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Immunological properties of a collagen-like synthetic polypeptide
314
BIOCHIMICA ET BIOPHYSI('A A('TA
I3BA 354O8 IMMUNOLOGICAL P R O P E R T I E S OF A C O L L A G E N - L I K E ~qYNTHETIC POLYPEPTIDE
F E L I X B O R E K * , J ( ) S E P H K U R T Z AND M I C H A E L SFA..k
Departments of Chemical hnmunologv and of Bzophystcs, The [l'ez.:mann lnstztutc ,~) Selene,, Rchovol (lsracl) (Received March 5th, rc~,q)
SUMMARY
A synthetic polypeptide (L-Pro Gly L-Pro)n with an ordered sequence, previously shown to have physical properties similar to those of collagen 2,v, was found to be immunogenic in guinea pigs and rabbits. Guinea pigs, immunized with (L-ProGly-L-Pro)n, produced antibodies which cross-reacted by passive cutaneous anaphylaxis with a random copolymer of similar composition (L-Pro66Glya4)n. However, no delayed-type skin cross-reactions between the two synthetic antigens were observed. Antibodies produced early in the course of inununization likewise failed to give cro.~sreactions. These results are interpreted as showing that the antigen nmst COlnplement the reactive moiety mediating delayed reactions or the early antibody to a higher degree than the late antibody. In a passive cutaneous anaphylaxis te~t cross-reaction~ were also observed between (L-Pro-Gly-L-Pro)n and fish, rat and guinea pig collagens. No cross-reaction~ between (L-Pro68Gly34)n and collagens or between gelatin and the ~ynthetic polypeptides were observed. The results suggest that the polymer of ordered sequence cross-reacts immunologically with collagen by virtue of the triple helix confi~rmation comnlon to both substances.
INTI/ODUCTION
The relationship between the chemical nature of antigens and their immunological properties has been a subject of numerous studies. An important contribution to the elucidation of this problem was made b y using synthetic polymers as antigens, reviewed recently by SELA1. However, due to the lack of suitable synthetic models, the role of protein conformation in immunogenicity and antigenic specificity could not be studied until quite recently, when the availability of polymers of tripeptide.,, which are synthetic polypeptides of ordered structure z,a, permitted an approach to * Present address" Department of Mmrobiology and hnmunology. Albert Einstein College of Medicine, Bronx, N Y., U.S A B~ockzm. Bu~phyq Acta, iS,~ (t()6O~ 314-323
COLLAGEN-LIKE SYNTHETIC POLYPEPTIDE
315
the study of this problem. Results of some of the experiments using this approach have been already published 4. An intriguing question, related to the above problem, has been the possibility of immunological cross-reactions between proteins of well-defined tertiary structure, such as collagen and synthetic polymers having similar composition and structure. .]ASIN AND GLYNN5 reported that a linear polymer of L-pr01ine, of average molecular weight 17 000 and having either Form I or Form II (right- or left-handed helix, respectively), induced immune response in guinea pigs. Subsequently they showed 6 that polyhydroxy-L-proline and its acetylated derivative (av. tool. wts. I i 000 and 16 000) were non-immunogenic in guinea pigs or rabbits, whereas a random linear copolymer of L-proline, glycine and hydroxy-t-proline ((L-Pro, Gly, L-Hyp)n) (av. mol. wt. above 4000), and its acetylated derivative ((L-Pro, Gly, L-AcPro)n) could be used to immunize guinea pigs though not rabbits. Guinea pigs immunized with (L-Pro, Gly, L-Hyp)n did not cross-react when skin-tested with calf collagen. Likewise, tanned erythrocytes coated with (L-Pro, Gly, L-Hyp)n failed to agglutinate when treated with anticollagen sera of guinea pigs or of rabbits. On the other hand, both types of cross-reactions were observed between (L-Pro, Gly, L-AcPro)n and acetylated calf collagen. The authors concluded that the structural differences between (L-Pro, Gly, L-Hyp)n and collagen were too great to permit cross-reactions between the two. The cross-reactions between (L-Pro, Gly, L-AcPro)n and acetylated collagen (in the absence of the cross-reactions with acetylated albumin) implicate the role of O-acetyl-hydroxyL-proline as a common antigenic determinant in these preparations. In view of the fact that a polymer of ordered sequence of the structure (L-ProGly-L-Pro) was synthesized in recent years 2 and was shown to have physical properties similar to those of collagen2, 7, it was of interest to find out whether this polymer is immunogenic and what would be the specificity of antibodies formed, particularly regarding their possible cross-reactions with collagen. Experiments aimed at these objectives are described in this paper. Some preliminary results have been published previously 4. MATERIALS AND METHODS
Antigens The preparation and physicochemical characterization of the polymer of ordered sequence (L-Pro-Gly-L-Pro)n was described by ENGEL et al. 2. In preliminary immunization experiments, two purified unfractionated preparations of the polymer of average molecular weights 4200 and 5500, respectively, were used. In subsequent experiments two fractions, eluted from Sephadex G-5o, of average molecular weights 7000 and 12 ooo, respectively, were used. The random copolymer of L-proline and glycine, (L-Pro66Gly34)n, was prepared from N-carboxy-L-proline anhydride s and N-carboxyglycine anhydride 9, using triethylamine as initiator and dioxane as solvent. Amino acid analysis showed the proline/glycine ratio to be 1.88 :I. Diffusion and sedimentation measurements gave an average molecular weight of 41oo. Fish collagen (ichthyocol) was obtained from Dr. S. Seifter (Albert Einstein College of Medicine, Bronx, N.Y.). Rat-skin collagen was a gift of Dr. G. Martin (National Institute of Dental Research, Bethesda, Md.). Acid-soluble calf-skin collagen was obtained from Dr. R. Timpl (Institut ftir ImmunoB~ochzm. B~ophys. Acta, i88 (1969) 314-323
316
F. B~)REK clct[.
logie der Universit~it Wien, Vienna). Guinea pig collagen was prepared by extracting ground guinea pig skin with I M NaC1 and then with o.5 M acetic acid, using the procedure of BORNSTEIN AND PIEZ1°. Only the acid-extracted traction was used in the studies described here. The amino acid composition of this preparation wa~ very similar to that found by GRASSMANNel al. 11 for calf-skin tropoeollagen. Mo~t collagen preparations were used at o.i°.i~ solutions in 0.5 M CaC12, buffered with o.o 5 M Trischloride (pH 7. I) a~ recornmended by GALLOPel al. v'. Some neutral collagen .-olutions were obtained by dialyzing o.i(!.() solutions in o.I M acetic acid againat o.5 M NaC1 with o.o2 M phosphate buffer, following the procedure devised by Dr. R Tnnpl (personal comnmnication). Gelatin (U.S.P. granular) was obtained t'v,ml Fisher Scientific Co., Pittsburgh, Pa. A ntisera Rabbit antiserum to acid-soluble calf-skin collagen was a gift of Dr. R. Timpl, (Institut fiir Immunologie der Universit~it Wien, Vienna). Adjuvants Complete Freund's adjuvant containing heat-killed mycobacteria, either Mvcobacterium tuberculoszs kominis or M~,cobacterbum bttlvrtcttm, w a s purchased from Difc~ Laboratories (Detroit). ,4 n imals The guinea pigs used were randomly bred albino and nonalbino male% bred at the Weizmann Institute, with an average initial weight of 2 kg. I m m u n i z a t i o n procedures Imnmnization of guinea pigs with tile synthetic polymers (Pro-Gly-Pro)n and (Pro66Gly34)n conaisted of injecting each animal with 0.2 ml of emulsion, containing equal volunles of a solution of o.5 nag or I nlg of the antigen in o.I5 M NaC1 and complete Freund's adjuvant containing M. tuberculosis, distributed between the hind fi)otpads. In one experiment two booster injection~ of I m g of antigen each in conlplete Freund's adjuvant containing M. butvricum were given on the I2th and ZlSt day after the primary inje'tion. The animals were tested by intradermal rejection ~f o.o 5 mg of antigen dissolved in o.I ml of o.15 M NaC1. Intradernlal injections of o.15 M NaC1 into immunized animals and injections of the test antigens into nonimmunized animals served as controls. Both immediate (2 h) and delayed (24 h) reactions were recorded. The skin testing started io days after immunization and was repeated at weekly intervals for 3-~) weeks. Some of the animals showing positive skin reactions, and later all the animals, were bled after each skin teat, and the sera were stored at - 2 o '~ until used. In some cases the sera, after preliminary teating, were pooled and fractionated by precipitation with halt-saturated (N H4).,SO4 solution. hnmunization of rabbits with the synthetic polymers was carried out by injecting each animal at multiple intradermal sites with 2.5 mg of the antigen, dissolved in o.5 ml of o.15 M NaCI and emulsified with I ml of complete Freund's adjuvant containing M . butvrwum. The animals (which had been pre-bled) were bled after 3 weeks and each was boosted a week later with 2.5 nag of antigen (injected in complete Freund's adjuvant at multiple intradermal sitea) and 3 m(mth* later with Bwcktm. l-lwpkys Acta, b~s (tO60) 314 ~-'3
COLLAGEN-LIKE SYNTHETIC POLYPEPTIDE
317
I0 mg of antigen (administered intramuscularly). The bleedings were repeated at 2-week intervals over the period of 6 months. The sera were stored and fractionated as described above. Procedures similar to those outlined above were followed in the immunizations of guinea pigs and rabbits with collagens, except that the antigens were dissolved in o.5 M CaC12, and the dose was 6oo/~g per guinea pig and I mg per rabbit. Complete Freund's adjuvant with M. butyricum was used. The course of immunization of guinea pigs was based on that described by STEFFEN13.
