Vol. 169, No. 3, 1990 June 29. 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 880-887
PREPARATION AND PARTIAL CHARACTERIZATION OF POLYCLONAL ANTIBODIES RAISED AGAINST A PRESELECIED SEQUENCE OF CALCIUM DEPENDENT LECTINS
0. BLANCK, V. lYIBAULT, C. GRANIER, J. VAN RIETSCHOTEN, J. COURAGEOT,and R. MIQUELIS* Laboratoire de Biochimie, C.N.R.S. UA 1179, Faculte de Mt?decinesecteurNord, Bd. Pierre-Dramard,13326MARSEILLE C&lex 15, France Received
April
20,
1990
SUMMARY : The synthetic-peptide strategy was used to generate antibodies raised against calcium-dependentlectinsof vertebrates.We demonstratethat a synthetic peptide predictedfrom the amino acid sequenceof the carbohydraterecognition domaincan induce blocking antibodieswhich would react with, or in closevicinity of, the binding site of the parent molecule. As the preselected sequencewas choosenin a consensussequenceregion, we alsopresentpreliminary investigations of the useof specific antiseraasa commonbiological probe againstcalcium dependentlectins. The availability of monospecific polyclonal sera opens new possibilities in biochemical and structural studiesaswell asimmunodectionof calcium dependent&tins. el990 Academic Press,Inc.
INTRODUCTION
: Calcium-dependent lectins belong to a growing family of animal lectins
involved in various cell functions such as cell migration and adhesion, immune recognition, intracellular routing of glycoproteins and cell regulation (1, 2). In addition to recognizing and selectively binding carbohydrates, these multipurpose molecules have effector domains for interacting with other cell componentssuchasplasmamembrane,cytoskeleton, extracellular matrix or membraneproteins (1,3). Conversely, the carbohydrate recognition domain (CRD) presentsa relatively high degreeof structural consistency: it is establishedthat it contains approximately 130 amino acid residueswith up to 50 % homology betweenlectins of vertebrates(4). In spite of thesehomologiesit wasobservedthat antibodiesprepared from different lectins do not cross react (5). This is probably becauseof low or lack of antigenicity of homologous regions. In order to circumvent these problems and to generate antibodies suitable for both prospectiveimmunodetectionof putative lectin molecules,aswell as for structural and biochemical studies,we decided to prepareantiseraagainsta protein consensussequencepre-establishedbefore the study. In this report we describe the selectionof the sequenceand the production and partial characterizationof derived antipeptideantibodies. * To whom all correspondenceshouldbe addressed. ABBREVIATIONS : Di-Tris-Lac:a, P-diaspartamideof Tris [(P-lactosyl-oxy)-methyl] (daminohexanamide)methane;SDS : sodiumdodecyl sulfate; PAGE : polyacrylamide gel electrophoresis; RHL :rabbit hepatic lectin; CHL :chicken hepatic lectin; CRD :carbohydrate recognition domain; BSA : bovine serumalbumin; KLH : keyhole limpet hemocyanin. 0006-291x/90 $1.50 Copyright 0 1990 by Academic Press, Inc. A11 rights of reproduction in any form reserved.
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Peptide synthesis and conjugation. Assembly of the peptide was performed by the solid phase technique (6) on the Applied Biosystem 430 A synthetiser. The crude peptide was applied to preparative Cl8 HPLC (Neosystem, France) and fractions were checked by analytical HPLC; only those containing the peptide with a purity greater than 95 % by integration at 230 nm were collected. Correct synthesis of the peptide was confirmed by amino acid analysis. Purified synthetic peptide was coupled to keyhole limpet hemocyanin (KLH) (Calbiochem) in the presence of glutaraldehyde (7). The stochiometry of each conjugate was determined by addition of 1251-labeledpeptide before the coupling reaction. In all cases the coupling efficiency was about 75-80 % and gave - 520 pg peptidelmg KLH. Antibodies production and purification. Male New Zealand rabbits were immunized by either intradermal or lymph node inoculation (8), using 125-150 pg of peptide conjugate or free peptide respectively emulsified in complete Freund’s adjuvant. The peptide preparation for booster injections were emulsified in Freund’s incomplete adjuvant. Fourteen days after the second booster injection, the sera were collected by bleeding of the ear vein and screened for antipeptide reactivity. For some experiments, antipeptide antibodies were further purified by affinity chromatography on immobilized peptide. Affinity columns were prepared by coupling peptide (6 mg) to CNBr activated sepharose 4B (3.5 ml) (Pharmacia) with a coupling efficiency of about 75 % as estimated by the coupling of 1251-labeled peptide. These affinity purified antipeptide antibodies (about 200 &ml serum) were found homogeneous as judged by SDS-PAGE. Preparation of cation dependent lectins. The galactose specific rat hepatic lectin (RHL),and the chicken hepatic lectin (CHL) were purified by established methods (9-10) with slight modifications. Immunoblotting. After separation on SDS-PAGE according to Laemmli (11). total tissue proteins or purified lectins were transblotted on a nitrocellulose sheet (0.45 pm) in a Biometrik Apparatus. The nitrocellulose sheet was blocked for 1 hour with 10 mM Tris-HCl pH 8.0, 150 mM NaCl containing 5 % (w/v) defatted milk and incubated for 1 hour with sera from immune or preimmune rabbit (l/100 in 0.05 % (w/v) Tween 20, 10 mM Tris HCl pH 8.0, 150 mM NaCl (TBST buffer)). After washing with TBST, the blots were incubated for 1 hour with goat antiiabbit