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Antibody combining site heterogeneity within the response to phosphocholine-keyhole limpet hemocyanin
0161.5890189 $3.00 +O.OO Pergamon Press plc
Molerulor hmunolog~~, Vol. 26, No. I, pp. 63-l Is 1989 Printed in Great Britam.
ANTIBODY COMBINING SITE HETEROGENEITY WITHIN THE RESPONSE TO PHOSPHOCHOLINEKEYHOLE LIMPET HEMOCYANIN* URS BRUDERER,~~~MARY P. STENZEL-POORE,? HANS PETER BACHINGER,~ JACK H. FELLMAN~ and MARVIN B. RITTENBERG? TDepartment of Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201, U.S.A. $Shriners Hospital for Crippled Children and the Department of Biochemistry, Sciences University, Portland, OR 97201, U.S.A. SDepartment of Biochemistry, Oregon University, Portland, OR 97201, U.S.A.
Oregon Health Health Sciences
(Firs1 received 30 March 1988; accepted 21 April 1988)
Abstract-The memory response to PC-KLH is dominated by two antibody populations differing in fine specificity. Group I antibodies show affinity for both phosphocholine (PC) and p-nitrophenyl phosphocholine (NPPC). Group II antibodies exhibit significant affinity only for NPPC. Here, we describe the binding site charactersitics of Group II antibodies and show that in recognizing NPPC these antibodies have a common requirement for the phenyl moiety, a negatively charged phosphate, and the trimethyl structure of the choline. However, Group II antibodies were found to differ in their requirement for the positively charged nitrogen of choline and thus could be divided into two subgroups. In contrast to Group II-A, G&up IIIB antibo&es recognize not only NPPC but also its analogp-&rophenyl-3,3-dimethyl but;1 nhosohate (NPDBP). which differs from NPPC by substituting a carbon for the positivelv charged hitrogen of the choline moiety. These results suggest that Group II-B antibodies do not require the posizve charge in order to bind, although the binding constant, Ka, was increased when the nitrogen was present. Furthermore, heterogeneity within Group II antibodies was characterized by differences in binding to
dinitrophenyl phosphocholine which has an additional phenyl ring and aminophenyl phosphocholine which has an amino group in place of the nitro group of NPPC. The results indicate that diversity in the memory response differ appreciably
to PC-KLH is reflected in the Group II antigen-binding phenotype in their recognition of various structural aspects of the hapten.
by antibodies
which
al., 1987) indicates that Group II antibodies use V genes different from those utilized by Group I antibodies, although some use V genes also found in Group I antibodies but in novel combinations. Thus, it appears that the diversification in the secondary response to PC-KLH is to a large extent the result of the recruitment of new gene combinations rather than of somatic mutations of Group I clonotypes. Group II antibodies all have in common that their binding to PC-conjugated proteins can be inhibited by NPPC. Here we analyze the contribution of different structural aspects of NPPC to the antigen-antibody We have compared the ability of interaction. Group II hybridoma proteins to recognize NPPC and analogs which differ from NPPC in both structure and charge (Table 1). Using p-nitrophenyl methyl phosphonate choline (NPMPC) we show that the negative charge in the phosphate moiety is crucial for the binding of all Group II antibodies tested. In contrast it is possible to distinguish two subsets of Group II antibodies by their different requirements for the positively charged nitrogen in the choline moiety. Group II-A antibodies require the presence of the positive charge since they are not inhibited by p-nitrophenyl 3,3-dimethyl butyl phosphate (NPDBP) in which a carbon replaces the positively charged nitrogen of the trimethylammonium moiety. Group II-B antibodies on the other hand recognize NPDBP although the binding constant, Ka, is in-
lNTRODUCTlON The memory response to phosphocholine-keyhole limpet hemocyanin (PC-KLH) is dominated by two antibody populations which differ in their fine specificity. Group I antibodies bind both phosphocholine (PC) and p-nitrophenyl-phosphocholine whereas Group II antibodies exhibit (NPPC), significant binding only to NPPC (Chang et al., 1982). Group I antibodies dominate the primary response, whereas Group II antibodies co-dominate in the memory response. Our previous work (Chang et al., 1984; Todd et al., 1984, 1985; Stenzel-Poore et
*Supported in part by Grant AI 14985 from the National Institutes of Health. IlCorresoondence should be sent to: U. Bruderer, Department of Microbiology and Immunology, L220, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201, U.S.A. U.B. was supported by a Swiss National Sciences Foundation Fellowship and a Tartar Foundation Research award. Abbreviations: APPC, p-aminophenyl phosphocholine; DPPC, N-(2,4-dinitrophenyl)-p-aminophenyl phosphocholine; ELISA, enzyme-linked immunosorbent assay; NPDBP, p-nitrophenyl-3,3-dimethyl butyl phosphate; NPMPC. p-nitrophenyl methyl phosphonate choline; NPEP. p-nitrophenyl ethyl phosphate; NPP, p-nitrophenyl phosphate; NPPC, p-nitrophenyl phosphocholine; PC, phosphocholine; PC-KLH, phosphocholine-keyhole limpet hemocyanin. 63
64
URS BRUDERERet Table
1. Chemical
structure
ul
of PC-ligands
KLH \
C=O
(HisMine)
Immunizing Form of PC Hapten
Phosphocholine-Proteln (PC-KLH)
Conjugate
Ligands choline
PC phosphocholine
? HO- POO-
cl$
CH3 Ct+CHs CH3
APPC paminophenyl
9
phosphocholine
NH*-
CH3
-O- P-0-Cl$-Ct@-CH3 & ;H
3
NPPC p-nitrophenyl
phosphccholine
-
?
0- PO-
&
CH3 CH; C,%-CHs
&, 3
NPDBP p-nitrophenyl-3,3 butyl phosphate
NPMPC pnitrophenyl phosphonate
NPEP p-nltrophenyl phosphate
dimethyl
NOT
methyl choline
ethyl
NPP p-nltrophenyl phosphate
DPPC (N-(2,4 dinitrophenygp-amlnophenyl phosphochollne
NOT
Fine specificity of anti-PC-KLH
creased when the nitrogen is present. So far all Group II-A antibodies (six) use kappa light chains and all Group II-B antibodies (seven) use lambda light chains. We also show that individual Group II antibodies differ in their ability to recognize the aminophenyl analog (APPC) as compared to NPPC. Moreover, we extend to hybridoma proteins our earlier observation (Chang et al., 1982) that serum Group II antibodies are not inhibited by p-nitrophenyl phosphate and show that p-nitrophenyl ethyl phosphate also failed to inhibit IO/13 monoclonal Group II proteins. This emphasizes the importance of the trimethyl structure of the choline moiety as an important feature of the overall antigenic architecture. These results suggest that the diversity of the Group II antibody population reflects variations in the intermolecular forces through which these antibody combining sites recognize various aspects of the haptenic structure.
MATERIALS
AND METHODS
antibodies The myelomas TEPCIS (TlS), McPC603 (M603) and MOPC167 (M 167) have been described (Andres et al., 1981; Crews et al., 1981; Gearhart et al., 1981). The hybridomas PCGl-1 and aPC 1 1 l- 1 are described by Chang et al. (1984); PCG2a-1 and PCGl-2 by Todd et al. (1985); PCG2b-3 and PCG3-3 by StenzelPoore et al. (1987). aPC104-8 and aPC56- 1 were derived from the same fusion as aPC1 I l- 1. PCG l-4 and PCGI-6 were derived from spleen cells of BALBic mice immunized with PC-KLH in CFA and fused 7 days after immunization as described (Chang et al., 1984). PCGl-9, PCGI-10, PCGI-11, PCGl-12, and PCGl-13 were produced in this laboratory by Dr T. Hall using the above protocol. Antibodies were purified on PC-Sepharose columns as described by Chang et al. (1984). Purified antibodies from the hyb~domas aPC56- 1, aPC I 11-I, and aPC 104-S were a generous gift of Dr C. Heusser. Ligands PC, NPPC, APPC, choline, and NPP were obtained from Sigma (St Louis, MO). DPPC was synthesized as described by Rodwell et al., 1983. p-Nitrophenyl-3,3dimethyl butyl phosphate (NPDBP) was synthesized by dissolving 2.56g of 4-nitrophenyl phosphodichloridate (Aldrich, Milwaukee, WI) in 25 ml of dry dioxane and 3. I ml of dry pryridine; 1.02 g of 3,3-dimethyl-l -butanol (Aldrich) dissolved in 5 ml dioxane was added dropwise over a period of 1 hr and a white pyridine hydrochloride precipitate appeared while the reaction mixture was stirred for another 2 hr. Then 3 ml of pyridine in 15 ml of water was added and stirred for a few minutes longer. The mixture was dried by rotary evaporation and the
antibodies
65
residue taken up in chloroform and extracted against water (2 parts CHCI, to 1 part water). The chloroform fraction which contained both mono- and diester was dried by rotary evaporation and the residue taken up in a minimum amount of water and adjusted to pH 7 with ammonium hydroxide. This was extracted twice with equal portions of l,2dichforoethane. The water fraction was lyophilized and shown by thin layer chromatography and direct probe mass spectroscopy to contain only pnitrophenyl-3,3-dimethyl butyl phosphate (NPDBP). (Parent ion: 303 m/z, base peak 219 m/z was attributed to the p-nitrophenyl phosphate fragment, 84m/z was attributed to the 3,3-dimethyl butyl fragment.) p-Nitrophenyl ethyl phosphate (NPEP) was synthesized by dissolving 703~1 of absolute ethanol and 2.164 ml of triethylamine in 25 ml of chlorofo~. To this solution 8.7g of 4-nitrophenyl phosphodichloridate dissolved in 25 ml of chloroform was added dropwise. The reaction mixture was stirred for 3 hr. White crystals of triethylamine hydrochloride formed and were removed by filtration. The chloroform phase was washed with water and dried by rotary evaporation. The residue was dissolved in water and extracted twice with ethyl acetate. The water phase was purified by HPLC on a Cl8 column using an n-propanol/water gradient. Three distinct fractions were isolated and shown by direct mass spectroscopy to be NPEP, the diethyl derivative and p-nitrophenol. The mass spectrum for NPEP lacking a parent ion exhibited a characteristic base peak at 139 m/z for the p-nitrophenol fragment and a major fragment at 109 m/z for the ethyl phosphate. p-Nitrophenyl methyl phosphonate choline (NPMPC) was synthesized by dissolving 5.2g of p-nitrophenol (Aldrich) in 35 ml chloroform by heating. To this, 5 g of methylphosphonic dichloridate (Aldrich) was added dropwise. After stirring for 2 hr, the mixture was rotary evaporated and 5.2g of choline (Sigma) dissolved in 50 ml of dry dioxane and 3.1 ml of dry pyridine were added. After stirring for 48 hr the solid phase was separated from the liquid phase by filtration. The solid phase was washed twice with dry ether, dissolved in water and the pH was adjusted with ammonium hydroxide to 7.2. This mixture was lyophilized, reacted with ethanol, and the unreacted choline (insoluble), was removed by filtration. The residue was shown to contain only NPMPC by thin layer chromatography and by direct probe mass spectroscopy. [Parent ion at 303 m/z, 244m/z was attributed to the ~-nitrophenyl ethyl phosphonate fragment (by loss of the trimethyl amino moiety), 166 m/z was attributed to the choline phosphonate fragment and 103 m/z M + 1 was attributed to choline.] Hapten inhibition assays ELISAs were performed er al., 1982.
as described by Chang
URS BRUDERER et al.
