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Antigenic structure of the aminoterminal region in type I procollagen
lmmunochemrstry, 1976, Vol. 13. pp. 967-974.
Pergamon
Press.
Printed in Great Britain
ANTIGENIC STRUCTURE OF THE AMINOTERMINAL REGION IN TYPE I PROCOLLAGEN CHARACTERIZATION OF SEQUENTIAL AND CONFORMATIONAL DETERMINANTS HEILWIG
ROHDE, UDO BECKER, HANS NOWACK
Max-Planck-Institut
fiir Biochemie, Abteilung Bindegewebsforschung,
and RUPERT TIMPL D-8033 Martinsried, Germany
(Received 25 June 1976) Abstract-Antibodies to calf or sheep skin procollagen, to its constituent pal(I) and pa2 chain or to peptide fragments from the aminoterminal region in pal were studied by radioimmune assays using various lZSI-labeled antigens..The highest antibody response was obtained against the globular region in pcrl(1). The adjacent precursor-specific collagenous and non-helical domains may modulate the antigenicity of the globular determinants but are themselves only weak antigens. No antigenic determinants were detected in the pct2 chain. Antibodies to pctl(1) chain did not cross-react with pa2 or pctl(II1) chains. Antisera against the globular procollagen peptide showed no difference between the peptide and procollagen indicating that the release of the globular region from pctl(1) by collagenase is not accompanied by large conformational alterations. Complete cross-reaction was observed between precursorspecific peptides obtained from calf or sheep procollagen. Reduction and S-carboxymethylation of the five disulfide bridges in the globular region largely destroyed antigenicity. However, a minor fraction of the antibodies against the native peptide could still react with the unfolded peptide. Determinants recognized in this reaction are apparently shared by the native and unfolded peptide and were stable towards trypsin. On the other hand, antibodies prepared against the reduced and alkylated procollagen peptide did not react with the native peptide. The data indicate that the globular regions in prl(1) chain possesses both conformational and sequential determinants which differ considerably in their immunogenic capacity.
INTRODUCTION
Collagens are synthesized in the form of precursors, the procollagens. These possess additional peptide segments at the ends of the c( chains. Specific procollagen peptidases are thought to remove these segments before the molecules are incorporated into the extracellular fibrils (Bornstein, 1974; Martin et al., 1975; Prockop et al., 1976). The most abundant type I collagen consists of two al(I) and one cr2 chain (mol. wts 95,000) which are assembled into a triple helical molecule. Normally, the levels of type I procollagen in tissues are low, but a form of this precursor accumulates in the skin of calves and sheep with the inherited disorder of connective tissue called dermatosparaxis (Lenaers et al., 1971; Fjolstad & Helle, 1974). This protein comprising the intact aminoterminal segment of procollagen plus collagen without the carboxyl portion of procollagen. Most of the chemical studies on procollagen have been carried out with the precursor form found in dermatosparactic tissue. The aminoterminal, precursor specific segment of dermatosparactic pal(I) chain consists of a globular, a collagen-like and a non-helical domain. The order of occurrence and the approximative sizes of these regions are shown in the diagram (Fig. 1). The whole precursor-specific segment plus a short portion from the aminoterminal end of al(I) chain can be obtained from the polypeptide by cleavage with cyanogen bromide. This peptide is called pc&CB 0.1 (Becker et al., 1976b). When the MM. 13/12--s
collagen-like sequence is destroyed by incubation with bacterial collagenase the globular region is released as a single peptide (Furthmayr et al., 1972). This peptide (poll-CB 0.1 Co1 1) has a size of 12,000 daltons and is folded into several loops by 5 disulfide bridges. The amino acid sequence of the region cleaved by procollagen peptidase has recently been determined (Fietzek & Kiihn, 1976). These studies indicate that there is a short non-helical segment on each side of the cleavage site. This segment is also released by collagenase and was isolated as a homogeneous peptide p&CB 0.1 Co1 2 (Hiirlein & Fietzek, unpublished). The aminoterminal portion of the pa2 chain is smaller than that found in pal(I) and consists mostly of a collagenous domain but may also contain short globular and non-helical sequences (Becker et al., 1976b; Becker, Fietzek and Timpl, unpublished). Antibodies have been produced in rabbits by injecting either dermatosparactic procollagen (Timpl et al., 1973; Kohn et al., 1974; Park et al., 1975) or the collagenase-derived aminoterminal peptide (Rohde et al., 1976). In both cases the antibodies produced react strongly with determinants in the globular region while little or no reaction was observed with collagen. Studies in inbred mice indicated that the procollagenspecific region possesses strong carrier determinants which can help to correct a low responder state against the triple-helix of collagen’(Hahn et al., 1975; Nowack et al., 1975). This observation agrees with other data showing that the antibody response to the 967
H. ROHDE, U. BECKER, H. NOWACK and R. TIMPL
968
precursor specific segment
collagen dl (I 1chain