Passive cutaneous anaphylaxis Whole and fractionated sera were examined b y the passive cutaneous anaphylaxis reaction 14 with modifications previously described 15, using I m g of test antigen per animal. Whole and fractionated sera of nonimmunized guinea pigs and rabbits served as controls. The guinea pig sera and fractions were tested in an isologous system, whereas the rabbit sera were tested in a heterologous system, i.e. in guinea pigs. Precipitin tests Precipitin tests on whole and fractionated rabbit anti-(Pro-Gly-Pro)n sera were carried out according to the procedure described b y SELA et al. TM, using (ProGly-Pro)n fraction of average molecular weight 12 ooo as test antigen. RESULTS
Immunization of guinea pigs with preparations of the polymer of ordered sequence (Pro--Gly-Pro)n after IO days resulted in the development of immediate and delayed skin sensitivity and in the formation of circulating antibodies, detectable by passive cutaneous anaphylaxis, in about 6o% of animals, as shown in Table I. In recording these results, no distinction was made among the various preparations of the polymer, since all of them had similar immunological properties, although the polymer fraction of higher molecular weight (I2 ooo) was somewhat more immunogenic than the other three preparations. No cross-reactions with the random copolymer of a similar composition, (Pro66Gly34)n, were observed in either delayed sensitivity or antibody system at this early stage of imnmnization. However, anti-(Pro-Gly-Pro)n sera, obtained 6 days later, did cross-react with (ProeeGly3a)n when tested b y passive cutaneous anaphylaxis. After one month the ratio of animals responding to (ProGly-Pro)n increased to 8O~o. The skin reactions were characterized by erythema and oedema; they were free of haemorrhagic or necrotic lesions. Repeated immunizing injections of the antigen did not alter the intensity or character of the response. Two experiments which were continued for longer than one month showed that the delayed sensitivity to (Pro-Gly-Pro)n became weaker with time and could not be observed 5 ° days after immunization, whereas the immediate reactions were characterized at this stage mainly by oedema, and the formation of passive cutaneous anaphylaxis detectable antibodies persisted at approximately the same level of intensity. Four animals sensitized to (Pro-Gly-Pro)n, were cross-skin tested on the 43rd day with a multichain copolymer p o l y P r o - - p o l y L y s 17 and all gave positive immediate reactions, with an average diameter 5o% of that of the homologous reactions. Biochzm. Biophys. Mcta, 188 (1969) 314-323
318
~,. I~(>REK et al.
TABLE i IMMUNE RESPONSES TO (Pro-Gly-Pro)n AND (PronnGlya~)n IN GUINEA Pros n (1. stands for not deternlmed. Numbers in parentheses give the average reaction dlanaeter lU 111111 Immun~zang anlzgen
NumDt't" of days after l
I l l DI I f
Test antzge~l
.qkln rcactwns* I mmcdmtc (92 k)
Dcla'~,~.d (21 It)
a ~1a / ~ ] I v -
~I l -
7allokl
(Pro-Gly-Pro)n (Pro-Gly-Pro) n (Pro-Gly-Pro)n (Pro-Gly Pro)n (Pro-Gly-Pro)n (Pro-Gly-Pro)n (Pro-Gly-Pro)n {Pro-Gly-Pro)n (Pro-Gly-Pro) n (Pro66Glya4)n (Pro68Glya4) n (Pro66Glya~) n (Pro~6Glyaa)n (Pro66Glyaa) n
IO Io 16 16 23 35 35 43 5° to IS 18 25 25
.qntlbody rrspons~ ( pa ~s*vc ~utamot~ [a.YlS/~,,Slllt't sFl a )
(Pro-Gly-Pro)n (l'ro~6Glya4) n (Pro-Gly-Pro)n (Pro66Glya4) n (Pro Gty Projn (Pro Gly-Pro)n (Pro~6Glya~)n (Pro Gly Pro),, {Pro-Gly-Pro)n (Pro6SGlya~)n (Pro86Glya4)n (Pro-Gly-Pro) n (Pro~6(;lya4)n (Pro Gly- Pr,~ln
23/35"* n.d. 27/35 n.d. 25/35 8!IO n d. 8/io ~/,S 3/t5 9/15 3/5 7/9 3/4
('9) (IO) (2o1 (25)
23/35 (,t~) o/5"** 25/35 (23) n.d. 28/35 (lo)
| 1/2o -t/5 27/35
()1IO (I¢))
,N/IO
(2o) 118) (26) (23) (I~J) (20)
nd 5/Io o/S 7/15 to/I5 o/5 5/~)
(15)
0/4
~,/Io (','5"**
7/]o
(15) (9) (2o) 12 J)
,~/io ~,/,"; ~'/5 n/9 I,,',) ¢,/u I)l')
* Reactions with an average diameter of 5 tnln or less were conMdered negatl\ t' *" Ratio of responding to total number of animals. *** All the animals tested gave positive reactions with the homologous t it gen
T h e i m m u n e r e s p o n s e of g u i n e a pigs to t h e r a n d o m c o p o l y m e r , (Pro6"(;lya4)n, s h o w n also in T a b l e I, was f o u n d to d e v e l o p m o r e s l o w l y t h a n t h a t to tile p o l y m e r of o r d e r e d s e q u e n c e w i t h a b o u t 6 o % of t h e a n i m a l s r e s p o n d i n g w h e n c h a l l e n g e d on t h e I 8 t h d a y a f t e r i m n m n i z a t i o n . C r o s s - r e a c t i o n s w i t h tile p o l y m e r of o r d e r e d s e q u e n c e w e r e s h o w n b y b o t h d i r e c t a n d p a s s i v e c u t a n e o u s a n a p h y l a x l s tests. F o u r o u t of 6 r a b b i t s i m m u n i z e d w i t h (Pro G l y - P r o ) n p r o d u c e d a n t i b o d i e s d e t e c t a b l e b y p a s s i v e c u t a n e o u s a n a p h y l a x i s . All t h e sera were n e g a t i v e in h o m o logous p r e c i p i t i n tests. T h e r e s u l t s of i m m u n o l o g i c a l c r o ~ s - r e a c t i o n e x p e r i m e n t s b e t w e e n collagen p r e p a r a t i o n s f r o m t h r e e d i f f e r e n t a n i m a l species a n d g u i n e a pig a n d r a b b i t a n t i s e r a to ( P r o - G l y Pro)n are s h o w n in T a b l e II. I t can be seen t h a t all t h e t h r e e c o l l a g e n p r e p a r a t i o n s c r o s s - r e a c t e d to s o m e e x t e n t b y t h e p a s s i v e c u t a n e o u s a n a p h y l a x i s t e s t w i t h a n t i - ( P r o - G l y - P r o ) n a n t i b o d i e s . All t h e r e a c t i o n s r e c o r d e d as p o s i t i v e w e r e of r e l a t i v e l y w e a k i n t e n s i t y a n d n o t h o m o g e n e o u s in a p p e a r a n c e . N e v e r t h e l e s s , t h e y c o u l d be c l e a r l y d i s t i n g u i s h e d f r o m t h e n e g a t i v e r e a c t i o n s o b t a i n e d w i t h sera of n o n i m m u n i z e d a n i m a l s . I n g e n e r a l t h e sera g i v i n g s t r o n g h o m o l o g o u s r e a c t i o n s ( a v e r a g e d i a m e t e r o f 3o m m a n d a b o v e ) g a v e also c r o s s - r e a c t i o n s . R a b b i t a n t i s e r a g a v e b e t t e r c r o s s - r e a c t i o n s t h a n d i d g u i n e a p i g a n t i s e r a . T h e use of m o r e c o n c e n t r a t e d i m n m n o g l o b u l i n f r a c t i o n s r e s u l t e d in m o r e i n t e n s e p a s s i v e c u t a n e o u s a n a p h y l a x i ~ r e a c t i o n s . O n t h e o t h e r h a n d , n o n e o f t h e g u i n e a p i g anti-(Pro°6Glya4)n sera t e s t e d cr,~.~s-reacted Bwchlm. Bwphys
Acta, iSb (1909) 3r4 32]
COLLAGEN-LIKE SYNTHETIC POLYPEPTIDE TABLE
319
II
CROSS-REACTIONS BY PASSIVE CUTANEOUS ANAPHYLAXIS IN GUINEA PIGS BETWEEN COLLAGEN PREPARATIONS AND ANTIBODIES TO ( P r o - G l y - P r o ) n
Collagen
lchthyocol Rat skin Guinea pig skin
N u m b e r of crossreactwns " Guinea p,g sera
Rabbit sera
I2/25"*
-I/7 3/4
2/I2 5/2o
I/I
* Homologous anti-collagen sera were used as positive controls whereas served as negative controls in all the tests. ** R a t i o o f t h e n u m b e r o f c r o s s - r e a c t i o n s t o t h e t o t a l n u m b e r o f t e s t s .
