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Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2959-2967
By
From the Departments of Pediatrics and Pathology, Johns Hopkins
University School of Medicine, Baltimore, MD.
The human platelet alloantigen 1 system (HPA-1) is determined by a
polymorphism at position 33 in the N-terminus of human glycoprotein
IIIa (GPIIIa). This naturally occurring substitution creates a
conformation in the HPA-1a allelic form that can be antigenic when
presented to an individual expressing the HPA-1b form. Anti-HPA-1a
antibodies generated by this immune response can lead to the
destruction of platelets, as seen in the clinical disorders, neonatal
alloimmune thrombocytopenia (NAIT) and posttransfusion purpura (PTP).
To understand better the structural requirements for recognition by
these pathogenic antibodies, we investigated the N-terminal 66 amino
acids from the HPA-1a form of human GPIIIa and the analogous amino
acids from the nonimmunogenic murine homolog. Our objectives were to
define further the boundaries of the HPA-1a epitope(s) in the
N-terminus of human GPIIIa, to isolate the murine 5' nucleotide
sequence and compare the deduced murine N-terminal sequence to that of
human, and to mutate the murine sequence systematically to include an
HPA-1a epitope(s). Murine amino acids that differed from human were
changed by site-directed mutagenesis to the analogous residues in the
HPA-1a form of human GPIIIa, starting and radiating from murine
position 33 (site of human polymorphism). This systematic approach
allowed us to pinpoint amino acids critical to a conformation recognized by anti-HPA-1a antibodies. Our results show that an HPA-1a
epitope can be created within the N-terminus of murine GPIIIa and raise
the possibility that murine models of HPA-1a sensitization can be developed.
HUMAN PLATELET alloantigen system 1 (HPA-1) involves a polymorphism in glycoprotein IIIa (GPIIIa), a
subunit of the fibrinogen receptor. In this human biallelic system, the
HPA-1a allele encodes a leucine at position 33 of GPIIIa and the allele for HPA-1b encodes a proline.1 In Caucasians from North
America and Europe, the gene frequency is estimated to be 83% for
HPA-1a and 17% for HPA-1b.2
Two bleeding disorders, neonatal alloimmune thrombocytopenia (NAIT) and
posttransfusion purpura (PTP), can be attributed to an immune response
of HPA-1b homozygous individuals to HPA-1a platelets.3 In
each disorder, antibodies are made against HPA-1a platelets, leading to
thrombocytopenia. Alloantibodies are made by HPA-1b homozygous mothers
against HPA-1a fetal platelets in NAIT. The thrombocytopenia in NAIT is
a leading cause of prenatal and perinatal intracranial hemorrhage. PTP
results from the transfusion of HPA-1a-positive platelets to
previously sensitized HPA-1b homozygous individuals.
Anti-HPA-1a antisera from patients with these disorders recognize a
three-dimensional structure in the HPA-1a form of human GPIIIa.4 This conformation is dependent on the leucine at
position 33.1 Disulfide bridges between cysteines (Cys) in
the protein are also important; alteration of the disulfide bonds by
reducing agents results in loss of HPA-1a antigenicity.5
The N-terminal region of GPIIIa contains seven Cys and the disulfide
bridges between Cys residues have been assigned.6 The
proposed Cys-pairing creates a cloverleaf-like structure with three
loops (see review by Newman7). This cloverleaf structure is
thought to be pulled into apposition with the highly Cys-rich core of
GPIIIa by a disulfide bond between Cys5 and
Cys435.6
A 66-amino acid segment of the N-terminus of human GPIIIa binds
anti-HPA-1a antisera from some individuals with NAIT or PTP. This
N-terminal segment includes the entire cloverleaf structure. Bowditch
et al8 first demonstrated the binding of this domain by
anti-HPA-1a antibodies and our laboratory confirmed his finding by the
production of soluble HPA-1 antigens.9 In the intact human
GPIIIa, it has been proposed that the
Cys5-Cys435 bridge between the N-terminal
domain and Cys-rich core may be necessary for optimal binding of
anti-HPA-1a antibodies.10 Alternatively, two types of
anti-HPA-1a antibodies have been proposed, one that binds solely the
N-terminus and another that binds the N-terminus bound to the Cys-rich
core by the Cys5-Cys435
bridge.11,12
To combat the specific antibodies that are problematic in NAIT and PTP,
