The Specificity of the Binding of Platelet Activating Factor (PAF) to Anti-PAF Antibodies Mary A. Smal, Brian A. Baldo*f and David G . Harlef Kolling Institute of Medical Research, Royal North Shore Hospital, St Leonards, NSW 2065, Australia Quantitative hapten inhibition experiments employing sheep anti-PAF antibodies and selected PAF analogues were undertaken with the aim of defining the antigenic determinant structures complementary to the antibody combining sites. The most important fine structural features for inhibition of antibody binding to PAF were shown to be an acetyl group at position 2 of the phospholipid glycerol backbone and an ether group at position 1. Of the naturally occurring compounds, C,6- and CI,,,-PAF proved to be the most potent inhibitors and more active than C,,-PAF while phospholipidswith a propionyl, butyryl or hexanoyl group at position 2 showed either weak or no inhibitory activity. The l-acyl, thioether and deoxy analogues proved inactive. Variations in the polar head group of PAF were found to be less critical with, for example, the dimethyl and ethanolamine derivatives retaining some activity. This antibody recognition pattern is very similar to that of the PAF receptor, although the antibodies appear to have a more specific requirement for an acyl linkage at position 2.
INTRODUCTION Platelet activating factor (PAF) (1-0-alkyl-2-O-acetylsn-glycero-3-phosphocholine) (Hanahan et af.,1980) is a biologically active phospholipid which induces aggregation of platelets at concentrations of 0.1 nM. The presence of the acetyl group at position 2 is an absolute requirement for biological activity of this phospholipid (Hanahan and Kumar, 1987). PAF has a number of physiological effects (Hanahan, 1986) which suggest that it may be involved in a variety of diseases including asthma (Barnes, 1988), anaphylactic shock (Vargaftig and Braquet, 1987), other allergic conditions (Barnes et al., 1988), kidney disease (Schlondorff and Neuwirth, 1986) and gastric ulceration (Stenson, 1988). If the role of PAF in health and disease is to be clarified, quantitative data on PAF levels in various fluids and tissues will be needed. A sensitive and specific radioimmunoassay (RIA) for PAF appears to have the greatest potential for rapidly quantitating this autacoid in large numbers of biological samples side-by-side in an assay that is easy and convenient to use. The specificity of the antiserum is critical to the accuracy and validity of such an assay. In two previous reports, the production of antibodies to PAF was described, however the specificity of these antibodies was not adequately defined (Nishihira et al., 1984; Karasawa et al., 1987). Recently, we have described the production of a PAF immunogen and an anti-PAF antiserum (Smal et al., 1989) and used the antiserum to develop a sensitive and specific RIA for PAF (Smal et af., 1990). In this paper, we present results of hapten inhibition studies designed to examine the fine structural specificity of the anti-PAF antibody combining sites by identifying
* t
Author to whom correspondence should be addressed. Also affiliated to: The Department of Medicine, University of Sydney, NSW 2006, Australia. Abbreviations used: PAF, platelet activating factor; RIA, radioimmunoassay.
the important structural features on the complementary phospholipids. These features were similar to those required for the pharmacological activity of PAF, indicating that the binding specificity of the antibodies is similar to the specificity of the PAF receptor.
EXPERIMENTAL Antiserum preparation. C,,-PAF ( 1 -0-(12', 1 T-dimethoxydodecyl)-2-0-acetyl-sn-glycero-3-phosphocholine)was prepared and coupled to methylated bovine serum albumin as described previously (Smal et af., 1989). Sheep were immunized with this material and the PAFacetylhydrolase activity in the antisera was destroyed by acid treatment as described recently (Smal el af.,1990). Hapten inhibition studies. Standard solutions of the com-
pounds to be tested were prepared in aqueous ethanol and then diluted in the assay buffer (0.05 M sodium acetate, pH 6.0, containing 0.05% Tween 20) to the appropriate concentration. Anti-PAF antiserum was used at 1 in 8000 diluted in the assay buffer supplemented with deactivated normal sheep serum (1 in 2000). The donkey anti-sheep immunoglobulin (Silenus Laboratories, Melbourne, Australia) (used as the second antibody) was diluted 1 in 250 in the assay buffer containing 6% poly(ethy1ene glycol) and [12SI]PAF (2200 Ci/mmol, New England Nuclear, Boston, MA, USA) was added to give approximately 40 000 counts/ min per 100 pL. Into duplicate polystyrene RIA tubes (Disposable Products, Adelaide, Australia), was placed 100 pL of each of the following: the solution to be tested for inhibition, anti-PAF serum, anti-sheep/tracer. The Bo tubes (zero binding) contained no inhibitor solution and the NSB tubes (nonspecific binding) contained only anti-sheep/tracer. The tubes were incubated at room temperature for 16 h, 4 mL of assay buffer was added and the tubes were centrifuged at 1900 x g for 25 min.