normal
sera
with the collagen preparations. No cross-reactions between commercial gelatin and anti-(Pro-Gly-Pro)n or anti-(ProeeGly34)n sera were observed. Sera of five guinea pigs immunized with ichthyocol, obtained 4 weeks after the last immunizing injection, reacted well in the passive cutaneous anaphylaxis test with the homologous antigen. No cross-reaction with guinea pig collagen was observed. The globulin fraction of pooled antisera gave a positive passive cutaneous anaphylaxis cross-reaction with (Pro-Gly-Pro)n, similar in appearance to those described above. Similarly, rabbit anti-ichthyocol and anti-guinea pig skin-collagen sera cross-reacted consistently in the passive cutaneous anaphylaxis test with (ProGly-Pro)n. No passive cutaneous anaphylaxis cross-reactions were observed between rabbit anti-ichthyocol sera and (ProeeGly34)n. In another experiment, (Pro-Gly-Pro)n gave a passive cutaneous anaphylaxis cross-reaction with rabbit anti-calf skin collagen sera. The blueing reaction was noticed only about 3o rain after the challenge with antigen, but it could be clearly distinguished from a transient nonspecific reaction obtained with normal rabbit serum in the same animal. A weak cross-reaction with anti-calf skin-collagen serum was elicited also by (Pro66Gly34)n, but in this case the reaction was only slightly more intense than the control reaction with normal serum. No converse cross-reactions between calf collagen and anti-(Pro-Gly-Pro)n sera were observed. DISCUSSION
The foregoing results indicate that four preparations of the polymer of ordered sequence (L-Pro--Sly--L-Pro), within the average molecular weight range of 4ooo12 ooo, were immunogenic in guinea pigs and rabbits. The guinea pigs responded both by antibody formation and skin sensitivity; the delayed sensitivity was found to be less persistent than the immediate sensitivity and antibody response. This observation is in accord with the findings in experiments with other synthetic antigens containing proline such as (L-Pro, Gly, L-AcPro)n (ref. 6) and the multichain polymer polyPro--polyLys (ref. 17). This is in contrast to other known types of synthetic antigens which induce equally strong immediate and delayed reactions or, in some cases, an exclusively delayed-type response1,1S in guinea pigs. Btochim. Bzophys. Acta, 188 (1969) 3 1 4 - 3 2 3
320
1,. BOREK el a/.
D a t a in Table I show that 2/3 of tile guinea pigs inmmnized with the polymer of ordered sequence, (Pro-Gly-Pro)n, responded on Day IO after inununization with delayed reactions to skin tests with the iminunizing antigen. On the other hand, none of the 5 animals which had been also tested with the random cot)olymer, (Pro66Glya4)n, gave any delayed cross-reaction.~ with this material. %imilarly, anti(Pro-Gly-Pro)n sera obtained on Day IO gave strong passive cutaneous anaphylaxi, reactions with the immunizing antigen, but they did not cross-react with the random copolymer. However, sera obtained from the ~ame animals () or 25 dav~ later cr,s~reacted well with the random copotymer. Animals, inmmnized with the random copolymer, cro~s-reactcd with the polymer of ordered sequence bv immediate, lint not by delayed, ,~kin reactions. Since no antibodies could be detected by passive cutaneous anaphylaxms in the sera of the,e animals on Day IO, no comparison could be made between the specifimties of the early and the late antibodies. The absence of delayed cross-reacti~ms between (Pro Gly Pro)n and (Pr~ ~a"(;lyal)n may be interpreted as being analogous to the 'carrier bpecificity' shown m delayedtype reactions of hapten-protein 1'~-21 and hapten-synthetie polypeptidO 8 conjugates. The same conjugates show 'hapten specificity' in antibody-mediated reactions. In our case, however, it is not possible to interpret the results in tern> of carrier z,s. hapten specificity but rather in terms of a larger antigenic determinant or of a higher degree of complementarity required between the antigen and the reactive moiety mediating delayed-type reactions, as compared to the complementarity between the antigen and the circulating antibody. The change of antibody specificity in the cour.~e of antibody production was previously observed in the case of hapten-protein conjugated antigens bv BOREK ANI) ,~ILVERSTEIN 22 wh() found that early antibodies, obtained q I I day, after immunization, did not cross-react with conjugates containing an unrelated carrier protein or a different type of hapten-protein linkage. Antibodieb, ,,btained on later days, cross-reacted with the c,mjugates mentioned above, thus showing 'hapten specificity'. In the ca~e of the anti-(Pro Gly-Pro)n response, the early antibodies, having presumably lower energies of interaction with antigen than t h , , e formed later2a, 24, apparently require a higher degree of complementarity with the antigen and interact to() weakly or not at all with the random polymer which probably ha~ rather few repeating Pro-(ilv-l~ro- .~e(tuences in its chain. BROWN :\Nil ('~I.YNN'~5 reported recently that guinea pigs imnmnized with random copolymers of prohne and glycine or of proline, glycine and O-acetyl-hydroxyproline, gave good immediate but no delayed cro~s-reaction~ with linear poly-l_ prolines. However, both immediate and delayed reactions were elicited with the immunizing antigens. These results are again consistent with the view that the determinants responsible for delayed sensitivity are larger than tho.~e inw~lved in antibody-mediated reaction. Data in Table I I show that the three collagen preparation~ tested cro~s-reacted to some extent b y passive cutaneous anaphylaxib with guinea pig and rabbit anti(Pro Gly-Pro)n antibodies*. Additional data, mentioned above, show that gainea pig and rabbit anti-collagen sera were capable of cross-reacting with (Pro (ilv Pro)n. No comparable cros,~-reactions were observed between the random copolymer (Pro66Glya4)n and the anti-collagen sera or between collagen and antl-(Pro"6(}lyaa)n l¢~ochtm Bu,phv~ .Iota, tsS {r<)~O) 3Jl 323
COLLAGEN-LIKE SYNTHETIC POLYPEPTIDE
321
sera. There was also a lack of cross-reactions between commercial gelatin and anti(Pro-Gly-Pro)n or anti-(ProSeGly~)u sera. The above results, together with the previously mentioned data showing immunological cross-reactions between (Pro-Gly-Pro)n and (ProeeGly34)n, suggest that the passive cutaneous anaphylaxis cross-reactions between the polymer of ordered sequence (Pro-Gly-Pro)n and collagen took place primarily by virtue of the triple-stranded helix conformation, common to both substances in questionZ, 7. The role of the triple helix in the immunology of collagen has never been clearly established. SCHMITTet al. 26 studied the impairment of the antigenic activity of calf collagen, as measured by complement fixation in the presence of rabbit antisera, by the prior treatment of collagen with pepsin or pronase. The authors concluded that the main antigenic sites of collagen reside in the telopeptides, peptide chains protruding from the triple-helix body of the molecule, readily split off by proteases under the conditions which leave intact the triple-helix conformation. The complement fixation capacity of collagen, however, was lost also on thermal denaturation which is known to disrupt the triple helix without affecting the attachment of telopeptides to the rest of the molecule. Thus, the final conclusion derived from the data was that both the telopeptides and the triple helix contained sites recognized by heterologous antibody, the first of the two being mainly responsible for the antigenic specificity. TIMPL et al. 27, employing haemagglutination-inhibition tests with denatured collagen (parent gelatin), found on calf, sheep, rat, guinea pig and rabbit collagen preparations the presence of both pepsin-labile and pepsin-stable antigenic determinants. Some of the latter kind were common to all the species studied, whereas others were restricted to two or three species. These determinants are presumably located on the chains of the triple helix ; after the