a better understanding of their structural requirements for binding
human GPIIIa is needed. In this investigation, we focused on the
requirements for recognition of the 66-amino acid segment of the
N-terminus of human GPIIIa by anti-HPA-1a antibodies. By deletion
analysis, we further truncated the N-terminal and C-terminal ends of
the 66-residue segment and found shorter fragments that could still
maintain a high degree of immunoreactivity. For comparison, we also
isolated the nucleotide sequence coding for the first 66 residues of
murine GPIIIa. The deduced amino acid sequence from mouse is highly
homologous to human GPIIIa. However, polyclonal human anti-HPA-1a
antibodies do not recognize the murine protein. To determine whether we
might identify residues that are critical for binding anti-HPA-1a
antibodies, residues within the murine GPIIIa N-terminus were
systematically changed to the human amino acid counterpart. We describe
herein the creation of an anti-HPA-1a binding domain within the
context of the nonimmunoreactive mouse protein. This accomplishment is
the first step towards the possible generation of a murine model of the
HPA-1 system.
Construction of Deletion Mutations of the Human GPIIIa N-Terminus
N-terminal deletions.
Using various 5' oligonucleotides that annealed to sequences
located between the initiating methionine codon and the site of the
human polymorphism, the position of the N-terminal truncation was
generated. The 3' amplification primer annealed to the original EcoRI cloning site. These primers contained restriction
endonuclease sites for directional cloning. After digestion with the
appropriate enzymes, the amplified fragments were cloned in frame into
pPROEX-1 expression system (Life Technologies, Gaithersburg, MD) and
subsequently moved into a pGST/HIS T2 prokaryotic expression system
(Pharmacia Biotech, Piscataway, NJ). This vector is essentially pGEX4T2
modified to include a 6xHis tag at the 3' end of the fusion
segment. In our deletion constructs, the fusion proteins generated
possess only a glutathione S-transferase (GST) fusion segment, because our inserts provide a stop codon before the 6xHis coding sequence. These N-terminal deletions include pGEXh3a9-66,
pGEXh3a17-66, and pGEXh3a23-66.
C-terminal deletions.
Similarly, 3' truncations were generated by PCR. In these
constructions, various 3' primers annealed to sequences
downstream of the polymorphism. The 5' oligonucleotide primer
annealed to a vector sequence 5' to the codon for the initiating
methionine of GPIIIa. Conveniently engineered restriction sites, one
previously made at the initiating methionine and the other in the
3' primer, were used to clone the PCR fragments into pGST/HIS T2.
The C-terminal truncations are pGEXh3a1-34,
pGEXh3a1-40, and pGEXh3a1-50.
Isolation and Sequencing of the 5' End of Murine GPIIIa cDNA
(BALB/c strain)
Isolation and Sequencing Fragments of the Murine GPIIIa Gene From Genetically Diverse Strains of Mice Genomic clones, containing the exon that codes for position 33 of mouse GPIIIa, from 10 genetically diverse strains of mice were also isolated and sequenced. DNA from mice with dissimilar ancestries was obtained, including that from strains A/J, BALB/cBy, C3H/He, C57BL/6J, C58/J, CBA/J, DBA/2J, SEC/1Gn, SM/J, and NZW (Jackson Laboratory, Bar Harbor, ME). In the region of interest, the genomic structure of the mouse GPIIIa gene was the same as that of the human GPIIIa gene.15 As in humans, a murine intron, approximately 9 kb in size (analogous to intron 1 of human), precedes an exon that begins coding at amino acid 30 (analogous to exon 2 of human). Since the coding sequence for residue 33 was located very close to the 5' end of the exon and the upstream intronic sequence was unknown, long PCR techniques were required to examine the residue coded for position 33. A forward primer that annealed to the end of the upstream exon (analogous to exon 1 in humans) and a reverse primer to the end of the exon in question (exon 2 in humans) were used to amplify the genomic sequence using a kit (Expand Long Template PCR System; Boehringer Mannheim, Indianapolis, IN). The resulting PCR fragments included a 9-kb intron and an exon encoding amino acids 30 to 84. The region encoding amino acids 30 to 84 from each fragment was sequenced.Construction of Substitution Mutations in the Murine GPIIIa N-Terminus Several techniques were used to generate the substitutions