ment of plasma membrane vesicles ofhuman macrophages to. Leishmania tropica promastigotes. J. Infect. Dis. 148:377-384. 9. Mosser, D. M., and P. J. Edelson.
Vol. 57, No. 2
INFECTION AND IMMUNITY, Feb. 1989, p. 630-632
0019-9567/89/020630-03$02.00/0 Copyright © 1989, American Society for Microbiology
NOTES
Antibodies Raised against Synthetic Peptides from the Arg-Gly-AspContaining Region of the Leishmania Surface Protein gp63 Cross-React with Human C3 and Interfere with gp63-Mediated Binding to Macrophages DAVID G. RUSSELL,* PATRICIA TALAMAS-ROHANA, AND JANIS ZELECHOWSKI
Department of Pathology, New York University Medical Center, 550 First Avenue, New York, New York 10016 Received 11 July 1988/Accepted 17 October 1988
The Leishmania surface glycoprotein gp63 binds to complement receptor type 3 on the macrophage surface. Antibody raised against a synthetic peptide containing the Arg-Gly-Asp region of the amino acid sequence of gp63 recognizes both gp63 and the ot-chain of human C3. Monovalent Fab fragments from this antibody block gp63-mediated binding to macrophages.
(RLLPGGLQQGRGDAVGPERGC) by D. Schlesinger of the New York University Medical Center. Figure 1 illustrates the effect of pep63 on attachment of various ligand-bearing particles to human MO. Soluble pep63 inhibited binding of both Bgp63 and EC3bi to MO. Half-maximal inhibition was observed at around 1 mg/ml (5
The attachment of Leishmania promastigotes to macrophages is a receptor-mediated event (5, 8). Recently, two abundant molecules on the promastigote surface, glycoprotein gp3 (4, 16, 17) and glycolipid lipophosphoglycan (7), have been shown to independently mediate binding to macrQphages. Studies conducted with intact promastigotes have implicated a range of receptors on the macrophage surface in attachment, including the mannosyl-fucosyl receptor (6, 18) and the receptor for the C3bi form of C3 (CR3) (1, 9). We have started matching ligands to their respective receptors, employing a particle capable of carrying individual parasite surface components (17). Experiments with gp63-coated beads (Bgp63) have shown that the parasite glycoprotein binds directly to complement receptor CR3 (17). CR3 recognizes similar Arg-Gly-Asp (RGD)-containing regions in both gp63 (3, 17) and its "natural" ligand, C3bi (20). The RGDcontaining region of gp63 has also been implicated by Rizvi and co-workers in attachment of intact promastigotes to macrophages (13). In this study we employ synthetic peptides based on this region of gp63 and antibodies raised against these peptides to examine the structural and functional relatedness of gp63 and C3. Leishmania mexicana MNYC/BZ/62/M379 promastigotes were cultured in semidefined medium 79 (2), and gp63 was isolated from stationary-phase cultures as described previously (15-17). Human monocyte-derived macrophages (MO) were purified from buffy coats, plated onto Terasaki plates, and used in attachment studies as detailed previously (17, 21). The ligand-bearing particles used in this study were prepared by dialysis, resulting in deposition of gp63 and phospholipid onto the surface of reverse-phase high-pressure liquid chromatography beads (17). Immunoglobulin G (IgG) beads were prepared by incubating Bgp63 in monoclonal anti-gp63 antibodies (17). Sheep erythrocytes coated with the C3bi from of C3 (EC3bi) were prepared as described previously (21). A synthetic peptide, pep63, was synthesized from residues 365 to 385 of the gp63 amino acid sequence
*
x
10' M) for both Bgp63 and EC3bi. No inhibition of
binding of IgG-coated beads was seen, showing that the effect was specific. This observation is consistent with the results of previous experiments in which we demonstrated that gp63 and C3bi are recognized by the same binding site on CR3 (17). Attachment of Bgp63 was inhibited only by RGD-containing peptides that blocked CR3 activity and was unaffected by peptides that inhibited the binding behavior of other integrins, such as the fibronectin receptor (17). This is in apparent contrast with the results of Rizvi et al. (13);
A
o 80
Bgp63
Ec3bi '10
1-
0
.0 C
am20
0
2Bg Conc. of pep63 (mg/ml)
FIG. 1. Competitive inhibition of binding of ligand-bearing beads using soluble synthetic peptide. Monolayers of MO were incubated with IgG-coated beads, Bgp63, or EC3bi in the presence of increasing concentrations of pep63. Both Bgp63 and EC3bi attachment was depressed by the peptide, whereas binding of IgG-coated beads remained unaffected. The vertical lines associated with each point indicate the standard deviations. Results are from a single experiment conducted in triplicate; similar results were obtained in three independent experiments. BIgG, IgG-coated beads.