disruption of the helical conformation they do not function in inducing species-specific antibodies 13 although they retain the capacity to react with the antibodies in the haemegglutination-inhibition assay. MICHAELI et al. ~8 reported recently that not only the native guinea pig collagen, but also its denatured form, gelatin, can show precipitin reactions with the rabbit antibodies. Thus, it seems that the relative importance of the triple-helix structure for the immunological functions of collagen depends on the type of assay used. The only other instance of an immunological cross-reaction between a synthetic peptide and unmodified collagen, reported thus far in literature, is that found by I~ETTMAN et al. 29 in which an octapeptide G l y - P r o - G l y - P r o - P r o - G l y - A l a - L y s showed specific precipitin reactions with rabbit antiserum to guinea-pig-skin collagen. The authors interpreted this phenomenon as resulting either from the aggregation of the peptide or from its adsorption to serum proteins with the formation of multivalent antigen. Specific binding of the 14C-labeled peptide to anti-collagen sera was also demonstrated. In an earlier search for the sites on gelatin molecule responsible for the antigenic specificity of the latter, SELA AND ARNON30 attempted to inhibit the specific gelatin anti-gelatin precipitin reaction by means of various random linear polymers and copolymers containing proline or hydroxyproline or both. Since none of the substances " I t is possible t h a t in those guinea pigs used for passive c u t a n e o u s a n a p h y l a x i s tests in which no cross-reactions were observed, the cross-reacting antibodies h a d been completely neutralized b y the native skin collagen before the test antigen was administered. However, no evidence is available in s u p p o r t of this possibihty.
Bioch,m. B,ophys. Acta, 188 (I969) 314-323
322
i, t3~)I¢E1,~ ~'l ell,
tested had any effect on tile homologous precipitin reaction, it was concluded that the antigenic sites on gelatin have chemical structures and conformations nmch more intricate than those present in the polymers used. Absence of precipitin cross-reaction~ was found by F u c g s AND SELAa1,32 between rabbit antisera to linear and multichain copolymers of tyrosine, glutamic acid, alanine and lysine--and ovalbumin, ede~tin or gelatin and between antisera to multichain copolymers of histidine, phenylalanine, leucine, methionine, glutanfic acid, alanine and lysine--and ovalbunun, bovine serum albumin and ribonuclease. K U N Z AND GILLaa reported that antisera to t)ara myosin failed to cross-react with various copolymers of glutamic acid, lysine, tvrosine and alanine. Neither did paramyosin cross-react with the anti-polypeptide sera MAVRERa4 recorded a lack of anaphylactic cross-reactions between a Glu I.vs copolymer and proteins; however, he found a passive cutaneous anaphylaxia crossreaction between an anti-Ala-Gly copolymer serum and gliadin aa. Rabbit anti-bovine y-globulin serum was shown by (~ILL AND MATTHEWSa6 not to cross-react with several copolymers of glut)talc acid, lyqne, phenylalanine and tyrosine, wherea~ high titer antisera to synthetic polypeptides crosa-reacted to a very slight extent with a wide variety of proteins. On the other hand, ~ A N D B E R G AND STOI.LARav dennmstrated exten.,ive cro.,sreactions bv the complement-fixation test between rabbit anti-poly-L-lysine sera and basic proteins such as histone and ribonuclease. WALSH et al. as reported that immunization of rabbits with (;ly-Lys--Ala or Glu Lys-Ala fyr copolymers induced the formation of helnolytic plaque-forming lymph node cells which croas-reacted with normal sheep erythrocytes. Antisera against some .~vnthetic copolymer~ containing D-alanine were reported recently to cross-react with a bacterial muc-peptidea'E In general, the results of the studies of inmmnological cross-reactions between synthetic polypeptides and proteins reflect not only the possible structural similarities between the two kinds of macromolecular substances but also the ~pecial conditions and sensitivity of the assay as well as the affinity for antigen of the antibodie~ under investigation In conclusion, the experilnents described in thia paper show a relatumship between the conformatl.n of the polymer of ordered sequence, (L-Pro (ily L-Pro)n, and its inmmnological propertie~. The immunological cross-reaction between the polymer and collagens of several species are most probably due to the conformational resemblance between the triple-stranded helix of collagen and that of the synthetic polypeptide. The weak, but positive, cross-reactions of guinea pig collagen with guinea pig antihodies against the polymer of ordered structure shows that in an animal it is possible to provoke formation of antibodies cross-reacting with a protein of the same animal species, by means of inmmnization with a synthetic antigen. ACKNOXVLE DGMENTS
This research has been sponsored in part by the Air Force Office of Scmntitic Research through the European Office of Aerospace Research (O.A.R.), U.S. Air Force under Grants EOAR-67-I 9 and EOOAR-6S-oo21. The excellent technical assistance of Miss N. Novik i~ gratefully acknowledged.
t?iochzm. Bzophvs .4cta, ISS (1969) 314 323
COLLAGEN-LIKE SYNTHETIC POLYPEPTIDE
323
REFERENCES I 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3° 31 32 33 34 35 36 37 38 39
]~{. SELA, Advan. Immunol., 5 (1966) 29. J. ENGEL, J. KURTZ, E. KATCHALSKI AND A. BERGER, J. Mol. B~ol., 17 (1966) 255. J. RAMACHANDRAN, Abstr. 7th Intern. Congr. Biochem. Japan, (1967) 982. M. SELA, B. SCHECHTER, I. SCHECHTER AND F. BOREK, Cold Sprzng Harbor Symp. Quant, Bzol., 32 (1967) 537. H. E. JASlN AND L. E. GLYNN, Immunology, 8 (1965) 95. H. E. JASIN AND L. E. GLYNN, Immunology, 8 (1965) 260. W. TRAUB AND A. YONATH, J. Mol. Btol., 16 (1966) 4o4 . A. BERGER, J. KURTZ AND E. KATCHALSKI, J. Am. Chem. Soc., 76 (1954) 5552. R. R. BECKER AND M. A. STAHMANN, J. Bzol. Chem., 204 (1953) 737P. BORNSTEIN AND 1{. A. PIEZ, J. Clin. Invest., 43 (1964) 1813. W. GRASSMAN, K. HANNIG AND M. SCHLEYER, Z. Physiol. Chem., 322 (196o) 71. P. M. GALLOP, S. SEIFTER AND E. MEILMAN, J. Blophys. Biochem. Cytol., 3 (1967) 345. C. STEFFEN, Ann. N . Y . Acad. Scz., 124 (1965) 57 o. Z. OVARY, Progr. Allergy, 5 (1958) 459. S. BEN-EFRAIM, S. FUCHS AND M. SELA, Immunology, 7 (1964) 142M. SELA, S. FUCHS AND R. ARNON, B,ochem. J., 85 (1962) 223. J.-C. JATON AND M. SELA, J. Biol. Chem., 243 (1968) 5616. F. BOREK, Current Topzcs ~4icrobiol. Immunol., 43 (1968) 126. n . BENACERRAF AND P. G. H. DELL, Immunology, 2 (1959) 53. P. G H. DELL AND A. M. SILVERSTEIN, J. Exptl. Med., I15 (1962) lO37. B. BENACERRAF AND B. B. LEVINE, J. Exptl. Med., 115 (1962). F. BOREK AND A. M. SILVERSTEIN, Nature, 205 (1965) 299. H. N. EISEN AND G. W. SISKIND, Bzochemistry, 3 (1964) 996. H. F. F u j I o AND F. KAROSH, Bzochem~stry, 5 (1966) 1856. P. C. BROWN AND L, E. GLYNN, Immunology, 15 (1968) 589. F. O. SCHMITT, L. LEVINE, M. P. DRAKE, A. L. RUBIN, D. PFAHL AND P. F. DAVISON, Proc. Natl. Acad. Sc,. U.S., 51 (1964) 493. R. TIMPL, I. WOLFF, G. WICK, H. FURTHMAYR AND C. STEFFEN, J. Immunol., IOI (1968) 725 . D. MICHAELI, H. KAMENECKA, E. BENJAMINI, J. R. KETTMAN, JR., D . Y . K . LEUNG AND R. C. MINER, Immunochem~stry, 5 (1968) 433J. R. KETTMAN, JR., E. BENJAMINI, D. MICHAELI AND D. Y. K. LEUNG, B*ochem. Biophys. Res Commun., 29 (1967) 623. M. SELA AND R. ARNON, Biochem. J., 77 (196o) 394. S. FUCHS AND M. SELA, B*ochem. J., 87 (1963) 7 o. S. FUCHS ANn M. SELA, Biochem. J., 93 (1964) 566. H. W. KUNZ AND T. J. GILL, I I I , Broch*m. B*ophys Aeta, 90 (1964) 318. P. H. MAURER, [. Immunol., 90 (1963) 493. P. H. MAURER, n. F. GERULAT AND P. PINCHUCK, J. Biol. Chem., 239 (1964) 922. T. J. GILL AND L. S. MATTHEWS, J. Bzol. Chem., 238 (1963) 1373. A. L. SANDBERG AND B. D. STOLLAR, J. Immunol., IOO (1968) 286. P. WALSH, P. H. ~AURER AND M. EGAN, ]. Immunol., i o o (1968) z66. W W KARAKAWA, P. H. MAURER, ~). WALStt AND R. M. KRAUSE, Federatzon Pyoc., 28 (1969) 633.