immunoglobulin antibodies conjugated to alkaline phosphatase (Promega Biotech.) diluted l/i’500 in TBST and then revealed with the bromochloroindolyl/nitroblue tetrazolium substrate (12). Radiolabeling immunoassays and binding assays. All labeled molecules were iodinated using the iodogen procedure (13) in the presence of l25I-Na (Amersham). The iodinated mixture was chromatographed on GlO Sephadex or PDlO columns (Phmacia) and the iodinated molecules were recovered with a specific activity of respectively : 9.0 pCi/nmole for the synthetic peptide, 320 Ki/nmol for Di-Tris-Lac (kindly provided by R.T. LEE, John Hopkins University, Baltimore, MD), 16-20 pCi/pg for RHL. Radioimmunoassays were performed as follows. Radioiodinated ligands were added to various dilutions of immune or non immune sera in 50 mM phosphate buffer, 1 % BSA, pH 7.4 (500 ~1 final volume) and incubated overnight at 4OC. After addition of non immune sera (50 ~1 of an l/50 dilution) and antirabbit immunoglobulin antibodies (50 ~1 of a l/4 dilution) and a second incubation (1 h 30 at 4”C), antibody ligand complexes were recovered as polyethylene glycol (PEG 8000, Sigma) pellets after PEG precipitation (400 pl of 12,5 % PEG (w/v) solution in binding buffer for 90 min at 4°C) and centrifugation (10,000 rpm for 10 min). The binding activity of RHL as well as the inhibitory capacity of antipeptide antibodies were as ligands and PEG to precipitate determined by an assay using 1251-asialofetuin or t=I-Di-Tris-Lac the ligand-lectin complexes. Each assay mixture contained 100-150 ng of lectin about 2.5x10-lo M and lmgfml BSA, 0.15 M sodium of 1251-asialofetuin or 3.4 x lo-10 M of t251-Di-Tris-Lac chloride, 20 mM calcium chloride, 20 mM Tris HCl pH 7.5,0.02 % sodium azide and 0.6 % (w/v) Triton X 100 in a final volume of 500 ~1 (1 hour at 20°C). Bovine gamma globulins (100 fl of 1.5 mg/ml solution) were then added and the mixture stored at 4’C for 30 min. The light precipitates were collected by vacuum filtration in Whatman GF/C filter disc. The assay tubes and filters were rinced twice with 5 ml of PEG solution (10% PEG) and the radioactivity on the filters was measured in a Packard autogamma counter. Non specific binding was determined by omission of the lectin or addition of a 200-600 fold excess of unlabeled ligand. 881
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Sequence comparison analysis. The RHL sequence was analyzed by the computer Software Package of B.I.S.A.N.C.E. (CITIZ, Centre Inter-Universitaire d’Informatique a Orientation BiomCdicale, Paris). Prediction of antigenic regions was assessed with the program 4-12 using the numeral values and method described by Parker et al. (14). Circular dichroism measurements. CD spectra were obtained on a Jobin-Yvon spectrophotometer (Longjumeau, France). Spectra were measured at 1 nm intervals with a constant time of 25 seconds at 25*C. Data were collected from five separate recordings and averaged using a microcomputer. A quartz cell of 0.5 mm path length with a sample concentration of 0.2 to 0.4 mg/ml was used in far ultraviolet spectra (260 to 182 nm). Data were expressed in variation of molar amino acid residue absorption coefficient (AE). RESULTS Peptide selection and analysis. Because their sequence and their lectin properties are well characterized, RHL (composed of RHLl and RHL2n monomers), CHL and the fucose hepatic lectin were chosen as models to determine a consensus sequence (5, 24). Since it is the most abundant monomer in hepatic lectin, RHLt was chosen as the reference monomer. The region located between amino acid residues 210-228 which presented a predicted immunogenic 0 turn (QNPG) and only 4 non-conserved residues among mammal lectins was thus selected to prepare a synthetic peptide. Two additional lysine residues served to orientate coupling to the carrier protein at the N-terminus rather than to the lysine in the middle of the sequence (Fig. 1). Fig. 2 shows that in aqueous solution the peptide presents a definite secondary structure different from an a helix conformation (lack of positive band near 190 nm and negative bands at
. .. . . .
. . . . . ..
WIGLTDOYGPYKW'JDGTD" ?Kl AWN0
AC0
RCSCWES
228 NUMBER
Figure 1 . hntigenicity prediction profile of the carbohydrate recognition domain of the major subunit of the galactose specific rat hepatic lectin (RHLt) constructed according to Parker a (14). A) carboxyl terminal sequence of the domain from amino acid residue 160 to 284; arrows indicate cysteine amino acid residues assumed to be involved in disulfide bridges (l), ( n ) represent invariant amino acid residues found among vertebrate calcium dependent lectins : RHLt (4) RHL2.3 (4), the chicken hepatic lectin (4), the fucosi hepatic fectin (24)).B) Enlarged view of 210-228 region of RHLt, in order to emphasize structural homologies between sequenced animal calcium dependent lectins : (*) invariant residues in the animal kingdom(l), ( a) highly conserved amino acid residues in aforesaid vertebrate membrane lectins. 882
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WAVELENGTH
Figure 2 .UItraviolet solution (300 @ml).
AND
BIOPHYSICAL
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-109 SERUM
[m)
circular dichroism
DILUTION
spectrum of the synthetic peptide ROV I in aqueous
I with serial dilution of rabbit sera immunized with Fi w-e 3 . lmmunoprecipitation of l%ROV 1 conjugated to KLH,( 0) free ROV I, (0) purified RLH. Preimmune rabbit sera was dim7 used to assess non specific binding (O*\.