/
I
\/--globular (-lOOAA1
I
collagen-like
1
(-4OA)
non-helical
(8AAI
1 (16AA)
\1 Ala-Gln
pdl-CB
-Met
9
0.1
Fig. 1. Schematic structure of the aminoterminal segment in pal(I) chain as based on data of Furthmayr et al. (1972), Fietzek & Kiihn (1976) and Becker et al. (19766). The assignment of the five disulfide bridges is arbitrary. The arrow indicates the site of cleavage by procollagen peptidase. The location of the fragments pal
globular procollagen peptide is regulated by T cells (Nowack et al., 19766). Due to its small size it may be possible with this peptide to establish the structure of both the haptenic and carrier determinants. In the present study we have determined the immunological importance of the different structural domains occurring within the aminoterminal region of type I procollagen including the importance of conformation. MATERIALS
Preparation
AND METHODS
and characterization
of antigens
Skins from dermatosparactic calves and sheep were kindly supplied by Dr. C. M. Lapiere, Liege, and Dr. 0. Helle, Oslo. Procollagen was extracted from ground tissue with 1 M NaCl and purified by precipitation using dialysis against 15’/, KCl, 0.02 M sodium phosphate pH 8.6. As jidged by electrophoresis these irocollagens consisted mostlv of ~crl(Il and oa2 chains with about 20-30X of a chins. fhe phi(I) and pa2 chains were extracted from the dermatosparactic sheep skin with 10 M urea and purified by chromatography on CM-cellulose and agarose (Becker et al., 1976b). The globular peptide segment of pal(I) chain (pal-CB 0.1 Col 1) was obtained by digesting isolated chains or the insoluble collagenous residue with bacteria1 collagenase (Furthmayr et al., 1972; Becker et al., 1976b). The aminoterminal cyanogen bromide peptide of pal(I) chain (palCB 0.1) was purified by ion-exchange chromatography (Becker et al., 1976b). Purified pal-CB 0.1 was dissolved in 8 M urea (2 mg/ml) and dialyzed for 4-5 days at 4°C against large volumes of 0.05 M sodium phosphate pH 7.0. During dialysis this peptide forms a trimeric aggregate as its collagenous domain folds into a triple-helical configuration. As judged by circular dichroism this triplehelical segment has a melting temperature of 45°C. Except when otherwise stated, all experiments were performed with trimeric pal
to prevent ,renaturation to the trimeric form (Bruckner & Engel, personal communication). The peptide fragment pal-CBO.l Co1 2 which consists of 11 amino acids from the precursor-specific part in pal(I) chain and 19 amino acids from the N-terminal end of al(I) chain (Fietzek & Kiihn, 1976) was prepared from a collagenase digest of calf procollagen. It was partially purified by chromatography on Bio-Gel P-10 and phosphocellulose (HGrlein, personal communication) and finally adsorbed to a DEAE-cellulose column equilibrated in 8 k urea, 5 mM Tris-HCl, pH 8.6. Application of a linear gradient to the column from 0 to 0.1 M NaCl eluted a peptide which resembled in its amino acid composition the sequence of oal-CB 0.1 Co1 2 (Fietzek & Kiihn, 1976). Type III procollagen was purified from fetal bovine skin (Timpl et al., 1975). The precursor-specific aminoterminal peptide pal(IIItCo1 l-3 was obtained by digestion with collagenase and was purified on Sephadex G-50 and DEAE-cellulose. It consists like pal-CBO.1, the comparable peptide from type I procollagen, of both a globular and a collagenous domain. Apparently, interchain disulfide bonds in this peptide prevent the degradation of the collagenous domain by collagenase (Nowack, Olsen & Timpl, 1976~). Reduction of peptides with dithioerythritol and S-carboxymethylation was carried out in 6 M guanidine (Furthmayr et al., 1972). Reduced peptides were digested with TPCK-trypsin (Worthington) in 0.2 M ammonium bicarbonate ~$7.9 at an enzyme-substrate ratio of 1:lOO for 24 hr at 37°C. Chromatograuhv on Bio-Gel P-10 showed that the digestion was c&pi&. The amino acid composition was determined after hydrolysis with 6 M HCI (24 hr, 110°C) on a Durrum D-500 analyser. The homogeneity of antigens was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate (Furthmayr &’ Timpl, 1971). Antisera
Rabbit antisera to procollagen and to the native or reduced peptide pal-CB0.1 Co1 1 were those used in previous studies (Timpl et al., 1973; Rohde et al., 1976). The same schedule was used to immunize rabbits with two 1 mg doses of pal(I) chains or with pa2 chain (2 x 1 mg)
969
Antigenicity of Procollagen or with peptide palCB0.1 (2 x 0.1 mg). Blood was collected from the animals over a period of several weeks starting 18 days after the booster injection. Antisera to collagen were prepared as described by Becker et al. (1976a). Female Sprague-Dawley rats were immunized with pa2 chains. In the tirst subcutaneous injection 0.2 mg pa2 was administered together with complete Freund’s adjuvant. An intraperitoneal booster with 0.1 mg pct2 but without adjuvants was given on day 23 and the animals were bled on day 42 by cardiac puncture. Antisera against rabbit immunoglobulin G were prepared in several goats (Lotter & Timpl, 1975). Radioimmune assays
Antigens were labeled with iodine-125 by the chloramine T method (McConahey & Dixon, 1966) following the procedure of Rohde et al. (1976). Usually, labeled peptides were separated from free iodine by dialysis. In the case of the small peptide pal_CB 0.1 Co1 2, separation of bound from unbound label was achieved on a Bio-Gel P-2 column which was equilibrated in phosphate-buffered
