Binding of anti-galactosyl antibodies to galactosylated liposomes

ImmunohJgv Letters. 8 (1984) 257 260.

Elsexier Imlet 513

BINDING

OF ANTI-GALACTOSYL GALACTOSYLATED

ANTIBODIES

TO

LIPOSOMES

l)ebi P R A S A D S A R K A R and Manoj K. DAS* I)~7~artment ~?/ k.)tzvme I:)Igim'ering. Indian hl~titute ~/ ( Twmical Bioh>g.l'. Jada~7~ur. ('ah utta 700 032, htdia

(Received 28 June 1984) (Modified version received 13 August 1984) (Accepted [4 August 1984) 1. Summary The binding characteristics of rabbit anti-galactosyl antibodies elicited through galactosylated liposomes were studied. The association constants (13 x 105 M a and 21 × 10-~ M i) of Fab~ and Fab fragments with the galactosylated liposomes were determined by measuring the binding of radioiodinated antibodies. The effect of free ligand (methyl-/3-1)-galactopyranoside) and the ligand coupled to the liposomal surface (galactosylated liposomes) on the structure of antibodies was investigated using circular dichroism (CD) measurements. Changes were observed in both the secondary and tertiary structure of antibodies upon binding to galactosylated liposomes. In contrast, the free ligand induced alterations only in the near-UV region of the C D spectra, indicating a change in tertiary structure only.

2. Introduction We have developed a method to elicit anti-carbohydrate specific antibodies using liposomes as both thc carrier and adjuvant [1 ]. The saccharide haptens were coupled to the surface of negatively charged, multilamellar, liposomes containing phosphatidylethanolamine. The glycosylated liposomes were found to induce a hapten-specific humoral im* 'rov, hom correspondenceshould be addressed. KeT wor~A."liposomes anticarbohydrate antibodies • conformational changes membrane antigens 0165 2478 . 84 " $3.00 © ElsevierScience Publishers B.V,

munc response in rabbits and the immunochemical characteristics of the antibodies have been studied [2]. Most of the antibodies belong to the lgM class but a minor proportion arc of lgG isotype. Haptenated liposomes could serve as a model for cell surface antigens (membrane antigens) and the binding of specific antibodies to the liposomes might be of considerable interest for a proper understanding of antibody interactions with cell surface antigens in vivo. it is possible, for example, that there are conformational alterations in antibodies during such interactions. Previously we have studied one such system comprising galactocerebroside liposomes and the antigalactocerebroside antibodies by CD measurements [3]. In the present report, the interaction of galactosylated liposomes with specific antibodies has been investigated. Measurements were also carried out with methyl-/3-D-galactopyranoside in order to compare the effect of free and liposome bound ligands on antibody structure. 3. Materials and Methods 3.1. M a w r i a l s

Galactosylated multilamellar liposomes were prepared as described earlier [1] and unilamellar vesicles were made following the procedure of Barenholz et al. [4]. Rabbit anti-galactosyl lgM and IgG antibodies were affinity purified [2]. The Fabta and Fab fragments of these antibodies were obtained as described previously [5,6]. These fragments were radiolabelled with 1251using chloramine-T [2]. All other reagents used were of analytical grade. 257

3.2. Binding (?/ Fah# and Fah with galacto.~3'lated

lipo.some.~ ] h i s was performed as described previously [2]. Multilamellar galactosylated liposomes (50 ~1, 6.3 nmol of galactose) were mixed with radiolabelled Fab# or Fab in 0.01 M phosphate bufti~r, 0.15 M NaC1, pH 7.4 (PBS)in polycarbonate tubes which had been saturated previously with bovine serum albumin (BSA). The tubes were incubated at 4 °C for 3 h and then centrifuged at 105,000 x g for 2 h. r h e radioactivity was measured in the supernatant to determine the amount of bound antibody. Control liposomes without galactose were used to determine the extent of non-specific binding and the results were corrected accordingly. 3.3. CI) Studies The C D of antibodies and the unilamellar liposome-antibody complex were measured in Jasco J20A recording spectropolarimeter. Ellipticity was calculated as mean residue ellipticity [-galactopyranoside (20 mM) approximately 70% saturation of IgM binding sites would occur [2].