205 and 222 nm (15-16)). strands hinging
Structure
on a p turn (residues
peptide. is in all p conformation
prediction
studies (17-18)
9-12, i.e. QNPG)
prompted
indicating
the presence
us to consider
of two p
that this synthetic
and well mimics the preselected sequence. This peptide (M.W.=
2508) was designated as ROV I.
--¤ \. \
2 3 -109 SERllM
4 5 DILUTION
RDV I CONCENTRATION
(-1ogM)
Figure 4 . Immunoprecipitation of 12%RLH with serial dilution of rabbit sera immunized with (D affinity purified RHL , (0) ROVt conjugated to KLH. (0) results with preimmune sera. Fimre 5 . Inhibition by ROV I of AS/ROV I binding to RHL. 883
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1
kDa
116 82
_ -
58 48.5-
-
36.5
_
2
34
5
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
67
2
t_ --
4
kDs
34 -116 -
58
-
36.5 26.6
26.6-
Ficure 6 .Immunoblots of calcium dependent lectins. Total membrane extract and affinity purified lectins were subjected to SDS-PAGE on a 10 % (A) or 14 % (B) resolving acrylamide gel. transferred to nitrocellulose and probed with antisera (l/l00 dilution, see Materials and Methods). A) RHL, lane 1 : silver nitrate stained gel; lane 2 : 5 ug of RHL probed with polyclonal anti RHLantiserum; lane 3 = 5 ug of RHL probed with AS/ROV I lane 4and 5 = RHL probed with preimmune serum (anti RHL and AS/ROV I respectively), lane 5 = RHL probed with polyclonal KLH antiserum; lane 7 = RHL probed with AS/ROV I incubated with ROV I(lO-5 M final concentration).B) CHL, lane 1 = silver nitrate stained gel of total liver protein, lane 2 = total liver protein probed with AS/ROV I, lane3 = purified CHL probedwith AS/ROV I, lane4 = CHL probedwith preimmune serum. f3-galactosidase (116 kDa), fructose-6-phosphatase (84 kDa), piruvate kinase (58 kDa ), fumarase (48.5 kDa), lactic dehydrogenase (36.5 kDa ) and triosephosphatase isomerase (26.6 kDa) served as standards.
Characterization
of polyclonal antisera raised against ROV I (AS/ROV I).
As shownin Fig. 3, both free and KLH conjugated ROV I were immunogenic,butthe best resultswere obtained with the conjugatedpeptide. Thus thesesera(AWROV I) and affinity purified monospeciflc antibodies (Ab/ROV I) were usedin the present study. Fig. 4 shows a significant immunoprecipitation of RHL with AS/ROV I and Fig. 5 that this immunoprecipitation was progressively abolished by increasing amounts of ROV I (Kn.5 = 0.5 x lo-*). In contrast, antibodies raised against RHL failed to recognize ROV I. (Fig. 3). Taken together theseresults indicate : 1) that the immunogenicityof the preselectedsequenceis probably poor, 2) that AS/ROV I doesrecognize the native lectin but only at the level of its preselectedsequence. Fig. 6 lane 1 shows the electrophoretic pattern of our RHL preparation with the three characteristic polypeptide bands near 41 KDa, 48.5 KDa and 58 KDa. Immunoblotting of this hepatic lectin indicated that AS/ROV I not only recognized the major polypeptide RHLt, but also detected two bandsnot previously visualized by the silver staining procedureat about 73 KDa and 84 KDa (Fig. 6A, lane 3). As the molecular weights of these polypeptides are close to those reported for the fucoselectin, a receptor whosespecificity alsoextends to galactoseresidues(5, 19), we speculatethat theserecognized polypeptides are the fucose lectins. AS/ROV I was also able to crossreact with CHL (Fig. 6B, lane 3); however antipeptide antibodiesfailed to detect RHL2 and RHL3 which are known to differ only in their glycoconjugate content (20). Controls showedthat the preselectedsequencewasnot widespread(recognition of only one polypeptide correspondingto the 26 kDa CHL monomersamongthe total chicken liver proteins, seeFig. 6B, lane 2) and was the only sequenceinvolved in AS/ROV I specific reactivity (inhibition of reactivity in the presenceof free peptides, seeFig. 6A, lane 7). Inhibition binding studies. The possibility that blocking antibodies were generated was investigated by binding experimentsusing RHL asthe receptor and 1251-asialofetuinasligand. As 884
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E : c O* 0
5
10
SERA Figure 7 . Inhibition of12%asialofetuin carried out as described under “Materials and ( q ) AWROV I.
ADDED
15
r
G
20°
(al)
binding to RHL. 12%asialofetuin and Methods” and challenged with
binding to RHL was (m) preimmune serum
shown in Fig. 7, asialofetuin binding was progessively inhibited by increasing amounts of antisera. To obtain stronger evidence for a possible direct involvement of the peptidic consensus sequence in the ligand association process, we performed a second set of experiments in which Ab/ROV I were first preincubated with RIIL and 1251-Dis-Tri-Lac, a small flexible neoglycoprotein able to reach free binding sites even in the presence of larger ligand (21-22) was used as a probe. Under these conditions, Ab/ROV I was also shown to block binding (Fig. 8). In both cases, and as expected, inhibition was not complete since specific antibodies do not recognize RHLm monomers. DISCUSSION
: The fact that AWROV
I does not recognize RHL2/3 is undoubtly a limiting factor
for its use as a probe to identify calcium dependent lectins. This shortcoming is likely linked to the
binding to RHL Fitzure 8 . Jnhibition of 1251-Di-Tris-Lac binding to RH. 12%Di-Tris-Lac carried out as described under “Materials and Methods” and challenged with ( W) preimmune and (0) Ab/ROV I .Results are mean values of two distinct experiments in duplicate. 885
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Table
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I ASIROV1
MOLECULE
SEOUENCE REACTIVITY
Aov
K 1
1
RHL-1
(210.229)
RHL-2/3
(255.303)
FucoasLect.
