sequence of this clone indicates that it spans the light-heavy chain junction of .... platelets but failed to immunoprecipitate the related endothelial cell cytoadhesin ...
Proc. Nati. Acad. Sci. USA Vol. 84, pp. 7114-7118, October 1987 Cell Biology
Molecular cloning and chemical synthesis of a region of platelet glycoprotein Hib involved in adhesive function (membrane proteins/cytoadhesins/conformational change/monoclonal antibody/cDNA)
J. C. LOFTUS*t, E. F. PLOW*, A. L. FRELINGER III*, S. E. D'SOUZA*, D. DIXON*, J. LACY*, J. SORGEt, AND M. H. GINSBERG* *Department of Immunology and tDepartment of Basic Clinical Research, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037
Communicated by Ernest Beutler, June 8, 1987 (received for review March 30, 1987)
ABSTRACT Membrane glycoprotein (GP) fIb-IIIa is a component of a platelet adhesive protein receptor. A region of the heavy chain of GPIIb, defined by the monoclonal antibody PMIH1, is involved in adhesion receptor function. We have localized and chemically synthesized this region of GPIIb. A cDNA clone that directs the synthesis of a fusion protein reactive with the PMI-1 antibody was isolated from a phage Xgtl1 expression library constructed with mRNA from an erythroleukemia (HEL) cell line. The deduced amino acid sequence of this clone indicates that it spans the light-heavy chain junction of GPIIb and contains a portion of the carboxyl terminus of the heavy chain and the amino terminus of the light chain. The PMI-1 epitope was found to be contained within a 9-kDa staphylococcal V8 protease fragment of GPIIb, and such a fragment was predicted within the putative heavy-chain sequence. A computerized antigen prediction program identifled a single sequence with a high probability of containing a continuous epitope. A synthetic 17-residue peptide containing this sequence binds PMI-1 and inhibits PMI-1 binding to GPIIb-IIIa. The peptide-antibody complex has an approximate Kd of 1.2 #uM, which compares to a Kd of 0.95 jzM for PMI-1 binding to GPIIb. The region containing the PMI-1 epitope shows no similarity to corresponding regions of two other adhesion receptors, indicating that this portion of GPIIb may function in activities unique to the platelet receptor.
13 subunit combines with a number of distinct a subunits. One group is found on leukocytes and has been referred to as the LFA-1, Mac-1, p150,95 family (10). The other is more widely distributed and has been referred to as the VLA family (13). The P subunit of the VLA family (13) in the chicken has been cloned and sequenced and was designated "integrin" (14). The sequence of integrin is similar to that of GPIIIa (15) and to the ,3 subunit of the leukocyte family (16). Moreover, partial sequences of several a subunits also indicate similarities (12, 17, 18). Thus, GPIIblIa is a member of a widely distributed superfamily of adhesion receptors. The sites on GPIIb-IIIa, or the other cytoadhesins, that are crucial for their functions as adhesion receptors are presently unknown. Several observations suggest that a functionally significant site on GPIIb-IIIa is near the epitope defined by the monoclonal antibody PMI-1. This antibody binds to the heavy chain of GPIIb (19) and defines a region of GPIIb that is associated with four distinct functional activities. First, PMI-1 inhibits platelet adhesion to collagen (19). Second, the surface orientation of this region is regulated by divalent cations as millimolar concentrations of calcium or magnesium suppress expression of the PMI-1 epitope (20). Third, abnormal divalent cation regulation of the conformation of this site is associated with a functional thrombasthenic state (20). Finally, the interaction of Arg-Gly-Asp-containing ligands with GPIIb-IIIa alters the conformation of this region, resulting in increased exposure of the PMI-1 epitope (21). We report here the molecular cloning and sequence of cDNA encoding the region of GPIIb containing the PMI-1 epitope. In addition, we report the chemical synthesis of a peptide containing the epitope. Finally, we report the predicted amino acid sequence of the light-heavy chain junction of GPIIb and statistically significant sequence homologies between this protein and the a subunits ofthe VnR and FnR.§ common
Cell adhesion generally involves recognition of specific adhesive proteins by cell-surface receptors. Recent evidence suggests that several adhesion receptors share the functional property of recognizing the three amino acid sequence Arg-Gly-Asp (1, 2). This group of related receptors includes the vitronectin receptor (VnR) and fibronectin receptor (FnR) (3, 4) and the platelet membrane glycoprotein Ilb-Ila (GPIIb-IIIa) complex. GPIIb-IIIa contributes to platelet function through interactions with Arg-Gly-Asp-containing proteins such as fibrinogen (5), fibronectin (6), and von Willebrand factor (7) and therefore is a component of a common platelet adhesive protein receptor (1, 8). GPIIb-IIIa is a noncovalent Ca2+-dependent heterodimer complex (9). The VnR (4) and other adhesion receptors (10) possess a similar gross subunit structure of noncovalently associated a and ,B subunits. The similar functional, structural, and antigenic properties of these proteins suggest they are members of an adhesion receptor family for which the designation "cytoadhesin" has been proposed (11). Within this family, distinct a subunits combine with a common or very similar l3 subunit, resulting in functionally distinguishable receptors (12). Strikingly, there exist at least two other groups of heterodimeric adhesion receptors in which a
