Connective Tissue Research, 1992, Vol. 28, pp. 13-28 Reprints available directly from the publisher Photocopying permitted by license only 0 1992 Gordon and Breach Science Publishers S.A. Printed in the United States of America
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MAPPING OF BINDING SITES FOR MONOCLdONAL ANTIBODIES TO CHICK TROPOELASTIN BY RECOMBINANT DNA TECHNIQUES VALERIA MARIGO, ARIANNA SITTA, DIN0 VOLPIN, and GIORGIO M. BRESSAN Institute of Histology and Embryology of the University of Padova, Via Trieste, 75, 35100 Padova, Italy (Received May 7 , 1991; in revised form October 8 , 1991; accepted October 16, 1991)
A fusion molecule consisting of the entire coding sequence of mature chicken tropoelastin preceded by 14 amino acids of the signal peptide and 9 amino acids of vector origin has been expressed in a recombinant bacterial system and purified. The molecule has been used as immunogen for the production of hybridomas. Monoclonal antibodies which bound specifically the immunogen were also reactive with tropoelastin purified from chick aorta and stained elastic fibers in aorta sections by immunofluorescence. The region of tropoelastin containing the antigenic determinant recognized by each antibody has been identified by a recombinant DNA expression strategy based on the use of cDNA clones spanning different portions of the coding sequence. It could be shown that several antibodies were directed against unique epitopes; among these, a group of antibodies bound specifically to the sequence (PGVGV),. Other antibodies were found to recognize antigenic determinants present more than once in the molecule. The monoclonal antibodies thus characterized will be useful reagents in studying the function of the different domains of tropoelastin. KEYWORDS: tropoelastin, recombinant protein, monoclonal antibodies, epitope mapping
INTRODUCTION As most extracellular matrix proteins, tropoelastin, the precursor of the main component of elastic fibers of connective tissues, is a multidomain protein. Initial sequence data of the protein have established the existence of two types of regions, one very hydrophobic, the other composed of stretches of alanines intercalated by lysine residues.' More recent sequence information from different species has shown that several different hydrophobic and lysine-containing repeats alternate in the whole molecule.2-5 In addition, a unique carboxyterminal domain, including two cysteine residues and terminating with the sequence arginine-lysine-arginine-lysine, has been detected in all tropoelastins. Finally, an unusual hydrophylic region has been identified in the human tropoelastin gene.4 Despite the wealth of data on the primary structure of tropoelastins, the knowledge of the function of the various repeats is only partial. The general agreement is that the alanine- and lysine-rich regions are involved in the formation of intermolecular crosslinks, whereas the hydrophobic regions confer elastic properties to the rnolecule.1.2 However, other functions could be performed by hydrophobic sequences, as indicated by the finding that one of these, formed by a repeating Address for correspondence: Dr. Din0 Volpin, Institute of Histology and Embryology, Via Trieste, 75, 35100 Padova, Italy. 13
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hexapeptide, is the target for a cellular receptor and is chemotactic for various cells.6.7 Moreover, it is possible that tropoelastin is involved in multiple interactions with itself and other molecular components of the elastic fiber.8 A significant contribution to the analysis of the different domains of some extracellular proteins has been obtained by the use of specific antibodies. The production of these antibodies has been achieved by two main procedures: polyclonal antibodies have been raised against defined oligopeptides chosen on the basis of sequence information of the molecu1e;g alternatively, monoclonal antibodies have been obtained by immunization of animals with the whole protein (or protein digests) and the regions recognized have been variously determined.10 In an attempt to develop multiple defined probes for chick tropoelastin, we have produced several monoclonal antibodies, and the region(s) they recognize on the molecule has been mapped with variable precision using a procedure based on the expression of different segments of the cDNA as fusion proteins in bacteria.
MATERIALS AND METHODS Purification of the Immunogen Recombinant chick tropoelastin has been used as immunogen. The BamHI fragment of Clone pEX1-TE72 was subcloned in the sense orientation in plasmid pCIPR (Fig. l), giving rise to the construct pCIPR-TE7. Expression in bacterial cultures was induced as described below. At the end of the induction period the cells were harvested by centrifugation, resuspended in 1/50volume of TEN buffer (50 mM Tris-HC1 pH 8.0,lOO mM NaC1,lO mM EDTA) and lysed by 20 cycles of sonication with a Soniprep 150 (Branson) set at % of maximal power. Each cycle lasted 30 seconds and was alternated with 1 min incubation of the sample on ice. The pellet obtained by centrifugation at 10,OOO x g for 30 min was resuspended in 1 M guanidine, 1 M NaCl, 1% Triton X-100 and centrifuged. This procedure was repeated, the pellet washed twice with TEN buffer and solubilized by boiling in 1-1.5 ml of 0.01M Na phosphate buffer pH 7.2,5% SDS, 5% P-mercaptoethanol. The sample was then fractionated by gel filtration through Sephacryl S-300 Superfine (Pharmacia) in 0,l M phosphate buffer pH 7.2 containing 1% SDS. The fractions obtained were analyzed by PAGE-SDS and those containing the immunogen were treated with acetone in order to remove SDS.11The precipitated protein was resuspended in a small volume (usually 0.2 ml) of 8 M urea and quantitated.'* Production of Hybridomas Hybridomas were produced as previously described.13 Just before injection, the urea concentration of the immunogen solution was lowered by dilution (10-20 x) with phosphate buffered saline. Initial screening of hybridomas was performed by reacting, in ELISA, the culture supernatants with the immunogen and, in parallel, with E. Coli extracts. Positive hybridomas were further confirmed by ELISA with tropoelastin purified from chick aortas.14 For preparation of E. Coli extracts, the cells from 800 ml of overnight culture were harvested by centrifugation at 5,000 X g for 20 min and resuspended in 8 ml of 50 mM TrisHCl pH 8.0,50 mM EDTA, 15% sucrose. After addition of 2 ml of 10 mg/ml lysozyme and
ng linker
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FIGURE 1 Physical map of bacterial expression vectors used in the present study. P,: right promoter of lambda phage; cI857: thermosensitive mutant of the CIgene of lambda phage; cro: region coding for the first 9 amino acids of the cro gene of lambda phage; Amp? ampicillin resistance gene.