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After removing the supernatant, the radioactivity remaining was measured and the percent of tracer bound (%B/B,) to the precipitate was calculated from the formula (B-NSB)/(B,-NSB) x 100. The binding of tracer to the antiserum was approximately 42% in the absence of inhibitors. Compounds were tested for inhibition at final concentrations up to 330 nM. Chemicals. C16-, Clx- and C,, ,-PAFs (1 -0-hexadecyl-,
1-0-octadecyl- and 1-0-octadec-9-cis-enyl-2-O-acetylsn-glycero-3-phosphocholines,respectively) were purchased from Bachem Feinchemikalien (Bubendorf, Switzerland). C,-PAF (l-O-(6',6'-dimethoxyhexyl)-20-acetyl-sn-glycero-3-phosphocholine)and racemic (f)C16-PAFwere prepared as described by Smal et al. (1989) and Hirth and Barner (1 982), respectively. Palmitoyl AGPC (1 -0-hexadecanoyl-2-0-acetyl-snglycero-3-phosphocholine) and ( h )thio-C,,-PAF (1-Soctadecyl-2-0-acetyl-1-thioglycero-3-phosphocholine) were generous gifts from Dr Tokumura (University of Tokushima, Japan) and Dr Muramatsu (Tokyo Medical and Dental University, Japan) respectively. ( I!= )DeoxyC,,-PAF ( 1-deoxy- 1-hexadecyl-2-0-acetyl-glycero-3phosphocholine) was from Lederle laboratories (Pearl River, NY, USA) and Ro 17-9461 (1-0-hexadecyl-2O-rnethylcarbamoyl-sn-glycero-3-phosphocholine),Ro 17-7826 ( 1 -0-octadecyl-2-deoxy-2-acetamido-snglycero-3-phosphocholine) and Ro 17-9777 (1-0-octadecyl - 2 - deoxy - 2 - (N-carbomethoxy)amino-glycero-3phosphocholine) were provided by Hoffmann-La Roche (Basel, Switzerland). PGEPC (1 -0-octadecyl-2-0propionyl-sn-glycero-3-phosphocholine),the corresponding 2-0-butyryl and 2-0-hexanoyl analogues (BuGEPC and HGEPC, respectively), AGEPE (10-alkyl-2-0-acetyl-sn-glycero-3-phosphoethanolamine), AGEPDME ( 1-0-octadecyl-2-0-acetyl-sn-glycero-3phospho-N,N-dimethylethanolamine), U66985 (1-0octadecyl-2-0-acetyl-sn-glycero-3-phosphoricacid-6't r i m e t h y l a m m o n i u m hexyl e s t e r ) a n d U66982 (1 -0-octadecyl - 2-0-acetyl-sn-glycero - 3 - phosphoric acid- 10'-trimethylammonium decyl ester) were generous gifts from Dr Hanahan (University of Texas Health Science Center, San Antonio, TX). The PAF antagonists CV 3988 (rac-3-(N-n-octadecyl-carbamoyloxy)-2-methoxypropyl-2-thiazolioethylphosphate),BN 52021 (3-tbutyl-hexahydro-2,4,7b, 1 1-trihydroxy-8-methyl-9H- I ,7a(epoxymethano)- 1H,6aH-cyclopenta[c]furo-[2,3-b]furo[3' 2':3,4]cyclopenta [ 1,2-d]furan-5,9,12(4H) and WEB 2086 (3-[4-(2-chlorophenyI)-9-methyl-6H-thieno[3,2-fl[ 1,2,4]triazolo-[4,3-a][ 1,4]-diazepin-2-y1]-1-(4-morpholinyl)- 1propanone) were from Takeda Chemical Industries (Osaka, Japan), Dr Pierre Braquet (Le Plessis Robinson, France) and Boehringer Ingelheim (Ingelheim am Rhein, FRG) respectively. Lyso-C,,-PAF (1 -0-hexadecylsn-glycero-3-phosphocholine), methyl lyso-PAF (1-0hexadecyl-2-0-methyl-sn-glycero-3-phosphocholine) and all other chemicals were purchased from the Sigma Chemical Co. (St Louis, MO, USA).
RESULTS AND DISCUSSION Inhibition studies with C-1 analogues of PAF. The inhibition profiles of the PAF analogues which showed significant 170 JOURNAL OF MOLECULAR RECOGNITION, VOL. 3, No. 4,1990
01
10
10
Inhibitor (prnolltube)
Figure 1. Recognition of PAF and some analogues by sheep anti-PAF antibodies. Inhibition dose responses obtained with: ( O ) , CIZ-PAF; ( 0 ) Ce-PAF; , (m),CTrj-PAF; ( O ) , Cie-PAF; (+), CieiPAF; ( 0 ) , racemic (f)CI6-PAF; (A), PGEPC; and (A), AG EPD M E.
binding are shown in Fig. 1 and the 50% inhibitory values for analogues with varying alkyl chain lengths and with nonether substituents at C-1 of the glycerol backbone, are listed in Table 1. The C-1 analogue with the greatest inhibitory activity is C,,-PAF (the twelve carbon long alkyl chain analog) which is not surprising since the antiserum was raised to this analogue coupled to the carrier. The shorter, six carbon analogue (C6PAF) was a slightly weaker inhibitor, as were the long chain analogues (cl6-9 C1,-and C,,,,-PAF) which are the predominant species in naturally occurring PAF. These results indicate that there is a broad specificity in the binding of this polyclonal antibody to the alkyl chain of PAF, although there is a preference for the medium length alkyl chain (i.e., C,,). In contrast, no inhibition was observed with ( f)deoxy-C,,-PAF, the analogue in which the ether oxygen was replaced by a methylene group, and very weak inhibition resulted with compounds in which the oxygen was replaced by either an acyl or a thioether linkage. These results demonstrated that there is an absolute requirement for an ether linkage at C-1 of the glycerol backbone, probably because of both the electronic distribution and the physical size of the grouping. Inhibition studies with C-2 analogues. The 50% inhibition values for these PAF analogues are shown in Table 1. Increasing the chain length of the acyl group decreased the binding dramatically; the propionyl analogue (PGEPC) was a weaker inhibitor than the acetyl analogue (C,,-PAF) (Fig. l), and higher homologues were not recognized. These results suggest that, along with the ether group, the acetyl group is specifically recognized by the antibody combining sites. Replacement of the acyl linkage with an amide (Ro 17-7826) or a N-linked carbamate (Ro 17-9777) abolished antibody binding, whereas a weak binding was observed with the 0-linked carbamate analogue (Ro 17-9461). These findings suggested that an acyl group at C-2, and particularly an acetyl, is necessary for antibody recognition. This is further substantiated by the failure of the hydroxy analogue (lyso-C,,-PAF) and the methoxy analogue (methyl lyso-PAF) to cause any inhibition.