4. Results

r h e binding of anti-galactosyl Fabp. and Fab with galactosylated liposomes was studied (Fig. 1). M ultilamellar vesicles were used in this case since it w.as noticed that the complexes of Fabp. or Fab with unilamellar liposomes could not be separated by centrifugation. The association constants for Fab# and Fab were found from the Scatchard plot to be 13 x l0 s M i and 21 x 105 M i respectively. A linear relationship was obtained in the Scatchard plot of the binding data (inset, Fig. I) indicating an apparent homogeneity of interaction. The effect of galactosylated liposomes on the conformation of antigalactosyl antibodies or its fragments was studied. The CD spectrum of anti-galactosyl IgM, Fab# and Fab are shown in Figs. 2, 3 258

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Fig. I. Binding cur',es ol Fab,u and Fab fragments ol antigalactosyl antibodies v, ith galactosylatcd liposome~,. Various anlounts of labelled Fab,u ( • ) and Fab ( O ) ",,sere added to 5() ul liposomcs (6.30 nrnol of galactosc) m 0.5 ml PBS. The inset depicb, the Scatchard analysis ol the binding data.

and 4. A negative band in the far-UV region (at 217 nm) was noticed in each case. In the near-UV region, a negative band around 295 nm was easily discernible using intact IgM but was not so clearly seen using the Fab# fragment. Addition of methyl-/3-t)-galactopyranoside to the antibodies caused changes in the near-UV CI) bands

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Fig. 2. C I ) spectra ot antigalactos.',l IgM antibodies. ('ur',e I lgM; Curve 2 - IgM in the presence of 20 mM methyl-/J-l>-galactop,,rano~ide. The concentration of IgM was 1.0 mg ml.

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than those observed following binding to methyl-/3-1rgalactopyranoside. ]'he changes observed in CD profiles of antibodies were specific since control liposomes without galactose or other sugars such as mannosc could not induce any change.

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Fig. 3. C D spectra of antigalactosyl F a b ~ . C u r v e 1 = Fab~; cur~c 2 - Fab,u ~ methyl-/3-i)-galactop~ranoside(20 mM): a n d cur~e 3 F a b ~ . galactos)lated unilamellar liposomes (25 nmol of ligand). The c o n c e n t r a t i o n of Fab/a was 0.5 rag: ml.

but not in far-UV region. The highest changes were observed using Fab/~ (Fig. 3) followed by IgM and Fab. However, with the fragments both the near-UV and far-UV regions ot the CD spectrum were affected following binding to the galactosylatcd liposomes. The [~b]217value of Fab# decreased from 2600 deg • cm -~. dmol i t o 1900deg. cm -~. dmol l a r e d u c tion of 27t~. Similarly a decrease in [~b]217value was also observed in the Fab spectrum ( 2000 deg • cm 2. dmol I t o 1700deg- c m Z . dmol ~).The galactosylated liposomes caused intermediate alteration in the near-UV regions especially around 295 nm in Fab/a or Fab. l h e changes were lower

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The results presented here suggest that antigalactosyl antibodies undergo considerable conformational alteration upon binding to galactosylated liposomes. The alterations involve the peptide backbone and the localizcd environments of aromatic residues and disulfide bonds. In contrast, free methyl-/J-D-galactopyranoside perturbs only the latter residues as changes are limited to the near-UV region ot the CD spectrum. I,igand coupled to the surfacc of liposomes appeared to induce a more conformational change in Fab/a or Fab fragments than did free ligand. This suggests that phospholipid bilayers of haptenated liposomcs might be able to interact with thc antibodics. Non-specific interactions together with the specific intcraction provided by the ligand might be responsible for the observed structural alterations in the antibodies. However. non-liganded liposomes themselves did not produce any change in the CD spectrum of the antibodies and other studies dealing with the events on cell surfaces are clearly required. It has been observed that hapten carriers play an important role in determining complement fixing activity [7,8]. The mechanisms underlying antibody binding to soluble and cell surface antigens are known to be different [9,10] and Kobayashi et al. observed diffcrences in the kinetics of antibody binding to a lipid hapten (free from liposomes) compared to unilamellar haptenated liposomes [11 ]. The compositions of liposomal membranes that determine the membrane fluidity may modulate the immune reactivity of haptens incorporated in liposomes [12].

320

Fig. 4. C D spectra of antigalactosyl Fab. C u r v e 1 = Fab; curve 2 - F a b • methyl-/J-l)-galactopyranoside (20 mM); a n d cur',e 3 -F a b + galactosylatcd unilamellar liposomes (25 n m o l of ligand). "lhe c o n c e n t r a t i o n of F a b was 0.5 r a g ml.

Acknowledgements We wish to thank Professor B. K. Bachhawat for his support and interest in this work. D.P.S. is the 259

recipient of a Scnior Research Fellowship of CSI R. India. The authors wish to thank Mr. Subodh Kumar Roy for his assistance in rccording the CI) spectra.

151 {61 17} [8]

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