of human amino acids into the murine GPIIIa N-terminus.Substituting amino acids at position 33 and 32. Initially, the mutations at positions 32 and 33 in combination were constructed by PCR. Two overlapping PCR fragments with convenient 5' and 3' restriction sites were generated. The forward oligonucleotide primer annealed to the initiating methionine codon and included a unique 5' BspHI restriction site. The reverse primers contained nucleotide substitutions that mutated the codons for positions 32 and 33 and, in addition, they silently mutated positions 34 and 35 to provide a unique BamHI restriction site. An overlapping 3' PCR fragment was generated using a forward primer that recreated the BamHI site and a reverse primer that included a unique NotI restriction site. After digesting the 5' fragment with BspHI and BamHI and the 3' fragment with BamHI and NotI, the two fragments were ligated together and inserted in-frame into pPROEX1 (Life Technologies). The insert with both positions 32 and 33 mutated was subsequently cloned into pGEX4T2 and named pGEXm3a32,33. Adding additional substitutions. Using a site-directed mutagenesis kit (Clontech, Palo Alto, CA) and the pGEXm3a32,33 template, nucleotide substitutions were introduced individually into the codons for residues 22, 30, and 39. The resulting plasmids were (1) pGEXm3a22,32,33, (2) pGEXm3a30,32,33, and (3) pGEXm3a32,33,39. Further combinations of mutations were constructed either by a megaprimer technique16,17 or by exchanging restriction fragments with mutations. The first technique used a megaprimer made by PCR using a mutagenic oligonucleotide for position 22, the 3' NotI primer, and the pGEXm3a30,32,33 template to generate pGEXm3a22,30,32,33. Further combinations were created by exchanging BamHI restriction fragments from pGEXm3a22,32,33, pGEXm3a30,32,33, and pGEXm3a22,30,32,33 with that from pGEXm3a32,33,39 which resulted in the addition of a mutation at position 39 to each construct. This exchange produced (1) pGEXm3a22,32,33,39, (2) pGEXm3a30,32,33,39, and (3) pGEXm3a22,30,32,33,39. Expression and Purification of Recombinant Proteins GST fusion proteins.
The fusion proteins were expressed in Escherichia coli DH5 Immunoassays The HPA-1a antisera tested were obtained from NAIT mothers or patients with PTP. The specificity of each antiserum was verified with a solid-phase enzyme-linked immunosorbent assay (ELISA) using isolated GPIIb/IIIa complexes derived from platelets of HPA-1a and 1b homozygotes.18 Previously described, anti-HPA-1a antibodies, affinity-purified against the N-terminal human GPIIIa (amino acids 1 to 66) fusion protein, were also used in some of the immunoassays,9 as well as the monoclonal antibody, SZ21 (Immunotech, Westbrook, ME).ELISA. Control and mutated proteins were assayed for reactivity to anti-HPA-1a antisera using an ELISA. Samples were tested in triplicate. Microtiter wells were coated with 200 ng purified fusion protein in 200 µL of 64 mmol/L carbonate buffer, pH 9.5 overnight at 4°C. After washing the wells with PBST, pH 7.2 (7.72 mmol/L Na2HPO4, 2 mmol/L NaH2PO4-H2O, 0.15 mol/L NaCl, and 0.05% Tween 20), each was blocked with 2% bovine serum albumin (BSA) in PBST and washed again with PBST. Antisera were diluted 1:40 and incubated with the immobilized antigen for 2 hours at 37°C. After washing, a 1:25,000 dilution of Protein A/G-horseradish peroxidase (HRP) conjugate (Pierce, Rockville, IL) was added to the well and the peroxidase was detected with tetramethylbenzidine (TMB) peroxidase substrate (Kirkegaard & Perry Labs, Gaithersburg, MD). Microtiter plates were read at optical density (OD) 450. Immunoblots. The reactivity of the clinical antibodies to the deletion and substitution mutations was assayed by immunoblots. Samples (5 to 10 µg) were electrophoresed on 10% to 15% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto nitrocellulose as previously described.9 Membranes were then blocked for 1 hour with 10% dried milk, washed with TBST (150 mmol/L NaCl, 0.05% Tween 20,10 mmol/L Tris-HCl, pH 8.0), and incubated with 5.0 µg/mL affinity-purified anti-HPA-1a antibody or 1:10 dilution of antisera. After washing with TBST, the blots were incubated with a 1:10,000 to 1:30,000 dilution of the Protein A/G-HRP conjugate. The immunoreactive proteins were detected by enhanced chemiluminescence (ECL) (Amersham, Arlington Heights, IL) or SuperSignal (Pierce).