(469.499)
CM.
K W 2345670
I
OLTDDN 9
10
a 11
PWKWVDOTDY 12 13 14 15
+ 16
17
II)
19
20
21
(139.150)
Comparison of the amino acid sequence of the synthetic peptide ROV I with homologous sequences of calcium dependent membrane lectins. Numbers in parentheses correspond to inclusive amino acid residues of each sequenceConserved amino acid residues are boxed in order to emphasize homology. (+) indirect evidence.
fact that the highest immunogenic
amino acidsare those forming
the p turn in ROV I, and that these
residuesare not highly conserved in the sequenceof calcium dependent lectins (Table I). Thus replacement of Q(9), N(lO) and P (12) in RHLl, by K, D and S, respectively in RHLm could account for the failure of AS/ROV
I to recognize
RI-Em.
On the other hand, that strict preservation
of the QNPG sequenceis not an absolutecondition for recognition by AS/ROV I is shown by the fact that the presenceof N(l0) alone can mediate its immunodetection on CHL. Similarly, the preservation of Q(9) in the lectin specific for fucose would probably account for its detection. Neverthelessour recent finding that AS/ROV I doesrecognize the N-acetylglucosamine thyroid lectin (23) (result not shown) hasprompted us to initiate investigations of its reactivity againsta larger spectrum of well defined lectins and to plan experiments for the determination of the minimum sequencerequiredto obtain a constantpositive reaction. As AS/ROVI demonstratedthat the preselectedsequenceis an accessibleregion of CRD, antipeptide antibodiescan be usedfor structural analysisand lend themselvesto the elucidation of the structure of the CRD binding site. Finally, as Ab/ROV I are blocking antibodies probably interacting directly on, or in closevicinity of, the true carbohydrate binding site, they could be used asa tool for the study of location and intracellular muting of “uncoupled” - i.e. ligand free &tins in biological systems. ACKNOWLEDGMENTS : We are greatly indebted to Pr. ROCHAT for his support and encouragement.We are grateful to Dr. REIKO T. LEE for providing us with Di-Tris-Lat. The authors greatly appreciate the help of E. LORET for circular dichroism determination of ROV I, J.C. FONTECILLA-CAMPS and Y. MALTHIERY for secondary structure and antigenicity prediction studies.The excellent animalhusbandryof M. ALVITRE wasalsoappreciated. REFERENCES 1. Drickamer, K. (1988) J. Biol. Chem,, a, 9557-9560. 2. Barondes, S.H.(1988) TIBS, u, 480-482. 3. Vega, M.A. and Strominger, J.L. (1989) Proc. Natl. Acad. Sci.USA, 86,26882692. 886
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4. 5. 6. 7. 8.
RESEARCH COMMUNICATIONS
Drickamer, K. (1987) Kidney Int., 2, S 167-S 180. Lehrman, M.A., Hill, R.L. (1986) J. Biol. Chem., 261,7419-7425. Merrifield, R.B., (1983) Science, m, 341-347. Avrameas, S., (1969) Immunochemistry, 6,43-52. Sigel, M.B., Sinha, Y.N. and Vanderlaan, W.P. (1983) in Methods in Enzymology, Vol. 93, Eds. J.J. Langone and H. Van Vu&is, Academic Press,p. 3-12. 9. Kawasaki, T. and Ashwell, G. (1977), J. Bioi. Chem. m, 6536-6543. 10. Kuhlenschmidt, T.B. and Lee, Y.C. (1984), Biochemistry, Z, 3569-3575. 11. Laemmli, N.K. (1970), Nature, 227, 680-685. 12. Harlow, E. and W. Lane (1988) in Antibodies, A Laboratory Manual, Cold Spring Harbor, p. 505. 13. Salacinski, P.R., McLean, C., Sykes, J.E.C., Clement-Jones,V.V. and Lowry, P.J. (1981) Anal. Biochem.,U, 136-146. 14. Parker, J.M.R., Guo, D. and Hodges, R.S. (1986) Biochemistry, 25, 5425-5432. 15. Brahms, S. and Brahms, J. (1980) J. Mol. Biol., m, 149-173. 16. Woody, R.W., (1985) in The Peptides(Hruby, V,. Ed.) Volume 7, pp 15-114, Academic PressInc. 17. Chou, P. and Fasman,G.D. (1977) J. Mol. Biol.,m, 135-175. 18. Dufton, M.J. and Hider, R.C. (1977) J. Mol. Biol., I& 177-190. 19. Lehrman, M.A., Haltiwanger, H.S. and Hill, R.L. (1986) J. Biol. Chem.,m, 7426-7432. 20. Halberg, D.F., Wager, R.E., Farell, D.C., Hildreth, IV, J., Quesenberry, M.S., Loeb, J.A., Holland, E.C. and Drickamer, K., (1987) J. Biol. Chem., =,98289838. 21. Lee, R.T.,Lin, P. and Lee, Y.C. (1984) Biochemistry, 21,4255-4261. 22. Hardy, M.R., Townsend, R.R., Parkhurst, S.M. and Lee, Y.C. (1985) Biochemistry, X 22-28. 23. Miquelis, R., Alquier, C., Monsigny, M. (1987) J. Biol. Chem., 262, 1529115298. 24. Hoyle, G.W. and R.L. Hill (1988) J. Biol. Chem., m, 7487-7492.