saline pH 7.2 containing 0.1% bovine serum albumin (PBS/BSA). The amount of label bound to peptide was determined by precipitation with trichloroacetic acid and was in the range of 8&90% except for pa2 chains where it was 40%. Labeled pal-CB 0.1 Co1 2 was not precipitated by trichloroacetic acid but binding to antibody (see Results, Fig. 4) indicated that at least 65% of its radioactivity was peptide bound. Usually, preparations of pa2 show a purity of 95-98% in electrophoresis and were mainly contaminated by p/l components (cross-linked pa chains). Examination of labeled pa2 by electrophoresis showed that about half of the label migrated with the mobility of pa2 while the rest was associated with the higher mol. wt components. Presumably, the low labeling efficiency reflects the low tyrosine content in pa2 chains (Becker et al., 1976b). Binding of labeled antigens by antibody was determined in duplicate in tubes containing 0.1 ml antiserum dilution, 0.1 ml antigen and 0.2 ml PBSBSA. Immune complexes were precipitated with goat antiserum to rabbit immunoglobulin G. The amount of labeled antigen added to the reaction was varied according to their mol. wts: 20 ng pa2 chain, 3 ng pal_CB 0.1, 2 ng pal-CB 0.1 Co1 1 and 0.2 ng pal-CB 0.1 Co1 2. Antigen binding capacity was calculated according to Minden & Farr (1973). In the inhibition tests antibodies were pre-incubated with unlabeled antigens prior to the addition of labeled antigen (Lotter & Timpl, 1975). Antigen concentrations were determined on the amino acid analyser after hydrolysis of aliquotes of the inhibitor solutions with 6 M HCI. In calculating molar equivalents, the mol. wts assumed for
the various peptides were those given by Becker et al. (1976b). RESULTS Antigenic properties pcrl(1) chain
of the precursor-spectfic
Pooled antisera to procollagen bound ‘251-labeled peptides (Table 1) obtained from the aminoterminal region of pal(I) chain either by cleavage with cyanogen bromide (pctl-CB0.1) or by digestion with collagenase (pc&CB 0.1 Co1 1). A similar reactivity was observed with antisera raised against pal(I) chains or the procollagen peptides. Reduction and alkylation of the procollagen peptide markedly decreased or even abolished its ability to combine with antibody. Previous studies have shown that these antisera show minimal or no reactions with collagen (Timpl et al., 1973; Rohde et al., 1976). The relative contribution of various regions in pal(I) to its antigenicity was studies by inhibition assays using antibodies against various antigens. These antibodies were reacted against the labeled peptide palLCB 0.1 which consists of the globular and collagen-like domain. As shown in Fig. 2 this peptide was a strong inhibitor both in trimeric, triple-helical and monomeric form. The smaller collagenase-derived peptide, pal-CB 0.1 Co1 1, comprising the globular domain also almost completely inhibited the reaction of antibodies. However, since the slope of the inhibition curve was less steep than observed with pctlLCB 0.1 it is likely that it has a lower affinity (Fig. 2b). Essentially no cross-reaction was observed with the precursor-specific peptide from type III procollagen although it also consists of a globular and collagenous domain. Since the globular region is the most important antigenic part but apparently not completely identical to the whole precursor-specific segment we also studied the reaction of antibodies to the peptide pal-CB 0.1 Co1 1 with labeled pal_CB 0.1 Co1 1 by inhibition assays (Fig. 3). In this system both peptides pal-CBO.l and pal-CB 0.1 Co1 1 as well as procollagen gave superimposable inhibition curves. Procollagen peptides derived from sheep or calf procollagen showed complete cross-reaction (Fig. 3a). pal(I)
Table 1. Binding by various antisera of 1251-labeled procollagen peptides
Antiserum to Procollagen pal(I) chain p&CB 0.1 palCB 0.1 co1 1 palCB 0.1 Co1 1, reduced
No. pool 709* 710* 728* 729b 667 670 707 713”
part of
pg Peptide bound per ml antiserum” palCB 0.1 pcrl_CB 0.1 co1 1, pal-CB O.lb co1 1 reduced 462.0 176.2 66.0 43.5 22.2 138.6 nd. nd. n.d.
’ Calculated according to Minden & Farr (1973). * Antigens from sheep; all others were of bovine origin. n.d. = not determined.
83.7 n.d. n.d. nd. n.d. 14.1 5.9 < 0.006 <0.006
0.29 < 0.006 0.014 < 0.006 < 0.006 0.47 0.23 0.13 0.30
H. ROHDE, U. BECKER, H. NOWACK and R. TIMPL
970 lCO-
0/-g-W
a)
,x X’
CL
x’
10’
,
/’
0x
I
I
10-2
o---+-o
I
102
100
10-2
100
102
Inhibitor [pMole]
Fig. 2. Comparison of various procollagen peptides by radioimmune inhibition assay. Antisera against peptide palCB 0.1 (No. 728, a) or against pal(I) chain (No. 709, b) were reacted with ‘251-labeled pal-CB 0.1. Inhibitors were peptides pctl-CB 0.1 (c+-o), monomeric pc+CB 0.1 (M), pal-CB 0.1 Co1 1 (x---x), and pal(II1) Co1 l-3 from bovine type III procollagen (Cl---O). All inhibitors except the last were from sheep.
observed with antisera to pctl-CB0.1 Co1 1. The binding capacity of the antisera except the latter one was in the range l-10 ng pal--CB 0.1 Co1 2/ml which on a molar basis is about lOOO-foldless binding activity than observed with the large procollagen peptides (see Table 1).