Amp'
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incubation on ice for 40 min, the cells were lysed by adding 24 ml of 0.1% Triton X-100 in 50 mM Tris-HC1pH 8.0 and leaving 15 min on ice. The supernatant recovered after centrifugation at 25,000 X g constituted the concentrated E. Coli extract.
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Isolation and Characterization of cDNA Clones Several clones for chick tropoelastin have been isolated from a random-primed cDNA aorta library in pEXl by antibody screening as previously described.2 Two additional clones spanning the amino terminal end of tropoelastin were obtained by cloning the BamHI-PstI and BamHI-Sau3A fragments of pEX1-TE7 into pEX1. The ends of all the clones were sequenced by the dideoxy-chain termination method15 and the polypeptide coded by each clone deduced from the published sequence of chick tropoelastin cDNA.2 Only clones whose deduced length was in agreement with the size determined by agarose gel electrophoresis were considered. The size expected for the encoded polypeptides was confirmed by PAGE-SDS analysis of expressed fusion proteins. Expression and Purification of Fusion Proteins The procedure for fusion protein expression followed that described by Stanley and collaborators16 and was similar for both the pCIPR-TE7 clone used to produce the immunogen and the different pEXl clones isolated from the cDNA library. The only difference concerned the type of E. Coli strain used, DH5 and pop 2136 for pCIPR-TE7 and pEXl clones respectively. Overnight cultures of the different clones grown at 30°C were diluted 1:lOO in fresh LBmedium (usually 400 ml for pCIPR-TE7 and 30 ml for pEXl clones) and incubated at 30°C. After 2 h, the temperature was shifted to 42°C to allow induction of expression and the cells incubated for additional 2 h. The incubation was terminated by cooling the cell suspension on ice for 10 min. Further manipulation of the cells containing pCIPR-TE7 has been already detailed above (see: purification of the immunogen). The partial purification of the proteins expressed by the pEXl clones was performed as follows. The cell pellet obtained by centrifugation at 6,000 X g was resuspended in 2 ml of TEN buffer, recentrifuged and recovered in 0.2 ml of the same buffer. The bacteria were then lysed by successive addition of i) 50 pl of lysozyme (10 mg/ml) and incubation on ice for 15 min; ii) 6 p1 of DNase I (10 mg/ml) and 2 p l of 1 M MgSO, and incubation for 15 min on ice; iii) 90 pl of 10 mM TrisHCl, pH 7.4 containing 0.1 M NaCl, 1% Triton X-100 and 0.5% Na deoxycolate and incubation for 20 min on ice. The pellet obtained by centrifugation, which contains mainly bacterial inclusion bodies, was then extracted twice with 0.6 ml of 1 M guanidine, 1 M NaCl, 1% Triton X-100, washed two times with TEN buffer (0.6 ml) and finally dissolved in 0.2 ml of 8 M urea and the protein concentration measured. Preparation of Medium Proteins From Cultured Aorta Cells Chick embryo aorta cells were prepared as described” and cultured in 90 mm dishes in Dulbecco modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum. When the culture was confluent, the medium was removed, the cells washed with DMEM and incubated with 1 ml of DMEM without serum for 16 h. The medium was harvested and proteins precipitated with cold 15% trichloroacetic acid (TCA). The precipitate was successively washed with 15% TCA, 80% ethanol, ether and finally dissolved in 100 p1 of 62.5
TROPOELASTIN EPITOPE MAPPING
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mM Tris-HC1 pH 6.8, 2% SDS, 10% glycerol, 5% P-mercaptoethanol and 0.01% bromophenol blue.
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Immunofluorescence Frozen sections, 5 Fm thick, were cut from aortas from 2 day old chicks. The sections were incubated for 2 h at room temperature with first antibody, consisting of either hybridoma supernatants, or a polyclonal antiserum to chick tropoelastinlg diluted 1:100, or a similarly diluted preimmune serum. After washing in phosphate buffered saline (PB:S), the slides were incubated for 1 h with the appropriate fluorescinated antibody (goat anti.-mouse IgG, Miles Laboratories Inc. ;goat anti-rabbit IgG, Diagnostic Pasteur), washed, mounted in 90% glycerol in PBS and examined in a Zeiss microscope equipped with epifluorescence optics. Other Immunologic Assays ELISA was performed following standard procedures.19 100 to 200 ng of protein were usually plated per well. For immunodetection of proteins blotted on nitrocellulose filters20 an alkaline phosphatase-based system was used: after incubation with alkaline phosphataseconjugated second antibody and washing, the filters were immersed in 100 mM:Tris-HC1 pH 9.5, 100 mM NaCl, 5 mM MgC1, containing 0.33 mg/ml nitroblue tetrazolium and 0.165 for a few minutes. mgiml 5-bromo-4-chloro-3-indonyl-phosphate
RESULTS Figure 1 shows the physical map of the expression vectors used in this study. pCIPR (Fig. l), an intermediate in the construction of pUEX vectors,21 allows the production of fusion proteins in which the contribution of vector sequences is limited to the first 9 amino acids, which are derived from the lambda cro gene. In contrast, a large portion of the fusion proteins synthesized from pEXl (Fig. 1) is encoded by plasmid sequences (cro + LacZ).16 Recombinant chick tropoelastin was obtained by expressing the previously characterized clone TE72 in pCIPR. The clone contains the entire sequence of mature tropoelastin and a portion of the signal peptide. The expected size of the recombinant protein is about 65 KD and was in good agreement with the electrophoretic migration of the majoir polypeptide produced after induction of expression (Fig. 2A). A direct proof that this band was a protein related to tropoelastin was obtained by Western blotting analysis of bacterial proteins, showing that the 65 KD component was recognized by an antiserum raised against tropoelastin purified from chick aortas (Fig. 2B). Purification of recombinant trolpoelastin was achieved by gel filtration chromatography as reported in Figure 3 . The final yield of protein with a degree of purity shown in the inset of Figure 3 was about 3 mg per liter of bacterial culture. The purified recombinant tropoelastin was used to raise monoclonal antibodies in mice. As a first step in the characterization of the antibodies, their reactivity with the: recombinant molecule and with tissue tropoelastin was compared. All the hybridomas which reacted specifically with the recombinant protein in ELISA were also positive when assayed against tropoelastin purified from chick tissues (data not shown). Moreover, as evaluated by inspection of Figure 4, the relative intensity of signal obtained in Western blots with the
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FIGURE 2 Analysis of recombinant tropoelastin expressed with the pCIPR vector. A: proteins solubilized by boiling the bacterial cell pellet in the presence of 2% SDS were resolved by electrophoresis in a 7.5% SDSpolyacrylamide gel and stained with Coomassie blue R 250. B: Western immunoblots of total protein reacted with an antiserum specific for chick tropoelastin. U: uninduced cells grown at 30°C; I: cells induced by incubation at 42°C. Arrows point out the position of recombinant tropoelastin. BSA: migration of bovine serum albumin.