0 1990 by John Wiley & Sons, Ltd.
SPECIFICITY OF ANTI-PAF ANTIBODIES
Table 1. Swcilici@ of anti-PAF antibodies: inhibition of PAF/anti-PAF interaction by C,,-PAF and somi structural analogues IC,b
Substituent at c,. c, or c,
IC,I
(pmol)
IC,, C,,-PAF
C16-PAF Cls-PAF Cis 1 -PAF
0.39 0.72 0.48
1.o 1.85 1.23
C6-PAF ClZ-PAF ( & ) Deoxy-Cl6-PAF ( ?c) Thio-Cls-PAF
0.28 0.1 4 NIC >lo@
0.72 0.36
> 256
Palrnitoyl- AGPC
>100
> 256
Compound'
c1
c2
NI NI
LYSO- C16- PA F Methyl lyso-PAF
4 H -0CH3 0
PGEPC
4 - C CH2CH3
I1
1.7
4.4
>loo
> 256
>loo
> 256
26
66.7
>100
> 256
> 100
> 256
2.3
5.9
AGEPE
21
53.8
U66985
8.3
21.3
U66982
>loo
> 256
0 I BuGEPC
4 - C ( CH2) 2C H3
0 HGEPC
-O-C(CHz),CH, 0
R O 17-9461
-0-CN HCH3
I
0 I1 Ro 17-7826
-N-CC H3 0
( 5 ) Ro 17-9777
-N-C-OCH
I 3
c3
0
I AGEPDME
+
-0-P-0 ( CH2)ZNH ( CH3)2
For ( * ) C16-PAF, ICw=0.84 and ICw/ICw C16-PAF=2.15. The PAF antagonists CV 3988, BN 52021 and WEB 2086 did not inhibit up to a concentration of 100 pmol per tube. "l-@alkyl group is C18 unless specified. bAmount (pmol) for 50% inhibition. "No inhibition observed up to 100 prnol per tube. dSorne inhibition observed up to 100 pmol per tube but 50% inhibition not achieved. ecl 6.
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MARY A. SMAL, BRIAN A. BALD0 A N D DAVID G. HARLE
Since the amount of ( *)C,,-PAF required for 50% inhibition was approximately double that of pure (-)C,,-PAF, this indicated that there was no significant binding of (+)C,,-PAF to the antibody binding sites and hence a specific requirement for the R configuration at c-2.
becomes weaker and is probably governed by the steric size of the substituents. The phosphocholine substituent appears to have a certain amount of rotational freedom when the molecule is bound to the polyclonal antibody, although there also appears to be some recognition of the ammonium group by the antibody combining region.
Inhibition studies with the C-3 analogues. The 50% inhibition values for analogues with different substituents at C-3 are shown in Table I . Removal of methyl groups from the nitrogen atom of the polar head group resulted in a decrease of binding of these analogues to the antibody combining site. On a molar basis, the dimethyl compound AGEPDME was approximately one sixth as active as CI,-PAF, while the demethylated analogue AGEPE had an ICso value nine times greater than AGEPDME (Fig. 1). This suggested that variations at C-3 are less important than changes at C-2 or C-1. The greater tolerance to change at C-3 was shown by the gradual reduction of recognition of analogues that have increasing distances between the nitrogen and phosphorus atoms. The IC,, of compound U66985, for example was 8.3 pmol compared to > 100 pmol for the U66982 compound.
Comparison of the antibody combining region and the PAF receptor. At first sight, the molecular recognition pattern
Overall view of the antibody combining region recognized by sheep antibodies. Although over 20 compounds were
examined for inhibitory activity, the panel is by no means exhaustive, particularly with the C-3 analogues. Nevertheless, there is already sufficient data to describe the combining region in some detail, and the required complementary structural features are illustrated in Fig. 2. Clear-cut and specific binding is indicated at the ether linkage at C-1 extending through to the ester linkage at C-2. This binding is most likely governed by charge rather than by hydrophobic interactions. Beyond this region, that is, extending into the hydrophobic C-1 alkyl chain and the C-2 acyl chain at the other end, the binding
for the PAF/antibody combining region shows a striking resemblance to that of the PAF receptor (Braquet and Godfroid, 1987). The order of antibody binding potency for the C-3 and the C-2 acyl analogues parallels the binding pattern of these compounds to the PAF receptor (Braquet and Godfroid, 1987); similarly, both the antibody and the receptor do not recognize PAF analogues where the C-1 ether linkage is replaced by other groups. The antibody recognition pattern, however, varies from the receptor in two domains of the PAF molecule. Firstly, antibody-binding is maximal for C- 1 analogues with a 12-carbon long alkyl chain (which is not unexpected since the immunogen was a 12 carbon long analogue), whereas receptor binding is maximal for the 1-0-hexadecyl PAF analogue. Secondly, the antibodybinding to the C-2 analogues is restricted to those with an 0-acyl-like linkage, that is, PAF, PGEPC and Ro 17-9461, whereas the receptor recognizes, in addition to these analogues, analogues containing ether, amide and carbamate linkages at C-2. Therefore, the common features of the receptor and antibody binding specificities are: an ether linkage at C-1, a C-2 substituent no bigger than a propionyl group, an sn configuration at C-2, and a C-3 polar head group with a specific distance between the phosphorous and nitrogen atoms. Lipophilicity in the 1-0-alkyl chain is required by antibody and receptor, but the degree and position is different. The greatest difference between antibody and receptor binding is in the nature of the linkage at C-2; the antibodies have a specific requirement for an acyl group, whereas the receptor will tolerate other linking groups. We also tested PAF antagonists that are structurally related to PAF (CV 3988) and some which are structurally unrelated (BN 52021 and WEB 2086) for inhibition of antibody binding, but all three compounds proved inactive. The antibodies, therefore, demonstrate, unlike the receptor, a strict requirement for the PAF structure, i.e., I-O-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine. These findings also lead to the clear conclusion that structure/antibody binding relationships do not necessarily reflect structure/receptor activity relationships. For the most biologically active PAF species, viz C,,, C,, and C,,,, the relative activities as agonists (i.e., for aggregation of platelets) and for inhibition of antibody binding were, respectively, 1.268:1:2 (Godfroid er al., 1987) and 1: 1.85: 1.23 (Table 1).
