecular Devices,
Menlo Park, CA). Each serum sample was also tested in
triplicate against wells coated only with blocking reagent.
The ELISA results were expressed as optical density at 490 nm
(OD,,,) with the antigen minus OD,,, without the antigen.
Reactivity of ACA+ sera with rCENP-A. The
heterologous expression of a eukaryotic protein is an
empirical problem. We were unsuccessful, in several
attempts using different expression vectors, to produce the antigen in Escherichiu coli (Sun D, Sullivan
KF: unpublished results). The approach that ultimately proved successful was to utilize the eukaryotic
baculovirus expression system in insect cells (Martinez et al: manuscript in preparation). This system has
a number of advantages, including the possibility of
certain posttranslational modifications not indigenous
to E coli. The protein produced is seen in Figure 1A. A
band of the expected size is clearly visible by
Coomassie blue staining of an SDS whole cell lysate
(Figure 1, lane 2). In comparison, a HeLa nuclear acid
extract, whose limited array of proteins includes the
core histones and CENP-A, exhibited no detectable
band by Coomassie blue staining (or silver staining) in
the same region above the histone H3 protein (Figure
1, lane 1). The recombinant protein was immunoreactive as assayed by the protein blot (Figure 1B). The
reactivity was specific, in that (a) the ACA+ serum did
not recognize a similar-sized insect cell host protein
when blotted against another baculovirus construct
expressing the Sm-B3(N) antigen (Figure 1, lane 3) and
‘1 2 3
a 3
2 3,
Figure 1. Baculovirus expression of CENP-A. Harvested cells were lysed in sodium dodecyl sulfate (SDS) incubation
buffer, fractionated by SDS-polyacrylamide gel electrophoresis, and immunoblotted. A, Coomassie blue stain. B,
Immunoblot with anticentromere antibody-positive serum. C, Immunoblot with anti-Sm-positive serum. Lane 1,
Acid-extracted HeLa nuclei. Lane 2, Insect cell lysate expressing the CENP-A antigen. Lane 3, Insect cell lysate
expressing the Sm-B3(N) antigen.
(b) an anti-Sm+ serum which reacted with the basic
Sm-B3 protein did not react with the basic CENP-A
Protein blot screen of ACA+ sera. We have
previously described experiments to design protein
blot conditions specifically for the CENP-A antigen.
The objective was to maximize the electrotransfer step
in the blot sequence for this basic protein (6). This
assay, as developed for the bona fide antigen, was now
utilized for the recombinant antigen, using SDS whole
cell lysates. Of the ACA+ sera tested, all reacted with
the 16-kd rCENP-A antigen (Figure 2). Included
among these sera was the Centers for Disease Control
and Prevention reference anticentromere serum no. 8
(Figure 2, lane 36). There was no reactivity exhibited
by normal human serum (Figure 2, lane 1) (of 5
samples tested) or by 3 ACA- control sera (Figure 2,
lanes 37-39). These same sera were also tested by
immunoblot against the antigen from HeLa cells, with
the same results (Hoch SO: unpublished observations). A pool of 9 anti-Scl-70+ sera also tested
negative against the rCENP-A (data not shown).
ELISA results. We used the FPLC-purified
baculovirus-expressed rCENP-A protein as antigen in an
ELISA to determine whether the recombinant protein
can be useful in developing diagnostic assays for detecting anticentromere antibodies. A total of 138 sera were
tested, including 38 ACA+ sera, 66 anti-Sm/RNP+ sera,
18 anti-Rob+ sera, 10 miscellaneous serum samples
(including 3 anti-Scl-70i sera, 1 pool of 9 anti-Scl-70+
sera, and 6 miscellaneous), and 6 normal sera. The
ELISA results are summarized in Figure 3. Of the 100
ACA- sera, 98 had OD,, values of <0.26. The mean
plus 4 standard deviations for these 98 sera was 0.28.
If the latter OD, value is used as the threshold for
the assay, then 95% of the ACA+ sera (36 of 38)
were positive in the ELISA system when tested for IgG
antibodies. Testing for other reactivities (IgM, IgA)
awaits a detailed analysis of the anti-CENP-A isotype
profile. The 2 false-positives among the ACA- sera
were originally characterized as antism+ and antiRNP t , respectively. When tested by immunoblot, both
sera reacted strongly with the rCENP-A, but not with
the CENP-A protein extracted from HeLa cells.
Figure 2. Protein blot assay of anticentromere antibody (ACA)-positive sera tested against recombinant CENP-A.
