Immunochemical properties of phosphatidyl cholesterol and its homologue

Chemistry and Physics of Lipids, 23 (1979) 2-12 © Elsevier/North-HollandScientific Publishers Ltd.

IMMUNOCHEMICAL PROPERTIES OF PHOSPHATIDYL CHOLESTEROL AND ITS HOMOLOGUE I. HARA, T. MURAMATSU, T. FUKUDA and J. SATO* Laboratory of Chemistry, Department of general Education, Tokyo Medical and Dental University, Kohnodai, IchikawashL Chiba Prefecture. *Department of Clinical Pathology, Dokkio University, School of Medicine, Mibu, Tochigi Prefecture (JapanJ Received November 25th, 1 9 7 7

accepted July 5th, 1978

1,2-Diapalmitoyl-rac-glycerol-3-phosphoryl-3'-cholesterol (PCH)and 1,2-dipalmitoyl-racglycerol-3-phosphoryl-20'-(3-hydroxynorpregn-5-ene) (PET) were combined with poly-L-lysine to form a PCH-poly-L-lysinecomplex and a PET-poly-L-lysinecomplex, respectively. These complexes were subcutaneously injected into rabbit foot pad with Freund's complete adjuvant. PCH antiserum showed specifieities against the phosphatidyl group, the cholesterol moiety and the side chain of cholesterol. PET antiserum contained the specific antibodies against the phosphatidyl group, the cholesterol moiety and the OH-group at the C3 position of the cholesterol molecule.

I. Introduction Antisera against acidic phospholipids, such as cardiolipin [1,3,5], phosphatidyl glycerol [1,5,6], phosphatidyl inisitol [2,4], phosphatidic acid [3], phosphatidyl ethanolamine [7] and phosphatidyl sefine [3] were obtained by immunizing with phospholipid-basic carrier protein complex. Similarly, antisera against cholesterol and cholesterol esters were prepared by inoculation of cholesteryl-3-formyl bovine serum albumin conjugate [8] and cholesterol semisebacate-human serum albumin complex [9]. By using etiocholenic acid-poly-L-lysine complex and cholesterol succinate-poly-L-lysine conjugate, antisera aghinst etiocholenic acid (ETIO) and that against cholesterol succinate (CHS) were obtained [10]. In latter cases, both anti-sera had two kinds of specificities. The first showed the specificities against the cholesterol moiety and OH group at C3 position of ring A, whereas the second exhibited the specificities towards the cholesterol moiety and the side chain of cholesterol. Recently, Muramatsu and Hara reported the synthesis of two phospholipidsterol conjugates, 1,2.dipalmitoyl-rac-glyceryl-3.phosphoryl-3'-cholesterol(PCH) Abbreviations: CHS, cholesterol succinate; ETIO, etiocholenic acid; PA, phosphatidic acid; PC, egg yolk phosphatidylcholine; PCH, 1,2-dipalmitoyl-rac-glyceryl-3-phosphoryl-3'-cholesterol; PET, 1,2-dipalmitoyl-rac-glyceryl-3-pbosphoryl-20'(3-hydroxynorpregn-5-ene).

8

L Hera et aL, Irnraunocheraistry ofphosphatidyl cholesterol

~H2-O-CO-R

CH2-O-CO-R

CH-O-CO-R , . ~ i CH2-O-P-O-t.v.t.~J OH

L..y,.. '

i

CH-O-CO-R I ,o, CH2-O-P-O-CH2 OH< ~

R= C15H31 PCH

OH PET

Fig. 1. Chemical structures of PCH (C6:H':t30',P)(1017.56) and PET (CseH~O~)(935.38). and 1,2-dipalmitoyl-¥ac-glycerol-3-phosphoryl-20'-(3-hydroxy as shown in Fig. 1 [ 11].

norpregn.5-ene) (PET)

II. Materials and Methods

PCH and PET [11] were prepared in our laboratory. Phosphatidic acid (PA) was derived from purified egg yolk phosphatidyl choline (PC) with phospholipase D (Boeringer, Mannheim, Germany) (EC 3144). Diglyceride was also obtained from purified egg yolk PC by the action of phospholipase C (Boeringer, Marmheim, Germany) (EC 31). CHS was prepared in our laboratory [10]. Etioeholenic acid (E. Merck, Darmstadt, Germany) was a commercial product, a.Napthyl phosphate (Tokyo Kasei, Co., Tokyo), 2-glycerophosphate (Tokyo Kasei, Co., Tokyo) and glycerol (Kanto Kagaku, Co., Tokyo) were commercial products. All of these products, except glycerol, were purified by recrystallization until they proved to be pure as judged by TLC. Poly-L-lysine (70 polymers) (Ajinomoto Co., Tokyo) was a commercial product.