CONCLUSION
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OF MOLECULAR RECOGNITION, VOL. 3,No. 4,1990
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SPECIFICITY OF ANTI-PAF ANTIBODIES
analogues reported to occur in various tissues and fluids are the various 1-0-alkyl homologues of PAF (Mueller et af., 1984; Mallet and Cunningham, 1985; Satouchi el af., 1985; Ramesha and Pickett, 1987; Tokumura et af., 1987) with C,,, C,,., and C,,,predominating. Hence, it seems likely that the RIA will be detecting only analogues that one would want to measure. However, since these analogues have slightly different inhibitory potencies, the PAF analogue(s) used as a standard in the RIA should be carefully chosen to reflect the molecular composition of the PAF fraction in the sample to be assayed. In summary, we have mapped the combining region
for the anti-PAF antibodies used in the PAF immunoassay and shown that they are highly specific for short chain acyl analogues of 1-0-alkyl-2-0-acyl-sn-glycero-3phosphocholine.
Acknowledgements We thank Drs D . J. Hanahan, T. Muramatsu and A. Tokumura for their generous donations of compounds and Drs S. Cooney, J. Czarnecki. A. McCaskill and J. Urban for technical help and/or advice. This work was supported by the National Health and Medical Research Council of Australia.
REFERENCES Barnes, P. J. (1988). Platelet activating factor and asthma. J. Allergy Clin. Immunol. 81, 152-1 60. Barnes, P. J., Chung, K. F., and Page, C. P. (1988). Platelet activating factor as a mediator of allergic disease. J. Allergy Clin. Immunol. 81, 91 9-934. Braquet, P., and Godfroid, J. J. (1987). Conformational properties of the PAF/acether receptor on platelets based on structure/ activity studies. In Platelet-activating factor and Related Lipid Mediators, ed. by F. Snyder, pp. 191-235. Plenum Press, New York. Godfroid, J. J., Broquet, C.,Jouquey, S., Lebbar, M., Heymans, F., Redeuilh, C., Steiner, E., Michel, E., Coeffier, E., Fichelle, J.. and Worcel, M. (1987). Structure/activity relationships in PAF/ acether. 3. Hydrophobic contribution to agonistic activity. J. Med. Chem. 30,792-797. Hanahan, D. J.. Demopoulos, C. A,, Liehr, J., and Pinckard, R. N. (1980). Identification of platelet activating factor isolated from rabbit basophils as acetyl glyceryl ether phosphorylcholine. J. Biol. Chem. 255, 5514 5 5 1 6. Hanahan, D. J . (1986). Platelet activating factor: a biologically active phosphoglyceride. Ann. Rev. Biochem. 55, 483-509. Hanahan, D. J., and Kumar, R. (1987). Platelet activating factor; chemical and biochemical characteristics. Prog. Lipid Res. 26, 1-28. Hirth, G., and Barner, R. (1982). Synthese von glycerylatherphosphatiden. Herstellung von 1-U-octadecyl-2-U-acetyl-snglyceryl-3-phosphorylcholine (platelet activating factor), des enantiomeren sowie einiger analoger verbindungen. Helv. Chim. Acta. 65,1059-1 084. Karasawa. K.. Fujita, K., Satoh, N, Hongo, T., Setaka, M., Ohno, M., and Nojima, S. (1987). Antibody to platelet activating factor (1- 0alkyl- 2-acetyl -sn- glycero- 3- phosphocholine; PA F). J. Biochem. 102,451-453. Mallet, A. I., and Cunningham, F. M. (1985). Structural identification of platelet activating factor in psoriatic scale. Biochem. Biophys. Res. Commun. 126, 192-1 98.