887
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 880-887
PREPARATION AND PARTIAL CHARACTERIZATION OF POLYCLONAL ANTIBODIES RAISED AGAINST A PRESELECIED SEQUENCE OF CALCIUM DEPENDENT LECTINS
0. BLANCK, V. lYIBAULT, C. GRANIER, J. VAN RIETSCHOTEN, J. COURAGEOT,and R. MIQUELIS* Laboratoire de Biochimie, C.N.R.S. UA 1179, Faculte de Mt?decinesecteurNord, Bd. Pierre-Dramard,13326MARSEILLE C&lex 15, France Received
April
20,
1990
SUMMARY : The synthetic-peptide strategy was used to generate antibodies raised against calcium-dependentlectinsof vertebrates.We demonstratethat a synthetic peptide predictedfrom the amino acid sequenceof the carbohydraterecognition domaincan induce blocking antibodieswhich would react with, or in closevicinity of, the binding site of the parent molecule. As the preselected sequencewas choosenin a consensussequenceregion, we alsopresentpreliminary investigations of the useof specific antiseraasa commonbiological probe againstcalcium dependentlectins. The availability of monospecific polyclonal sera opens new possibilities in biochemical and structural studiesaswell asimmunodectionof calcium dependent&tins. el990 Academic Press,Inc.
INTRODUCTION
: Calcium-dependent lectins belong to a growing family of animal lectins
involved in various cell functions such as cell migration and adhesion, immune recognition, intracellular routing of glycoproteins and cell regulation (1, 2). In addition to recognizing and selectively binding carbohydrates, these multipurpose molecules have effector domains for interacting with other cell componentssuchasplasmamembrane,cytoskeleton, extracellular matrix or membraneproteins (1,3). Conversely, the carbohydrate recognition domain (CRD) presentsa relatively high degreeof structural consistency: it is establishedthat it contains approximately 130 amino acid residueswith up to 50 % homology betweenlectins of vertebrates(4). In spite of thesehomologiesit wasobservedthat antibodiesprepared from different lectins do not cross react (5). This is probably becauseof low or lack of antigenicity of homologous regions. In order to circumvent these problems and to generate antibodies suitable for both prospectiveimmunodetectionof putative lectin molecules,aswell as for structural and biochemical studies,we decided to prepareantiseraagainsta protein consensussequencepre-establishedbefore the study. In this report we describe the selectionof the sequenceand the production and partial characterizationof derived antipeptideantibodies. * To whom all correspondenceshouldbe addressed. ABBREVIATIONS : Di-Tris-Lac:a, P-diaspartamideof Tris [(P-lactosyl-oxy)-methyl] (daminohexanamide)methane;SDS : sodiumdodecyl sulfate; PAGE : polyacrylamide gel electrophoresis; RHL :rabbit hepatic lectin; CHL :chicken hepatic lectin; CRD :carbohydrate recognition domain; BSA : bovine serumalbumin; KLH : keyhole limpet hemocyanin. 0006-291x/90 $1.50 Copyright 0 1990 by Academic Press, Inc. A11 rights of reproduction in any form reserved.
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Peptide synthesis and conjugation. Assembly of the peptide was performed by the solid phase technique (6) on the Applied Biosystem 430 A synthetiser. The crude peptide was applied to preparative Cl8 HPLC (Neosystem, France) and fractions were checked by analytical HPLC; only those containing the peptide with a purity greater than 95 % by integration at 230 nm were collected. Correct synthesis of the peptide was confirmed by amino acid analysis. Purified synthetic peptide was coupled to keyhole limpet hemocyanin (KLH) (Calbiochem) in the presence of glutaraldehyde (7). The stochiometry of each conjugate was determined by addition of 1251-labeledpeptide before the coupling reaction. In all cases the coupling efficiency was about 75-80 % and gave - 520 pg peptidelmg KLH. Antibodies production and purification. Male New Zealand rabbits were immunized by either intradermal or lymph node inoculation (8), using 125-150 pg of peptide conjugate or free peptide respectively emulsified in complete Freund’s adjuvant. The peptide preparation for booster injections were emulsified in Freund’s incomplete adjuvant. Fourteen days after the second booster injection, the sera were collected by bleeding of the ear vein and screened for antipeptide reactivity. For some experiments, antipeptide antibodies were further purified by affinity chromatography on immobilized peptide. Affinity columns were prepared by coupling peptide (6 mg) to CNBr activated sepharose 4B (3.5 ml) (Pharmacia) with a coupling efficiency of about 75 % as estimated by the coupling of 1251-labeled peptide. These affinity purified antipeptide antibodies (about 200 &ml serum) were found homogeneous as judged by SDS-PAGE. Preparation of cation dependent lectins. The galactose specific rat hepatic lectin (RHL),and the chicken hepatic lectin (CHL) were purified by established methods (9-10) with slight modifications. Immunoblotting. After separation on SDS-PAGE according to Laemmli (11). total tissue proteins or purified lectins were transblotted on a nitrocellulose sheet (0.45 pm) in a Biometrik Apparatus. The nitrocellulose sheet was blocked for 1 hour with 10 mM Tris-HCl pH 8.0, 150 mM NaCl containing 5 % (w/v) defatted milk and incubated for 1 hour with sera from immune or preimmune rabbit (l/100 in 0.05 % (w/v) Tween 20, 10 mM Tris HCl pH 8.0, 150 mM NaCl (TBST buffer)). After washing with TBST, the blots were incubated for 1 hour with goat antiiabbit