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FIGURE 3 Purification of recombinant tropoelastin by gel filtration through Sephacryl S-300. Individual fractions were analyzed by SDS gel electrophoresis in order to identify the fractions containing tropoelastin (horizontal bar). V, = void volume and V, = total volume of the column. Inset: electrophoretic pattern of proteins of two of the fractions in which tropoelastin (arrow) was the predominant band. Numbers on the right indicate the mobility of molecular weight markers.
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FIGURE 4 Comparison of the pattern of reactivity of the different monoclonal antibodies against recombinant and native tropoelastin by Western immunoblot analysis. Aliquots of the cell extract obtained from induced bacterial cells as described in the legend to Figure 2 (A), or of the TCA precipitate of the medium from cultured chick embryo aortic cells (B) were subjected to SDS gel electrophoresis, blotted onto nitrocellulose filters and discrete strips reacted with individual monoclonal antibodies as indicated. BSA: migration of bovine serum albumin.
different antibodies was similar when either the recombinant protein or tropoelastin released in the medium by cultured chick embryo aorta cells was used. When assayed by immunofluorescence on chick aorta sections, all the antibodies stained fibrils in a way similar to a rabbit polyclonal antibody produced against purified chick aorta tropoelastinls (Fig. 5). Further characterization of the monoclonal antibodies was obtained by delimiting the region of the tropoelastin molecule containing the epitope recognized. This was effected by determining the reactivity of each antibody against the recombinant protein expressed by cDNA clones spanning different portions of the coding region. As shown in Figure 6 for some of the cDNA clones, the recombinant proteins were efficiently expressed by the pEX vector. The proteins were the major polypeptide species present in bacterial extracts, and significant proteolysis was not found in most clones, or, when present, it gave rise to only a few discrete bands just below the undegraded product.
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TROPOELASTIN EPITOPE MAPPING
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FIGURE 5 Localization of immunoreactive sites in 2 day old chick aorta by immunofluorescence. The first antibody used was: a rabbit antiserum against chick tropoelastin (A); rabbit preimmune serum (B); monoclonal antibody V95D6 (C); monoclonal antibody V94G12 (D). The patterns of immunofluorescence of C and D are representative of the distribution observed with the different monoclonal antibodies. Bar = 10 pm.
Examples of mapping of the regions recognized by the antibodies are reported in Figure 7. The pattern of Figure 7A was identical for a group of seven antibodies out of 17 analyzed. The region common to all the cDNA clones which reacted with the antibodies, could be easily delimited between the end of clone TE24 and the beginning of clone TE29, which were both negative. This region includes the coding sequence of amino acids 42.7-451 of the mature protein as deduced from clone TE7,2 and consists of five repeats of the lpentapeptide PGVGV Formal proof that this was indeed the sequence recognized by the antibodies was obtained by an ELISA in which the synthetic polypentapeptide (PGVGV), was used as substrate: only the seven antibodies previously identified produced a significant signal (Table I). The region recognized by antibody V91B1O could be restricted within a short unique sequence, delimited by the region of overlapping of clones 4A12 and 6B21 (amino acids 622-632) (Fig. 7B). However, this was not the case for several antibodies, which gave a positive signal also with the protein expressed by cDNA clones which did not overlap. A typical example is shown in Figure 7C: the limits at the amino- and carboxy-terminal end are given by the end of clone TE13 and the beginning of clone TE29 respectively l(amino acids 281-450); two positive clones spanning partially this region, TE19 and TE24, did not share common sequences. A more complex reactivity was manifested by the antibody of Figure
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-116KD
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FIGURE 6 Analysis of protein expressed by a group of cDNA clones. After induction at 42”C, inclusion bodies were prepared as described under “Methods”, run in a 6% SDS-polyacrylamide gel and protein bands visualized by Coomassie blue staining. Dots at the right of the lanes mark the undegraded fusion protein, whose size was in agreement with the value expected from the coding sequence contained in the clone. Migration of molecular weight standards is indicated on the right.
7D. In this case, two discontinuities between positive clones could be identified: between TE19 and TE24 and between TE24 and TE29. Additional characterization of the antibodies was obtained by investigating species specificity by indirect ELISA using alpha elastin from chick and human aorta and from bovine legamentum nucae (Fig. 8). The majority of the antibodies reacted with bovine and human alpha elastin; some, however, were specifically inhibited only by the chicken protein.