Corresponding author. 630
NOTES
VOL. 57, 1989 a--l-
-b
1g
F(ab)
A
631
B
18000
4
anti-pepSi
Im r
L1co ",,-
control~
~~~~~control
"I
S
VT2
1
200[
2
FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel of purified L. mexicana gp 63 (lanes 1) and semipurified human C3 (lanes 2), separated on a 9% polyacrylamide gel. The gel was either stained in Coomassie blue (a) or transferred to a nitrocellulose membrane and probed with affinity-purified anti-pep63 antibody (b). The anti-pep63 antibody recognized both gp63 (-*) and the a-chain of human C3
(V).
however, their experiments were conducted with intact promastigotes and not with isolated gp63. pep63 was coupled to keyhole limpet hemocyanin by glutaraldehyde and was inoculated into rabbits. Anti-pep63 antibody was affinity purified on a pep63-activated CH Sepharose (Pharmacia, Inc.) column. gp63 and partially purified human C3 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 2) and transferred by electroblot to nitrocellulose membrane. The membrane was then probed first with affinity-purified anti-pep63 antibody and then with alkaline phosphatase-conjugated anti-rabbit IgG antibody. When the blot was developed, it was found that both gp63 and the ox-chain of human C3, which contains the RGD tripeptide (20), had reacted with anti-pep63 antibody (Fig. 2). This result demonstrates that the RGD-containing regions of gp63 and C3 show structural similarities capable of generating cross-reactive antibodies. The binding of anti-pep63 antibody to these two proteins can be titrated out with increasing concentrations of free peptide (not illustrated), confirming the specificity of anti-pep63 recognition of C3 and gp63. The effect of anti-pep63 antibody on the binding of Bgp63 to human MO was examined by incubating Bgp63 with MO in the presence of either intact or univalent Fab fragments from the anti-pep63 antibody (Fig. 3). Intact anti-pep63 antibody caused a slight increase in the attachment of Bgp63 to MO (Fig. 3A). However, although there was only a small quantitative difference between the binding of Bgp63 in the presence of nonimmune antibody and binding in the presence of anti-pep63 antibody, there was a marked qualitative difference in the binding behavior. Bgp63 in anti-pep63 antibody were avidly phagocytosed, whereas Bgp63 in nonimmune antibody remained bound to the MO surface (not illustrated). This result is consistent with observations that CR3 on resting phagocytes mediates binding but not phagocytosis, whereas the Fc receptor is a strong stimulator of internalization (17, 21). This result also indicates that Bgp63 in the presence of anti-pep63 antibody were being opsonized. In contrast, anti-pep63 Fab fragments inhibited the binding of Bgp63 to MO (Fig. 3B), suggesting that binding to CR3 relies on the accessibility of the RGD-containing region of gp63. Similar experiments conducted with anti-pep63 Fab fragments and EC3bi were unsuccessful, indicating that
40
0
100
200
0
300
100
200
300
Antibody Concentration (pg/mi)
FIG. 3. Effect of anti-pep63 antibody and Fab fragments on the binding of Bgp63 to human MO. Control preparations contained equivalent concentrations of nonimmune antibody or Fab fragments. Intact anti-pep63 antibody caused a slight enhancement in binding of Bgp63 to MO (A) over the nonimmune controls. Interestingly, Bgp63 in the presence of intact anti-pep63 antibody were internalized, but the control preparations in nonimmune antibody remained attached to the cell surface. Fab fragments from nonimmune antibody did not affect Bgp63 binding (B). However, Fab fragments from anti-pep63 antibody mediated strong inhibition of Bgp63 attachment (B). The vertical lines associated with each point indicate the standard deviations. These results were confirmed in three independent experiments.