66
AfJinity determinations The affinities of monoclonal antibodies for NPPC, NPDBP, and DPPC were determined by fluorescence quenching using an SLM 8000 fluorescence spectrometer (SLM Instruments, Urbana, IL). The excitation and emission wave lengths were 295 and 345 nm, respectively, at 25’C maintained with a Lauda K-21/R circulating waterbath (Brinkman Instruments, Westbury, NY). Antibody concn were calculated by using E :fj = 14.0. Antibodies were diluted in PBS and all binding sites were assumed to be active. Twenty-five microlitre aliquots from a NPPC or NPDBP stock solution of 1Om~4 M were added with stirring in 10 steps to 2.9 ml antibody samples which ranged in their concn from 3.95 x lOmy to 1.22 x 10m8M. The concn of DPPC added in 10 aliquots of 10 ~1 varied from 2.72 x 10m4 to 1.36 x 10m5 M depending on the affinity of the antibody sample. Nonspecific quenching by NPDBP was accounted for by subtracting the nonspecific quenching obtained with the titration of NPDBP in the presence of irrelevant monoclonal or mouse IgG antibodies (Sigma) with concn identical to those in the titrations with the relevant antibodies. Q,,, was obtained by linear regression analysis of the reciprocal of quenching vs the reciprocal of ligand concn (l/Q vs l/[ligand]) extrapolated to (l/[ligand] = 0). Binding parameters were determined using Scatchard plots (Scatchard, 1949) and were confirmed by Hill (Hill, 1910) and Steward-Petty plots (Steward and Petty, 1972). Calculations were performed on an IBM PC/XT computer with the Asyst program environment (McMillan Software Company, New York, NY).
RESULTS
In order to characterize the epitope(s) recognized by Group II antibodies, we analyzed a panel of Group II hybridoma antibodies in hapten inhibition assays and these were compared with Group I antibodies (Table 2). Table 1 shows the chemical structures of PC-ligands used to characterize hybridoma antibodies and the structure of PC in the context of a PCprotein conjugate which was used to elicit them. As described earlier (Chang et al., 1982) Group I antibodies are distinguished from Group II by differences in fine specificity. The binding of Group I antibodies to PC-protein conjugates (PC-histone) can be inhibited by PC as well as by NPPC, whereas the binding of Group II antibodies is inhibited by NPPC but not PC (Table 2). None of the hybridoma antibodies recognize NPP (results not shown) as reported previously for serum Group II antibodies (Chang et a/., 1982). While the binding of the Group I antibodies PCGl-4 and PCGl-6 and the binding of the Group I1 antibodies PCGl-1, aPClll-1, and aPC104-8 is inhibited by high concn of NPEP. the
remaining antibodies show no detectable recognition of this ligand. Choline is only recognized by the Group I antibodies T15, M167, PCGl-4, and PCGl-6. The antibodies which do recognize NPEP or choline exhibit significantly lower Iso values for NPPC than for NPEP or choline. Taken together these results suggest that the analyzed Group II antibodies recognize an epitope which consists of the PC and the phenyl moieties. Furthermore, we found differences in the recognition of NPPC and APPC. These two molecules differ only in the functional group at the para position of the phenyl ring. NPPC has a nitro group, while APPC has an amino group (Table 1). As shown in Table 2, there are antibodies with preferential binding for APPC, e.g. Group I antibodies expressing Vrc22 light chains (T15, PCGI-4, PCGl-6) and antibodies with preferential binding for NPPC, e.g. PCGI-10 and PCGl-11. PCGl-6 and PCGl-IO have comparable I,, values for NPPC (0.02 and 0.06) whereas their Iso values for APPC differ by more than 160-fold. There is no correlation between preferential binding of NPPC or APPC and Group II-A or Group II-B binding characteristics. In order to analyze the importance of ionic interactions, we synthesized two analogs of NPPC. NPDBP is identical to NPPC except that it has a carbon in place of the positively charged nitrogen in the choline moiety. NPMPC is identical to NPPC except that it has a methyl group in place of the negatively charged oxygen in the phosphate moiety (Table 1). Because nitrogen and carbon on the one hand and oxygen and the methyl group on the other hand do not differ significantly in size, all three molecules are isosteric. In Table 3 we show that the negative charge in the phosphate moiety is crucial for binding of Group II antibodies. In contrast all Group I antibodies recognize NPMPC. T15 and M603 exhibit much higher 1,” values for NPMPC than for NPPC whereas M167, PCGl-4, and PCGl-6 have comparable I,,, values for these two ligands. Group II antibodies can be divided into two subgroups based on recognition of NPDBP. Group II-B antibodies recognize this ligand whereas Group II-A antibodies do not. Inhibition of Group I antibodies by NPDBP is weak (PCGl-4, PCGl-6) or undetectable. These findings suggest that although Group II-A and Group II-B both recognize NPPC, their binding is the result of different interactions. For all Group II hybridoma antibodies tested SO far, there is an association between the Group II-A characteristic and K light chain expression and the Group II-B characteristic and i light chain usage (Table 3). The fact that Group II-A contains antibodies with VKI-3 as well as with Vk-24 light chains demonstrates that the association of Group II-A and K light chain utilization is not restricted to a particular VK-gene. Group II-A and Group 11-B antibodies can each utilize members of three VH families. Furthermore. the same VH gent (M141) is
Fine
specificity
Table 2. Soecificitv
of anti-PC-KLH
of monoclonal
antibodies
Grow
I and Grow
II antibodies
Inhibitors Antibody*
GKJUtJ
Tl5 M603 Ml67 PCG l-4 PCG l-h
I
o.owt
I I I I
PCGI-I aPCl1 I-J PCG2a-I aPCl04-8 PCGI-2 PCGI-13 PCG2b-3 PCG3-3 aPC56- I PCGI-9 PCGI-IO PCGI-1 I PCGI-I2
NPEP
Choline
0.03 0.13 0.002 0.004
0.041 0.27 0.05 0.01 0.02
0.005$ 0.31 0.084 0.005 0.004
>lO$ >I0 > IO 2.0 3.5
I .4f > IO 0.6 0.3 I.5
Ii II II II I1 II
210 210 > IO > IO > IO >I0
0.074 2.1 0.54 0.026 5.3 6.6
0.88 3.6 0.40 0.24 5.2 z IO
3.2 9.4 >I0 3.6 >I0 >I0
>I0 1 IO > IO >I0 210 >I0
II II II II II II II
> IO > 10 > 10 >I0 >lO >I0 > IO
0.53 0.44 0.063 0.8 0.06 0.16 0.087
0.59 1.5 0.46 6.1 0.67 6.3 0.5 I
>I0 >I0 >I0 >iO > to >I0 >I0
>I0 >I0 >I0 210 > IO >I0 210
PCP
*The origin of the antibodies is referenced tThe I,, values for PC and NPPC of Tl5, Chang ef al., 1984; PCG2b-3, PCG3-3, $ls,, values: concentrations of hapten (mM)
in Materials and Methods. McPC603, Ml67, PCGI-I, aPCl I l-l, PCGI-2 aPC56-I (Stenzel-Poore et ol., 1987). needed to inhibit antibody binding by 50%.