chains were strongly inhibitory but differed by a factor of 2-5 in inhibiting potency when compared with pal-CB0.1 Co1 1. No inhibitory activity could be detected on pa2 chains even when tested in 5&100-fold higher concentration than pal(I) chains. The possible occurrence of minor populations of antibody directed against precursor specific regions in pal(I) other than the globular domain was examined by direct binding studies. The collagen-like part has yet not been isolated as a distinct fragment and could therefore not be used in this study. However, another small fragment pul
Lack of antibody response to pu2 chain
Since pu2 chains did not react with antibody to the aminoterminal segment in pal(I) chain (Fig. 3) it was of interest to determine the response of rabbits or rats after injection with pa2 chains. Incubation of undiluted antisera to pu2 chain with 1251-labeled pa2 showed that less than 10% of the peptide-bound radioactivity was bound. However, some of the anti-pa2 chain antisera showed a distinct although weak reaction with labeled peptides from pal(I) chain. Presumably, the pa2 preparation used for immunization contained sufficient amounts of contaminating
two tyrosine residues and could therefore be labeled with lz51. Binding studies with labeled pc&CB 0.1 Co1 2 showed a distinct reaction with antisera to procollagen or pal(I) chain and also with antisera to type I collagen (Fig. 4). Essentially no binding was
bl
al
I
I
to-’
____
I
10’
I
103
I
I
10-Z
I
I
100
I
I
102
Inhibitor [ pMole]
Fig. 3. Radioimmune inhibition assay with antisera to the globular region in procollagen (peptide pal-CB 0 1 Co1 1) and ‘251-labeled pal
971
Antigenicity of Procollagen
-60
\
Antiserum
dilution
Fig. 4. Binding of ‘ZSI-labeled peptide pctlCB 0.1 Co1 2 by antisera to procollagen ( x ~ x ), collagen (M), pal(I) chain (Cl---U) and peptide pctl-CB 0.1 Co1 1 (A-A). Immune complexes were precipitated by goat antiserum to rabbit IgG. pal(I) to provoke these antibodies. These contaminants were probably cross-linked p/l components or disulfide-linked procollagen both of which chromatograph near the region of pa2 chains on CM cellulose. No binding of pz2 chains was also observed with antisera to procollagen, pal(I) chain and the peptides pal-CB 0.1 and pa-CB0.1 Co1 1. Identification of antigenic determinants shared by the natioe and unfolded globular region of pal(I) chain The effect of reducing all disulfide bonds in the globular region of pal(I) chain on inhibiting activity is shown in Fig. 5. Reduced and alkylated peptides did not inhibit the reaction of most of the antisera with native peptides except when used at 5000-10,000-fold higher concentrations than native peptides (Fig. 5a). A few antisera have been found which show a weak reaction with the labeled. un-
folded peptide (Table 1). These particular antisera could be distinctly inhibited by low concentrations of unfolded peptide but the extent of inhibition did not exceed 10% (Fig. 5b). The data may indicate that native and unfolded peptides have antigenic determinants in common but the antibody levels directed toward these determinants are far lower than those reacting with conformational determinants. Alternatively, sufficient levels of unfolded peptide may occur in the supposedly native preparation to elicit antibodies. To distinguish between these possibilities we examined the binding of antibodies to procollagen or pal-CB 0.1 Co1 1 with labeled reduced pal-CB 0.1 Co1 1 by inhibition assays. In the case of shared determinants we should observe a similar inhibition with both the native and unfolded peptide. On the other hand, an antibody reaction specific for the unfolded peptide alone should be inhibited much better by this peptide (Young & Leung, 1970). The examples shown in Fig. 6 support the first interpretation. Both the native and unfolded peptide completely inhibit the reaction even though there were distinct differences in the amount of antigen required to achieve 50% inhibition as well as in the slopes of the inhibition curves. Most likely these differences may reflect the relative proportions of different antibodies in the antisera. While unfolded peptide only reacts with a minor population of antibody, native peptide is able to react with all antibodies and so higher amounts of peptides are required to reach equivalent levels in inhibition. Antibodies against determinants shared by native and unfolded peptide could also be efficiently inhibited by a tryptic digest prepared from reduced palCB 0.1 Co1 1 (Fig. 6). Therefore, it should be possible to locate some of these determinants by using individual tryptic peptides. Antibodies to the shared determinants could also be inhibited by the reduced and alkylated precursorspecific peptide from type III procollagen (Fig. 6). About lOO-fold greater amounts were required for a significant reaction when compared with the unfolded peptide from type I procollagen. Considerably less
./’
b)
/ .
I
102
10-l
Inhibitor [ pMok]
Fig. 5. Comparison of native and reduced and alkylated procollagen peptides by radioimmune assay. Antisera against native peptides pal-CB 0.1 (a) and pal-CB 0.1 Co1 1 (b) were reacted against 1251-labeled native peptides pal-CBO.l and pal-CB 0.1 Co1 1, respectively. Inhibitors of these reactions were native pal-CB 0.1 (M), reduced and alkylated pal-CB 0.1 (A---A), native palCB 0.1 Co1 1 (M) and reduced and alkylated palCB 0.1 Co1 1 (A---A).