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Mueller, H. W., O’Flaherty, J. T., and Wykle, R. L. (1984). The molecular species distribution of platelet-activating factor synthesized by rabbit and human neutrophils. J. Biol. Chem. 23, 14554-1 4559. Nishihira, J., Ishibashi, T., and Imai, Y. (1984). Production and characterization of specific antibodies against 1 - U-alkyl-2acetyl-sn-glycero-3-phosphocholine (a potent hypotensive and platelet-activating ether-linked phospholipid). J. Biochem. 95. 1247-1 251. Ramesha, C. S., and Pickett, W. C. (1987). Species-specific variations in the molecular heterogeneity of the platelet activating factor. J. Immunol. 138, 1559-1 563. Satouchi, K., Oda, M., Yasunaga, K., and Saito, K. (1985). Evidence for production of 1 -acyl-2-acetyl-sn-glyceryl-3-phosphorylcholine concomitantly with platelet activating factor. Biochem. Biophys. Res. Commun. 128, 1409-1 41 7. Schlondorff, D., and Neuwirth, R. (1986). Platelet activating factor and the kidney. Am. J. Physiol. 251, F1-F11. Smal, M. A,, Baldo, B. A., and Redmond, J. W. (1989). Production of antibodies to platelet activating factor. Mol. Immunol. 26, 71 1-71 9. Smal, M. A., Baldo, B. A., and McCaskill, A. (1990). A specific sensitive radioimmunoassay for platelet-activating factor (PAF). J. Immunol. Methods 128, 183-1 88. Stenson, W. F. (1988). Platelet-activating factor and inflammatory bowel disease. Gastroenterol. 95, 1416-1 421. Tokumura, A., Kamiyasu, K.,Takauchi, K., and Tsukatani, H (1987). Evidence for existence of various hornologues and analogues of platelet activating factor in a lipid extract of bovine brain. Biochem. Biophys. Res. Commun. 145,415-425. Vargafiig, B. B.. and Braquet, P. G. (1987). PAF/acether todayrelevance for acute experimental anaphylaxis. In Britkh Medical BulletinVol. 43, ed. by D. A. Willoughby, pp. 31 2-335. Churchill Livingstone, London. Received 5 March 1990; accepted (revised) 2 April 1990.
JOURNAL OF MOLECULAR RECOGNITION, VOL. 3, No. 4, 1990 173
INTRODUCTION Platelet activating factor (PAF) (1-0-alkyl-2-O-acetylsn-glycero-3-phosphocholine) (Hanahan et af.,1980) is a biologically active phospholipid which induces aggregation of platelets at concentrations of 0.1 nM. The presence of the acetyl group at position 2 is an absolute requirement for biological activity of this phospholipid (Hanahan and Kumar, 1987). PAF has a number of physiological effects (Hanahan, 1986) which suggest that it may be involved in a variety of diseases including asthma (Barnes, 1988), anaphylactic shock (Vargaftig and Braquet, 1987), other allergic conditions (Barnes et al., 1988), kidney disease (Schlondorff and Neuwirth, 1986) and gastric ulceration (Stenson, 1988). If the role of PAF in health and disease is to be clarified, quantitative data on PAF levels in various fluids and tissues will be needed. A sensitive and specific radioimmunoassay (RIA) for PAF appears to have the greatest potential for rapidly quantitating this autacoid in large numbers of biological samples side-by-side in an assay that is easy and convenient to use. The specificity of the antiserum is critical to the accuracy and validity of such an assay. In two previous reports, the production of antibodies to PAF was described, however the specificity of these antibodies was not adequately defined (Nishihira et al., 1984; Karasawa et al., 1987). Recently, we have described the production of a PAF immunogen and an anti-PAF antiserum (Smal et al., 1989) and used the antiserum to develop a sensitive and specific RIA for PAF (Smal et af., 1990). In this paper, we present results of hapten inhibition studies designed to examine the fine structural specificity of the anti-PAF antibody combining sites by identifying
* t
Author to whom correspondence should be addressed. Also affiliated to: The Department of Medicine, University of Sydney, NSW 2006, Australia. Abbreviations used: PAF, platelet activating factor; RIA, radioimmunoassay.
the important structural features on the complementary phospholipids. These features were similar to those required for the pharmacological activity of PAF, indicating that the binding specificity of the antibodies is similar to the specificity of the PAF receptor.
EXPERIMENTAL Antiserum preparation. C,,-PAF ( 1 -0-(12', 1 T-dimethoxydodecyl)-2-0-acetyl-sn-glycero-3-phosphocholine)was prepared and coupled to methylated bovine serum albumin as described previously (Smal et af., 1989). Sheep were immunized with this material and the PAFacetylhydrolase activity in the antisera was destroyed by acid treatment as described recently (Smal el af.,1990). Hapten inhibition studies. Standard solutions of the com-
pounds to be tested were prepared in aqueous ethanol and then diluted in the assay buffer (0.05 M sodium acetate, pH 6.0, containing 0.05% Tween 20) to the appropriate concentration. Anti-PAF antiserum was used at 1 in 8000 diluted in the assay buffer supplemented with deactivated normal sheep serum (1 in 2000). The donkey anti-sheep immunoglobulin (Silenus Laboratories, Melbourne, Australia) (used as the second antibody) was diluted 1 in 250 in the assay buffer containing 6% poly(ethy1ene glycol) and [12SI]PAF (2200 Ci/mmol, New England Nuclear, Boston, MA, USA) was added to give approximately 40 000 counts/ min per 100 pL. Into duplicate polystyrene RIA tubes (Disposable Products, Adelaide, Australia), was placed 100 pL of each of the following: the solution to be tested for inhibition, anti-PAF serum, anti-sheep/tracer. The Bo tubes (zero binding) contained no inhibitor solution and the NSB tubes (nonspecific binding) contained only anti-sheep/tracer. The tubes were incubated at room temperature for 16 h, 4 mL of assay buffer was added and the tubes were centrifuged at 1900 x g for 25 min.