Sodium dodecyl sulfate (SDS) whole cell lysates containing baculovirus-expressed human CENP-A were fractionated on
block gels by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Individual
strips were blotted with the following sera: lane 1, normal human serum; lanes 2-36, ACA-positive sera (including the
Centers for Disease Control and Prevention reference ACA serum [lane 81); lanes 37-39, ACA- scleroderma sera.
under denaturing- conditions. But it was the
- ELISA that provided a vehicle for direct antibody
The clinical assay of choice for anticentromere
of a discrete CENP polypeptide, specifically,
antibodies has been immunofluorescence staining of
forms of CENP-B (7-9). Our experience
selected cell lines. The immunoblot technique proved
of human CENP-A from HeLa cells
very effective in discriminating individual CENP polyindicates that it is also not practical to design an
ELISA for the cellular form of this CENP antigen
because its natural abundance is minuscule (Martinez
et a]: manuscript in preparation). Thus, we have
turned to the recombinant expression of CENP-A.
We chose as our template the human source of
the antigen, to maximize potential ACA epitope recognition. By analogy to CENP-B, several groups have
reported the generation of rabbit or murine antibodies
specific to sequences unique to the human CENP-B
polypeptide (10,ll). Likewise, the choice of the eukaryotic baculovirus expression system was made, to
facilitate the production of recombinant antigen that
closely mimics the bona fide source. We produced a
full-length form of the antigen that spans the natural
: I
residues 1-140 of the human CENP-A protein. All of
the ACA+ sera tested by immunoblot were reactive
with the rCENP-A, as were 95% of the ACA+ sera
Normals Anti-SmlRNP Anti-Ro/La Others ACA
tested by ELISA. Moreover, the assay showed eviFigure 3. Results of enzyme-linked immunosofient assay screen of
dence of specificity in that only 2 of 100 ACAanticentromere antibody (ACAtpositive sera against recombinant
CENP-A. Individual sera were tested in triplicate against baculovhsrheumatic disease patient and normal sera reacted
expressed CENP-A, purified by cation exchange chromatography.
with rCENP-A.
The sera (diluted 1:2,OOO)were tested with antigen (A) and without
Why include the CENP-A antigen in the deantigen (B). Dashed line represents the mean value (A - B) + 4 SD
of a quantitative assay for ACA reacof the control sera (ACA negative). See Materials and Methods for
the relative importance of the
details of the assay.
. .......
major CENP polypeptides was argued on the basis of
immunoblot assays, with conflicting results. Such observations are not surprising, since a number of factors
will influence blot results. In particular, solubilization
conditions prior to SDS-PAGE fractionation have an
effect. A variety of protocols to extract the CENP
polypeptides from the nucleus have been reported.
But, as we have shown for CENP-A, there are varying
losses of the antigen to the insoluble cellular debris,
depending on the extraction conditions (6). This is not
unexpected, in that the centromere antigens as a whole
have proven to be recalcitrant to solubilization in
physiologic buffers, a property which has been used to
describe the kinetochore. Furthermore, blot results
are influenced by the fact that the optimal immunoblot
parameters, i.e., the choice of gel conditions, electrotransfer buffer, blotting membrane, etc., will not necessarily be the same for proteins that differ in size and
charge, as do CENP-A, CENP-B, and CENP-C.
Thus, it is premature, based on protein blot
data, to single out just one of the major CENP antigens
as the basis for the development of an improved (i.e.,
rapid and quantitative) ACA assay. Rather, one could
consider the analogy of the Sm antigens associated
with systemic lupus erythematosus. For that disease,
assay development is focusing on all of the major Sm
B and D polypeptides. Likewise, for the ACA assays,
it is certainly practical to develop an ELISA for
CENP-A as an adjunct to the CENP-B ELISAs, which
have already demonstrated potential utility as ACA
monitors (7-9). And in contrast to the blot assay, the
ELISA can be used to substantially increase the number
of sera tested and resolve the issue of the specificity of
ACA+ sera in general for CENP-A, relative to the
incidence reported for CENP-B (7).
Why develop a quantitative assay for anticentromere antibodies? The obvious answer is to facilitate
the diagnosis of a major clinical subgroup of scleroderma patients, with the attendant implications regarding treatment. But there are other, less general, possibilities. Interesting questions have been raised as to
the utility of ACA in predicting disease progression,
e.g., the potential for ACA+ patients who present
only with Raynaud’s phenomenon to develop CREST
syndrome (12,13), and the associated risk factor for
loss of 1 or more digits in ACA t patients (14,15). The
extension of such observations to individual polypeptides is a natural outgrowth of these assays.
T h e a u t h o r s t h a n k Dr. April Chang-Miller f o r her
generous gift of antibodies, and the National Cell C u l t u r e
facility for large-scale production of HeLa cells. T h e a u t h o r s
a l s o express their appreciation to La Vonne Melheim for
expert secretarial assistance in preparing t h e manuscript.
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