912
H. ROHDE, U. BECKER, H. NOWACK and R. TIMPL
10-l
10'
Inhibitor [pMole]
Fig. 6. Demonstration of antibodies to sequential antigenic determinants in the globular peptide palCB 0.1 Co1 1. Antisera No. 670 (a) and 667 (b) to peptide pal-CB 0.1 Co1 1, and to procollagen (c) were reacted against iz51-labeled, reduced and alkylated pal-CB 0.1 Co1 1. Inhibitors were native peptide palCB 0.1 Co1 1 (M), reduced and alkylated pal-CB 0.1 Co1 1 (x--x ), a tryptic digest of reduced pal-CBO.1 Co1 1 (O---O), and reduced and alkylated peptide pal(II1) Co1 l-3 from type III procollagen (A---A). cross-reactivity has been observed between the native peptides (Fig. 2). The data resemble therefore previous observations (Arnon & Maron, 1971) that sequential determinants in proteins show higher cross-reactivity than conformational determinants. Specificity of the antibody response to the unfolded globular region Antisera prepared against reduced and alkylated p&CB 0.1 Co1 1 bound to the unfolded but not to the native peptide (Table 1). The specificity of the antibodies was further studied by inhibition assay (Fig. 7). While reduced p&CB 0.1 Co1 1 was an effeceven concentrations, low at tive inhibitor 5000-lO,OO@fold greater amounts of native peptide did not completely inhibit the reaction. DISCUS!SION
Previous studies have shown that the precursorspecific segment of procollagen from dermatosparac'OQr
tic animals is a globular peptide whose conformation is stabilized by intra-chain disulfide bridges (Furthmayr et al., 1972). More recent studies demonstrated an additional segment with a typical collagen sequence (Becker et al., 1976b) and the presence of a short non-helical segment in this region (Fietzek & Kuhn, 1976). It was therefore of interest to compare the contribution of each domain to the immunological properties of procollagen. The present study confirms and extends previous observations (Timpl et al., 1973) that for antigenic activity the globular domain of pal(I) chain is the most important region in this preparation of procollagen. The collagenous domain is apparently only weakly immunogenic but may modulate to some extent antigenic expression of the determinants in the globular part (Fig. 2). Similar differences between the cyanogen bromide and collagenase-derived procollagen peptides could also be detected by hemagglutination-inhibition (Becker et al., 1976b) and have also been reported in a study on chick procollagen (von
x
al
T8o 2 5 :E 60 .g
/ x
C 40-
/
1.1 -Lo4o I
16'
10'
I
I
103
I
I
10-2
100
102
Inhibitor [pMole]
Fig. 7. Radioimmune inhibit& assay with antisera Nos. 713 (a) and 707 (b) against reduced and alkylated peptide palCB 0.1 Co1 1. The ‘251-labeled antigen was reduced and alkylated pal-CB 0.1. Co1 1. Inhibitors were native peptide palCB 0.1 Co1 1 (o----O) and reduced and alkylated pal-CB 0.1 Co1 1 ( x -x ).
913
Antigenicity of Procollagen der Mark et al., 1973). Since the major antigenic determinants in the globular region are conformation dependent it is likely that the influence of the collagenous domain is related to conformational alterations. However, antisera produced against the globular procollagen peptide did not distinguish between the peptide and procollagen. Apparently, the conformational changes produced by removing the collagenous from ‘the globular domain are very small and are not recognized regularly in the antibody response. A weak reaction was detected against the non-helical segment (peptide pctl-CB 0.1 Co1 2) which joins the precursor-specific portion of pal(I) with the aminoterminal end of ml(I) chain. An antigenic determinant of collagen has been previously located in this region (Rauterberg et al., 1972) which explains why antibodies to collagen react with pcz_CB 0.1 Co1 2. The presence in this peptide of determinants unique to procollagen remains to be determined. There was at least no cross-reaction with antibodies to the globular procollagen peptide pc&CB 0.1 Co1 1. The pa2 chain did not react with antibody prepared against pal(I) and in addition showed essentially no immunogenic activity. The precursor-specific, aminoterminal segment of pa2 mainly consists of a collagenlike sequence (Becker et al., 1976; Becker, Fietzek and Timpl, unpublished). It is however, still possible that an intact pct2 chain containing the carboxyl segment possesses different immunogenic properties. Antibodies to determinants of pal(I) chain showed also no or only little reaction with a peptide unique to the aminoterminal region in pctl(II1) chain of type III procollagen. A strong antibody response can be produced against type III procollagen and these antibodies also distinguish between type I and type III procollagen (Nowack et al., 1976a). The precursorspecific part of type III procollagen consists, like in pal(I) chain, of a large globular, a collagenous and a non-helical domain but showed significant differences in its amino acid composition (Nowack et al., 1976~).Thus, globular determinants are unique to distinct types of procollagen and determine their strong immunogenic activity. Two types of antigenic determinants could be demonstrated in the globular region. One type is unique for the native, disulfide-bonded structure, the other is shared by the native and by the reduced and alkylated peptide. Both sites differ in their immunogenic capacity and usually less than 10% of the total antibodies produced are directed toward unfolded determinants. The immunological findings agree with recent circular dichroism studies on the globular procollagen peptide (Bruckner & Engel, unpublished). The data suggest that the native peptide possesses regions which assume a distinct structure while other regions have an extended, linear conformation. Reduction and alkylation of all disulfide bonds destroys completely the conformation. The two kinds of determinants in the globular region of procollagen correspond to the two categories of conformational and sequential determinants to which Sela et al. (1967) have attributed the antigenicity of proteins. Whereas there is no doubt on the existence of conformational determinants in globular proteins, serious objections have been raised against evidence for sequential determinants (Crump-
ton, 1974; Sachs et al., 1972). Young and Leung (1970), for example, compared antibodies against native lysozyme with those against the unfolded protein and found no difference in their reaction patterns with unfolded lysozyme. Antigenicity detected in unfolded globular proteins may therefore be altematively explained by antibodies against denatured antigen which was present in the native preparation used for immunization. This explanation appears unlikely for procollagen since considerable differences were observed by inhibition assays between antibodies to sequential determinants in the native peptide and antibodies to the unfolded procollagen peptide. Rather the data indicate that denaturation unmasks determinants which are ordinarily buried in the region of the disulfide bridges. Trypsin treatment did not significantly change antigenie activity of the sequential determinants in the procollagen peptide. More recent studies with isolated tryptic fragments showed antigenicity in regions as small as a hexapeptide sequence (Rohde & Timpl, unpublished). It is unlikely that such small fragments exhibit a distinct conformation but it cannot be excluded that they become folded when bound to the antibody combining site (Crumpton, 1974; Sachs et a/., 1972). Since the precise localization of these determinants is feasible, information on the three-dimensional structure of the procollagen peptide should clarify conformation of those sites which are operationally defined here as sequential determinants. Acknowledgements-These studies were supported by Grants Ti 95/l and Ti 95/2 from the Deutsche Forschungs-
gemeinschaft.