A. Chemical analysis Phosphorus was determined by King's method and poly-L-lysine by Folin's procedure [ 13]. B. Preparation of antigen PCH or PET was combined with poly-L-lysine as follows: PCH or PET in alcohol solution was added to an equal volume of water and stirred vigorously to obtain a fine suspension. One volume of the suspension (1 mg/ml) and one volume of the water solution ofpoly-L-lysine (5 mg/ml) were mixed and stirred at room temperature for 2 h. The precipitate formed was separated by centdfugation at 300 rev./min for 10 min and washed several times with 0.85% NaCt solution.

L Hara et al., Immunochemistry ofphosphatidyl cholesterol

9

C. Preparation o f sheep red blood cells sensitized with hapten Sheep red blood cells were treated with 3.05% formalin solution according to Cox [11]. To one volume of form alinized suspension of sheep red blood cells in 1/15 M phosphate buffer-0.85% NaCi solution, pH 7.2, was added an equal volume of a methanol solution of hapten (10 raM) containing 2 mg/ml of dicyclohexylcarbodiimide. The resulting mixture was incubated at 4°C for 30 min. Thereafter, the sensitized sheep red blood cells were washed several times with 1/15 M phosphate buffer-0.85% NaC1 solution, and a 0.5% suspension of the sentisized sheep red blood cells in buffer solution containing 1% normal rabbit serum was prepared. D. Preparation o f antiserum Hapten-poly-L-lysine complex, 10 mg, was subcutaneously injected into rabbit foot pad together with Freund's complete adjuvant. After one month, the injection was repeated using with the same dose level. When the titre did not rise, a further injection with the same dose was carried out. Two weeks after the last injection, the rabbits were bled and antiserum was obtained. E. Passive hemagglutination reaction and passive hemagglutination inhibition reaction Passive hemagglutination reaction was carried out according to the method of Herbert [15] using a micro-technique with phosphate buffer-0.85% NaC1 solution containing 1% normal rabbit serum as diluent. 25 ~ul of sensitized sheep red blood cell suspension (0.25-0.50%) was added to 25/al of a serial dilution of antiserum. After mixing, the reaction mixture was moderately shaken for a few rain, incubated at room temperature for 18-20 h, and the degree ofhemagglutination was measured. In the case of the hemagglutination inhibition reaction, to 25/al of the serial dilution of hapten suspension in 1/15 M phosphate buffer solution -0.85% NaC1 solution, pH 7.2, was added 25/al of the diluted antiserum solution. The reaction mixture was incubated at 37°C for 30 min. After incubation, 25 /zl of the sensitized sheep red blood cell suspension was added to the mixture, the degree of hemagglutination was estimated by the method o f Herbert [15].

III. Results The chemical composition of the PCH-poly-L-lysine complex and the PET-poly-Llysine complex were found in assuming the molecular weight of poly-L-lysine as average 8000 as follows: PCH-poly-L-lysine complex : PCH :poly-L-iysine (2.41 : l)(mol/mol) PET-poly-L-lysine complex : PET:poly-L-lysine (2.11 : 1)(mol/mol) In order to examine the reactivities of PCH, PET and other haptens, a series of passive hemagglutination reactions were carried out as shown in Table 1. According to Table 1, PCH, PET and other haptens yielded positive reactions in higher titre with homologous antisera. PCH reacted more strongly with antiserum

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L Ham et aL, Immunochemistry of phosphatidyl cholesterol

Table 1 Hemagglutination reactions of sheep red blood cells sensitized by PCH and PET with various antisera. Antiserum against

Sensitized by

Antiserum titre a

PA

PA PCH PET CHS PCH PET ETIO PCH PET

1 1 1 1 1 1 1 1 1

CHS

ETIO

: 1024" : 32 :4 : 1024 : 128 : 64 : 4096 : 16 : 256

aAntiserum titre was determined by the last dilution of antiserum showing the hemagglutination degree of 1. against CHS t h a n P E T , and PET reacted m o r e strongly w i t h antiserum against E T I O t h a n PCH. F o r d e t e r m i n i n g the structural adaptability and t h e essential structure for antigenicity o f the lipid h a p t e n , passive h e m a g g l u t i n a t i o n inhibition reactions using P e r t - a n t i PCH antiserum and PET-anti PET antiserum systems were carried out as s h o w n in Tables 2 and 3. A c c o r d i n g to Table 2, PCH strongly inhibited the PCH-anti-PCH antiserum system b u t b o t h P E T and P A showed i n h i b i t o r y activities o f the same order. Table 2 Inhibitory activities of various lipidic haptens to the hemagglutination reaction between PCH and anti-PCH antiserum. Inhibitor

PCH PET PA a-naphthyl phosphate cholesterol phosphate 2-glycerophosphate glycerol diglyceride

Dilution of inhibitor (0.1 tool) 1:2

1:4

1:8

1 : 16

1:32

control

0 1 1 2 2 2 4 4

0 2 1 2 3 3 4 4

1 3 2 2 4 4 4 4

1 4 2 3 4 4 4 4

3 4 4 4 4 4 4 4

4a 4 4 4 4 4 4 4

alnhibitory activity was described as follows: 0 = complete inhibition, 1 = strong inhibition, 2 = medium inhibition, 3 = slight inhibition and 4 = no inhibition.