Bzochim. Bzophys. Acta, I88 (I969) 314-323
BIOCHIMICA ET BIOPHYSI('A A('TA
I3BA 354O8 IMMUNOLOGICAL P R O P E R T I E S OF A C O L L A G E N - L I K E ~qYNTHETIC POLYPEPTIDE
F E L I X B O R E K * , J ( ) S E P H K U R T Z AND M I C H A E L SFA..k
Departments of Chemical hnmunologv and of Bzophystcs, The [l'ez.:mann lnstztutc ,~) Selene,, Rchovol (lsracl) (Received March 5th, rc~,q)
SUMMARY
A synthetic polypeptide (L-Pro Gly L-Pro)n with an ordered sequence, previously shown to have physical properties similar to those of collagen 2,v, was found to be immunogenic in guinea pigs and rabbits. Guinea pigs, immunized with (L-ProGly-L-Pro)n, produced antibodies which cross-reacted by passive cutaneous anaphylaxis with a random copolymer of similar composition (L-Pro66Glya4)n. However, no delayed-type skin cross-reactions between the two synthetic antigens were observed. Antibodies produced early in the course of inununization likewise failed to give cro.~sreactions. These results are interpreted as showing that the antigen nmst COlnplement the reactive moiety mediating delayed reactions or the early antibody to a higher degree than the late antibody. In a passive cutaneous anaphylaxis te~t cross-reaction~ were also observed between (L-Pro-Gly-L-Pro)n and fish, rat and guinea pig collagens. No cross-reaction~ between (L-Pro68Gly34)n and collagens or between gelatin and the ~ynthetic polypeptides were observed. The results suggest that the polymer of ordered sequence cross-reacts immunologically with collagen by virtue of the triple helix confi~rmation comnlon to both substances.
INTI/ODUCTION
The relationship between the chemical nature of antigens and their immunological properties has been a subject of numerous studies. An important contribution to the elucidation of this problem was made b y using synthetic polymers as antigens, reviewed recently by SELA1. However, due to the lack of suitable synthetic models, the role of protein conformation in immunogenicity and antigenic specificity could not be studied until quite recently, when the availability of polymers of tripeptide.,, which are synthetic polypeptides of ordered structure z,a, permitted an approach to * Present address" Department of Mmrobiology and hnmunology. Albert Einstein College of Medicine, Bronx, N Y., U.S A B~ockzm. Bu~phyq Acta, iS,~ (t()6O~ 314-323
COLLAGEN-LIKE SYNTHETIC POLYPEPTIDE
315
the study of this problem. Results of some of the experiments using this approach have been already published 4. An intriguing question, related to the above problem, has been the possibility of immunological cross-reactions between proteins of well-defined tertiary structure, such as collagen and synthetic polymers having similar composition and structure. .]ASIN AND GLYNN5 reported that a linear polymer of L-pr01ine, of average molecular weight 17 000 and having either Form I or Form II (right- or left-handed helix, respectively), induced immune response in guinea pigs. Subsequently they showed 6 that polyhydroxy-L-proline and its acetylated derivative (av. tool. wts. I i 000 and 16 000) were non-immunogenic in guinea pigs or rabbits, whereas a random linear copolymer of L-proline, glycine and hydroxy-t-proline ((L-Pro, Gly, L-Hyp)n) (av. mol. wt. above 4000), and its acetylated derivative ((L-Pro, Gly, L-AcPro)n) could be used to immunize guinea pigs though not rabbits. Guinea pigs immunized with (L-Pro, Gly, L-Hyp)n did not cross-react when skin-tested with calf collagen. Likewise, tanned erythrocytes coated with (L-Pro, Gly, L-Hyp)n failed to agglutinate when treated with anticollagen sera of guinea pigs or of rabbits. On the other hand, both types of cross-reactions were observed between (L-Pro, Gly, L-AcPro)n and acetylated calf collagen. The authors concluded that the structural differences between (L-Pro, Gly, L-Hyp)n and collagen were too great to permit cross-reactions between the two. The cross-reactions between (L-Pro, Gly, L-AcPro)n and acetylated collagen (in the absence of the cross-reactions with acetylated albumin) implicate the role of O-acetyl-hydroxyL-proline as a common antigenic determinant in these preparations. In view of the fact that a polymer of ordered sequence of the structure (L-ProGly-L-Pro) was synthesized in recent years 2 and was shown to have physical properties similar to those of collagen2, 7, it was of interest to find out whether this polymer is immunogenic and what would be the specificity of antibodies formed, particularly regarding their possible cross-reactions with collagen. Experiments aimed at these objectives are described in this paper. Some preliminary results have been published previously 4. MATERIALS AND METHODS
Antigens The preparation and physicochemical characterization of the polymer of ordered sequence (L-Pro-Gly-L-Pro)n was described by ENGEL et al. 2. In preliminary immunization experiments, two purified unfractionated preparations of the polymer of average molecular weights 4200 and 5500, respectively, were used. In subsequent experiments two fractions, eluted from Sephadex G-5o, of average molecular weights 7000 and 12 ooo, respectively, were used. The random copolymer of L-proline and glycine, (L-Pro66Gly34)n, was prepared from N-carboxy-L-proline anhydride s and N-carboxyglycine anhydride 9, using triethylamine as initiator and dioxane as solvent. Amino acid analysis showed the proline/glycine ratio to be 1.88 :I. Diffusion and sedimentation measurements gave an average molecular weight of 41oo. Fish collagen (ichthyocol) was obtained from Dr. S. Seifter (Albert Einstein College of Medicine, Bronx, N.Y.). Rat-skin collagen was a gift of Dr. G. Martin (National Institute of Dental Research, Bethesda, Md.). Acid-soluble calf-skin collagen was obtained from Dr. R. Timpl (Institut ftir ImmunoB~ochzm. B~ophys. Acta, i88 (1969) 314-323