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MARY A. SMAL, BRIAN A. BALD0 AND DAVID G. HARLE
After removing the supernatant, the radioactivity remaining was measured and the percent of tracer bound (%B/B,) to the precipitate was calculated from the formula (B-NSB)/(B,-NSB) x 100. The binding of tracer to the antiserum was approximately 42% in the absence of inhibitors. Compounds were tested for inhibition at final concentrations up to 330 nM. Chemicals. C16-, Clx- and C,, ,-PAFs (1 -0-hexadecyl-,
1-0-octadecyl- and 1-0-octadec-9-cis-enyl-2-O-acetylsn-glycero-3-phosphocholines,respectively) were purchased from Bachem Feinchemikalien (Bubendorf, Switzerland). C,-PAF (l-O-(6',6'-dimethoxyhexyl)-20-acetyl-sn-glycero-3-phosphocholine)and racemic (f)C16-PAFwere prepared as described by Smal et al. (1989) and Hirth and Barner (1 982), respectively. Palmitoyl AGPC (1 -0-hexadecanoyl-2-0-acetyl-snglycero-3-phosphocholine) and ( h )thio-C,,-PAF (1-Soctadecyl-2-0-acetyl-1-thioglycero-3-phosphocholine) were generous gifts from Dr Tokumura (University of Tokushima, Japan) and Dr Muramatsu (Tokyo Medical and Dental University, Japan) respectively. ( I!= )DeoxyC,,-PAF ( 1-deoxy- 1-hexadecyl-2-0-acetyl-glycero-3phosphocholine) was from Lederle laboratories (Pearl River, NY, USA) and Ro 17-9461 (1-0-hexadecyl-2O-rnethylcarbamoyl-sn-glycero-3-phosphocholine),Ro 17-7826 ( 1 -0-octadecyl-2-deoxy-2-acetamido-snglycero-3-phosphocholine) and Ro 17-9777 (1-0-octadecyl - 2 - deoxy - 2 - (N-carbomethoxy)amino-glycero-3phosphocholine) were provided by Hoffmann-La Roche (Basel, Switzerland). PGEPC (1 -0-octadecyl-2-0propionyl-sn-glycero-3-phosphocholine),the corresponding 2-0-butyryl and 2-0-hexanoyl analogues (BuGEPC and HGEPC, respectively), AGEPE (10-alkyl-2-0-acetyl-sn-glycero-3-phosphoethanolamine), AGEPDME ( 1-0-octadecyl-2-0-acetyl-sn-glycero-3phospho-N,N-dimethylethanolamine), U66985 (1-0octadecyl-2-0-acetyl-sn-glycero-3-phosphoricacid-6't r i m e t h y l a m m o n i u m hexyl e s t e r ) a n d U66982 (1 -0-octadecyl - 2-0-acetyl-sn-glycero - 3 - phosphoric acid- 10'-trimethylammonium decyl ester) were generous gifts from Dr Hanahan (University of Texas Health Science Center, San Antonio, TX). The PAF antagonists CV 3988 (rac-3-(N-n-octadecyl-carbamoyloxy)-2-methoxypropyl-2-thiazolioethylphosphate),BN 52021 (3-tbutyl-hexahydro-2,4,7b, 1 1-trihydroxy-8-methyl-9H- I ,7a(epoxymethano)- 1H,6aH-cyclopenta[c]furo-[2,3-b]furo[3' 2':3,4]cyclopenta [ 1,2-d]furan-5,9,12(4H) and WEB 2086 (3-[4-(2-chlorophenyI)-9-methyl-6H-thieno[3,2-fl[ 1,2,4]triazolo-[4,3-a][ 1,4]-diazepin-2-y1]-1-(4-morpholinyl)- 1propanone) were from Takeda Chemical Industries (Osaka, Japan), Dr Pierre Braquet (Le Plessis Robinson, France) and Boehringer Ingelheim (Ingelheim am Rhein, FRG) respectively. Lyso-C,,-PAF (1 -0-hexadecylsn-glycero-3-phosphocholine), methyl lyso-PAF (1-0hexadecyl-2-0-methyl-sn-glycero-3-phosphocholine) and all other chemicals were purchased from the Sigma Chemical Co. (St Louis, MO, USA).
RESULTS AND DISCUSSION Inhibition studies with C-1 analogues of PAF. The inhibition profiles of the PAF analogues which showed significant 170 JOURNAL OF MOLECULAR RECOGNITION, VOL. 3, No. 4,1990
01
10
10
Inhibitor (prnolltube)
Figure 1. Recognition of PAF and some analogues by sheep anti-PAF antibodies. Inhibition dose responses obtained with: ( O ) , CIZ-PAF; ( 0 ) Ce-PAF; , (m),CTrj-PAF; ( O ) , Cie-PAF; (+), CieiPAF; ( 0 ) , racemic (f)CI6-PAF; (A), PGEPC; and (A), AG EPD M E.