Truncations of the Human GPIIIa N-Terminus Using recombinant proteins, we have further defined the binding domain of an HPA-1a epitope by systematically dissecting the immunoreactive region of the N-terminal 66-residue segment of human GPIIIa (Fig 1A). Three N-terminal and three C-terminal truncations of the 66-residue segment were constructed and tested for anti-HPA-1a recognition. By ELISA (Fig 1B), we showed a progressive reduction in immunoreactivity from fusion proteins with sequential deletions from either direction. Removal of residues 1 to 8 from the human GPIIIa N-terminus reduces the reactivity approximately 30% relative to the intact segment. This deleted segment includes Cys5 which is believed to be joined via a disulfide bond to Cys435 in the intact protein. When residues 1 to 17 are deleted, an approximate 53% decrease from the intact segment is observed. This missing segment extends into the first loop of the proposed cloverleaf structure and includes Cys16 that pairs with Cys49 to generate the third loop of the structure. Loss of six additional amino acids within the first loop in the deletion mutation m3a1-22 results in a 75% reduction in signal from the control. Truncations from the C-terminal direction show that removal of residues 51 to 66, not contained within the cloverleaf, decreases reactivity approximately 30% from control. Deletions of residues 41 to 66 and 35 to 66, which interrupt the second and third loops, drop the signal 73% and 86% from control, respectively.
Murine N-Terminal GPIIIa Sequence To compare the amino acid sequence of the immunoreactive human GPIIIa N-terminus to the nonimmunoreactive murine GPIIIa N-terminus, the 5' end of the murine (BALB/c) GPIIIa cDNA (Genbank accession no. U83167) was isolated and sequenced. The nucleotide sequence of the segment that codes for amino acids 1 to 66 is 83% identical to the analogous sequence in human. The degree of identity is similar to the 86% DNA homology determined for the remainder of the cDNA by Cieutat et al14 (amino acid sequence begins at position 79, Genbank accession no. 386151). The high degree of nucleotide conservation translates into an overall amino acid identity of 89% between the full-length murine and human proteins.
Reactivity of Anti-HPA-1a Antibodies and Antisera to Substitution
Mutations in the Murine GPIIIa N-Terminus
Competition With the Human Recombinant Epitope To determine how similar the epitope generated in the murine N-terminal GPIIIa sequence is to that of the human epitope, we tested the ability of the mutated murine proteins to compete with the human recombinant protein for antibody binding. After pretreating the anti-HPA-1a antisera with increasing concentrations of the modified murine GPIIIa N-termini, antisera were assayed by ELISA for their ability to bind the corresponding human 66-amino acid segment. The results of a competition ELISA using an antiserum are shown in Fig 3; similar results were observed with two additional anti-HPA-1a antisera (data not shown).