associated with Group II-A and Group II-B antibodies (Table 3). Thus it seems that the generation of Group II-A/II-B binding phenotypes is not linked to heavy chain usage. In order to determine the intrinsic affinities of Groups I and II antibodies, we used NPPC, NPDBP, and DPPC in fluorescence quenching experiments. APPC could not be used because it quenched the antibody fluorescence nonspecifically. DPPC was initially included because it has been used previously to determine the affinity of Group I proteins (Rodweli et al., 1983; Gearhart ef al., 1989); NPPC and NPDBP are used for the first time in this study. All three ligands are believed to reflect the diazophenyl linkage in the PC-protein conjugate Table 3. Charge dependency Antibody Tl5 M603 Ml67 PCGI -4 PCGI-6
Group
APPC
NPPCt
(Table I). Group I and Group II-B antibodies exhibit detectable affinities for NPPC and DPPC whereas Group II-A antibodies show low or no detectable affinities for these ligands (Table 4) despite the fact that NPPC is an effective inhibitor in the ELISA. There is considerable heterogeneity even within a group or subgroup, most evident in Group II-B where the affinities for NPPC and DPPC vary by more than 140-fold and 350”fold, respectively. Group I antibodies have comparable affinities for both ligands or higher affinity for DPPC. Within the Group II-B there are antibodies with preferential binding for NPPC (PCG2b-3, PCG3-3, aPCG56-1, PCGl-12) exhibiting two to four times higher affinities for NPPC than for DPPC. In contrast,
of monoclonal
Heavy chain* Isotype VH Family
are taken from
Group
I and Group
Light chaint NPPCf
II antibodies Inhibitors NPDBP
NPMPC
SlO?(VHl) S107(VHl) SI07(VHl) 5107(VHl) Sl07(VHI)
VK22 ViC8 VK24 vu22 VK22
0.W 0.27 0.05 0.01 0.02
>l@ >lO z IO 4.6 6.3
4.w 1.8 0.09 0.04 0.05
>’I P y 2a ”3 )‘I E’1
QS2 (Ml41) S107 (VHI) 5107 SlO7(VHI) J558fVH-12) J558
VKI-3 VKI-3 VKI-3 Vul-3 V~24 V~24
0.074 2.1 0.54 0.026 5.3 6.6
> IO > IO > IO >I0 > IO > IO
> IO > IO > 10 >I0 >I0 > 10
:I 2b Y3 Yl
452 (M14lf Q52 (M141) Q52(M141) 7183
0.53 0.44 0.063 0.8 0.06 0.16 0.087
0.75 0.40 0.28 0.77 I.9 6.5 0.42
>lO >I0 >I0 > IO >I0 >I0 >I0
I
a
I I I I
* ti .,jI .,II
PCGI-I aPCIII-I PCG2a-I aPC 104-R PCGI-2 PCGl-I3
II-A II-A 11-A II-A II-A II-A
PCG2b-3 PCG3-3 aPC56-I PCG l-9 PCGI-IO PCGI-I I PCGI-I2
II-B II-B II-B II-B II-B II-B II-B
VI
vl vl Yl
~52 Q52 5558
‘VH family assignments were made by one or more of the following methods: Northern blot analysis (Stenzel-Poore et al., 1988). Southern blot analysis (Stenzel-Poore et al., 1987; Rittenberg et nl., 1986). N-terminal amino acid sequencing (Gearhart PI ai., 1981; Rittenberg ef al., 1986) and Tl5 idiotype expression. The VH gene where known is given in parentheses. flight chain assignments were made by N-terminal amino acid sequencing, Tl5 idiotype expression and/or isoelectric focusing (Todd ef al., 1984, 1985). ISee footnote t of Table 2. pl,, values (mM).
URS BRUDERER et al.
68 Table 4. Binding constants
Antlbodq
Group
TI5 McPC603 Ml67 PCGI -4 PCG l-6
I I I I I
PCG I LlPCll I-I PCG?a I aP(‘lO4.X PCG?b-? PCGJ-I 3PC56I f’<‘G I -Y PCG I IO PCGI-I1 PC‘G-I?
of monoclonal Group I and Group fluorescence quenching ?iPPC
II antibodies
Ka x lO’(M NPDBP
I 8 z 0.25* <0.5+ 5x 103 I.1 ’ 0.2 0.0 r 0.03
N.T.: N.T. N.T. N.T. N.T.
II-A II-A II-A II-4
0 77 f 0.04
N.T. N.T. N.T. N.T.
II-H II-R 11-B II-B II-B 11-B II-B
‘Y7 i 1.0 YI oixo 25.1) 2.5 2.4 : (I.? 5.2 : 0.3 IS.9 T 0.9 0.64 i 0.08
1.x 0.2 I I.? i_ + 2.7 4.5 * 0.7 10 St 0.74 t 0.08
co.5
*R pi standard dcviatwn of two determmatlons. tAHinity b&w the It‘wzl of detectmn :N.T. not te\tcd becaue NPDBP was unable to inhibit ELISA.
PCGI-IO, PCGl-11 and the more extreme PCGl-Y show up to 43-fold higher affinity for DPPC than for NPPC (Table 4). Group II-B antibodies have lower affinity constants for NPDBP than for NPPC and DPPC (Table 4). Although the binding of all Group II-B antibodies to PC-conjugated protein can be inhibited with NPDBP (Table 3). some exhibit no detectable affinity for this ligand in the fluorescence quenching assay, suggesting that the charge in the trimethylammonium group is not essential for binding but can add to the strength of the interaction. DISCUSSION
We previously described that diversity in the memory response to PC-KLH is acquired through antibodies differing in their fine specificity [Group I vs Group 11 (Chang et d., 1984; Todd et al., 1984. 1985; Stenzel-Poore rt al., 1987)]. Furthermore, we reported that V-gene heterogeneity within the Group II response is much less restricted than that of the pauciclonal Group 1 response (Crews et al.. 1981: Perlmutter, 1984). Here, we analyze combining site heterogeneity of Group II antibodies by characterizing the antigen-antibody interactions responsible for the generation of specificity and affinity. Binding of Group II antibodies to PC-conjugated proteins can be inhibited by NPPC but not unconjugated protein, NPP, NPEP, or PC suggesting that the epitope recognized by these antibodies is comprised of the PC and the phenyl moieties. In agreement with our findings that PC is an integral part of the epitope recognized by Group II. Wicker et al. (1982) found that Group II antibodies bound to APPC and PC conjugated to bovine serum albumin to p-diazophenylarsonate (PC-BSA) but not conjugated to BSA (Ars-BSA). Since both Ars-BSA and PC-BSA contain the same diazophenyl ring and
the binding
determined
by
‘) DPPC 5.1 1.9 5.x 3.0 30
tn.1 kO.1 2 0.3 i_ 0. I i 0.04
0.x0 z 0 01 <0.25t 1025 <025 22 32.1 12.7 102.7 I?.‘) 47.1 0.29
0 04 *1 I4 _i 7.2 _t 5.0 t 0.5 * 7.5 * 0.04
of these antlhodxs
m
tyrosyl and/or hystidyl carrier determinants, their studies suggest that neither the carrier nor the linkage is sufficient for binding. Our experiments with NPMPC and NPDBP demonstrate the importance of ionic interactions. Group 11 antibodies do not recognize NPMPC showing that the negative charge in the phosphate moiety is absolutely required. NPDBP, which differs from NPPC by the lack of charge in the structure corresponding to the trimethylammonium moiety of choline is recognized by Group II-B, but not by Group II-A antibodies which demonstrates that Group II specificity can be achieved in at least two ways. This finding indicates that the choline is in fact a necessary part of the epitope recognized by Group II-A antibodies and that the binding is charge dependent. It is likely therefore that ionic interactions with the trimethylammonium moiety are essential for these antibodies to bind. Although the correponding affinities are several fold lower for NPDBP than for NPPC, ionic interactions seem to be much less important for Group II-B than for Group II-A antibodies. Because the energy of interaction of juxtaposed groups is inversely proportional to the distance between the oppositely charged ions of antibodies and antigens (Absolom and van Oss. lY86), the energy of charge interactions is highly distance dependent. Thus, Group II-A antibodies which only recognize the positively charged hapten appear to have a negatively charged contact residue that is critical to the interaction. Group II-B on the other hand may lack the corresponding residue entirely or have it positioned where it is not in close proximity to the trimethylammonium and thus, it would contribute less to the energy of the interaction. Hydrophobic interactions between apolar groups have been described to be of major importance among the bonds of immunological significance (Karush, 1962; Tsutsui et ~1.. 1977: Absolom and van
Fine specificity
of anti-PC-KLH
Oss, 1986). Therefore, it seems likely that, in contrast to Group II-A antibodies, hydrophobic interactions between the 3,3-dimethyl butyl moiety and the Group II-B antibody combining site are a dominant feature in binding to the latter. The finding that some Group II-A antibodies (3/6) weakly recognize NPEP but not the uncharged NPDBP may indicate that the ionic interaction between these antibodies and the choline moiety is dominant and that the hydrophobic character of the choline moiety in the absence of a charge would result in repulsion which would explain the failure to bind NPDBP. Group I antibodies are easier to inhibit with analogs of NPPC than Group II antibodies which may be due to the smaller epitope recognized by Group I antibodies. T15 and M603 are much more easily inhibited by NPPC than by either choline or NPMPC indicating that the phosphate group itself as well as the negative charge are essential for binding. In contrast M 167, PCG l-4, and PCG l-6 have comparable I,, values for NPMPC and NPPC but were inhibited less well by choline. Thus, it seems that the phosphate moiety is a crucial component of the epitope recognized by these antibodies but that this requirement need not extend to the negative charge. These findings are in agreement with the results of Goetze and Richards (1977) who deduced from NMR analysis that for Ml67 the strength of the interaction with the choline subsite is much stronger than with the phosphate subsite whereas both subsites contributed comparably to the binding of T15 and M603. Furthermore, they showed that for antibodies with the same light chain the importance of the two subsites could differ. Whether the difference between T 15 and PCG 1-4 and PCG 1-6 can be explained by somatic mutations remains to be determined. VH genes from four VH families (452, S107, 5558, 7183) have been found to be expressed in the Group II response (Stenzel-Poore rt al., 1989; Table 3). Different VH genes can be used within the same subgroup, e.g. the Group II-A antibody PCGI-2 expresses a VH-12 gene product (Rittenberg et al., 1986) aPCl l l-I and aPC104-8 utilize the VH-1 gene product (Stenzel-Poore et al., 1989) and PCGI-I uses the VH gene Ml41 (Stenzel-Poore et al., 1987). The Ml41 VH gene is also associated with the Group II-B proteins PCG2b-3, PCG3-3 and aPC56-1 (Stenzel-Poore et al., 1987), suggesting that the heavy chain may not be the critical factor in the generation of II-A or II-B binding sites. This is consistent with our observation that the II-A/II-B distinction thus far has been linked to the expression of K(Group II-A) and i(Group II-B) light chains. The ionic interactions associated with Group II-A binding properties are not restricted to a particular K light chain in that antibodies with either VKI-3 or V~24 light chains are found in this group. In contrast to the Group II response, the Group I response is dominated by antibodies expressing one
antibodies
69
particular VH gene (VHI) in combination with V~22 or to a lesser extent, VK 8 or V~24 light chains (Crews et a/., 1981; Gearhart et al., 1981). Crystallographic analysis of the Group I myeloma protein M603 with PC in the active site (Segal et al., 1974; Padlan et al., 1976) revealed that the positively charged trimethylammonium moiety of the hapten interacts predominantly with the heavy chain [Glu 35H and Glu 53H (M603 numbering of Rudikoff and Potter, 1974)], suggesting that the heavy chain of Group I antibodies is dominantly responsible for the binding to PC. Since four distinct VH gene families can be used to generate Group II binding sites (Table 3) our data suggest that the light chain may be of more importance in the generation of the Group II combining site. Rodwell et al. (1983) who first determined affinities of Group I antibodies for DPPC, analyzed the contribution of the phenyl ring to the binding constant. They showed that some Group I antibodies exhibited up to 80-fold higher affinity for DPPC than for PC itself. They concluded from their data that DPPC rather than PC was the more appropriate ligand for these antibodies and as a consequence, the phenyl group makes a substantial contribution to the binding constants. Furthermore, Mandel et al. (1984) tested the contribution of a phenyl structure in the anti-lactose system. By comparing the affinities for p-nitrophenyl-p-D-lactoside and for methyl+o-lactoside, they showed that the phenyl moiety made a significant contribution to the affinity. However, they concluded that it was not possible to determine whether the phenyl group was part of the epitope or whether it made a nonspecific contribution to the affinity due to its hydrophobic character and proximity to the antigen binding site. However, they provided evidence for the latter by demonstrating that not only antibodies elicited by p-aminophenylb-lactoside but also by a vaccine prepared with Streptococcus faecalis, which lacks the phenyl linkage, exhibited higher affinity for p-nitrophenyl-B-Dlactoside as compared with methyl-/?-o-lactoside. it is not possible to discriminate Accordingly, between these two possibilities in the case of Group I antibodies. For Group II antibodies, at least the phenyl ring of NPPC seems to be part of the epitope because these antibodies show, in contrast to Group I antibodies, no detectable affinity for PC itself. We found that specific fluorescence quenching by NPPC is not inhibited even by 1OO-fold excess of PC (results not shown) and that binding to PC does not lead to fluorescence enhancement of Group II antibodies (results not shown) as has been reported for Group I antibodies (Pollet and Edelhoch, 1973). Our results also indicate that there is heterogeneity in the binding preference for NPPC and DPPC within Group II-B. Three of the seven Group II-B antibodies exhibited two to four times higher affinity for NPPC whereas the other three showed the reverse pattern, e.g. PCGl-9 exhibits >40-fold higher
70
URS BRUDERER r~ al.