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H. ROHDE, U. BECKER, H. NOWACK and R. TIMPL
Nowack H., Gay S., Wick G., Becker U. & Timpl R. (1976a) J. Immun. Meth. 12, 117. Nowack H., Rohde H., Gijtze D. & Timpl R. (1976b) Immunogenetics, in press. Nowack H., Olsen B. R. & Timpl R. (1976~) Eur. J. Biothem. To be published. Park E., Church R. L. & Tanzer M. L. (1975) Immunology 28, 781. Prockop D. J., Berg R. A., Kivirikko K. I. & Uitto J. (1976) In Biochemistry of Collagen, (Edited by Ramachandran G. N. & Reddi A. H.) Plenum, NY, in press. Rauterberg J., Timpl R. & Furthmayr H. (1972) Eur. J. Biochem. 21. 231.
Rohde H., Nowack H., Becker U. & Timpl R. (1976) J. Immun. Meth. 11, 135. Sachs D. H., Schechter A. N., Eastlake A. & Anfinsen C. B. (1972) Proc. natn. Acad. Sci., U.S.A. 69, 3790. Sela M., Schechter B., Schechter M. & Borek F. (1967) Cold Surina Harbor Svmu. auant. Biol. 32, 537.
Timpl R.: W&k G., Furthmayr H., Lapiere C. M. & Kuhn K. (1973) Eur. J. Biochem. 32, 584. Timpl R., Glanville R. W., Nowack H., Wiedemann H., Fietzek P. P. & Kuhn K. (1975) Hoppe-Seyler’s Z. physiol. Chem. 356, 1783. Von de? Mark K., Click E. M. & Bornstein P. (1973) Archs. Biochem. Biophys. 156, 356. Young J. D. & Leung C. Y. (1970) Biochemistry 9, 2755.
Pergamon
Press.
Printed in Great Britain
ANTIGENIC STRUCTURE OF THE AMINOTERMINAL REGION IN TYPE I PROCOLLAGEN CHARACTERIZATION OF SEQUENTIAL AND CONFORMATIONAL DETERMINANTS HEILWIG
ROHDE, UDO BECKER, HANS NOWACK
Max-Planck-Institut
fiir Biochemie, Abteilung Bindegewebsforschung,
and RUPERT TIMPL D-8033 Martinsried, Germany
(Received 25 June 1976) Abstract-Antibodies to calf or sheep skin procollagen, to its constituent pal(I) and pa2 chain or to peptide fragments from the aminoterminal region in pal were studied by radioimmune assays using various lZSI-labeled antigens..The highest antibody response was obtained against the globular region in pcrl(1). The adjacent precursor-specific collagenous and non-helical domains may modulate the antigenicity of the globular determinants but are themselves only weak antigens. No antigenic determinants were detected in the pct2 chain. Antibodies to pctl(1) chain did not cross-react with pa2 or pctl(II1) chains. Antisera against the globular procollagen peptide showed no difference between the peptide and procollagen indicating that the release of the globular region from pctl(1) by collagenase is not accompanied by large conformational alterations. Complete cross-reaction was observed between precursorspecific peptides obtained from calf or sheep procollagen. Reduction and S-carboxymethylation of the five disulfide bridges in the globular region largely destroyed antigenicity. However, a minor fraction of the antibodies against the native peptide could still react with the unfolded peptide. Determinants recognized in this reaction are apparently shared by the native and unfolded peptide and were stable towards trypsin. On the other hand, antibodies prepared against the reduced and alkylated procollagen peptide did not react with the native peptide. The data indicate that the globular regions in prl(1) chain possesses both conformational and sequential determinants which differ considerably in their immunogenic capacity.
INTRODUCTION
Collagens are synthesized in the form of precursors, the procollagens. These possess additional peptide segments at the ends of the c( chains. Specific procollagen peptidases are thought to remove these segments before the molecules are incorporated into the extracellular fibrils (Bornstein, 1974; Martin et al., 1975; Prockop et al., 1976). The most abundant type I collagen consists of two al(I) and one cr2 chain (mol. wts 95,000) which are assembled into a triple helical molecule. Normally, the levels of type I procollagen in tissues are low, but a form of this precursor accumulates in the skin of calves and sheep with the inherited disorder of connective tissue called dermatosparaxis (Lenaers et al., 1971; Fjolstad & Helle, 1974). This protein comprising the intact aminoterminal segment of procollagen plus collagen without the carboxyl portion of procollagen. Most of the chemical studies on procollagen have been carried out with the precursor form found in dermatosparactic tissue. The aminoterminal, precursor specific segment of dermatosparactic pal(I) chain consists of a globular, a collagen-like and a non-helical domain. The order of occurrence and the approximative sizes of these regions are shown in the diagram (Fig. 1). The whole precursor-specific segment plus a short portion from the aminoterminal end of al(I) chain can be obtained from the polypeptide by cleavage with cyanogen bromide. This peptide is called pc&CB 0.1 (Becker et al., 1976b). When the MM. 13/12--s