although the antibody recognized denatured C3, it was unable to bind to this region of the intact, native protein. Bgp63 were incubated with MO and 250 ,ug of either nonimmune or anti-pep63 Fab fragments per ml in the
.0 *BgpS3 2SOug/ml control Flab)
200
ABgp63*250ug/ml anti-pep63 Flab)
0
100
200
300
400
pepC3 Conc. (pg/ml)
FIG. 4. Effect of soluble pep63 on the anti-pep63 Fab fragmentmediated inhibition of Bgp63. Bgp63 were incubated with monolayers of MO and 250 FLg of either nonimmune or anti-pep63 Fab fragments per ml in the presence of increasing concentrations of soluble pep63. In the presence of nonimmune Fab fragments, the binding of Bgp63 remained high and constant until the concentration of pep63 exceeded 200 ,ug/ml, from which point binding was inhibited. However, in the presence of anti-pep63 Fab fragments, the binding of Bgp63 was initially low, but it increased with the concentration of pep63 until a threshold point was reached above which binding was again inhibited.
632
NOTES
presence of increasing amounts of pep63 (Fig. 4). In the control preparation, which contained nonimmune Fab fragments, the attachment of Bgp63 remained relatively constant until the concentration of pep63 exceeded 200 ,ug/ml, from which point binding was inhibited. In contrast, the preparation containing anti-pep63 Fab fragments showed low binding of Bgp63 in the absence of free peptide. However, as the concentration of pep63 increased, so did attachment of Bgp63 to the MO, indicating that free pep63 competes for anti-pep63 Fab fragments and thus exposes the RGD-containing region of gp63 on the particle surface and promotes binding to MO. The attachment of Bgp63 in the presence of anti-pep63 antibody was also inhibited at higher concentrations of soluble peptide. The significance of these results is twofold. First, the behavior of the anti-pep63 antibody in blocking the attachment of Bgp63 to CR3 confirms the earlier proposal that gp63 interacts with CR3 via an RGD-containing region in its primary structure (3, 17). Second, the existence of crossreactive epitopes on gp63 and C3 is relevant to previous observations concerning the role of complement in the infection of macrophages by Leishmania promastigotes. Other investigators have demonstrated that CR3 plays a role in the binding of promastigotes to MO (1, 9). To explain the ability of anti-C3 Fab fragments to inhibit parasite attachment in the absence of complement, Blackwell and coworkers proposed that the MO secretes complement components that opsonize the promastigotes (1, 19). However, since gp63 and the a-chain of C3 contain cross-reactive epitopes, this result could be explained by antibody crossreactivity. In our laboratory, we examined the relative C3 acceptor activity of the two macrophage-binding ligands, gp63 and lipophosphoglycan (14). We used polyclonal antigp63 antibody to precipitate radiolabeled C3-gp63 complexes. Because of the presence of cross-reactive epitopes, these complexes may have produced artificially high values for the amount of C3 deposited on gp63, compared with more recent results for Leishmania major lipophosphoglycan (12). The results described here demonstrate that the structural relatedness of gp63 and C3 is responsible not only for their sharing of the same receptor (CR3) on MO (17), but also for their generating antibodies that recognize both proteins. This result is similar to the finding of Rizvi and colleagues, who showed that antifibronectin antibody could precipitate Leishmania chagasi gp63 (13) and postulated, on the basis of RGD-peptide inhibition data, that gp63 binds to the fibronectin receptor. The arguments for and against the involvement of CR3 versus the fibronectin receptor in the binding of gp63 to macrophages are given in greater detail in two recent review articles (11; D. G. Russell, Immunol. Today, in press). The ability of anti-pep63 Fab fragments to inhibit gp63-mediated attachment to MO confirms the earlier contention (17) that the RGD-containing region of the protein recognizes CR3. However, the biological significance of the gp63-CR3 interaction during the invasion of macrophages by promastigotes is still far from clear and will remain so until the receptor(s) for lipophosphoglycan has been identified and the possible role of serum opsonins has been more fully assessed (7, 10, 12). This study was supported by grants from the MacArthur Foundation and the Rockefeller Foundation and by Public Health Service grants BRSG S07 and RR5399-26 from the National Institutes of Health. We thank V. Nussenzweig and S. D. Wright for their encouragement and critical comments.
INFECT. IMMUN.
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