DISCUSSION The purpose of this work was the production of monoclonal antibodies with specificity against different regions of the tropoelastin molecule. We also wanted to explore the possibility of substituting native tropoelastin with recombinant tropoelastin in immunological investigations, given the fact that the latter represents an easy and efficient source of
TROPOELASTIN EPITOPE MAPPING lE7
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FIGURE 7 Mapping of antibody binding sites in tropoelastin. The protein expressed by the cDNA clones indicated was reacted with the monoclonal antibodies in direct ELISA. Positive clones (thick lines, p < 0.1) were identified by comparing triplicate values obtained with each antibody with those obtained with a control monoclonal antibody (specific for human chorionic gonadotropin) using the Student’st test. The figure reports only a few representative examples. The pattern of reactivity shown in A is common to the 7 monoclonal antibodies which bind the polypentapeptide repeat of tropoelastin (see Table I). B: reactivity of antibody V91B10. C: reactivity of antibodies V94G12 and V95B3. D: Reactivity of antibody V81A5. The scale is calibrated in amino acids, where number 1 is the first residue of the mature protein.
protein. The results indicate that, at least for the type of assays that have been performed in this study, native and recombinant tropoelastin are, indeed, interchangeable. Thus, an antiserum specific for chick aorta tropoelastin recognized the recombinant protein and, conversely, the monoclonal antibodies raised against the bacterial product reacted in very similar ways with the two proteins. In addition, all the monoclonal antibodies gave the immunofluorescence pattern expected for aorta elastic fibers, indicating that they bind to native elastin. The data also indicate that the presence of 23 additional amino acids (9 from the cro sequence and 14 from the signal peptide of tropoelastin) at the amino-terminal end of the mature protein does not grossly alter the immunologic properties of recombinant versus native tropoelastin. Definition of the regions containing the epitope for the monoclonal antibodies has been based on the expression of recombinant cDNA clones. This strategy has been shown to be very effective in mapping antigenic determinants of other pr0teins.22~23In order to be reliable, the technique requires that the protein are expressed very efficiently and that they
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TABLE I Reactivity of the monoclonal antibodies against the synthetic polypentapeptide (PGVGV), as determined by ELISA
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Antibody Controlc V81A5 V87F7 V90F9* V91B10 V92B4 V92B8 V93B5 V93B6 V93C2* V94B8* V94G 12 v95c5* V95D6* V95E11* V95G6 V92B2* V96C4
Mean O.D.,
2 sda
0.011 2 0.008 0.024 t 0.005 0.027 2 0.005 0.149 2 0.013 0.019 ? 0.005 0.063 2 0.050 0.026 2 0.005 0.016 t 0.015 0.009 2 0.012 0.269 ? 0.027 0.172 ? 0.005 0.033 t 0.006 0.381 2 0.021 1.938 ? 0.016 0.369 2 0.010 0.018 ? 0.006 0.168 ? 0.006 0.028 ? 0.011
Significanceb p > 0.1 p > 0.1 p < 0.001 p > 0.1 p > 0.1 p > 0.1 p > 0.1 p > 0.1 p < 0.001 p < 0.001 p > 0.1 p < 0.001 p < 0.001 p < 0.001 p > 0.1 p < 0.001 p > 0.1
%obtained from triplicate determinations. sd = standard deviation bvalues obtained with the test and the control antibody were compared by the Student’s t-test =the control antibody was a monoclonal against human chorionic gonadotropin *antibodiespredicted to react with the polypentapeptide sequence of tropoelastin on the basis of the analysis shown in Figure 7
are not degraded by bacterial proteases: both conditions have been verified in our experiments. In addition, applicability of the technique in the case of tropoelastin was assessed with antibodies which recognize a region containing the repeating sequence (PGVGV),, by showing that only the group of antibodies identified by the analysis with recombinant clones reacted with the synthetic polypentapeptide. Our results indicate that the different domains of tropoelastin are not similarly antigenic. Thus, the group of antibodies specific for the polypentapeptide accounts for about one third of total antibodies, a very high proportion considering that the sequence (PGVGV), constitutes only about 7% of tropoelastin. This suggests that the polypentapeptide sequence is a major antigenic determinant of chick tropoelastin. On the contrary, no antibodies directed against the amino-terminus of the molecule were obtained. These differences could be due to either a variability of intrinsic antigenicity of the various sequences or to the preferential exposure of some regions at the surface of the molecule. Excepting the regions mentioned above, the epitopes for the remaining antibodies were apparently evenly distributed in the molecule. Figure 9 summarizes the results of antigenic region mapping obtained with several monoclonal antibodies. It is apparent that different classes of antibodies could be distinguished. One class comprised the antibodies recognizing an epitope which is certainly unique in tropoelastin. Examples of this class were the antibodies directed against the polypentapeptide repeat region (amino acids 425-477). The sequence (PGVGV),, which should be long enough to accommodate for a linear epitope (unfoldon) sequence,24 is present in two other positions in the molecule (amino acids 338-347 and 606-615); nevertheless,
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FIGURE 8 Species reactivity of the monoclonal antibodies. Inhibition ELISA was performed using chick alpha elastin as immobilized substrate and alpha elastin from chick (A), human (B) and bovine (C) as inhibitor protein. V95G6: A; V92B4: A. V91B10: 0 ; V95Ell: 0 ; V94G12: 0 ; V95D6:
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FIGURE 9 Map of the regions of tropoelastin containing the antigenic sites identified by different monoclonal antibodies. Scale is defined as in Figure 7.