316
F. B~)REK clct[.
logie der Universit~it Wien, Vienna). Guinea pig collagen was prepared by extracting ground guinea pig skin with I M NaC1 and then with o.5 M acetic acid, using the procedure of BORNSTEIN AND PIEZ1°. Only the acid-extracted traction was used in the studies described here. The amino acid composition of this preparation wa~ very similar to that found by GRASSMANNel al. 11 for calf-skin tropoeollagen. Mo~t collagen preparations were used at o.i°.i~ solutions in 0.5 M CaC12, buffered with o.o 5 M Trischloride (pH 7. I) a~ recornmended by GALLOPel al. v'. Some neutral collagen .-olutions were obtained by dialyzing o.i(!.() solutions in o.I M acetic acid againat o.5 M NaC1 with o.o2 M phosphate buffer, following the procedure devised by Dr. R Tnnpl (personal comnmnication). Gelatin (U.S.P. granular) was obtained t'v,ml Fisher Scientific Co., Pittsburgh, Pa. A ntisera Rabbit antiserum to acid-soluble calf-skin collagen was a gift of Dr. R. Timpl, (Institut fiir Immunologie der Universit~it Wien, Vienna). Adjuvants Complete Freund's adjuvant containing heat-killed mycobacteria, either Mvcobacterium tuberculoszs kominis or M~,cobacterbum bttlvrtcttm, w a s purchased from Difc~ Laboratories (Detroit). ,4 n imals The guinea pigs used were randomly bred albino and nonalbino male% bred at the Weizmann Institute, with an average initial weight of 2 kg. I m m u n i z a t i o n procedures Imnmnization of guinea pigs with tile synthetic polymers (Pro-Gly-Pro)n and (Pro66Gly34)n conaisted of injecting each animal with 0.2 ml of emulsion, containing equal volunles of a solution of o.5 nag or I nlg of the antigen in o.I5 M NaC1 and complete Freund's adjuvant containing M. tuberculosis, distributed between the hind fi)otpads. In one experiment two booster injection~ of I m g of antigen each in conlplete Freund's adjuvant containing M. butvricum were given on the I2th and ZlSt day after the primary inje'tion. The animals were tested by intradermal rejection ~f o.o 5 mg of antigen dissolved in o.I ml of o.15 M NaC1. Intradernlal injections of o.15 M NaC1 into immunized animals and injections of the test antigens into nonimmunized animals served as controls. Both immediate (2 h) and delayed (24 h) reactions were recorded. The skin testing started io days after immunization and was repeated at weekly intervals for 3-~) weeks. Some of the animals showing positive skin reactions, and later all the animals, were bled after each skin teat, and the sera were stored at - 2 o '~ until used. In some cases the sera, after preliminary teating, were pooled and fractionated by precipitation with halt-saturated (N H4).,SO4 solution. hnmunization of rabbits with the synthetic polymers was carried out by injecting each animal at multiple intradermal sites with 2.5 mg of the antigen, dissolved in o.5 ml of o.15 M NaCI and emulsified with I ml of complete Freund's adjuvant containing M . butvrwum. The animals (which had been pre-bled) were bled after 3 weeks and each was boosted a week later with 2.5 nag of antigen (injected in complete Freund's adjuvant at multiple intradermal sitea) and 3 m(mth* later with Bwcktm. l-lwpkys Acta, b~s (tO60) 314 ~-'3
COLLAGEN-LIKE SYNTHETIC POLYPEPTIDE
317
I0 mg of antigen (administered intramuscularly). The bleedings were repeated at 2-week intervals over the period of 6 months. The sera were stored and fractionated as described above. Procedures similar to those outlined above were followed in the immunizations of guinea pigs and rabbits with collagens, except that the antigens were dissolved in o.5 M CaC12, and the dose was 6oo/~g per guinea pig and I mg per rabbit. Complete Freund's adjuvant with M. butyricum was used. The course of immunization of guinea pigs was based on that described by STEFFEN13.
Passive cutaneous anaphylaxis Whole and fractionated sera were examined b y the passive cutaneous anaphylaxis reaction 14 with modifications previously described 15, using I m g of test antigen per animal. Whole and fractionated sera of nonimmunized guinea pigs and rabbits served as controls. The guinea pig sera and fractions were tested in an isologous system, whereas the rabbit sera were tested in a heterologous system, i.e. in guinea pigs. Precipitin tests Precipitin tests on whole and fractionated rabbit anti-(Pro-Gly-Pro)n sera were carried out according to the procedure described b y SELA et al. TM, using (ProGly-Pro)n fraction of average molecular weight 12 ooo as test antigen. RESULTS
Immunization of guinea pigs with preparations of the polymer of ordered sequence (Pro--Gly-Pro)n after IO days resulted in the development of immediate and delayed skin sensitivity and in the formation of circulating antibodies, detectable by passive cutaneous anaphylaxis, in about 6o% of animals, as shown in Table I. In recording these results, no distinction was made among the various preparations of the polymer, since all of them had similar immunological properties, although the polymer fraction of higher molecular weight (I2 ooo) was somewhat more immunogenic than the other three preparations. No cross-reactions with the random copolymer of a similar composition, (Pro66Gly34)n, were observed in either delayed sensitivity or antibody system at this early stage of imnmnization. However, anti-(Pro-Gly-Pro)n sera, obtained 6 days later, did cross-react with (ProeeGly3a)n when tested b y passive cutaneous anaphylaxis. After one month the ratio of animals responding to (ProGly-Pro)n increased to 8O~o. The skin reactions were characterized by erythema and oedema; they were free of haemorrhagic or necrotic lesions. Repeated immunizing injections of the antigen did not alter the intensity or character of the response. Two experiments which were continued for longer than one month showed that the delayed sensitivity to (Pro-Gly-Pro)n became weaker with time and could not be observed 5 ° days after immunization, whereas the immediate reactions were characterized at this stage mainly by oedema, and the formation of passive cutaneous anaphylaxis detectable antibodies persisted at approximately the same level of intensity. Four animals sensitized to (Pro-Gly-Pro)n, were cross-skin tested on the 43rd day with a multichain copolymer p o l y P r o - - p o l y L y s 17 and all gave positive immediate reactions, with an average diameter 5o% of that of the homologous reactions. Biochzm. Biophys. Mcta, 188 (1969) 314-323
318
~,. I~(>REK et al.