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I. Hara et al., Immunochemistry o f phosphatidyi cholesterol

Table 3 Inh~itory activities of various lipidic haptens to the hemagglutination reaction between PET and anti-PET antiserum. Inhibitor

PCH PET PA a-naphthyl phosphate cholesterol phosphate 2-glycerophosphate glycerol diglyceride

Dilution of inhibitor (0.1 mol) 1:2

1:4

1:8

1:16

1:32

control

1 0 1 1 1 2 4 4

1 0 1 I 2 3 4 4

3 1 4 4 3 4 4 4

4 1 4 4 4 4 4 4

4 1 4 4 4 4 4 4

4a 4 4 4 4 4 4 4

alnhibitory activity was described as follows: 0 = complete inhibition, 1 = strong inhibition, 2 = medium inhibition, 3 = slight inhibition and 4 = no inhibition. From Table 3, it can be seen, that PET strongly inhibited the PET-anti PET antiserum system, and PCH and PA showed also inhibitory activities o f the same order. a-Naphthyl phosphate, cholesteryl phosphate and 2-glycerophosphate bearing a phosphoryl radical in c o m m o n structure slightly inhibited both systems.

IV. Discussion Inoue and Nojima [1] Kataoka and Nojima [2], Tamamura et al. [3], Schiefer [4], Radung [5], and Schiefer et al. [6] have obtained antisera against acidic phospholipids. Tamamura et al. [3] found the cross reactions among PA, cardiolipin and phosphatidyl serine. From these experiments, the antigenic determinant o f acidic phospholipids has been assumed to be the phosphatidyl moiety. We have found that PCH and PET reacted with antiserum against PA in spite of different reactivity, as shown in Table 1. Moreover, PA inhibited both PCH-anti PCH antiserum system and PET-anti PET antiserum system to the same degree, as shown in Tables 2 and 3. From these results, it can be concluded that one o f the determinants in PCH and PET might be the phosphatidyl moiety. Indeed, the phosphatidyl group showed stronger inhibitory activity than a phosphoryl radical in a-Naphthyl phosphate, cholesteryl phosphate and 2-glycerophosphate. This means that the acyl groups in the phosphatidyl group played some role in strengthening the antigenic activity o f the phosphatidyl moiety. The interaction o f PCH and PET with anti-CHS antiserum and anti-ETIO antiserum demonstrated clearly the difference o f the combining site o f steroid moiety with a

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L Hara et al., Immunochemlstry of phosphatidyl cholesterol

phosphoryl radical or the difference between a free OH group at C3 position and the side chain at C2o position of the steroid moiety. According to our experiments [ 10], CHS produces specific antibodies against the cholesterol moiety and the side chain of cholesterol, and ETI0 forms specific antibodies against the cholesterol moiety and the free OH group at its C3 position. The results of these experiments provided evidence that PCH and PET produce three kinds of antibodies, two of which correspond to the common structures of PCH and PET, that is, the phosphatidyl group and the cholesterol moiety. The third of antibody corresponds to that free OH group at C3 position, in the case of PET, and to the side chain of cholesterol, in the case of PCH.

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [ 15]

K. Inoue and S. Nojima, Biochem. Biophys. Acta 144 (1967) 409 T. Kataoka and S. Nojima, J. lmmunol. 105 (1970) 502 S. Tamamura, T. Hashimoto and L Hara, Jap. J. Expl. Med. 41 (I971) 31 H.G. Schiefer, Hoppe-Seyler's Z. Physiol. Chem. 354 (1973) 722,726 A. Radunz, Z. Naturforsch. 26 b (1971) 916 H. Schiefer, U. Gerhardt and H. Brunner, Hoppe-Seyler's Z. Physiol. Chem. 356 (1975) 559 K. Uemura and S.C. Kinsky, Biochemistry 11 (1972) 4085 A. Klopstock, M. Pinto and A. Rimon, J. Immunol. 92 (1964) 515 J.M. Bailey, B. Bright and R. Tomer, Nature 201 (1964) 407 J. Sato, T. Fukuda and I. Hara, Jap. J. Expl. Med. 46 (1976) 213 T. Muramatsu, I. Hara and M. Hayashi. Chem. Phys. Livids 20 (1977) 131 E.J. King, Biochem. J. 26 (1932) 292 O.H. Lowry, N.J. Rosenbrough, A.L. Farr and R.J. Randall, J. Biol. Chem. 193 (1951) 265 C.D. Cox and S.D. Vermilion, J. Lab. Clin. Med. 48 (1956) 298 W.J.Herbert, "Handbook of Experimental Immunology" (D.M. Weir, ed.) Blackwell Scientific Publication, Oxford, (1973) p. 20.11