affinity for DPPC than for NPPC. Except that DPPC has an additional dinitrophenyl (DNP) moiety linked to the phenyl ring of NPPC (see Table l), these two ligands share the same structures. The addition of the DNP moiety thus appears to contribute dramatically to the binding by PCGI-9. Accordingly, as demonstrated by Mandel et al. (1984) for anti-lactose antibodies, the second phenyl group may also be part of the epitope which is recognized, suggesting that the active site of PCGl-9 may differ appreciably from those Group II-B antibodies with preferential binding to NPPC. Alternatively, as discussed above, the DNP moiety may serve to stabilize the antigenantibody combination in PCGl-9 through an interaction outside of the combining site. Individual antibodies differed by more than IOO-fold in their preferential binding to either NPPC or APPC, a ligand used previously (Wicker et al., 1982) to discriminate between Group I and Group II antibodies. Preference for the nitro or amino derivative does not seem to be associated with either of the sub-populations but rather is a characteristic of a particular antibody. Nitro and amino groups have an inductive effect on rc electrons of the aromatic ring, with nitro diminishing and amino enhancing. The nature of the interaction of the aromatic site of the hgand with the antibody is likely to involve 71electron interaction and hydrophobic interaction. Thus, it is conceivable that antibodies which show preferential binding to APPC depend on interactions with rt electrons as well as on hydrophobic interactions. In contrast, for antibodies which show a binding preference to NPPC, the n electrons may contribute relatively little to the interaction compared to hydrophobic interactions in the phenyl region. Although studies of serum Group II antibodies suggest that VK-I-3 usage would be most relevant (Todd et al., 1985; Bruderer et ul.. 1989). the results of hapten-inhibition ELISA suggest that Groups II-A and II-B would have a comparable distribution of affinities for NPPC but as shown in Table 4, Group II-A antibodies have very low binding constants. This is consistent with previous reports that Is,, values do not necessarily reflect intrinsic affinities (Foiles et al., 1983; Peterfy et al., 1983; Friguet et al., 1984). The main difference between fluorescence quenching which measures the primary interaction between hapten and antibody and hapten inhibition ELISA is that in the latter the antibodies can bind to the PCprotein complex. Since all of the antibodies described were selected on the basis of their ability to bind to PC-protein it is possible that neither NPPC nor DPPC adequately reflects the immunizing form of PC in PC-KLH. Thus, Group II-A and II-B could be subject to the same antigenic selective pressure although this is not revealed by their intrinsic affinities for the haptens. Previous assessments of Group I (Rodwell et al., 1983; Gearhart et al., 1989) indicated that somatic mutation accounts for increased affinity of IgG antibodies; it remains to be
determined whether the higher affinity of Group II-B antibodies compared to Group II-A results from somatic mutation, the use of particular V genes or a combination of both mechanisms. Acknowledgements-We thank P. Bresnahan, M. Brown, A. Buenafe, Q. Chen, and Dr T. Tittle for critical review of the manuscript and Dr C. H. Heusser for providing hyhridoma proteins aPC56-1, aPClll-I. and aPC104-X.
REFERENCES Ahsolom D. R. and van Oss C. J. (1986) The nature of the antigen-antibody bond and the factors affecting its association and dissociation. Crit. Rev. Immun. 6, I 46. Andres C. M., Maddalena A., Hudak S., Young N. M. and Claflin J. L. (1981) Anti-phosphocholine hyhridoma antibodies--II. Functional analysis of binding sites within three antibody families. J. cup. Med. 154, 15841598. Bruderer U., Aehersold R., Blaser K. and Heusser C. H. (1989) Characterization of the Group I and Group II antibodies against PC-KLH in normal and T15 idiotype suppressed BALB/c mice. Immunology (in press). Chang S. P., Brown M. and Rittenberg M. B. (1982) Immunologic memory to phosphorylcholine-II. PCKLH induces two antibody populations that dominate different isotypes. /. Immun. 128, 702-706. Chang S. P., Perlmutter R. M., Brown M., Heusser C. H.. Hood L. and Rittenherg M. B. (1984) Immunologic memory to phosphocholine-IV. Hyhridomas representative of Group I (T15-like) and Group II (non TIS-like) antibodies utilize distinct VH genes. J. Immun. 132, 155&1555. Crews S., Griffin J., Huang H., Calame K. and Hood L. (1981) A single VH gene segment encodes the immune response to phosphorylchohne: somatic mutation is correlated with-the class-of the antibody. Cell 25, 59 66. Foiles P. G.. Todd I. and Rittenberg M. B. (1983) Comparison of three methods for determining antibody affinity using monoclonal antibodies. Fed. Proc. 42, 932. Friguet B., Djavadi-Ohaniance L. and Goldberg M. E. (1984) Some monoclonal antibodies raised with a native protein bind preferentially to the denatured antigen. Molec. Immun. 21, 673 -677. Gearhart P. J., Johnson N. D., Douglas R. and Hood L. (1981) IgG antibodies to phosphorylcholine exhibit more diversity than their IgM counterparts. .Vururr 291, 29 34. GearhartP. J., Caron D. M.. Douglas R. H.. Bruderer II.. Rittenberg M. B. and Hood L. (1989) Maior role of somatic mutation is to increase affinity of antibodies. Proc. natn. Acad. Sci. USA (in press). Goetze A. M. and Richards J. H. (1977) Structure-function relations in phosphorylcholine-binding mouse myeloma proteins. Proc. natn. Acad. Sri. USA 74, 2109%2112. Hill A. V. (1910) A new mathematical treatment of changes of ionic concentration in muscle and nerve under action of electric currents with a theory as to their mode of excitation. J. Physiol. XL, 19&224. Karush F. (1962) Immunological specificity and molecular structure. In Advances in Immunology (Edited by Dixon F. J. and Humphry J. H.). Vol. 2. pp. I 40. Academic Press, New York. Mandal C., Mandal C. and Karush I. (lYX4) Kestriction m IgM expression-V. Fine structure analysis in the antilactose system. Moler. Immun. 21, 8355900. Padlan E. A., Davis D. R., Rudikoff S. and Potter M. (1976) Structural basis for the specificity of phosphorylcholinebinding immunoglobulins. Immunochemistry 13,945-~949. Perlmutter R. M. (1984) The molecular genetics of phosphocholine-binding antibodies. In The Biology o/ Idiotypes (Edited by Green M. R. and Nisonoff A.), p. 59. Plenum Press, New York.