TABLE i IMMUNE RESPONSES TO (Pro-Gly-Pro)n AND (PronnGlya~)n IN GUINEA Pros n (1. stands for not deternlmed. Numbers in parentheses give the average reaction dlanaeter lU 111111 Immun~zang anlzgen
NumDt't" of days after l
I l l DI I f
Test antzge~l
.qkln rcactwns* I mmcdmtc (92 k)
Dcla'~,~.d (21 It)
a ~1a / ~ ] I v -
~I l -
7allokl
(Pro-Gly-Pro)n (Pro-Gly-Pro) n (Pro-Gly-Pro)n (Pro-Gly Pro)n (Pro-Gly-Pro)n (Pro-Gly-Pro)n (Pro-Gly-Pro)n {Pro-Gly-Pro)n (Pro-Gly-Pro) n (Pro66Glya4)n (Pro68Glya4) n (Pro66Glya~) n (Pro~6Glyaa)n (Pro66Glyaa) n
IO Io 16 16 23 35 35 43 5° to IS 18 25 25
.qntlbody rrspons~ ( pa ~s*vc ~utamot~ [a.YlS/~,,Slllt't sFl a )
(Pro-Gly-Pro)n (l'ro~6Glya4) n (Pro-Gly-Pro)n (Pro66Glya4) n (Pro Gty Projn (Pro Gly-Pro)n (Pro~6Glya~)n (Pro Gly Pro),, {Pro-Gly-Pro)n (Pro6SGlya~)n (Pro86Glya4)n (Pro-Gly-Pro) n (Pro~6(;lya4)n (Pro Gly- Pr,~ln
23/35"* n.d. 27/35 n.d. 25/35 8!IO n d. 8/io ~/,S 3/t5 9/15 3/5 7/9 3/4
('9) (IO) (2o1 (25)
23/35 (,t~) o/5"** 25/35 (23) n.d. 28/35 (lo)
| 1/2o -t/5 27/35
()1IO (I¢))
,N/IO
(2o) 118) (26) (23) (I~J) (20)
nd 5/Io o/S 7/15 to/I5 o/5 5/~)
(15)
0/4
~,/Io (','5"**
7/]o
(15) (9) (2o) 12 J)
,~/io ~,/,"; ~'/5 n/9 I,,',) ¢,/u I)l')
* Reactions with an average diameter of 5 tnln or less were conMdered negatl\ t' *" Ratio of responding to total number of animals. *** All the animals tested gave positive reactions with the homologous t it gen
T h e i m m u n e r e s p o n s e of g u i n e a pigs to t h e r a n d o m c o p o l y m e r , (Pro6"(;lya4)n, s h o w n also in T a b l e I, was f o u n d to d e v e l o p m o r e s l o w l y t h a n t h a t to tile p o l y m e r of o r d e r e d s e q u e n c e w i t h a b o u t 6 o % of t h e a n i m a l s r e s p o n d i n g w h e n c h a l l e n g e d on t h e I 8 t h d a y a f t e r i m n m n i z a t i o n . C r o s s - r e a c t i o n s w i t h tile p o l y m e r of o r d e r e d s e q u e n c e w e r e s h o w n b y b o t h d i r e c t a n d p a s s i v e c u t a n e o u s a n a p h y l a x l s tests. F o u r o u t of 6 r a b b i t s i m m u n i z e d w i t h (Pro G l y - P r o ) n p r o d u c e d a n t i b o d i e s d e t e c t a b l e b y p a s s i v e c u t a n e o u s a n a p h y l a x i s . All t h e sera were n e g a t i v e in h o m o logous p r e c i p i t i n tests. T h e r e s u l t s of i m m u n o l o g i c a l c r o ~ s - r e a c t i o n e x p e r i m e n t s b e t w e e n collagen p r e p a r a t i o n s f r o m t h r e e d i f f e r e n t a n i m a l species a n d g u i n e a pig a n d r a b b i t a n t i s e r a to ( P r o - G l y Pro)n are s h o w n in T a b l e II. I t can be seen t h a t all t h e t h r e e c o l l a g e n p r e p a r a t i o n s c r o s s - r e a c t e d to s o m e e x t e n t b y t h e p a s s i v e c u t a n e o u s a n a p h y l a x i s t e s t w i t h a n t i - ( P r o - G l y - P r o ) n a n t i b o d i e s . All t h e r e a c t i o n s r e c o r d e d as p o s i t i v e w e r e of r e l a t i v e l y w e a k i n t e n s i t y a n d n o t h o m o g e n e o u s in a p p e a r a n c e . N e v e r t h e l e s s , t h e y c o u l d be c l e a r l y d i s t i n g u i s h e d f r o m t h e n e g a t i v e r e a c t i o n s o b t a i n e d w i t h sera of n o n i m m u n i z e d a n i m a l s . I n g e n e r a l t h e sera g i v i n g s t r o n g h o m o l o g o u s r e a c t i o n s ( a v e r a g e d i a m e t e r o f 3o m m a n d a b o v e ) g a v e also c r o s s - r e a c t i o n s . R a b b i t a n t i s e r a g a v e b e t t e r c r o s s - r e a c t i o n s t h a n d i d g u i n e a p i g a n t i s e r a . T h e use of m o r e c o n c e n t r a t e d i m n m n o g l o b u l i n f r a c t i o n s r e s u l t e d in m o r e i n t e n s e p a s s i v e c u t a n e o u s a n a p h y l a x i ~ r e a c t i o n s . O n t h e o t h e r h a n d , n o n e o f t h e g u i n e a p i g anti-(Pro°6Glya4)n sera t e s t e d cr,~.~s-reacted Bwchlm. Bwphys
Acta, iSb (1909) 3r4 32]
COLLAGEN-LIKE SYNTHETIC POLYPEPTIDE TABLE
319
II
CROSS-REACTIONS BY PASSIVE CUTANEOUS ANAPHYLAXIS IN GUINEA PIGS BETWEEN COLLAGEN PREPARATIONS AND ANTIBODIES TO ( P r o - G l y - P r o ) n
Collagen
lchthyocol Rat skin Guinea pig skin
N u m b e r of crossreactwns " Guinea p,g sera
Rabbit sera
I2/25"*
-I/7 3/4
2/I2 5/2o
I/I
* Homologous anti-collagen sera were used as positive controls whereas served as negative controls in all the tests. ** R a t i o o f t h e n u m b e r o f c r o s s - r e a c t i o n s t o t h e t o t a l n u m b e r o f t e s t s .
normal
sera
with the collagen preparations. No cross-reactions between commercial gelatin and anti-(Pro-Gly-Pro)n or anti-(ProeeGly34)n sera were observed. Sera of five guinea pigs immunized with ichthyocol, obtained 4 weeks after the last immunizing injection, reacted well in the passive cutaneous anaphylaxis test with the homologous antigen. No cross-reaction with guinea pig collagen was observed. The globulin fraction of pooled antisera gave a positive passive cutaneous anaphylaxis cross-reaction with (Pro-Gly-Pro)n, similar in appearance to those described above. Similarly, rabbit anti-ichthyocol and anti-guinea pig skin-collagen sera cross-reacted consistently in the passive cutaneous anaphylaxis test with (ProGly-Pro)n. No passive cutaneous anaphylaxis cross-reactions were observed between rabbit anti-ichthyocol sera and (ProeeGly34)n. In another experiment, (Pro-Gly-Pro)n gave a passive cutaneous anaphylaxis cross-reaction with rabbit anti-calf skin collagen sera. The blueing reaction was noticed only about 3o rain after the challenge with antigen, but it could be clearly distinguished from a transient nonspecific reaction obtained with normal rabbit serum in the same animal. A weak cross-reaction with anti-calf skin-collagen serum was elicited also by (Pro66Gly34)n, but in this case the reaction was only slightly more intense than the control reaction with normal serum. No converse cross-reactions between calf collagen and anti-(Pro-Gly-Pro)n sera were observed. DISCUSSION
The foregoing results indicate that four preparations of the polymer of ordered sequence (L-Pro--Sly--L-Pro), within the average molecular weight range of 4ooo12 ooo, were immunogenic in guinea pigs and rabbits. The guinea pigs responded both by antibody formation and skin sensitivity; the delayed sensitivity was found to be less persistent than the immediate sensitivity and antibody response. This observation is in accord with the findings in experiments with other synthetic antigens containing proline such as (L-Pro, Gly, L-AcPro)n (ref. 6) and the multichain polymer polyPro--polyLys (ref. 17). This is in contrast to other known types of synthetic antigens which induce equally strong immediate and delayed reactions or, in some cases, an exclusively delayed-type response1,1S in guinea pigs. Btochim. Bzophys. Acta, 188 (1969) 3 1 4 - 3 2 3
320
1,. BOREK el a/.