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MAPPING OF BINDING SITES FOR MONOCLdONAL ANTIBODIES TO CHICK TROPOELASTIN BY RECOMBINANT DNA TECHNIQUES VALERIA MARIGO, ARIANNA SITTA, DIN0 VOLPIN, and GIORGIO M. BRESSAN Institute of Histology and Embryology of the University of Padova, Via Trieste, 75, 35100 Padova, Italy (Received May 7 , 1991; in revised form October 8 , 1991; accepted October 16, 1991)
A fusion molecule consisting of the entire coding sequence of mature chicken tropoelastin preceded by 14 amino acids of the signal peptide and 9 amino acids of vector origin has been expressed in a recombinant bacterial system and purified. The molecule has been used as immunogen for the production of hybridomas. Monoclonal antibodies which bound specifically the immunogen were also reactive with tropoelastin purified from chick aorta and stained elastic fibers in aorta sections by immunofluorescence. The region of tropoelastin containing the antigenic determinant recognized by each antibody has been identified by a recombinant DNA expression strategy based on the use of cDNA clones spanning different portions of the coding sequence. It could be shown that several antibodies were directed against unique epitopes; among these, a group of antibodies bound specifically to the sequence (PGVGV),. Other antibodies were found to recognize antigenic determinants present more than once in the molecule. The monoclonal antibodies thus characterized will be useful reagents in studying the function of the different domains of tropoelastin. KEYWORDS: tropoelastin, recombinant protein, monoclonal antibodies, epitope mapping
INTRODUCTION As most extracellular matrix proteins, tropoelastin, the precursor of the main component of elastic fibers of connective tissues, is a multidomain protein. Initial sequence data of the protein have established the existence of two types of regions, one very hydrophobic, the other composed of stretches of alanines intercalated by lysine residues.' More recent sequence information from different species has shown that several different hydrophobic and lysine-containing repeats alternate in the whole molecule.2-5 In addition, a unique carboxyterminal domain, including two cysteine residues and terminating with the sequence arginine-lysine-arginine-lysine, has been detected in all tropoelastins. Finally, an unusual hydrophylic region has been identified in the human tropoelastin gene.4 Despite the wealth of data on the primary structure of tropoelastins, the knowledge of the function of the various repeats is only partial. The general agreement is that the alanine- and lysine-rich regions are involved in the formation of intermolecular crosslinks, whereas the hydrophobic regions confer elastic properties to the rnolecule.1.2 However, other functions could be performed by hydrophobic sequences, as indicated by the finding that one of these, formed by a repeating Address for correspondence: Dr. Din0 Volpin, Institute of Histology and Embryology, Via Trieste, 75, 35100 Padova, Italy. 13
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hexapeptide, is the target for a cellular receptor and is chemotactic for various cells.6.7 Moreover, it is possible that tropoelastin is involved in multiple interactions with itself and other molecular components of the elastic fiber.8 A significant contribution to the analysis of the different domains of some extracellular proteins has been obtained by the use of specific antibodies. The production of these antibodies has been achieved by two main procedures: polyclonal antibodies have been raised against defined oligopeptides chosen on the basis of sequence information of the molecu1e;g alternatively, monoclonal antibodies have been obtained by immunization of animals with the whole protein (or protein digests) and the regions recognized have been variously determined.10 In an attempt to develop multiple defined probes for chick tropoelastin, we have produced several monoclonal antibodies, and the region(s) they recognize on the molecule has been mapped with variable precision using a procedure based on the expression of different segments of the cDNA as fusion proteins in bacteria.
MATERIALS AND METHODS Purification of the Immunogen Recombinant chick tropoelastin has been used as immunogen. The BamHI fragment of Clone pEX1-TE72 was subcloned in the sense orientation in plasmid pCIPR (Fig. l), giving rise to the construct pCIPR-TE7. Expression in bacterial cultures was induced as described below. At the end of the induction period the cells were harvested by centrifugation, resuspended in 1/50volume of TEN buffer (50 mM Tris-HC1 pH 8.0,lOO mM NaC1,lO mM EDTA) and lysed by 20 cycles of sonication with a Soniprep 150 (Branson) set at % of maximal power. Each cycle lasted 30 seconds and was alternated with 1 min incubation of the sample on ice. The pellet obtained by centrifugation at 10,OOO x g for 30 min was resuspended in 1 M guanidine, 1 M NaCl, 1% Triton X-100 and centrifuged. This procedure was repeated, the pellet washed twice with TEN buffer and solubilized by boiling in 1-1.5 ml of 0.01M Na phosphate buffer pH 7.2,5% SDS, 5% P-mercaptoethanol. The sample was then fractionated by gel filtration through Sephacryl S-300 Superfine (Pharmacia) in 0,l M phosphate buffer pH 7.2 containing 1% SDS. The fractions obtained were analyzed by PAGE-SDS and those containing the immunogen were treated with acetone in order to remove SDS.11The precipitated protein was resuspended in a small volume (usually 0.2 ml) of 8 M urea and quantitated.'* Production of Hybridomas Hybridomas were produced as previously described.13 Just before injection, the urea concentration of the immunogen solution was lowered by dilution (10-20 x) with phosphate buffered saline. Initial screening of hybridomas was performed by reacting, in ELISA, the culture supernatants with the immunogen and, in parallel, with E. Coli extracts. Positive hybridomas were further confirmed by ELISA with tropoelastin purified from chick aortas.14 For preparation of E. Coli extracts, the cells from 800 ml of overnight culture were harvested by centrifugation at 5,000 X g for 20 min and resuspended in 8 ml of 50 mM TrisHCl pH 8.0,50 mM EDTA, 15% sucrose. After addition of 2 ml of 10 mg/ml lysozyme and
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el miiig 1inker
PEX 5783 bp
FIGURE 1 Physical map of bacterial expression vectors used in the present study. P,: right promoter of lambda phage; cI857: thermosensitive mutant of the CIgene of lambda phage; cro: region coding for the first 9 amino acids of the cro gene of lambda phage; Amp? ampicillin resistance gene.
Amp'
..
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incubation on ice for 40 min, the cells were lysed by adding 24 ml of 0.1% Triton X-100 in 50 mM Tris-HC1pH 8.0 and leaving 15 min on ice. The supernatant recovered after centrifugation at 25,000 X g constituted the concentrated E. Coli extract.