immunoglobulin antibodies conjugated to alkaline phosphatase (Promega Biotech.) diluted l/i’500 in TBST and then revealed with the bromochloroindolyl/nitroblue tetrazolium substrate (12). Radiolabeling immunoassays and binding assays. All labeled molecules were iodinated using the iodogen procedure (13) in the presence of l25I-Na (Amersham). The iodinated mixture was chromatographed on GlO Sephadex or PDlO columns (Phmacia) and the iodinated molecules were recovered with a specific activity of respectively : 9.0 pCi/nmole for the synthetic peptide, 320 Ki/nmol for Di-Tris-Lac (kindly provided by R.T. LEE, John Hopkins University, Baltimore, MD), 16-20 pCi/pg for RHL. Radioimmunoassays were performed as follows. Radioiodinated ligands were added to various dilutions of immune or non immune sera in 50 mM phosphate buffer, 1 % BSA, pH 7.4 (500 ~1 final volume) and incubated overnight at 4OC. After addition of non immune sera (50 ~1 of an l/50 dilution) and antirabbit immunoglobulin antibodies (50 ~1 of a l/4 dilution) and a second incubation (1 h 30 at 4”C), antibody ligand complexes were recovered as polyethylene glycol (PEG 8000, Sigma) pellets after PEG precipitation (400 pl of 12,5 % PEG (w/v) solution in binding buffer for 90 min at 4°C) and centrifugation (10,000 rpm for 10 min). The binding activity of RHL as well as the inhibitory capacity of antipeptide antibodies were as ligands and PEG to precipitate determined by an assay using 1251-asialofetuin or t=I-Di-Tris-Lac the ligand-lectin complexes. Each assay mixture contained 100-150 ng of lectin about 2.5x10-lo M and lmgfml BSA, 0.15 M sodium of 1251-asialofetuin or 3.4 x lo-10 M of t251-Di-Tris-Lac chloride, 20 mM calcium chloride, 20 mM Tris HCl pH 7.5,0.02 % sodium azide and 0.6 % (w/v) Triton X 100 in a final volume of 500 ~1 (1 hour at 20°C). Bovine gamma globulins (100 fl of 1.5 mg/ml solution) were then added and the mixture stored at 4’C for 30 min. The light precipitates were collected by vacuum filtration in Whatman GF/C filter disc. The assay tubes and filters were rinced twice with 5 ml of PEG solution (10% PEG) and the radioactivity on the filters was measured in a Packard autogamma counter. Non specific binding was determined by omission of the lectin or addition of a 200-600 fold excess of unlabeled ligand. 881
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Sequence comparison analysis. The RHL sequence was analyzed by the computer Software Package of B.I.S.A.N.C.E. (CITIZ, Centre Inter-Universitaire d’Informatique a Orientation BiomCdicale, Paris). Prediction of antigenic regions was assessed with the program 4-12 using the numeral values and method described by Parker et al. (14). Circular dichroism measurements. CD spectra were obtained on a Jobin-Yvon spectrophotometer (Longjumeau, France). Spectra were measured at 1 nm intervals with a constant time of 25 seconds at 25*C. Data were collected from five separate recordings and averaged using a microcomputer. A quartz cell of 0.5 mm path length with a sample concentration of 0.2 to 0.4 mg/ml was used in far ultraviolet spectra (260 to 182 nm). Data were expressed in variation of molar amino acid residue absorption coefficient (AE). RESULTS Peptide selection and analysis. Because their sequence and their lectin properties are well characterized, RHL (composed of RHLl and RHL2n monomers), CHL and the fucose hepatic lectin were chosen as models to determine a consensus sequence (5, 24). Since it is the most abundant monomer in hepatic lectin, RHLt was chosen as the reference monomer. The region located between amino acid residues 210-228 which presented a predicted immunogenic 0 turn (QNPG) and only 4 non-conserved residues among mammal lectins was thus selected to prepare a synthetic peptide. Two additional lysine residues served to orientate coupling to the carrier protein at the N-terminus rather than to the lysine in the middle of the sequence (Fig. 1). Fig. 2 shows that in aqueous solution the peptide presents a definite secondary structure different from an a helix conformation (lack of positive band near 190 nm and negative bands at
. .. . . .
. . . . . ..
WIGLTDOYGPYKW'JDGTD" ?Kl AWN0
AC0
RCSCWES
228 NUMBER
Figure 1 . hntigenicity prediction profile of the carbohydrate recognition domain of the major subunit of the galactose specific rat hepatic lectin (RHLt) constructed according to Parker a (14). A) carboxyl terminal sequence of the domain from amino acid residue 160 to 284; arrows indicate cysteine amino acid residues assumed to be involved in disulfide bridges (l), ( n ) represent invariant amino acid residues found among vertebrate calcium dependent lectins : RHLt (4) RHL2.3 (4), the chicken hepatic lectin (4), the fucosi hepatic fectin (24)).B) Enlarged view of 210-228 region of RHLt, in order to emphasize structural homologies between sequenced animal calcium dependent lectins : (*) invariant residues in the animal kingdom(l), ( a) highly conserved amino acid residues in aforesaid vertebrate membrane lectins. 882
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WAVELENGTH
Figure 2 .UItraviolet solution (300 @ml).
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-109 SERUM
[m)
circular dichroism
DILUTION
spectrum of the synthetic peptide ROV I in aqueous
I with serial dilution of rabbit sera immunized with Fi w-e 3 . lmmunoprecipitation of l%ROV 1 conjugated to KLH,( 0) free ROV I, (0) purified RLH. Preimmune rabbit sera was dim7 used to assess non specific binding (O*\.