MATERIALS AND METHODS Materials. The human erythroleukemia, HEL, cell line (22) was obtained from T. Papayanopoulou (University of Washington, Seattle, WA). The polyclonal anti GPIIb-IIIa antiserum was prepared as described (11). Production of the monoclonal antibody PMI-1 has been described in detail (19). The monoclonal antibody Tab (23) was generously provided by Rodger McEver (University of Texas, San Antonio). EcoRI-predigested Xgtll arms and high-efficency X phage Abbreviations: VnR, vitronectin receptor; FnR, fibronectin receptor.
tTo whom reprint requests should be addressed.
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. §1734 solely to indicate this fact.
§This sequence is being deposited in the EMBL/GenBank data base (Bolt, Beranek, and Newman Laboratories, Cambridge, MA, and Eur. Mol. Biol. Lab., Heidelberg) (accession no. J02963).
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packaging extracts (Gigapack) were obtained from Stratagene Cloning Systems (San Diego, CA). cDNA Library Construction and Antibody Screening. Total cellular RNA was prepared from HEL cells by the guanidinium isothiocyanate/cesium chloride method (24). Double-stranded cDNA was synthesized from 10 ug of HEL poly(A)+ RNA using avian myeloblastosis virus reverse transcriptase (Life Sciences, St. Petersburg, FL) and random oligodeoxynucleotide primers according to standard procedures (25). cDNA was ligated into the EcoRI site of Xgtll (26) and packaged into phage according to the manufacturer's protocol. Screening of the library with the rabbit polyclonal GPIIb-IIIa antiserum was performed as described (27) using the Vectastain ABC method (Vector Laboratories, Burlingame, CA). Affinity purification of antibodies on insert-encoded polypeptides was performed according to the method of Weinberger et al. (28). Surface labeling of cells and immunoprecipitations were performed as described (11). The immunoreactivity of the PMI-1 with the fusion proteins of positive clones was determined by immunoblotting nitrocellulose transfers (29). DNA Sequencing and Sequence Analysis. Recombinant phage DNA from positive clones was digested with EcoRI and subcloned into M13mpl8. The DNA sequence for each strand was determined by the method of Sanger et al. (30) using the specific primer-directed DNA sequencing method (31). DNA sequences and the amino acid sequences were analyzed with the University of Wisconsin Genetics Computer Group (UWGCG) Sequence Analysis Software Package (32). The degree of similarity in the amino acid sequences of GPIlb and VnR a (17) was examined using the Relate program for detecting distant relationships (33). Sequence searches were performed using the National Institutes of Health Genbank and European Molecular Biology Laboratory (EMBL) nucleotide sequence data bases, and the National Biomedical Research Foundation (NBRF) protein sequence data base. 11 Peptides. Based on the amino acid sequence deduced from the nucleotide sequence, synthetic peptides were synthesized on an ABI 430A peptide synthesizer. Amino acid analysis of each peptide was consistent with the desired sequences. Predictions of antigenicity from protein primary structure were calculated using the algorithm of Wolf et al. (34). Anti-peptide antibodies were elicited by immunization of rabbits with peptides coupled with glutaraldehyde to thyroglobulin (bovine type 1; Sigma) (35). Anti-peptide antibodies were detected in an ELISA according to the method of Engvall (36). Specificity of the anti-peptide antibodies for GPIIb was determined by immunoblotting (29) purified GPIIb-IIIa. Protease Digestion. GPIIb-IIIa was purified from solubilized platelet membranes by HPLC on a TSK4000 column in a buffer containing 1% NaDodSO4, 10 mM thioglycolate, and 10 mM sodium acetate (pH 7.3). Samples containing GPIIb were acetone-precipitated and reconstituted to 0.4 mg/ml in 0.01 M phosphate/0.15 M NaCl containing 1% NaDodSO4. Staphylococcal V8 protease (Cooper Biomedical, Malvern, PA) was added to 20 tkg/ml and the enzyme-substrate mixture was incubated at 37°C. At various time points, aliquots were removed, mixed with reducing gel sample buffer, electrophoresed on a 10-20% gradient NaDodSO4/ polyacrylamide gel, transferred to nitrocellulose (29), and probed with PMI-1.