Fine specificity
of anti-PC-KLH
Peterfy F., Kunsela P. and Makehi 0. (1983) Affinity requirements for antibody assays mapped by monoclonal antibodies. J. Immun. 130, 18d9-1813. Pollet R. and Edelhoch H. (1973) . , The binding __pronerties of anti-phosphorylcholine mouse myeloma proteins as measured by protein fluorescence. J. biol. Chem. 248, 5443-5447. Rittenberg M. B., Glanville R. W., Aebersold R. H., Chang S. P. and Brown M. (1986) Immunologic memory to phosphorylcholine (PC~VIII.,Expression of the VH-12 gene product in the response to PC-keyhole limpet hemocyanin. Eur. J. Immun. 16, 503-507. Rodwell J. D., Gearhart P. J. and Karush F. (1983) Restriction in IgM expression-IV. Affinity analysis of monoclonal anti-phosphorylcholine antibodies. J. Immum 130, 313-316. Rudikoff S. and Potter M. (1974) Variable region sequence of the heavy chain from a phosphorylcholine binding myeloma protein. ~~~c~ern~~~r~ 13, 4033-4038. Scatchard G. (1949) The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51, 660672. Segal D. M., Padlan E. A., Cohen 6. H., Rudikoff S., Potter M. and Davies D. R. (1974) The three-dimensional structure of a phosphorylcholine-binding mouse immunoglobulin Fab and the nature of the antigen binding site. Proc. mtn. Acad. Sci. USA 71, 4298-4302. Stenzel-Poore M., Hall T. J., Heusser C. H., Faust C. H.
antibodies
71
and Rittenberg M. B. (1987) Immunologic memory to PC-KLH: Participation of the 452 VH gene family. J. Immun. 139, 1698S1703. Stenzel-Poore M., Keller N., Heusser C. H. and Rittenberg M. B. (1989) Antibody diversity in the memory response to PC-KLH: expression of the S107, Q52,5558 and 7183 VH families. (submitted). Steward M. W. and Petty R. E. (1972) The antigen-binding characteristics of antibody pools of different relative affinity. immunology 23, 881-887. Todd I.1 Chang S. P., Perlmutter R. M., Aebersold R., Heusser C. H.. Hood L. and Rittenbere M. B. (1984) Immunologic memory to phosphocholice-V. Hybrid: omas representative of Group I1 antibodies utilize VK l-3 gene(s). J. Immun. 132, 1556-1560. Todd I., Brown M. and Rittenberg M. B. (1985) Immunologic memory to phosphocholine-VI. Heterogeneity in light chain gene expression. Eur. J. Imm~n. IS, 177-183. Tsutsui K., Koide N.. Tomoda J., Hayashi H., Hatase 0. and Oda T. (1977) Role of hvdronhobic interaction in hapten-antibody binding. A& Med. Okqvama 31, 2899294. Wicker L. S., Guelde G., Scher I. and Kenny J. J. (1982) Antibodies from the Lyb-5. cell subset predominate in the secondary IgG response to phosphocholine. J. Immun. 129,950-953.
Molerulor hmunolog~~, Vol. 26, No. I, pp. 63-l Is 1989 Printed in Great Britam.
ANTIBODY COMBINING SITE HETEROGENEITY WITHIN THE RESPONSE TO PHOSPHOCHOLINEKEYHOLE LIMPET HEMOCYANIN* URS BRUDERER,~~~MARY P. STENZEL-POORE,? HANS PETER BACHINGER,~ JACK H. FELLMAN~ and MARVIN B. RITTENBERG? TDepartment of Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201, U.S.A. $Shriners Hospital for Crippled Children and the Department of Biochemistry, Sciences University, Portland, OR 97201, U.S.A. SDepartment of Biochemistry, Oregon University, Portland, OR 97201, U.S.A.
Oregon Health Health Sciences
(Firs1 received 30 March 1988; accepted 21 April 1988)
Abstract-The memory response to PC-KLH is dominated by two antibody populations differing in fine specificity. Group I antibodies show affinity for both phosphocholine (PC) and p-nitrophenyl phosphocholine (NPPC). Group II antibodies exhibit significant affinity only for NPPC. Here, we describe the binding site charactersitics of Group II antibodies and show that in recognizing NPPC these antibodies have a common requirement for the phenyl moiety, a negatively charged phosphate, and the trimethyl structure of the choline. However, Group II antibodies were found to differ in their requirement for the positively charged nitrogen of choline and thus could be divided into two subgroups. In contrast to Group II-A, G&up IIIB antibo&es recognize not only NPPC but also its analogp-&rophenyl-3,3-dimethyl but;1 nhosohate (NPDBP). which differs from NPPC by substituting a carbon for the positivelv charged hitrogen of the choline moiety. These results suggest that Group II-B antibodies do not require the posizve charge in order to bind, although the binding constant, Ka, was increased when the nitrogen was present. Furthermore, heterogeneity within Group II antibodies was characterized by differences in binding to
dinitrophenyl phosphocholine which has an additional phenyl ring and aminophenyl phosphocholine which has an amino group in place of the nitro group of NPPC. The results indicate that diversity in the memory response differ appreciably
to PC-KLH is reflected in the Group II antigen-binding phenotype in their recognition of various structural aspects of the hapten.
by antibodies
which
al., 1987) indicates that Group II antibodies use V genes different from those utilized by Group I antibodies, although some use V genes also found in Group I antibodies but in novel combinations. Thus, it appears that the diversification in the secondary response to PC-KLH is to a large extent the result of the recruitment of new gene combinations rather than of somatic mutations of Group I clonotypes. Group II antibodies all have in common that their binding to PC-conjugated proteins can be inhibited by NPPC. Here we analyze the contribution of different structural aspects of NPPC to the antigen-antibody We have compared the ability of interaction. Group II hybridoma proteins to recognize NPPC and analogs which differ from NPPC in both structure and charge (Table 1). Using p-nitrophenyl methyl phosphonate choline (NPMPC) we show that the negative charge in the phosphate moiety is crucial for the binding of all Group II antibodies tested. In contrast it is possible to distinguish two subsets of Group II antibodies by their different requirements for the positively charged nitrogen in the choline moiety. Group II-A antibodies require the presence of the positive charge since they are not inhibited by p-nitrophenyl 3,3-dimethyl butyl phosphate (NPDBP) in which a carbon replaces the positively charged nitrogen of the trimethylammonium moiety. Group II-B antibodies on the other hand recognize NPDBP although the binding constant, Ka, is in-
lNTRODUCTlON The memory response to phosphocholine-keyhole limpet hemocyanin (PC-KLH) is dominated by two antibody populations which differ in their fine specificity. Group I antibodies bind both phosphocholine (PC) and p-nitrophenyl-phosphocholine whereas Group II antibodies exhibit (NPPC), significant binding only to NPPC (Chang et al., 1982). Group I antibodies dominate the primary response, whereas Group II antibodies co-dominate in the memory response. Our previous work (Chang et al., 1984; Todd et al., 1984, 1985; Stenzel-Poore et
*Supported in part by Grant AI 14985 from the National Institutes of Health. IlCorresoondence should be sent to: U. Bruderer, Department of Microbiology and Immunology, L220, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201, U.S.A. U.B. was supported by a Swiss National Sciences Foundation Fellowship and a Tartar Foundation Research award. Abbreviations: APPC, p-aminophenyl phosphocholine; DPPC, N-(2,4-dinitrophenyl)-p-aminophenyl phosphocholine; ELISA, enzyme-linked immunosorbent assay; NPDBP, p-nitrophenyl-3,3-dimethyl butyl phosphate; NPMPC. p-nitrophenyl methyl phosphonate choline; NPEP. p-nitrophenyl ethyl phosphate; NPP, p-nitrophenyl phosphate; NPPC, p-nitrophenyl phosphocholine; PC, phosphocholine; PC-KLH, phosphocholine-keyhole limpet hemocyanin. 63
64
URS BRUDERERet Table
1. Chemical
structure
ul
of PC-ligands
KLH \
C=O
(HisMine)
Immunizing Form of PC Hapten
Phosphocholine-Proteln (PC-KLH)
Conjugate
Ligands choline
PC phosphocholine
? HO- POO-
cl$
CH3 Ct+CHs CH3
APPC paminophenyl
9
phosphocholine
NH*-
CH3
-O- P-0-Cl$-Ct@-CH3 & ;H
3
NPPC p-nitrophenyl
phosphccholine
-
?
0- PO-
&
CH3 CH; C,%-CHs
&, 3
NPDBP p-nitrophenyl-3,3 butyl phosphate
NPMPC pnitrophenyl phosphonate
NPEP p-nltrophenyl phosphate
dimethyl
NOT
methyl choline
ethyl
NPP p-nltrophenyl phosphate
DPPC (N-(2,4 dinitrophenygp-amlnophenyl phosphochollne
NOT
Fine specificity of anti-PC-KLH
creased when the nitrogen is present. So far all Group II-A antibodies (six) use kappa light chains and all Group II-B antibodies (seven) use lambda light chains. We also show that individual Group II antibodies differ in their ability to recognize the aminophenyl analog (APPC) as compared to NPPC. Moreover, we extend to hybridoma proteins our earlier observation (Chang et al., 1982) that serum Group II antibodies are not inhibited by p-nitrophenyl phosphate and show that p-nitrophenyl ethyl phosphate also failed to inhibit IO/13 monoclonal Group II proteins. This emphasizes the importance of the trimethyl structure of the choline moiety as an important feature of the overall antigenic architecture. These results suggest that the diversity of the Group II antibody population reflects variations in the intermolecular forces through which these antibody combining sites recognize various aspects of the haptenic structure.