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Isolation and Characterization of cDNA Clones Several clones for chick tropoelastin have been isolated from a random-primed cDNA aorta library in pEXl by antibody screening as previously described.2 Two additional clones spanning the amino terminal end of tropoelastin were obtained by cloning the BamHI-PstI and BamHI-Sau3A fragments of pEX1-TE7 into pEX1. The ends of all the clones were sequenced by the dideoxy-chain termination method15 and the polypeptide coded by each clone deduced from the published sequence of chick tropoelastin cDNA.2 Only clones whose deduced length was in agreement with the size determined by agarose gel electrophoresis were considered. The size expected for the encoded polypeptides was confirmed by PAGE-SDS analysis of expressed fusion proteins. Expression and Purification of Fusion Proteins The procedure for fusion protein expression followed that described by Stanley and collaborators16 and was similar for both the pCIPR-TE7 clone used to produce the immunogen and the different pEXl clones isolated from the cDNA library. The only difference concerned the type of E. Coli strain used, DH5 and pop 2136 for pCIPR-TE7 and pEXl clones respectively. Overnight cultures of the different clones grown at 30°C were diluted 1:lOO in fresh LBmedium (usually 400 ml for pCIPR-TE7 and 30 ml for pEXl clones) and incubated at 30°C. After 2 h, the temperature was shifted to 42°C to allow induction of expression and the cells incubated for additional 2 h. The incubation was terminated by cooling the cell suspension on ice for 10 min. Further manipulation of the cells containing pCIPR-TE7 has been already detailed above (see: purification of the immunogen). The partial purification of the proteins expressed by the pEXl clones was performed as follows. The cell pellet obtained by centrifugation at 6,000 X g was resuspended in 2 ml of TEN buffer, recentrifuged and recovered in 0.2 ml of the same buffer. The bacteria were then lysed by successive addition of i) 50 pl of lysozyme (10 mg/ml) and incubation on ice for 15 min; ii) 6 p1 of DNase I (10 mg/ml) and 2 p l of 1 M MgSO, and incubation for 15 min on ice; iii) 90 pl of 10 mM TrisHCl, pH 7.4 containing 0.1 M NaCl, 1% Triton X-100 and 0.5% Na deoxycolate and incubation for 20 min on ice. The pellet obtained by centrifugation, which contains mainly bacterial inclusion bodies, was then extracted twice with 0.6 ml of 1 M guanidine, 1 M NaCl, 1% Triton X-100, washed two times with TEN buffer (0.6 ml) and finally dissolved in 0.2 ml of 8 M urea and the protein concentration measured. Preparation of Medium Proteins From Cultured Aorta Cells Chick embryo aorta cells were prepared as described” and cultured in 90 mm dishes in Dulbecco modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum. When the culture was confluent, the medium was removed, the cells washed with DMEM and incubated with 1 ml of DMEM without serum for 16 h. The medium was harvested and proteins precipitated with cold 15% trichloroacetic acid (TCA). The precipitate was successively washed with 15% TCA, 80% ethanol, ether and finally dissolved in 100 p1 of 62.5
TROPOELASTIN EPITOPE MAPPING
17
mM Tris-HC1 pH 6.8, 2% SDS, 10% glycerol, 5% P-mercaptoethanol and 0.01% bromophenol blue.
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Immunofluorescence Frozen sections, 5 Fm thick, were cut from aortas from 2 day old chicks. The sections were incubated for 2 h at room temperature with first antibody, consisting of either hybridoma supernatants, or a polyclonal antiserum to chick tropoelastinlg diluted 1:100, or a similarly diluted preimmune serum. After washing in phosphate buffered saline (PB:S), the slides were incubated for 1 h with the appropriate fluorescinated antibody (goat anti.-mouse IgG, Miles Laboratories Inc. ;goat anti-rabbit IgG, Diagnostic Pasteur), washed, mounted in 90% glycerol in PBS and examined in a Zeiss microscope equipped with epifluorescence optics. Other Immunologic Assays ELISA was performed following standard procedures.19 100 to 200 ng of protein were usually plated per well. For immunodetection of proteins blotted on nitrocellulose filters20 an alkaline phosphatase-based system was used: after incubation with alkaline phosphataseconjugated second antibody and washing, the filters were immersed in 100 mM:Tris-HC1 pH 9.5, 100 mM NaCl, 5 mM MgC1, containing 0.33 mg/ml nitroblue tetrazolium and 0.165 for a few minutes. mgiml 5-bromo-4-chloro-3-indonyl-phosphate
RESULTS Figure 1 shows the physical map of the expression vectors used in this study. pCIPR (Fig. l), an intermediate in the construction of pUEX vectors,21 allows the production of fusion proteins in which the contribution of vector sequences is limited to the first 9 amino acids, which are derived from the lambda cro gene. In contrast, a large portion of the fusion proteins synthesized from pEXl (Fig. 1) is encoded by plasmid sequences (cro + LacZ).16 Recombinant chick tropoelastin was obtained by expressing the previously characterized clone TE72 in pCIPR. The clone contains the entire sequence of mature tropoelastin and a portion of the signal peptide. The expected size of the recombinant protein is about 65 KD and was in good agreement with the electrophoretic migration of the majoir polypeptide produced after induction of expression (Fig. 2A). A direct proof that this band was a protein related to tropoelastin was obtained by Western blotting analysis of bacterial proteins, showing that the 65 KD component was recognized by an antiserum raised against tropoelastin purified from chick aortas (Fig. 2B). Purification of recombinant trolpoelastin was achieved by gel filtration chromatography as reported in Figure 3 . The final yield of protein with a degree of purity shown in the inset of Figure 3 was about 3 mg per liter of bacterial culture. The purified recombinant tropoelastin was used to raise monoclonal antibodies in mice. As a first step in the characterization of the antibodies, their reactivity with the: recombinant molecule and with tissue tropoelastin was compared. All the hybridomas which reacted specifically with the recombinant protein in ELISA were also positive when assayed against tropoelastin purified from chick tissues (data not shown). Moreover, as evaluated by inspection of Figure 4, the relative intensity of signal obtained in Western blots with the
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B
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A
U
I
U
I
FIGURE 2 Analysis of recombinant tropoelastin expressed with the pCIPR vector. A: proteins solubilized by boiling the bacterial cell pellet in the presence of 2% SDS were resolved by electrophoresis in a 7.5% SDSpolyacrylamide gel and stained with Coomassie blue R 250. B: Western immunoblots of total protein reacted with an antiserum specific for chick tropoelastin. U: uninduced cells grown at 30°C; I: cells induced by incubation at 42°C. Arrows point out the position of recombinant tropoelastin. BSA: migration of bovine serum albumin.