D a t a in Table I show that 2/3 of tile guinea pigs inmmnized with the polymer of ordered sequence, (Pro-Gly-Pro)n, responded on Day IO after inununization with delayed reactions to skin tests with the iminunizing antigen. On the other hand, none of the 5 animals which had been also tested with the random cot)olymer, (Pro66Glya4)n, gave any delayed cross-reaction.~ with this material. %imilarly, anti(Pro-Gly-Pro)n sera obtained on Day IO gave strong passive cutaneous anaphylaxi, reactions with the immunizing antigen, but they did not cross-react with the random copolymer. However, sera obtained from the ~ame animals () or 25 dav~ later cr,s~reacted well with the random copotymer. Animals, inmmnized with the random copolymer, cro~s-reactcd with the polymer of ordered sequence bv immediate, lint not by delayed, ,~kin reactions. Since no antibodies could be detected by passive cutaneous anaphylaxms in the sera of the,e animals on Day IO, no comparison could be made between the specifimties of the early and the late antibodies. The absence of delayed cross-reacti~ms between (Pro Gly Pro)n and (Pr~ ~a"(;lyal)n may be interpreted as being analogous to the 'carrier bpecificity' shown m delayedtype reactions of hapten-protein 1'~-21 and hapten-synthetie polypeptidO 8 conjugates. The same conjugates show 'hapten specificity' in antibody-mediated reactions. In our case, however, it is not possible to interpret the results in tern> of carrier z,s. hapten specificity but rather in terms of a larger antigenic determinant or of a higher degree of complementarity required between the antigen and the reactive moiety mediating delayed-type reactions, as compared to the complementarity between the antigen and the circulating antibody. The change of antibody specificity in the cour.~e of antibody production was previously observed in the case of hapten-protein conjugated antigens bv BOREK ANI) ,~ILVERSTEIN 22 wh() found that early antibodies, obtained q I I day, after immunization, did not cross-react with conjugates containing an unrelated carrier protein or a different type of hapten-protein linkage. Antibodieb, ,,btained on later days, cross-reacted with the c,mjugates mentioned above, thus showing 'hapten specificity'. In the ca~e of the anti-(Pro Gly-Pro)n response, the early antibodies, having presumably lower energies of interaction with antigen than t h , , e formed later2a, 24, apparently require a higher degree of complementarity with the antigen and interact to() weakly or not at all with the random polymer which probably ha~ rather few repeating Pro-(ilv-l~ro- .~e(tuences in its chain. BROWN :\Nil ('~I.YNN'~5 reported recently that guinea pigs imnmnized with random copolymers of prohne and glycine or of proline, glycine and O-acetyl-hydroxyproline, gave good immediate but no delayed cro~s-reaction~ with linear poly-l_ prolines. However, both immediate and delayed reactions were elicited with the immunizing antigens. These results are again consistent with the view that the determinants responsible for delayed sensitivity are larger than tho.~e inw~lved in antibody-mediated reaction. Data in Table I I show that the three collagen preparation~ tested cro~s-reacted to some extent b y passive cutaneous anaphylaxib with guinea pig and rabbit anti(Pro Gly-Pro)n antibodies*. Additional data, mentioned above, show that gainea pig and rabbit anti-collagen sera were capable of cross-reacting with (Pro (ilv Pro)n. No comparable cros,~-reactions were observed between the random copolymer (Pro66Glya4)n and the anti-collagen sera or between collagen and antl-(Pro"6(}lyaa)n l¢~ochtm Bu,phv~ .Iota, tsS {r<)~O) 3Jl 323
COLLAGEN-LIKE SYNTHETIC POLYPEPTIDE
321
sera. There was also a lack of cross-reactions between commercial gelatin and anti(Pro-Gly-Pro)n or anti-(ProSeGly~)u sera. The above results, together with the previously mentioned data showing immunological cross-reactions between (Pro-Gly-Pro)n and (ProeeGly34)n, suggest that the passive cutaneous anaphylaxis cross-reactions between the polymer of ordered sequence (Pro-Gly-Pro)n and collagen took place primarily by virtue of the triple-stranded helix conformation, common to both substances in questionZ, 7. The role of the triple helix in the immunology of collagen has never been clearly established. SCHMITTet al. 26 studied the impairment of the antigenic activity of calf collagen, as measured by complement fixation in the presence of rabbit antisera, by the prior treatment of collagen with pepsin or pronase. The authors concluded that the main antigenic sites of collagen reside in the telopeptides, peptide chains protruding from the triple-helix body of the molecule, readily split off by proteases under the conditions which leave intact the triple-helix conformation. The complement fixation capacity of collagen, however, was lost also on thermal denaturation which is known to disrupt the triple helix without affecting the attachment of telopeptides to the rest of the molecule. Thus, the final conclusion derived from the data was that both the telopeptides and the triple helix contained sites recognized by heterologous antibody, the first of the two being mainly responsible for the antigenic specificity. TIMPL et al. 27, employing haemagglutination-inhibition tests with denatured collagen (parent gelatin), found on calf, sheep, rat, guinea pig and rabbit collagen preparations the presence of both pepsin-labile and pepsin-stable antigenic determinants. Some of the latter kind were common to all the species studied, whereas others were restricted to two or three species. These determinants are presumably located on the chains of the triple helix ; after the disruption of the helical conformation they do not function in inducing species-specific antibodies 13 although they retain the capacity to react with the antibodies in the haemegglutination-inhibition assay. MICHAELI et al. ~8 reported recently that not only the native guinea pig collagen, but also its denatured form, gelatin, can show precipitin reactions with the rabbit antibodies. Thus, it seems that the relative importance of the triple-helix structure for the immunological functions of collagen depends on the type of assay used. The only other instance of an immunological cross-reaction between a synthetic peptide and unmodified collagen, reported thus far in literature, is that found by I~ETTMAN et al. 29 in which an octapeptide G l y - P r o - G l y - P r o - P r o - G l y - A l a - L y s showed specific precipitin reactions with rabbit antiserum to guinea-pig-skin collagen. The authors interpreted this phenomenon as resulting either from the aggregation of the peptide or from its adsorption to serum proteins with the formation of multivalent antigen. Specific binding of the 14C-labeled peptide to anti-collagen sera was also demonstrated. In an earlier search for the sites on gelatin molecule responsible for the antigenic specificity of the latter, SELA AND ARNON30 attempted to inhibit the specific gelatin anti-gelatin precipitin reaction by means of various random linear polymers and copolymers containing proline or hydroxyproline or both. Since none of the substances " I t is possible t h a t in those guinea pigs used for passive c u t a n e o u s a n a p h y l a x i s tests in which no cross-reactions were observed, the cross-reacting antibodies h a d been completely neutralized b y the native skin collagen before the test antigen was administered. However, no evidence is available in s u p p o r t of this possibihty.
Bioch,m. B,ophys. Acta, 188 (I969) 314-323
322
i, t3~)I¢E1,~ ~'l ell,
tested had any effect on tile homologous precipitin reaction, it was concluded that the antigenic sites on gelatin have chemical structures and conformations nmch more intricate than those present in the polymers used. Absence of precipitin cross-reaction~ was found by F u c g s AND SELAa1,32 between rabbit antisera to linear and multichain copolymers of tyrosine, glutamic acid, alanine and lysine--and ovalbumin, ede~tin or gelatin and between antisera to multichain copolymers of histidine, phenylalanine, leucine, methionine, glutanfic acid, alanine and lysine--and ovalbunun, bovine serum albumin and ribonuclease. K U N Z AND GILLaa reported that antisera to t)ara myosin failed to cross-react with various copolymers of glutamic acid, lysine, tvrosine and alanine. Neither did paramyosin cross-react with the anti-polypeptide sera MAVRERa4 recorded a lack of anaphylactic cross-reactions between a Glu I.vs copolymer and proteins; however, he found a passive cutaneous anaphylaxia crossreaction between an anti-Ala-Gly copolymer serum and gliadin aa. Rabbit anti-bovine y-globulin serum was shown by (~ILL AND MATTHEWSa6 not to cross-react with several copolymers of glut)talc acid, lyqne, phenylalanine and tyrosine, wherea~ high titer antisera to synthetic polypeptides crosa-reacted to a very slight extent with a wide variety of proteins. On the other hand, ~ A N D B E R G AND STOI.LARav dennmstrated exten.,ive cro.,sreactions bv the complement-fixation test between rabbit anti-poly-L-lysine sera and basic proteins such as histone and ribonuclease. WALSH et al. as reported that immunization of rabbits with (;ly-Lys--Ala or Glu Lys-Ala fyr copolymers induced the formation of helnolytic plaque-forming lymph node cells which croas-reacted with normal sheep erythrocytes. Antisera against some .~vnthetic copolymer~ containing D-alanine were reported recently to cross-react with a bacterial muc-peptidea'E In general, the results of the studies of inmmnological cross-reactions between synthetic polypeptides and proteins reflect not only the possible structural similarities between the two kinds of macromolecular substances but also the ~pecial conditions and sensitivity of the assay as well as the affinity for antigen of the antibodie~ under investigation In conclusion, the experilnents described in thia paper show a relatumship between the conformatl.n of the polymer of ordered sequence, (L-Pro (ily L-Pro)n, and its inmmnological propertie~. The immunological cross-reaction between the polymer and collagens of several species are most probably due to the conformational resemblance between the triple-stranded helix of collagen and that of the synthetic polypeptide. The weak, but positive, cross-reactions of guinea pig collagen with guinea pig antihodies against the polymer of ordered structure shows that in an animal it is possible to provoke formation of antibodies cross-reacting with a protein of the same animal species, by means of inmmnization with a synthetic antigen. ACKNOXVLE DGMENTS
This research has been sponsored in part by the Air Force Office of Scmntitic Research through the European Office of Aerospace Research (O.A.R.), U.S. Air Force under Grants EOAR-67-I 9 and EOOAR-6S-oo21. The excellent technical assistance of Miss N. Novik i~ gratefully acknowledged.
t?iochzm. Bzophvs .4cta, ISS (1969) 314 323
COLLAGEN-LIKE SYNTHETIC POLYPEPTIDE
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Bzochim. Bzophys. Acta, I88 (I969) 314-323