MATERIALS
AND METHODS
antibodies The myelomas TEPCIS (TlS), McPC603 (M603) and MOPC167 (M 167) have been described (Andres et al., 1981; Crews et al., 1981; Gearhart et al., 1981). The hybridomas PCGl-1 and aPC 1 1 l- 1 are described by Chang et al. (1984); PCG2a-1 and PCGl-2 by Todd et al. (1985); PCG2b-3 and PCG3-3 by StenzelPoore et al. (1987). aPC104-8 and aPC56- 1 were derived from the same fusion as aPC1 I l- 1. PCG l-4 and PCGI-6 were derived from spleen cells of BALBic mice immunized with PC-KLH in CFA and fused 7 days after immunization as described (Chang et al., 1984). PCGl-9, PCGI-10, PCGI-11, PCGl-12, and PCGl-13 were produced in this laboratory by Dr T. Hall using the above protocol. Antibodies were purified on PC-Sepharose columns as described by Chang et al. (1984). Purified antibodies from the hyb~domas aPC56- 1, aPC I 11-I, and aPC 104-S were a generous gift of Dr C. Heusser. Ligands PC, NPPC, APPC, choline, and NPP were obtained from Sigma (St Louis, MO). DPPC was synthesized as described by Rodwell et al., 1983. p-Nitrophenyl-3,3dimethyl butyl phosphate (NPDBP) was synthesized by dissolving 2.56g of 4-nitrophenyl phosphodichloridate (Aldrich, Milwaukee, WI) in 25 ml of dry dioxane and 3. I ml of dry pryridine; 1.02 g of 3,3-dimethyl-l -butanol (Aldrich) dissolved in 5 ml dioxane was added dropwise over a period of 1 hr and a white pyridine hydrochloride precipitate appeared while the reaction mixture was stirred for another 2 hr. Then 3 ml of pyridine in 15 ml of water was added and stirred for a few minutes longer. The mixture was dried by rotary evaporation and the
antibodies
65
residue taken up in chloroform and extracted against water (2 parts CHCI, to 1 part water). The chloroform fraction which contained both mono- and diester was dried by rotary evaporation and the residue taken up in a minimum amount of water and adjusted to pH 7 with ammonium hydroxide. This was extracted twice with equal portions of l,2dichforoethane. The water fraction was lyophilized and shown by thin layer chromatography and direct probe mass spectroscopy to contain only pnitrophenyl-3,3-dimethyl butyl phosphate (NPDBP). (Parent ion: 303 m/z, base peak 219 m/z was attributed to the p-nitrophenyl phosphate fragment, 84m/z was attributed to the 3,3-dimethyl butyl fragment.) p-Nitrophenyl ethyl phosphate (NPEP) was synthesized by dissolving 703~1 of absolute ethanol and 2.164 ml of triethylamine in 25 ml of chlorofo~. To this solution 8.7g of 4-nitrophenyl phosphodichloridate dissolved in 25 ml of chloroform was added dropwise. The reaction mixture was stirred for 3 hr. White crystals of triethylamine hydrochloride formed and were removed by filtration. The chloroform phase was washed with water and dried by rotary evaporation. The residue was dissolved in water and extracted twice with ethyl acetate. The water phase was purified by HPLC on a Cl8 column using an n-propanol/water gradient. Three distinct fractions were isolated and shown by direct mass spectroscopy to be NPEP, the diethyl derivative and p-nitrophenol. The mass spectrum for NPEP lacking a parent ion exhibited a characteristic base peak at 139 m/z for the p-nitrophenol fragment and a major fragment at 109 m/z for the ethyl phosphate. p-Nitrophenyl methyl phosphonate choline (NPMPC) was synthesized by dissolving 5.2g of p-nitrophenol (Aldrich) in 35 ml chloroform by heating. To this, 5 g of methylphosphonic dichloridate (Aldrich) was added dropwise. After stirring for 2 hr, the mixture was rotary evaporated and 5.2g of choline (Sigma) dissolved in 50 ml of dry dioxane and 3.1 ml of dry pyridine were added. After stirring for 48 hr the solid phase was separated from the liquid phase by filtration. The solid phase was washed twice with dry ether, dissolved in water and the pH was adjusted with ammonium hydroxide to 7.2. This mixture was lyophilized, reacted with ethanol, and the unreacted choline (insoluble), was removed by filtration. The residue was shown to contain only NPMPC by thin layer chromatography and by direct probe mass spectroscopy. [Parent ion at 303 m/z, 244m/z was attributed to the ~-nitrophenyl ethyl phosphonate fragment (by loss of the trimethyl amino moiety), 166 m/z was attributed to the choline phosphonate fragment and 103 m/z M + 1 was attributed to choline.] Hapten inhibition assays ELISAs were performed er al., 1982.
as described by Chang
URS BRUDERER et al.
66
AfJinity determinations The affinities of monoclonal antibodies for NPPC, NPDBP, and DPPC were determined by fluorescence quenching using an SLM 8000 fluorescence spectrometer (SLM Instruments, Urbana, IL). The excitation and emission wave lengths were 295 and 345 nm, respectively, at 25’C maintained with a Lauda K-21/R circulating waterbath (Brinkman Instruments, Westbury, NY). Antibody concn were calculated by using E :fj = 14.0. Antibodies were diluted in PBS and all binding sites were assumed to be active. Twenty-five microlitre aliquots from a NPPC or NPDBP stock solution of 1Om~4 M were added with stirring in 10 steps to 2.9 ml antibody samples which ranged in their concn from 3.95 x lOmy to 1.22 x 10m8M. The concn of DPPC added in 10 aliquots of 10 ~1 varied from 2.72 x 10m4 to 1.36 x 10m5 M depending on the affinity of the antibody sample. Nonspecific quenching by NPDBP was accounted for by subtracting the nonspecific quenching obtained with the titration of NPDBP in the presence of irrelevant monoclonal or mouse IgG antibodies (Sigma) with concn identical to those in the titrations with the relevant antibodies. Q,,, was obtained by linear regression analysis of the reciprocal of quenching vs the reciprocal of ligand concn (l/Q vs l/[ligand]) extrapolated to (l/[ligand] = 0). Binding parameters were determined using Scatchard plots (Scatchard, 1949) and were confirmed by Hill (Hill, 1910) and Steward-Petty plots (Steward and Petty, 1972). Calculations were performed on an IBM PC/XT computer with the Asyst program environment (McMillan Software Company, New York, NY).
RESULTS
In order to characterize the epitope(s) recognized by Group II antibodies, we analyzed a panel of Group II hybridoma antibodies in hapten inhibition assays and these were compared with Group I antibodies (Table 2). Table 1 shows the chemical structures of PC-ligands used to characterize hybridoma antibodies and the structure of PC in the context of a PCprotein conjugate which was used to elicit them. As described earlier (Chang et al., 1982) Group I antibodies are distinguished from Group II by differences in fine specificity. The binding of Group I antibodies to PC-protein conjugates (PC-histone) can be inhibited by PC as well as by NPPC, whereas the binding of Group II antibodies is inhibited by NPPC but not PC (Table 2). None of the hybridoma antibodies recognize NPP (results not shown) as reported previously for serum Group II antibodies (Chang et a/., 1982). While the binding of the Group I antibodies PCGl-4 and PCGl-6 and the binding of the Group I1 antibodies PCGl-1, aPClll-1, and aPC104-8 is inhibited by high concn of NPEP. the
remaining antibodies show no detectable recognition of this ligand. Choline is only recognized by the Group I antibodies T15, M167, PCGl-4, and PCGl-6. The antibodies which do recognize NPEP or choline exhibit significantly lower Iso values for NPPC than for NPEP or choline. Taken together these results suggest that the analyzed Group II antibodies recognize an epitope which consists of the PC and the phenyl moieties. Furthermore, we found differences in the recognition of NPPC and APPC. These two molecules differ only in the functional group at the para position of the phenyl ring. NPPC has a nitro group, while APPC has an amino group (Table 1). As shown in Table 2, there are antibodies with preferential binding for APPC, e.g. Group I antibodies expressing Vrc22 light chains (T15, PCGI-4, PCGl-6) and antibodies with preferential binding for NPPC, e.g. PCGI-10 and PCGl-11. PCGl-6 and PCGl-IO have comparable I,, values for NPPC (0.02 and 0.06) whereas their Iso values for APPC differ by more than 160-fold. There is no correlation between preferential binding of NPPC or APPC and Group II-A or Group II-B binding characteristics. In order to analyze the importance of ionic interactions, we synthesized two analogs of NPPC. NPDBP is identical to NPPC except that it has a carbon in place of the positively charged nitrogen in the choline moiety. NPMPC is identical to NPPC except that it has a methyl group in place of the negatively charged oxygen in the phosphate moiety (Table 1). Because nitrogen and carbon on the one hand and oxygen and the methyl group on the other hand do not differ significantly in size, all three molecules are isosteric. In Table 3 we show that the negative charge in the phosphate moiety is crucial for binding of Group II antibodies. In contrast all Group I antibodies recognize NPMPC. T15 and M603 exhibit much higher 1,” values for NPMPC than for NPPC whereas M167, PCGl-4, and PCGl-6 have comparable I,,, values for these two ligands. Group II antibodies can be divided into two subgroups based on recognition of NPDBP. Group II-B antibodies recognize this ligand whereas Group II-A antibodies do not. Inhibition of Group I antibodies by NPDBP is weak (PCGl-4, PCGl-6) or undetectable. These findings suggest that although Group II-A and Group II-B both recognize NPPC, their binding is the result of different interactions. For all Group II hybridoma antibodies tested SO far, there is an association between the Group II-A characteristic and K light chain expression and the Group II-B characteristic and i light chain usage (Table 3). The fact that Group II-A contains antibodies with VKI-3 as well as with Vk-24 light chains demonstrates that the association of Group II-A and K light chain utilization is not restricted to a particular VK-gene. Group II-A and Group 11-B antibodies can each utilize members of three VH families. Furthermore. the same VH gent (M141) is
Fine
specificity
Table 2. Soecificitv
of anti-PC-KLH
of monoclonal
antibodies
Grow
I and Grow
II antibodies
Inhibitors Antibody*
GKJUtJ
Tl5 M603 Ml67 PCG l-4 PCG l-h
I
o.owt
I I I I
PCGI-I aPCl1 I-J PCG2a-I aPCl04-8 PCGI-2 PCGI-13 PCG2b-3 PCG3-3 aPC56- I PCGI-9 PCGI-IO PCGI-1 I PCGI-I2
NPEP
Choline
0.03 0.13 0.002 0.004
0.041 0.27 0.05 0.01 0.02
0.005$ 0.31 0.084 0.005 0.004
>lO$ >I0 > IO 2.0 3.5
I .4f > IO 0.6 0.3 I.5
Ii II II II I1 II
210 210 > IO > IO > IO >I0
0.074 2.1 0.54 0.026 5.3 6.6
0.88 3.6 0.40 0.24 5.2 z IO
3.2 9.4 >I0 3.6 >I0 >I0
>I0 1 IO > IO >I0 210 >I0
II II II II II II II
> IO > 10 > 10 >I0 >lO >I0 > IO
0.53 0.44 0.063 0.8 0.06 0.16 0.087
0.59 1.5 0.46 6.1 0.67 6.3 0.5 I
>I0 >I0 >I0 >iO > to >I0 >I0
>I0 >I0 >I0 210 > IO >I0 210
PCP
*The origin of the antibodies is referenced tThe I,, values for PC and NPPC of Tl5, Chang ef al., 1984; PCG2b-3, PCG3-3, $ls,, values: concentrations of hapten (mM)
in Materials and Methods. McPC603, Ml67, PCGI-I, aPCl I l-l, PCGI-2 aPC56-I (Stenzel-Poore et ol., 1987). needed to inhibit antibody binding by 50%.