binding are shown in Fig. 1 and the 50% inhibitory values for analogues with varying alkyl chain lengths and with nonether substituents at C-1 of the glycerol backbone, are listed in Table 1. The C-1 analogue with the greatest inhibitory activity is C,,-PAF (the twelve carbon long alkyl chain analog) which is not surprising since the antiserum was raised to this analogue coupled to the carrier. The shorter, six carbon analogue (C6PAF) was a slightly weaker inhibitor, as were the long chain analogues (cl6-9 C1,-and C,,,,-PAF) which are the predominant species in naturally occurring PAF. These results indicate that there is a broad specificity in the binding of this polyclonal antibody to the alkyl chain of PAF, although there is a preference for the medium length alkyl chain (i.e., C,,). In contrast, no inhibition was observed with ( f)deoxy-C,,-PAF, the analogue in which the ether oxygen was replaced by a methylene group, and very weak inhibition resulted with compounds in which the oxygen was replaced by either an acyl or a thioether linkage. These results demonstrated that there is an absolute requirement for an ether linkage at C-1 of the glycerol backbone, probably because of both the electronic distribution and the physical size of the grouping. Inhibition studies with C-2 analogues. The 50% inhibition values for these PAF analogues are shown in Table 1. Increasing the chain length of the acyl group decreased the binding dramatically; the propionyl analogue (PGEPC) was a weaker inhibitor than the acetyl analogue (C,,-PAF) (Fig. l), and higher homologues were not recognized. These results suggest that, along with the ether group, the acetyl group is specifically recognized by the antibody combining sites. Replacement of the acyl linkage with an amide (Ro 17-7826) or a N-linked carbamate (Ro 17-9777) abolished antibody binding, whereas a weak binding was observed with the 0-linked carbamate analogue (Ro 17-9461). These findings suggested that an acyl group at C-2, and particularly an acetyl, is necessary for antibody recognition. This is further substantiated by the failure of the hydroxy analogue (lyso-C,,-PAF) and the methoxy analogue (methyl lyso-PAF) to cause any inhibition.
0 1990 by John Wiley & Sons, Ltd.
SPECIFICITY OF ANTI-PAF ANTIBODIES
Table 1. Swcilici@ of anti-PAF antibodies: inhibition of PAF/anti-PAF interaction by C,,-PAF and somi structural analogues IC,b
Substituent at c,. c, or c,
IC,I
(pmol)
IC,, C,,-PAF
C16-PAF Cls-PAF Cis 1 -PAF
0.39 0.72 0.48
1.o 1.85 1.23
C6-PAF ClZ-PAF ( & ) Deoxy-Cl6-PAF ( ?c) Thio-Cls-PAF
0.28 0.1 4 NIC >lo@
0.72 0.36
> 256
Palrnitoyl- AGPC
>100
> 256
Compound'
c1
c2
NI NI
LYSO- C16- PA F Methyl lyso-PAF
4 H -0CH3 0
PGEPC
4 - C CH2CH3
I1
1.7
4.4
>loo
> 256
>loo
> 256
26
66.7
>100
> 256
> 100
> 256
2.3
5.9
AGEPE
21
53.8
U66985
8.3
21.3
U66982
>loo
> 256
0 I BuGEPC
4 - C ( CH2) 2C H3
0 HGEPC
-O-C(CHz),CH, 0
R O 17-9461
-0-CN HCH3
I
0 I1 Ro 17-7826
-N-CC H3 0
( 5 ) Ro 17-9777
-N-C-OCH
I 3
c3
0
I AGEPDME
+
-0-P-0 ( CH2)ZNH ( CH3)2
For ( * ) C16-PAF, ICw=0.84 and ICw/ICw C16-PAF=2.15. The PAF antagonists CV 3988, BN 52021 and WEB 2086 did not inhibit up to a concentration of 100 pmol per tube. "l-@alkyl group is C18 unless specified. bAmount (pmol) for 50% inhibition. "No inhibition observed up to 100 prnol per tube. dSorne inhibition observed up to 100 pmol per tube but 50% inhibition not achieved. ecl 6.
0 1990 by John Wiley & Sons, Ltd.
JOURNAL OF MOLECULAR RECOGNITION, VOL. 3, No. 4,1990
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MARY A. SMAL, BRIAN A. BALD0 A N D DAVID G. HARLE
Since the amount of ( *)C,,-PAF required for 50% inhibition was approximately double that of pure (-)C,,-PAF, this indicated that there was no significant binding of (+)C,,-PAF to the antibody binding sites and hence a specific requirement for the R configuration at c-2.
becomes weaker and is probably governed by the steric size of the substituents. The phosphocholine substituent appears to have a certain amount of rotational freedom when the molecule is bound to the polyclonal antibody, although there also appears to be some recognition of the ammonium group by the antibody combining region.
Inhibition studies with the C-3 analogues. The 50% inhibition values for analogues with different substituents at C-3 are shown in Table I . Removal of methyl groups from the nitrogen atom of the polar head group resulted in a decrease of binding of these analogues to the antibody combining site. On a molar basis, the dimethyl compound AGEPDME was approximately one sixth as active as CI,-PAF, while the demethylated analogue AGEPE had an ICso value nine times greater than AGEPDME (Fig. 1). This suggested that variations at C-3 are less important than changes at C-2 or C-1. The greater tolerance to change at C-3 was shown by the gradual reduction of recognition of analogues that have increasing distances between the nitrogen and phosphorus atoms. The IC,, of compound U66985, for example was 8.3 pmol compared to > 100 pmol for the U66982 compound.