0
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Nu~BER
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-116KD
80
FIGURE 3 Purification of recombinant tropoelastin by gel filtration through Sephacryl S-300. Individual fractions were analyzed by SDS gel electrophoresis in order to identify the fractions containing tropoelastin (horizontal bar). V, = void volume and V, = total volume of the column. Inset: electrophoretic pattern of proteins of two of the fractions in which tropoelastin (arrow) was the predominant band. Numbers on the right indicate the mobility of molecular weight markers.
E0
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;
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B
4
BSA
FIGURE 4 Comparison of the pattern of reactivity of the different monoclonal antibodies against recombinant and native tropoelastin by Western immunoblot analysis. Aliquots of the cell extract obtained from induced bacterial cells as described in the legend to Figure 2 (A), or of the TCA precipitate of the medium from cultured chick embryo aortic cells (B) were subjected to SDS gel electrophoresis, blotted onto nitrocellulose filters and discrete strips reacted with individual monoclonal antibodies as indicated. BSA: migration of bovine serum albumin.
different antibodies was similar when either the recombinant protein or tropoelastin released in the medium by cultured chick embryo aorta cells was used. When assayed by immunofluorescence on chick aorta sections, all the antibodies stained fibrils in a way similar to a rabbit polyclonal antibody produced against purified chick aorta tropoelastinls (Fig. 5). Further characterization of the monoclonal antibodies was obtained by delimiting the region of the tropoelastin molecule containing the epitope recognized. This was effected by determining the reactivity of each antibody against the recombinant protein expressed by cDNA clones spanning different portions of the coding region. As shown in Figure 6 for some of the cDNA clones, the recombinant proteins were efficiently expressed by the pEX vector. The proteins were the major polypeptide species present in bacterial extracts, and significant proteolysis was not found in most clones, or, when present, it gave rise to only a few discrete bands just below the undegraded product.
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TROPOELASTIN EPITOPE MAPPING
21
FIGURE 5 Localization of immunoreactive sites in 2 day old chick aorta by immunofluorescence. The first antibody used was: a rabbit antiserum against chick tropoelastin (A); rabbit preimmune serum (B); monoclonal antibody V95D6 (C); monoclonal antibody V94G12 (D). The patterns of immunofluorescence of C and D are representative of the distribution observed with the different monoclonal antibodies. Bar = 10 pm.
Examples of mapping of the regions recognized by the antibodies are reported in Figure 7. The pattern of Figure 7A was identical for a group of seven antibodies out of 17 analyzed. The region common to all the cDNA clones which reacted with the antibodies, could be easily delimited between the end of clone TE24 and the beginning of clone TE29, which were both negative. This region includes the coding sequence of amino acids 42.7-451 of the mature protein as deduced from clone TE7,2 and consists of five repeats of the lpentapeptide PGVGV Formal proof that this was indeed the sequence recognized by the antibodies was obtained by an ELISA in which the synthetic polypentapeptide (PGVGV), was used as substrate: only the seven antibodies previously identified produced a significant signal (Table I). The region recognized by antibody V91B1O could be restricted within a short unique sequence, delimited by the region of overlapping of clones 4A12 and 6B21 (amino acids 622-632) (Fig. 7B). However, this was not the case for several antibodies, which gave a positive signal also with the protein expressed by cDNA clones which did not overlap. A typical example is shown in Figure 7C: the limits at the amino- and carboxy-terminal end are given by the end of clone TE13 and the beginning of clone TE29 respectively l(amino acids 281-450); two positive clones spanning partially this region, TE19 and TE24, did not share common sequences. A more complex reactivity was manifested by the antibody of Figure
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-116KD
-68KD
-45KD
FIGURE 6 Analysis of protein expressed by a group of cDNA clones. After induction at 42”C, inclusion bodies were prepared as described under “Methods”, run in a 6% SDS-polyacrylamide gel and protein bands visualized by Coomassie blue staining. Dots at the right of the lanes mark the undegraded fusion protein, whose size was in agreement with the value expected from the coding sequence contained in the clone. Migration of molecular weight standards is indicated on the right.
7D. In this case, two discontinuities between positive clones could be identified: between TE19 and TE24 and between TE24 and TE29. Additional characterization of the antibodies was obtained by investigating species specificity by indirect ELISA using alpha elastin from chick and human aorta and from bovine legamentum nucae (Fig. 8). The majority of the antibodies reacted with bovine and human alpha elastin; some, however, were specifically inhibited only by the chicken protein.