associated with Group II-A and Group II-B antibodies (Table 3). Thus it seems that the generation of Group II-A/II-B binding phenotypes is not linked to heavy chain usage. In order to determine the intrinsic affinities of Groups I and II antibodies, we used NPPC, NPDBP, and DPPC in fluorescence quenching experiments. APPC could not be used because it quenched the antibody fluorescence nonspecifically. DPPC was initially included because it has been used previously to determine the affinity of Group I proteins (Rodweli et al., 1983; Gearhart ef al., 1989); NPPC and NPDBP are used for the first time in this study. All three ligands are believed to reflect the diazophenyl linkage in the PC-protein conjugate Table 3. Charge dependency Antibody Tl5 M603 Ml67 PCGI -4 PCGI-6
Group
APPC
NPPCt
(Table I). Group I and Group II-B antibodies exhibit detectable affinities for NPPC and DPPC whereas Group II-A antibodies show low or no detectable affinities for these ligands (Table 4) despite the fact that NPPC is an effective inhibitor in the ELISA. There is considerable heterogeneity even within a group or subgroup, most evident in Group II-B where the affinities for NPPC and DPPC vary by more than 140-fold and 350”fold, respectively. Group I antibodies have comparable affinities for both ligands or higher affinity for DPPC. Within the Group II-B there are antibodies with preferential binding for NPPC (PCG2b-3, PCG3-3, aPCG56-1, PCGl-12) exhibiting two to four times higher affinities for NPPC than for DPPC. In contrast,
of monoclonal
Heavy chain* Isotype VH Family
are taken from
Group
I and Group
Light chaint NPPCf
II antibodies Inhibitors NPDBP
NPMPC
SlO?(VHl) S107(VHl) SI07(VHl) 5107(VHl) Sl07(VHI)
VK22 ViC8 VK24 vu22 VK22
0.W 0.27 0.05 0.01 0.02
>l@ >lO z IO 4.6 6.3
4.w 1.8 0.09 0.04 0.05
>’I P y 2a ”3 )‘I E’1
QS2 (Ml41) S107 (VHI) 5107 SlO7(VHI) J558fVH-12) J558
VKI-3 VKI-3 VKI-3 Vul-3 V~24 V~24
0.074 2.1 0.54 0.026 5.3 6.6
> IO > IO > IO >I0 > IO > IO
> IO > IO > 10 >I0 >I0 > 10
:I 2b Y3 Yl
452 (M14lf Q52 (M141) Q52(M141) 7183
0.53 0.44 0.063 0.8 0.06 0.16 0.087
0.75 0.40 0.28 0.77 I.9 6.5 0.42
>lO >I0 >I0 > IO >I0 >I0 >I0
I
a
I I I I
* ti .,jI .,II
PCGI-I aPCIII-I PCG2a-I aPC 104-R PCGI-2 PCGl-I3
II-A II-A 11-A II-A II-A II-A
PCG2b-3 PCG3-3 aPC56-I PCG l-9 PCGI-IO PCGI-I I PCGI-I2
II-B II-B II-B II-B II-B II-B II-B
VI
vl vl Yl
~52 Q52 5558
‘VH family assignments were made by one or more of the following methods: Northern blot analysis (Stenzel-Poore et al., 1988). Southern blot analysis (Stenzel-Poore et al., 1987; Rittenberg et nl., 1986). N-terminal amino acid sequencing (Gearhart PI ai., 1981; Rittenberg ef al., 1986) and Tl5 idiotype expression. The VH gene where known is given in parentheses. flight chain assignments were made by N-terminal amino acid sequencing, Tl5 idiotype expression and/or isoelectric focusing (Todd ef al., 1984, 1985). ISee footnote t of Table 2. pl,, values (mM).
URS BRUDERER et al.
68 Table 4. Binding constants
Antlbodq
Group
TI5 McPC603 Ml67 PCGI -4 PCG l-6
I I I I I
PCG I LlPCll I-I PCG?a I aP(‘lO4.X PCG?b-? PCGJ-I 3PC56I f’<‘G I -Y PCG I IO PCGI-I1 PC‘G-I?
of monoclonal Group I and Group fluorescence quenching ?iPPC
II antibodies
Ka x lO’(M NPDBP
I 8 z 0.25* <0.5+ 5x 103 I.1 ’ 0.2 0.0 r 0.03
N.T.: N.T. N.T. N.T. N.T.
II-A II-A II-A II-4
0 77 f 0.04
N.T. N.T. N.T. N.T.
II-H II-R 11-B II-B II-B 11-B II-B
‘Y7 i 1.0 YI oixo 25.1) 2.5 2.4 : (I.? 5.2 : 0.3 IS.9 T 0.9 0.64 i 0.08
1.x 0.2 I I.? i_ + 2.7 4.5 * 0.7 10 St 0.74 t 0.08
co.5
*R pi standard dcviatwn of two determmatlons. tAHinity b&w the It‘wzl of detectmn :N.T. not te\tcd becaue NPDBP was unable to inhibit ELISA.
PCGI-IO, PCGl-11 and the more extreme PCGl-Y show up to 43-fold higher affinity for DPPC than for NPPC (Table 4). Group II-B antibodies have lower affinity constants for NPDBP than for NPPC and DPPC (Table 4). Although the binding of all Group II-B antibodies to PC-conjugated protein can be inhibited with NPDBP (Table 3). some exhibit no detectable affinity for this ligand in the fluorescence quenching assay, suggesting that the charge in the trimethylammonium group is not essential for binding but can add to the strength of the interaction. DISCUSSION
We previously described that diversity in the memory response to PC-KLH is acquired through antibodies differing in their fine specificity [Group I vs Group 11 (Chang et d., 1984; Todd et al., 1984. 1985; Stenzel-Poore rt al., 1987)]. Furthermore, we reported that V-gene heterogeneity within the Group II response is much less restricted than that of the pauciclonal Group 1 response (Crews et al.. 1981: Perlmutter, 1984). Here, we analyze combining site heterogeneity of Group II antibodies by characterizing the antigen-antibody interactions responsible for the generation of specificity and affinity. Binding of Group II antibodies to PC-conjugated proteins can be inhibited by NPPC but not unconjugated protein, NPP, NPEP, or PC suggesting that the epitope recognized by these antibodies is comprised of the PC and the phenyl moieties. In agreement with our findings that PC is an integral part of the epitope recognized by Group II. Wicker et al. (1982) found that Group II antibodies bound to APPC and PC conjugated to bovine serum albumin to p-diazophenylarsonate (PC-BSA) but not conjugated to BSA (Ars-BSA). Since both Ars-BSA and PC-BSA contain the same diazophenyl ring and
the binding
determined
by
‘) DPPC 5.1 1.9 5.x 3.0 30
tn.1 kO.1 2 0.3 i_ 0. I i 0.04
0.x0 z 0 01 <0.25t 1025 <025 22 32.1 12.7 102.7 I?.‘) 47.1 0.29
0 04 *1 I4 _i 7.2 _t 5.0 t 0.5 * 7.5 * 0.04
of these antlhodxs
m
tyrosyl and/or hystidyl carrier determinants, their studies suggest that neither the carrier nor the linkage is sufficient for binding. Our experiments with NPMPC and NPDBP demonstrate the importance of ionic interactions. Group 11 antibodies do not recognize NPMPC showing that the negative charge in the phosphate moiety is absolutely required. NPDBP, which differs from NPPC by the lack of charge in the structure corresponding to the trimethylammonium moiety of choline is recognized by Group II-B, but not by Group II-A antibodies which demonstrates that Group II specificity can be achieved in at least two ways. This finding indicates that the choline is in fact a necessary part of the epitope recognized by Group II-A antibodies and that the binding is charge dependent. It is likely therefore that ionic interactions with the trimethylammonium moiety are essential for these antibodies to bind. Although the correponding affinities are several fold lower for NPDBP than for NPPC, ionic interactions seem to be much less important for Group II-B than for Group II-A antibodies. Because the energy of interaction of juxtaposed groups is inversely proportional to the distance between the oppositely charged ions of antibodies and antigens (Absolom and van Oss. lY86), the energy of charge interactions is highly distance dependent. Thus, Group II-A antibodies which only recognize the positively charged hapten appear to have a negatively charged contact residue that is critical to the interaction. Group II-B on the other hand may lack the corresponding residue entirely or have it positioned where it is not in close proximity to the trimethylammonium and thus, it would contribute less to the energy of the interaction. Hydrophobic interactions between apolar groups have been described to be of major importance among the bonds of immunological significance (Karush, 1962; Tsutsui et ~1.. 1977: Absolom and van
Fine specificity
of anti-PC-KLH
Oss, 1986). Therefore, it seems likely that, in contrast to Group II-A antibodies, hydrophobic interactions between the 3,3-dimethyl butyl moiety and the Group II-B antibody combining site are a dominant feature in binding to the latter. The finding that some Group II-A antibodies (3/6) weakly recognize NPEP but not the uncharged NPDBP may indicate that the ionic interaction between these antibodies and the choline moiety is dominant and that the hydrophobic character of the choline moiety in the absence of a charge would result in repulsion which would explain the failure to bind NPDBP. Group I antibodies are easier to inhibit with analogs of NPPC than Group II antibodies which may be due to the smaller epitope recognized by Group I antibodies. T15 and M603 are much more easily inhibited by NPPC than by either choline or NPMPC indicating that the phosphate group itself as well as the negative charge are essential for binding. In contrast M 167, PCG l-4, and PCG l-6 have comparable I,, values for NPMPC and NPPC but were inhibited less well by choline. Thus, it seems that the phosphate moiety is a crucial component of the epitope recognized by these antibodies but that this requirement need not extend to the negative charge. These findings are in agreement with the results of Goetze and Richards (1977) who deduced from NMR analysis that for Ml67 the strength of the interaction with the choline subsite is much stronger than with the phosphate subsite whereas both subsites contributed comparably to the binding of T15 and M603. Furthermore, they showed that for antibodies with the same light chain the importance of the two subsites could differ. Whether the difference between T 15 and PCG 1-4 and PCG 1-6 can be explained by somatic mutations remains to be determined. VH genes from four VH families (452, S107, 5558, 7183) have been found to be expressed in the Group II response (Stenzel-Poore rt al., 1989; Table 3). Different VH genes can be used within the same subgroup, e.g. the Group II-A antibody PCGI-2 expresses a VH-12 gene product (Rittenberg et al., 1986) aPCl l l-I and aPC104-8 utilize the VH-1 gene product (Stenzel-Poore et al., 1989) and PCGI-I uses the VH gene Ml41 (Stenzel-Poore et al., 1987). The Ml41 VH gene is also associated with the Group II-B proteins PCG2b-3, PCG3-3 and aPC56-1 (Stenzel-Poore et al., 1987), suggesting that the heavy chain may not be the critical factor in the generation of II-A or II-B binding sites. This is consistent with our observation that the II-A/II-B distinction thus far has been linked to the expression of K(Group II-A) and i(Group II-B) light chains. The ionic interactions associated with Group II-A binding properties are not restricted to a particular K light chain in that antibodies with either VKI-3 or V~24 light chains are found in this group. In contrast to the Group II response, the Group I response is dominated by antibodies expressing one