Proc. Nat. Acad. Sci. USA 84 (1987)
Platelet Preparation and Binding Assays. Platelets were isolated from acid citrate dextrose anticoagulated human blood as described (37). PMI-1 was radioiodinated as described (38). The effect of peptides on the binding of PMI-1 to platelets was determined in a competitive binding assay (39). Direct binding ofPMI-1 to the synthetic peptides and the effect of the peptides on the binding of PMI-1 to purified GPIIb-IIIa in detergent solution was determined by arn ELISA (36). Microtiter plates were coated for 1 hr at 37°C with GPIIb-IIIa (20 ,ug/ml), peptides V-41 or V-43 (20 ,uM), or bovine serum albumin blocked with excess albumin (5%, wt/vol), and underwent reaction with either PMI-1 or the monoclonal antibody Tab. For competitive ELISA, the competing protein or peptide was added to the well before adding the primary antibody. Binding was assayed in the presence of either 5 mM EDTA or 2 mM calcium.
RESULTS Identification of cDNA Clones. Initial screening of 200,000 recombinants with the rabbit polyclonal GPIIb-IIIa antiserum identified six immunoreactive clones. One of these clones, HEL41, contained a 1.1-kilobase insert, which directed the synthesis of a bacterial fusion protein -140 kDa with characteristics consistent with it possessing GPIIb epitopes. These characteristics included (i) reactivity of the polyclonal serum with the fusion protein encoded by this clone was blocked by prior absorption with purified GPIIb-IIIa: (ii) antibodies affinity-purified on the fusion protein reacted specifically with GPIIb in immunoblots of platelet lysates and -purified GPIIb-IIIa but failed to react with VnR a (12); and (iii) the affinity-purified antiserum immunoprecipitated GPIIb-IIIa from surface-labeled platelets but failed to immunoprecipitate the related endothelial cell cytoadhesin (Fig. 1). In addition, the fusion protein encoded by HEL41 was immunoreactive with the monoclonal antibody PMI-1 (Fig. 2). Determination of Nucleotide Sequence. The cDNA sequence (Fig. 3) consists of an open reading frame of 682 bases with a TAG termination codon at position 683 followed by 419 bases of 3' untranslated sequence. The first 120 nucleotides at the 5' end of the clone possess the characteristics of the consensus Alu repetitive sequence (40). Computer search revealed no significant identity between HEL41 coding sequence and other nucleotide sequences present in the GenBank and EMBL data bases as of March 1987. Amino Acid Sequence. The cDNA sequence of clone HEL41 encodes an open reading frame of 227 amino acids (Fig. 3). The determined N-terminal sequence for the light chain of GPIIb, as reported by Charo et al. (18), can be identified within the clone as the sequence between L(157) and T(171). Note that the consensus Alu repetitive sequence translates in frame and accounts for the first 40 amino acids encoded by HEL41. To investigate whether this sequence
a&-M~a aflb
aft-a Ab FIG. 1. Lack of reactivity of endothelial cell cytoadhesion with anti-GPIIb. Lysates of surface radioiodinated platelets or endothelial cells were immunoprecipitated with polyclonal anti-GPIIb-IlIa (alib-II1a) or antibodies affinity purified on the HEL41-encoded fusion protein (aIlb). Immunoprecipitates were analyzed on NaDod-
IEMBL/GenBank Genetic Sequence
Database (1986) GenBank (Bolt, Beranek, and Newman Laboratories, Cambridge, MA), Tape Release 3.0. 1Protein Identification Resource (1986) Protein Sequence Database (Natl. Biomed. Res. Found., Washington, DC), Release 2.0.
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der reducing conditions. Autoradiograms are shown.
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Proc. Natl. Acad. Sci. USA 84 (1987)
portion of GPIIIb and the VnR a subunit (17) detected regions of similarity, a.llowing for conservative substitutions. Evaluation of the sstatistical significance of the relationship betweoen the