DISCUSSION The purpose of this work was the production of monoclonal antibodies with specificity against different regions of the tropoelastin molecule. We also wanted to explore the possibility of substituting native tropoelastin with recombinant tropoelastin in immunological investigations, given the fact that the latter represents an easy and efficient source of
TROPOELASTIN EPITOPE MAPPING lE7
23
TE7
TE3 TES
TEP TE33
TE3 TES
-TE32
-
TED
TE10
El0
El3
TE13
TE19
TEl9
TEP
TEZ
TE23 TE24
TE23
TE29 TE30
TE24
TE29 TE30
TEWl TEUIZ
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TE6Wl
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TEZZ
TE23 TE24
TES TEJO
TE23 TE24
TEWl TEUl2
TE682l
TE3 TES
TE29 TE30
TE3ASl
-
TE,IA12
C
TE6B21
D
FIGURE 7 Mapping of antibody binding sites in tropoelastin. The protein expressed by the cDNA clones indicated was reacted with the monoclonal antibodies in direct ELISA. Positive clones (thick lines, p < 0.1) were identified by comparing triplicate values obtained with each antibody with those obtained with a control monoclonal antibody (specific for human chorionic gonadotropin) using the Student’st test. The figure reports only a few representative examples. The pattern of reactivity shown in A is common to the 7 monoclonal antibodies which bind the polypentapeptide repeat of tropoelastin (see Table I). B: reactivity of antibody V91B10. C: reactivity of antibodies V94G12 and V95B3. D: Reactivity of antibody V81A5. The scale is calibrated in amino acids, where number 1 is the first residue of the mature protein.
protein. The results indicate that, at least for the type of assays that have been performed in this study, native and recombinant tropoelastin are, indeed, interchangeable. Thus, an antiserum specific for chick aorta tropoelastin recognized the recombinant protein and, conversely, the monoclonal antibodies raised against the bacterial product reacted in very similar ways with the two proteins. In addition, all the monoclonal antibodies gave the immunofluorescence pattern expected for aorta elastic fibers, indicating that they bind to native elastin. The data also indicate that the presence of 23 additional amino acids (9 from the cro sequence and 14 from the signal peptide of tropoelastin) at the amino-terminal end of the mature protein does not grossly alter the immunologic properties of recombinant versus native tropoelastin. Definition of the regions containing the epitope for the monoclonal antibodies has been based on the expression of recombinant cDNA clones. This strategy has been shown to be very effective in mapping antigenic determinants of other pr0teins.22~23In order to be reliable, the technique requires that the protein are expressed very efficiently and that they
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TABLE I Reactivity of the monoclonal antibodies against the synthetic polypentapeptide (PGVGV), as determined by ELISA
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Antibody Controlc V81A5 V87F7 V90F9* V91B10 V92B4 V92B8 V93B5 V93B6 V93C2* V94B8* V94G 12 v95c5* V95D6* V95E11* V95G6 V92B2* V96C4
Mean O.D.,
2 sda
0.011 2 0.008 0.024 t 0.005 0.027 2 0.005 0.149 2 0.013 0.019 ? 0.005 0.063 2 0.050 0.026 2 0.005 0.016 t 0.015 0.009 2 0.012 0.269 ? 0.027 0.172 ? 0.005 0.033 t 0.006 0.381 2 0.021 1.938 ? 0.016 0.369 2 0.010 0.018 ? 0.006 0.168 ? 0.006 0.028 ? 0.011
Significanceb p > 0.1 p > 0.1 p < 0.001 p > 0.1 p > 0.1 p > 0.1 p > 0.1 p > 0.1 p < 0.001 p < 0.001 p > 0.1 p < 0.001 p < 0.001 p < 0.001 p > 0.1 p < 0.001 p > 0.1
%obtained from triplicate determinations. sd = standard deviation bvalues obtained with the test and the control antibody were compared by the Student’s t-test =the control antibody was a monoclonal against human chorionic gonadotropin *antibodiespredicted to react with the polypentapeptide sequence of tropoelastin on the basis of the analysis shown in Figure 7
are not degraded by bacterial proteases: both conditions have been verified in our experiments. In addition, applicability of the technique in the case of tropoelastin was assessed with antibodies which recognize a region containing the repeating sequence (PGVGV),, by showing that only the group of antibodies identified by the analysis with recombinant clones reacted with the synthetic polypentapeptide. Our results indicate that the different domains of tropoelastin are not similarly antigenic. Thus, the group of antibodies specific for the polypentapeptide accounts for about one third of total antibodies, a very high proportion considering that the sequence (PGVGV), constitutes only about 7% of tropoelastin. This suggests that the polypentapeptide sequence is a major antigenic determinant of chick tropoelastin. On the contrary, no antibodies directed against the amino-terminus of the molecule were obtained. These differences could be due to either a variability of intrinsic antigenicity of the various sequences or to the preferential exposure of some regions at the surface of the molecule. Excepting the regions mentioned above, the epitopes for the remaining antibodies were apparently evenly distributed in the molecule. Figure 9 summarizes the results of antigenic region mapping obtained with several monoclonal antibodies. It is apparent that different classes of antibodies could be distinguished. One class comprised the antibodies recognizing an epitope which is certainly unique in tropoelastin. Examples of this class were the antibodies directed against the polypentapeptide repeat region (amino acids 425-477). The sequence (PGVGV),, which should be long enough to accommodate for a linear epitope (unfoldon) sequence,24 is present in two other positions in the molecule (amino acids 338-347 and 606-615); nevertheless,
25
TROPOELASTIN EPITOPE MAPPING
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50
0 100
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0 0
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Inhibitor protein (pghell)
.;
FIGURE 8 Species reactivity of the monoclonal antibodies. Inhibition ELISA was performed using chick alpha elastin as immobilized substrate and alpha elastin from chick (A), human (B) and bovine (C) as inhibitor protein. V95G6: A; V92B4: A. V91B10: 0 ; V95Ell: 0 ; V94G12: 0 ; V95D6:
~
~
I
400 ~
_
I
600
I m
_
_
V91810
V81A5
anti-(PGVGV),
187F7
V95G6
V94G 1 2
V9284
V9288
FIGURE 9 Map of the regions of tropoelastin containing the antigenic sites identified by different monoclonal antibodies. Scale is defined as in Figure 7.
~~
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I ~
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