antibodies
69
particular VH gene (VHI) in combination with V~22 or to a lesser extent, VK 8 or V~24 light chains (Crews et a/., 1981; Gearhart et al., 1981). Crystallographic analysis of the Group I myeloma protein M603 with PC in the active site (Segal et al., 1974; Padlan et al., 1976) revealed that the positively charged trimethylammonium moiety of the hapten interacts predominantly with the heavy chain [Glu 35H and Glu 53H (M603 numbering of Rudikoff and Potter, 1974)], suggesting that the heavy chain of Group I antibodies is dominantly responsible for the binding to PC. Since four distinct VH gene families can be used to generate Group II binding sites (Table 3) our data suggest that the light chain may be of more importance in the generation of the Group II combining site. Rodwell et al. (1983) who first determined affinities of Group I antibodies for DPPC, analyzed the contribution of the phenyl ring to the binding constant. They showed that some Group I antibodies exhibited up to 80-fold higher affinity for DPPC than for PC itself. They concluded from their data that DPPC rather than PC was the more appropriate ligand for these antibodies and as a consequence, the phenyl group makes a substantial contribution to the binding constants. Furthermore, Mandel et al. (1984) tested the contribution of a phenyl structure in the anti-lactose system. By comparing the affinities for p-nitrophenyl-p-D-lactoside and for methyl+o-lactoside, they showed that the phenyl moiety made a significant contribution to the affinity. However, they concluded that it was not possible to determine whether the phenyl group was part of the epitope or whether it made a nonspecific contribution to the affinity due to its hydrophobic character and proximity to the antigen binding site. However, they provided evidence for the latter by demonstrating that not only antibodies elicited by p-aminophenylb-lactoside but also by a vaccine prepared with Streptococcus faecalis, which lacks the phenyl linkage, exhibited higher affinity for p-nitrophenyl-B-Dlactoside as compared with methyl-/?-o-lactoside. it is not possible to discriminate Accordingly, between these two possibilities in the case of Group I antibodies. For Group II antibodies, at least the phenyl ring of NPPC seems to be part of the epitope because these antibodies show, in contrast to Group I antibodies, no detectable affinity for PC itself. We found that specific fluorescence quenching by NPPC is not inhibited even by 1OO-fold excess of PC (results not shown) and that binding to PC does not lead to fluorescence enhancement of Group II antibodies (results not shown) as has been reported for Group I antibodies (Pollet and Edelhoch, 1973). Our results also indicate that there is heterogeneity in the binding preference for NPPC and DPPC within Group II-B. Three of the seven Group II-B antibodies exhibited two to four times higher affinity for NPPC whereas the other three showed the reverse pattern, e.g. PCGl-9 exhibits >40-fold higher
70
URS BRUDERER r~ al.
affinity for DPPC than for NPPC. Except that DPPC has an additional dinitrophenyl (DNP) moiety linked to the phenyl ring of NPPC (see Table l), these two ligands share the same structures. The addition of the DNP moiety thus appears to contribute dramatically to the binding by PCGI-9. Accordingly, as demonstrated by Mandel et al. (1984) for anti-lactose antibodies, the second phenyl group may also be part of the epitope which is recognized, suggesting that the active site of PCGl-9 may differ appreciably from those Group II-B antibodies with preferential binding to NPPC. Alternatively, as discussed above, the DNP moiety may serve to stabilize the antigenantibody combination in PCGl-9 through an interaction outside of the combining site. Individual antibodies differed by more than IOO-fold in their preferential binding to either NPPC or APPC, a ligand used previously (Wicker et al., 1982) to discriminate between Group I and Group II antibodies. Preference for the nitro or amino derivative does not seem to be associated with either of the sub-populations but rather is a characteristic of a particular antibody. Nitro and amino groups have an inductive effect on rc electrons of the aromatic ring, with nitro diminishing and amino enhancing. The nature of the interaction of the aromatic site of the hgand with the antibody is likely to involve 71electron interaction and hydrophobic interaction. Thus, it is conceivable that antibodies which show preferential binding to APPC depend on interactions with rt electrons as well as on hydrophobic interactions. In contrast, for antibodies which show a binding preference to NPPC, the n electrons may contribute relatively little to the interaction compared to hydrophobic interactions in the phenyl region. Although studies of serum Group II antibodies suggest that VK-I-3 usage would be most relevant (Todd et al., 1985; Bruderer et ul.. 1989). the results of hapten-inhibition ELISA suggest that Groups II-A and II-B would have a comparable distribution of affinities for NPPC but as shown in Table 4, Group II-A antibodies have very low binding constants. This is consistent with previous reports that Is,, values do not necessarily reflect intrinsic affinities (Foiles et al., 1983; Peterfy et al., 1983; Friguet et al., 1984). The main difference between fluorescence quenching which measures the primary interaction between hapten and antibody and hapten inhibition ELISA is that in the latter the antibodies can bind to the PCprotein complex. Since all of the antibodies described were selected on the basis of their ability to bind to PC-protein it is possible that neither NPPC nor DPPC adequately reflects the immunizing form of PC in PC-KLH. Thus, Group II-A and II-B could be subject to the same antigenic selective pressure although this is not revealed by their intrinsic affinities for the haptens. Previous assessments of Group I (Rodwell et al., 1983; Gearhart et al., 1989) indicated that somatic mutation accounts for increased affinity of IgG antibodies; it remains to be
determined whether the higher affinity of Group II-B antibodies compared to Group II-A results from somatic mutation, the use of particular V genes or a combination of both mechanisms. Acknowledgements-We thank P. Bresnahan, M. Brown, A. Buenafe, Q. Chen, and Dr T. Tittle for critical review of the manuscript and Dr C. H. Heusser for providing hyhridoma proteins aPC56-1, aPClll-I. and aPC104-X.
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Fine specificity
of anti-PC-KLH
Peterfy F., Kunsela P. and Makehi 0. (1983) Affinity requirements for antibody assays mapped by monoclonal antibodies. J. Immun. 130, 18d9-1813. Pollet R. and Edelhoch H. (1973) . , The binding __pronerties of anti-phosphorylcholine mouse myeloma proteins as measured by protein fluorescence. J. biol. Chem. 248, 5443-5447. Rittenberg M. B., Glanville R. W., Aebersold R. H., Chang S. P. and Brown M. (1986) Immunologic memory to phosphorylcholine (PC~VIII.,Expression of the VH-12 gene product in the response to PC-keyhole limpet hemocyanin. Eur. J. Immun. 16, 503-507. Rodwell J. D., Gearhart P. J. and Karush F. (1983) Restriction in IgM expression-IV. Affinity analysis of monoclonal anti-phosphorylcholine antibodies. J. Immum 130, 313-316. Rudikoff S. and Potter M. (1974) Variable region sequence of the heavy chain from a phosphorylcholine binding myeloma protein. ~~~c~ern~~~r~ 13, 4033-4038. Scatchard G. (1949) The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51, 660672. Segal D. M., Padlan E. A., Cohen 6. H., Rudikoff S., Potter M. and Davies D. R. (1974) The three-dimensional structure of a phosphorylcholine-binding mouse immunoglobulin Fab and the nature of the antigen binding site. Proc. mtn. Acad. Sci. USA 71, 4298-4302. Stenzel-Poore M., Hall T. J., Heusser C. H., Faust C. H.
antibodies
71
and Rittenberg M. B. (1987) Immunologic memory to PC-KLH: Participation of the 452 VH gene family. J. Immun. 139, 1698S1703. Stenzel-Poore M., Keller N., Heusser C. H. and Rittenberg M. B. (1989) Antibody diversity in the memory response to PC-KLH: expression of the S107, Q52,5558 and 7183 VH families. (submitted). Steward M. W. and Petty R. E. (1972) The antigen-binding characteristics of antibody pools of different relative affinity. immunology 23, 881-887. Todd I.1 Chang S. P., Perlmutter R. M., Aebersold R., Heusser C. H.. Hood L. and Rittenbere M. B. (1984) Immunologic memory to phosphocholice-V. Hybrid: omas representative of Group I1 antibodies utilize VK l-3 gene(s). J. Immun. 132, 1556-1560. Todd I., Brown M. and Rittenberg M. B. (1985) Immunologic memory to phosphocholine-VI. Heterogeneity in light chain gene expression. Eur. J. Imm~n. IS, 177-183. Tsutsui K., Koide N.. Tomoda J., Hayashi H., Hatase 0. and Oda T. (1977) Role of hvdronhobic interaction in hapten-antibody binding. A& Med. Okqvama 31, 2899294. Wicker L. S., Guelde G., Scher I. and Kenny J. J. (1982) Antibodies from the Lyb-5. cell subset predominate in the secondary IgG response to phosphocholine. J. Immun. 129,950-953.