205 and 222 nm (15-16)). strands hinging
Structure
on a p turn (residues
peptide. is in all p conformation
prediction
studies (17-18)
9-12, i.e. QNPG)
prompted
indicating
the presence
us to consider
of two p
that this synthetic
and well mimics the preselected sequence. This peptide (M.W.=
2508) was designated as ROV I.
--¤ \. \
2 3 -109 SERllM
4 5 DILUTION
RDV I CONCENTRATION
(-1ogM)
Figure 4 . Immunoprecipitation of 12%RLH with serial dilution of rabbit sera immunized with (D affinity purified RHL , (0) ROVt conjugated to KLH. (0) results with preimmune sera. Fimre 5 . Inhibition by ROV I of AS/ROV I binding to RHL. 883
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1
kDa
116 82
_ -
58 48.5-
-
36.5
_
2
34
5
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I
67
2
t_ --
4
kDs
34 -116 -
58
-
36.5 26.6
26.6-
Ficure 6 .Immunoblots of calcium dependent lectins. Total membrane extract and affinity purified lectins were subjected to SDS-PAGE on a 10 % (A) or 14 % (B) resolving acrylamide gel. transferred to nitrocellulose and probed with antisera (l/l00 dilution, see Materials and Methods). A) RHL, lane 1 : silver nitrate stained gel; lane 2 : 5 ug of RHL probed with polyclonal anti RHLantiserum; lane 3 = 5 ug of RHL probed with AS/ROV I lane 4and 5 = RHL probed with preimmune serum (anti RHL and AS/ROV I respectively), lane 5 = RHL probed with polyclonal KLH antiserum; lane 7 = RHL probed with AS/ROV I incubated with ROV I(lO-5 M final concentration).B) CHL, lane 1 = silver nitrate stained gel of total liver protein, lane 2 = total liver protein probed with AS/ROV I, lane3 = purified CHL probedwith AS/ROV I, lane4 = CHL probedwith preimmune serum. f3-galactosidase (116 kDa), fructose-6-phosphatase (84 kDa), piruvate kinase (58 kDa ), fumarase (48.5 kDa), lactic dehydrogenase (36.5 kDa ) and triosephosphatase isomerase (26.6 kDa) served as standards.
Characterization
of polyclonal antisera raised against ROV I (AS/ROV I).
As shownin Fig. 3, both free and KLH conjugated ROV I were immunogenic,butthe best resultswere obtained with the conjugatedpeptide. Thus thesesera(AWROV I) and affinity purified monospeciflc antibodies (Ab/ROV I) were usedin the present study. Fig. 4 shows a significant immunoprecipitation of RHL with AS/ROV I and Fig. 5 that this immunoprecipitation was progressively abolished by increasing amounts of ROV I (Kn.5 = 0.5 x lo-*). In contrast, antibodies raised against RHL failed to recognize ROV I. (Fig. 3). Taken together theseresults indicate : 1) that the immunogenicityof the preselectedsequenceis probably poor, 2) that AS/ROV I doesrecognize the native lectin but only at the level of its preselectedsequence. Fig. 6 lane 1 shows the electrophoretic pattern of our RHL preparation with the three characteristic polypeptide bands near 41 KDa, 48.5 KDa and 58 KDa. Immunoblotting of this hepatic lectin indicated that AS/ROV I not only recognized the major polypeptide RHLt, but also detected two bandsnot previously visualized by the silver staining procedureat about 73 KDa and 84 KDa (Fig. 6A, lane 3). As the molecular weights of these polypeptides are close to those reported for the fucoselectin, a receptor whosespecificity alsoextends to galactoseresidues(5, 19), we speculatethat theserecognized polypeptides are the fucose lectins. AS/ROV I was also able to crossreact with CHL (Fig. 6B, lane 3); however antipeptide antibodiesfailed to detect RHL2 and RHL3 which are known to differ only in their glycoconjugate content (20). Controls showedthat the preselectedsequencewasnot widespread(recognition of only one polypeptide correspondingto the 26 kDa CHL monomersamongthe total chicken liver proteins, seeFig. 6B, lane 2) and was the only sequenceinvolved in AS/ROV I specific reactivity (inhibition of reactivity in the presenceof free peptides, seeFig. 6A, lane 7). Inhibition binding studies. The possibility that blocking antibodies were generated was investigated by binding experimentsusing RHL asthe receptor and 1251-asialofetuinasligand. As 884
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E : c O* 0
5
10
SERA Figure 7 . Inhibition of12%asialofetuin carried out as described under “Materials and ( q ) AWROV I.
ADDED
15
r
G
20°
(al)
binding to RHL. 12%asialofetuin and Methods” and challenged with
binding to RHL was (m) preimmune serum
shown in Fig. 7, asialofetuin binding was progessively inhibited by increasing amounts of antisera. To obtain stronger evidence for a possible direct involvement of the peptidic consensus sequence in the ligand association process, we performed a second set of experiments in which Ab/ROV I were first preincubated with RIIL and 1251-Dis-Tri-Lac, a small flexible neoglycoprotein able to reach free binding sites even in the presence of larger ligand (21-22) was used as a probe. Under these conditions, Ab/ROV I was also shown to block binding (Fig. 8). In both cases, and as expected, inhibition was not complete since specific antibodies do not recognize RHLm monomers. DISCUSSION
: The fact that AWROV
I does not recognize RHL2/3 is undoubtly a limiting factor
for its use as a probe to identify calcium dependent lectins. This shortcoming is likely linked to the
binding to RHL Fitzure 8 . Jnhibition of 1251-Di-Tris-Lac binding to RH. 12%Di-Tris-Lac carried out as described under “Materials and Methods” and challenged with ( W) preimmune and (0) Ab/ROV I .Results are mean values of two distinct experiments in duplicate. 885
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Table
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I ASIROV1
MOLECULE
SEOUENCE REACTIVITY
Aov
K 1
1
RHL-1
(210.229)
RHL-2/3
(255.303)
FucoasLect.