collagen-like sequence is destroyed by incubation with bacterial collagenase the globular region is released as a single peptide (Furthmayr et al., 1972). This peptide (poll-CB 0.1 Co1 1) has a size of 12,000 daltons and is folded into several loops by 5 disulfide bridges. The amino acid sequence of the region cleaved by procollagen peptidase has recently been determined (Fietzek & Kiihn, 1976). These studies indicate that there is a short non-helical segment on each side of the cleavage site. This segment is also released by collagenase and was isolated as a homogeneous peptide p&CB 0.1 Co1 2 (Hiirlein & Fietzek, unpublished). The aminoterminal portion of the pa2 chain is smaller than that found in pal(I) and consists mostly of a collagenous domain but may also contain short globular and non-helical sequences (Becker et al., 1976b; Becker, Fietzek and Timpl, unpublished). Antibodies have been produced in rabbits by injecting either dermatosparactic procollagen (Timpl et al., 1973; Kohn et al., 1974; Park et al., 1975) or the collagenase-derived aminoterminal peptide (Rohde et al., 1976). In both cases the antibodies produced react strongly with determinants in the globular region while little or no reaction was observed with collagen. Studies in inbred mice indicated that the procollagenspecific region possesses strong carrier determinants which can help to correct a low responder state against the triple-helix of collagen’(Hahn et al., 1975; Nowack et al., 1975). This observation agrees with other data showing that the antibody response to the 967
H. ROHDE, U. BECKER, H. NOWACK and R. TIMPL
968
precursor specific segment
collagen dl (I 1chain
/
I
\/--globular (-lOOAA1
I
collagen-like
1
(-4OA)
non-helical
(8AAI
1 (16AA)
\1 Ala-Gln
pdl-CB
-Met
9
0.1
Fig. 1. Schematic structure of the aminoterminal segment in pal(I) chain as based on data of Furthmayr et al. (1972), Fietzek & Kiihn (1976) and Becker et al. (19766). The assignment of the five disulfide bridges is arbitrary. The arrow indicates the site of cleavage by procollagen peptidase. The location of the fragments pal
globular procollagen peptide is regulated by T cells (Nowack et al., 19766). Due to its small size it may be possible with this peptide to establish the structure of both the haptenic and carrier determinants. In the present study we have determined the immunological importance of the different structural domains occurring within the aminoterminal region of type I procollagen including the importance of conformation. MATERIALS
Preparation
AND METHODS
and characterization
of antigens
Skins from dermatosparactic calves and sheep were kindly supplied by Dr. C. M. Lapiere, Liege, and Dr. 0. Helle, Oslo. Procollagen was extracted from ground tissue with 1 M NaCl and purified by precipitation using dialysis against 15’/, KCl, 0.02 M sodium phosphate pH 8.6. As jidged by electrophoresis these irocollagens consisted mostlv of ~crl(Il and oa2 chains with about 20-30X of a chins. fhe phi(I) and pa2 chains were extracted from the dermatosparactic sheep skin with 10 M urea and purified by chromatography on CM-cellulose and agarose (Becker et al., 1976b). The globular peptide segment of pal(I) chain (pal-CB 0.1 Col 1) was obtained by digesting isolated chains or the insoluble collagenous residue with bacteria1 collagenase (Furthmayr et al., 1972; Becker et al., 1976b). The aminoterminal cyanogen bromide peptide of pal(I) chain (palCB 0.1) was purified by ion-exchange chromatography (Becker et al., 1976b). Purified pal-CB 0.1 was dissolved in 8 M urea (2 mg/ml) and dialyzed for 4-5 days at 4°C against large volumes of 0.05 M sodium phosphate pH 7.0. During dialysis this peptide forms a trimeric aggregate as its collagenous domain folds into a triple-helical configuration. As judged by circular dichroism this triplehelical segment has a melting temperature of 45°C. Except when otherwise stated, all experiments were performed with trimeric pal
to prevent ,renaturation to the trimeric form (Bruckner & Engel, personal communication). The peptide fragment pal-CBO.l Co1 2 which consists of 11 amino acids from the precursor-specific part in pal(I) chain and 19 amino acids from the N-terminal end of al(I) chain (Fietzek & Kiihn, 1976) was prepared from a collagenase digest of calf procollagen. It was partially purified by chromatography on Bio-Gel P-10 and phosphocellulose (HGrlein, personal communication) and finally adsorbed to a DEAE-cellulose column equilibrated in 8 k urea, 5 mM Tris-HCl, pH 8.6. Application of a linear gradient to the column from 0 to 0.1 M NaCl eluted a peptide which resembled in its amino acid composition the sequence of oal-CB 0.1 Co1 2 (Fietzek & Kiihn, 1976). Type III procollagen was purified from fetal bovine skin (Timpl et al., 1975). The precursor-specific aminoterminal peptide pal(IIItCo1 l-3 was obtained by digestion with collagenase and was purified on Sephadex G-50 and DEAE-cellulose. It consists like pal-CBO.1, the comparable peptide from type I procollagen, of both a globular and a collagenous domain. Apparently, interchain disulfide bonds in this peptide prevent the degradation of the collagenous domain by collagenase (Nowack, Olsen & Timpl, 1976~). Reduction of peptides with dithioerythritol and S-carboxymethylation was carried out in 6 M guanidine (Furthmayr et al., 1972). Reduced peptides were digested with TPCK-trypsin (Worthington) in 0.2 M ammonium bicarbonate ~$7.9 at an enzyme-substrate ratio of 1:lOO for 24 hr at 37°C. Chromatograuhv on Bio-Gel P-10 showed that the digestion was c&pi&. The amino acid composition was determined after hydrolysis with 6 M HCI (24 hr, 110°C) on a Durrum D-500 analyser. The homogeneity of antigens was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate (Furthmayr &’ Timpl, 1971). Antisera
Rabbit antisera to procollagen and to the native or reduced peptide pal-CB0.1 Co1 1 were those used in previous studies (Timpl et al., 1973; Rohde et al., 1976). The same schedule was used to immunize rabbits with two 1 mg doses of pal(I) chains or with pa2 chain (2 x 1 mg)
969