Comparison of the antibody combining region and the PAF receptor. At first sight, the molecular recognition pattern
Overall view of the antibody combining region recognized by sheep antibodies. Although over 20 compounds were
examined for inhibitory activity, the panel is by no means exhaustive, particularly with the C-3 analogues. Nevertheless, there is already sufficient data to describe the combining region in some detail, and the required complementary structural features are illustrated in Fig. 2. Clear-cut and specific binding is indicated at the ether linkage at C-1 extending through to the ester linkage at C-2. This binding is most likely governed by charge rather than by hydrophobic interactions. Beyond this region, that is, extending into the hydrophobic C-1 alkyl chain and the C-2 acyl chain at the other end, the binding
for the PAF/antibody combining region shows a striking resemblance to that of the PAF receptor (Braquet and Godfroid, 1987). The order of antibody binding potency for the C-3 and the C-2 acyl analogues parallels the binding pattern of these compounds to the PAF receptor (Braquet and Godfroid, 1987); similarly, both the antibody and the receptor do not recognize PAF analogues where the C-1 ether linkage is replaced by other groups. The antibody recognition pattern, however, varies from the receptor in two domains of the PAF molecule. Firstly, antibody-binding is maximal for C- 1 analogues with a 12-carbon long alkyl chain (which is not unexpected since the immunogen was a 12 carbon long analogue), whereas receptor binding is maximal for the 1-0-hexadecyl PAF analogue. Secondly, the antibodybinding to the C-2 analogues is restricted to those with an 0-acyl-like linkage, that is, PAF, PGEPC and Ro 17-9461, whereas the receptor recognizes, in addition to these analogues, analogues containing ether, amide and carbamate linkages at C-2. Therefore, the common features of the receptor and antibody binding specificities are: an ether linkage at C-1, a C-2 substituent no bigger than a propionyl group, an sn configuration at C-2, and a C-3 polar head group with a specific distance between the phosphorous and nitrogen atoms. Lipophilicity in the 1-0-alkyl chain is required by antibody and receptor, but the degree and position is different. The greatest difference between antibody and receptor binding is in the nature of the linkage at C-2; the antibodies have a specific requirement for an acyl group, whereas the receptor will tolerate other linking groups. We also tested PAF antagonists that are structurally related to PAF (CV 3988) and some which are structurally unrelated (BN 52021 and WEB 2086) for inhibition of antibody binding, but all three compounds proved inactive. The antibodies, therefore, demonstrate, unlike the receptor, a strict requirement for the PAF structure, i.e., I-O-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine. These findings also lead to the clear conclusion that structure/antibody binding relationships do not necessarily reflect structure/receptor activity relationships. For the most biologically active PAF species, viz C,,, C,, and C,,,, the relative activities as agonists (i.e., for aggregation of platelets) and for inhibition of antibody binding were, respectively, 1.268:1:2 (Godfroid er al., 1987) and 1: 1.85: 1.23 (Table 1).
CONCLUSION
172 JOURNAL
OF MOLECULAR RECOGNITION, VOL. 3,No. 4,1990
0 1990 by John Wiley & Sons, Ltd.
SPECIFICITY OF ANTI-PAF ANTIBODIES
analogues reported to occur in various tissues and fluids are the various 1-0-alkyl homologues of PAF (Mueller et af., 1984; Mallet and Cunningham, 1985; Satouchi el af., 1985; Ramesha and Pickett, 1987; Tokumura et af., 1987) with C,,, C,,., and C,,,predominating. Hence, it seems likely that the RIA will be detecting only analogues that one would want to measure. However, since these analogues have slightly different inhibitory potencies, the PAF analogue(s) used as a standard in the RIA should be carefully chosen to reflect the molecular composition of the PAF fraction in the sample to be assayed. In summary, we have mapped the combining region
for the anti-PAF antibodies used in the PAF immunoassay and shown that they are highly specific for short chain acyl analogues of 1-0-alkyl-2-0-acyl-sn-glycero-3phosphocholine.
Acknowledgements We thank Drs D . J. Hanahan, T. Muramatsu and A. Tokumura for their generous donations of compounds and Drs S. Cooney, J. Czarnecki. A. McCaskill and J. Urban for technical help and/or advice. This work was supported by the National Health and Medical Research Council of Australia.
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Mueller, H. W., O’Flaherty, J. T., and Wykle, R. L. (1984). The molecular species distribution of platelet-activating factor synthesized by rabbit and human neutrophils. J. Biol. Chem. 23, 14554-1 4559. Nishihira, J., Ishibashi, T., and Imai, Y. (1984). Production and characterization of specific antibodies against 1 - U-alkyl-2acetyl-sn-glycero-3-phosphocholine (a potent hypotensive and platelet-activating ether-linked phospholipid). J. Biochem. 95. 1247-1 251. Ramesha, C. S., and Pickett, W. C. (1987). Species-specific variations in the molecular heterogeneity of the platelet activating factor. J. Immunol. 138, 1559-1 563. Satouchi, K., Oda, M., Yasunaga, K., and Saito, K. (1985). Evidence for production of 1 -acyl-2-acetyl-sn-glyceryl-3-phosphorylcholine concomitantly with platelet activating factor. Biochem. Biophys. Res. Commun. 128, 1409-1 41 7. Schlondorff, D., and Neuwirth, R. (1986). Platelet activating factor and the kidney. Am. J. Physiol. 251, F1-F11. Smal, M. A,, Baldo, B. A., and Redmond, J. W. (1989). Production of antibodies to platelet activating factor. Mol. Immunol. 26, 71 1-71 9. Smal, M. A., Baldo, B. A., and McCaskill, A. (1990). A specific sensitive radioimmunoassay for platelet-activating factor (PAF). J. Immunol. Methods 128, 183-1 88. Stenson, W. F. (1988). Platelet-activating factor and inflammatory bowel disease. Gastroenterol. 95, 1416-1 421. Tokumura, A., Kamiyasu, K.,Takauchi, K., and Tsukatani, H (1987). Evidence for existence of various hornologues and analogues of platelet activating factor in a lipid extract of bovine brain. Biochem. Biophys. Res. Commun. 145,415-425. Vargafiig, B. B.. and Braquet, P. G. (1987). PAF/acether todayrelevance for acute experimental anaphylaxis. In Britkh Medical BulletinVol. 43, ed. by D. A. Willoughby, pp. 31 2-335. Churchill Livingstone, London. Received 5 March 1990; accepted (revised) 2 April 1990.
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