(469.499)
CM.
K W 2345670
I
OLTDDN 9
10
a 11
PWKWVDOTDY 12 13 14 15
+ 16
17
II)
19
20
21
(139.150)
Comparison of the amino acid sequence of the synthetic peptide ROV I with homologous sequences of calcium dependent membrane lectins. Numbers in parentheses correspond to inclusive amino acid residues of each sequenceConserved amino acid residues are boxed in order to emphasize homology. (+) indirect evidence.
fact that the highest immunogenic
amino acidsare those forming
the p turn in ROV I, and that these
residuesare not highly conserved in the sequenceof calcium dependent lectins (Table I). Thus replacement of Q(9), N(lO) and P (12) in RHLl, by K, D and S, respectively in RHLm could account for the failure of AS/ROV
I to recognize
RI-Em.
On the other hand, that strict preservation
of the QNPG sequenceis not an absolutecondition for recognition by AS/ROV I is shown by the fact that the presenceof N(l0) alone can mediate its immunodetection on CHL. Similarly, the preservation of Q(9) in the lectin specific for fucose would probably account for its detection. Neverthelessour recent finding that AS/ROV I doesrecognize the N-acetylglucosamine thyroid lectin (23) (result not shown) hasprompted us to initiate investigations of its reactivity againsta larger spectrum of well defined lectins and to plan experiments for the determination of the minimum sequencerequiredto obtain a constantpositive reaction. As AS/ROVI demonstratedthat the preselectedsequenceis an accessibleregion of CRD, antipeptide antibodiescan be usedfor structural analysisand lend themselvesto the elucidation of the structure of the CRD binding site. Finally, as Ab/ROV I are blocking antibodies probably interacting directly on, or in closevicinity of, the true carbohydrate binding site, they could be used asa tool for the study of location and intracellular muting of “uncoupled” - i.e. ligand free &tins in biological systems. ACKNOWLEDGMENTS : We are greatly indebted to Pr. ROCHAT for his support and encouragement.We are grateful to Dr. REIKO T. LEE for providing us with Di-Tris-Lat. The authors greatly appreciate the help of E. LORET for circular dichroism determination of ROV I, J.C. FONTECILLA-CAMPS and Y. MALTHIERY for secondary structure and antigenicity prediction studies.The excellent animalhusbandryof M. ALVITRE wasalsoappreciated. REFERENCES 1. Drickamer, K. (1988) J. Biol. Chem,, a, 9557-9560. 2. Barondes, S.H.(1988) TIBS, u, 480-482. 3. Vega, M.A. and Strominger, J.L. (1989) Proc. Natl. Acad. Sci.USA, 86,26882692. 886
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4. 5. 6. 7. 8.
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Drickamer, K. (1987) Kidney Int., 2, S 167-S 180. Lehrman, M.A., Hill, R.L. (1986) J. Biol. Chem., 261,7419-7425. Merrifield, R.B., (1983) Science, m, 341-347. Avrameas, S., (1969) Immunochemistry, 6,43-52. Sigel, M.B., Sinha, Y.N. and Vanderlaan, W.P. (1983) in Methods in Enzymology, Vol. 93, Eds. J.J. Langone and H. Van Vu&is, Academic Press,p. 3-12. 9. Kawasaki, T. and Ashwell, G. (1977), J. Bioi. Chem. m, 6536-6543. 10. Kuhlenschmidt, T.B. and Lee, Y.C. (1984), Biochemistry, Z, 3569-3575. 11. Laemmli, N.K. (1970), Nature, 227, 680-685. 12. Harlow, E. and W. Lane (1988) in Antibodies, A Laboratory Manual, Cold Spring Harbor, p. 505. 13. Salacinski, P.R., McLean, C., Sykes, J.E.C., Clement-Jones,V.V. and Lowry, P.J. (1981) Anal. Biochem.,U, 136-146. 14. Parker, J.M.R., Guo, D. and Hodges, R.S. (1986) Biochemistry, 25, 5425-5432. 15. Brahms, S. and Brahms, J. (1980) J. Mol. Biol., m, 149-173. 16. Woody, R.W., (1985) in The Peptides(Hruby, V,. Ed.) Volume 7, pp 15-114, Academic PressInc. 17. Chou, P. and Fasman,G.D. (1977) J. Mol. Biol.,m, 135-175. 18. Dufton, M.J. and Hider, R.C. (1977) J. Mol. Biol., I& 177-190. 19. Lehrman, M.A., Haltiwanger, H.S. and Hill, R.L. (1986) J. Biol. Chem.,m, 7426-7432. 20. Halberg, D.F., Wager, R.E., Farell, D.C., Hildreth, IV, J., Quesenberry, M.S., Loeb, J.A., Holland, E.C. and Drickamer, K., (1987) J. Biol. Chem., =,98289838. 21. Lee, R.T.,Lin, P. and Lee, Y.C. (1984) Biochemistry, 21,4255-4261. 22. Hardy, M.R., Townsend, R.R., Parkhurst, S.M. and Lee, Y.C. (1985) Biochemistry, X 22-28. 23. Miquelis, R., Alquier, C., Monsigny, M. (1987) J. Biol. Chem., 262, 1529115298. 24. Hoyle, G.W. and R.L. Hill (1988) J. Biol. Chem., m, 7487-7492.
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