Recognition of the Panel of Murine Substitution Mutations by the Monoclonal Antibody SZ21 We also tested the murine monoclonal antibody SZ21 with the mutated murine proteins to determine whether it recognized the same amino acids as the clinical antibodies. This monoclonal antibody SZ21 can distinguish between the HPA-1a and HPA-1b forms of GPIIIa under certain conditions. We used the immunoblot conditions of Weiss et al19 to discriminate the HPA-1a form from the HPA-1b form of human GPIIIa. When compared with clinical antibodies, our immunoblot results show that the SZ21 monoclonal antibody has a similar pattern of recognition of the substitution mutations with one notable difference (Fig 4A). The substitution at position 39 is not important in the antigenic determinant of this antibody. Unlike the clinical antibodies (Fig 4B), substitution mutations m3a32,33,39 (lane e) and m3a22,32,33,39 (lane g) show no immunoreactivity with SZ21 (Fig 4A). Furthermore, the SZ21 signal (Fig 4A) from mutation m3a30,32,33 (lane d) is as intense as any of the reactive proteins, indicating that neither position 22 nor 39 is key to this epitope. This finding is consistent with published reports of the SZ21 binding site (residues 28 to 35 of human GPIIIa) for this antibody.10
The objective of this study was to identify critical elements in human GPIIIa that generate a conformation recognized by anti-HPA-1a antibodies. By revealing the binding requirements of the anti-HPA-1a antibodies, we may be able to regulate or neutralize those antibodies that are problematic in NAIT and PTP in the future. We have taken advantage of the fact that murine GPIIIa is similar to the human protein, yet not reactive to anti-HPA-1a antibodies. By comparing the two sequences, residues that are important in the development of the antigen conformation were identified. Having narrowed the immunoreactive region in the human GPIIIa N-terminus to 42 amino acids that include residues 9 to 50, we concentrated on the six differences between the mouse and human sequence that occur within this epitope-containing domain.
We thank Ruth M. Graupera for subcloning the deletion mutations into the pGEX expression vector.
Submitted July 27, 1998; accepted December 21, 1998.
Supported by a National Blood Foundation grant, American Heart Established Investigator Grant No. 9740238N, and National Institutes of Health Grant No. HL-59955. G.N. was supported by a training fellowship from the University of Milan, Italy.
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.
Presented in part at the American Society of Hematology meeting, December 5-9, 1997, San Diego, CA. Address reprint requests to Emily A. Barron-Casella, PhD, Division of Hematology/Department of Pediatrics, Johns Hopkins University School of Medicine, 720 Rutland Ave, Ross Bldg, Room 1129, Baltimore, MD 21205; e-mail: ebcasell{at}jhmi.edu.
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23:4530, 1995 17. Aiyar A, Leis J: Modification of the megaprimer method of PCR mutagenesis: Improved amplification of the final product. Biotechniques 14:366, 1993[Medline] [Order article via Infotrieve] 18. Kim HO, Kennedy SD, Kickler TS: Studies using immobilized platelet glycoproteins for detection of platelet alloantibodies. Am J Clin Pathol 104:258, 1995[Medline] [Order article via Infotrieve] 19. Weiss EJ, Goldschmidt-Clermont PJ, Grigoryev D, Jin Y, Kickler TS, Bray PF: A monoclonal antibody (SZ21) specific for platelet GPIIIa distinguishes PlA1 from PlA2. Tissue Antigens 46:374, 1995[Medline] [Order article via Infotrieve]
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  N. A. Watkins, E. Schaffner-Reckinger, D. L. Allen, G. J. Howkins, N. H. C. Brons, G. A. Smith, P. Metcalfe, M. F. Murphy, N. Kieffer, and W. H. Ouwehand HPA-1a phenotype-genotype discrepancy reveals a naturally occurring Arg93Gln substitution in the platelet beta 3 integrin that disrupts the HPA-1a epitope Blood, March 1, 2002; 99(5): 1833 - 1839. [Abstract] [Full Text] [PDF]
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TOP
ABSTRACT
INTRODUCTION
and purified under the standard, nondenaturing protocol given for pGEX
expression systems (GST Gene Fusion System manual; Pharmacia Biotech,
Piscataway, NJ). In the midst of these experiments, we found that the
amount of contaminating GST protein decreased and the amount of
specific fusion protein increased, when the bacterial cultures were
grown at 30°C. To insure the proper folding of the proteins, the
purified proteins were incubated in a mixture of oxidized and reduced
glutathione as previously described.
3.
Blood
86:234, 1995