Antigenicity of Procollagen or with peptide palCB0.1 (2 x 0.1 mg). Blood was collected from the animals over a period of several weeks starting 18 days after the booster injection. Antisera to collagen were prepared as described by Becker et al. (1976a). Female Sprague-Dawley rats were immunized with pa2 chains. In the tirst subcutaneous injection 0.2 mg pa2 was administered together with complete Freund’s adjuvant. An intraperitoneal booster with 0.1 mg pct2 but without adjuvants was given on day 23 and the animals were bled on day 42 by cardiac puncture. Antisera against rabbit immunoglobulin G were prepared in several goats (Lotter & Timpl, 1975). Radioimmune assays
Antigens were labeled with iodine-125 by the chloramine T method (McConahey & Dixon, 1966) following the procedure of Rohde et al. (1976). Usually, labeled peptides were separated from free iodine by dialysis. In the case of the small peptide pal_CB 0.1 Co1 2, separation of bound from unbound label was achieved on a Bio-Gel P-2 column which was equilibrated in phosphate-buffered
saline pH 7.2 containing 0.1% bovine serum albumin (PBS/BSA). The amount of label bound to peptide was determined by precipitation with trichloroacetic acid and was in the range of 8&90% except for pa2 chains where it was 40%. Labeled pal-CB 0.1 Co1 2 was not precipitated by trichloroacetic acid but binding to antibody (see Results, Fig. 4) indicated that at least 65% of its radioactivity was peptide bound. Usually, preparations of pa2 show a purity of 95-98% in electrophoresis and were mainly contaminated by p/l components (cross-linked pa chains). Examination of labeled pa2 by electrophoresis showed that about half of the label migrated with the mobility of pa2 while the rest was associated with the higher mol. wt components. Presumably, the low labeling efficiency reflects the low tyrosine content in pa2 chains (Becker et al., 1976b). Binding of labeled antigens by antibody was determined in duplicate in tubes containing 0.1 ml antiserum dilution, 0.1 ml antigen and 0.2 ml PBSBSA. Immune complexes were precipitated with goat antiserum to rabbit immunoglobulin G. The amount of labeled antigen added to the reaction was varied according to their mol. wts: 20 ng pa2 chain, 3 ng pal_CB 0.1, 2 ng pal-CB 0.1 Co1 1 and 0.2 ng pal-CB 0.1 Co1 2. Antigen binding capacity was calculated according to Minden & Farr (1973). In the inhibition tests antibodies were pre-incubated with unlabeled antigens prior to the addition of labeled antigen (Lotter & Timpl, 1975). Antigen concentrations were determined on the amino acid analyser after hydrolysis of aliquotes of the inhibitor solutions with 6 M HCI. In calculating molar equivalents, the mol. wts assumed for
the various peptides were those given by Becker et al. (1976b). RESULTS Antigenic properties pcrl(1) chain
of the precursor-spectfic
Pooled antisera to procollagen bound ‘251-labeled peptides (Table 1) obtained from the aminoterminal region of pal(I) chain either by cleavage with cyanogen bromide (pctl-CB0.1) or by digestion with collagenase (pc&CB 0.1 Co1 1). A similar reactivity was observed with antisera raised against pal(I) chains or the procollagen peptides. Reduction and alkylation of the procollagen peptide markedly decreased or even abolished its ability to combine with antibody. Previous studies have shown that these antisera show minimal or no reactions with collagen (Timpl et al., 1973; Rohde et al., 1976). The relative contribution of various regions in pal(I) to its antigenicity was studies by inhibition assays using antibodies against various antigens. These antibodies were reacted against the labeled peptide palLCB 0.1 which consists of the globular and collagen-like domain. As shown in Fig. 2 this peptide was a strong inhibitor both in trimeric, triple-helical and monomeric form. The smaller collagenase-derived peptide, pal-CB 0.1 Co1 1, comprising the globular domain also almost completely inhibited the reaction of antibodies. However, since the slope of the inhibition curve was less steep than observed with pctlLCB 0.1 it is likely that it has a lower affinity (Fig. 2b). Essentially no cross-reaction was observed with the precursor-specific peptide from type III procollagen although it also consists of a globular and collagenous domain. Since the globular region is the most important antigenic part but apparently not completely identical to the whole precursor-specific segment we also studied the reaction of antibodies to the peptide pal-CB 0.1 Co1 1 with labeled pal_CB 0.1 Co1 1 by inhibition assays (Fig. 3). In this system both peptides pal-CBO.l and pal-CB 0.1 Co1 1 as well as procollagen gave superimposable inhibition curves. Procollagen peptides derived from sheep or calf procollagen showed complete cross-reaction (Fig. 3a). pal(I)
Table 1. Binding by various antisera of 1251-labeled procollagen peptides
Antiserum to Procollagen pal(I) chain p&CB 0.1 palCB 0.1 co1 1 palCB 0.1 Co1 1, reduced
No. pool 709* 710* 728* 729b 667 670 707 713”
part of
pg Peptide bound per ml antiserum” palCB 0.1 pcrl_CB 0.1 co1 1, pal-CB O.lb co1 1 reduced 462.0 176.2 66.0 43.5 22.2 138.6 nd. nd. n.d.
’ Calculated according to Minden & Farr (1973). * Antigens from sheep; all others were of bovine origin. n.d. = not determined.
83.7 n.d. n.d. nd. n.d. 14.1 5.9 < 0.006 <0.006
0.29 < 0.006 0.014 < 0.006 < 0.006 0.47 0.23 0.13 0.30
H. ROHDE, U. BECKER, H. NOWACK and R. TIMPL
970 lCO-
0/-g-W
a)
,x X’
CL
x’
10’
,
/’
0x
I
I
10-2
o---+-o
I
102
100
10-2
100
102
Inhibitor [pMole]
Fig. 2. Comparison of various procollagen peptides by radioimmune inhibition assay. Antisera against peptide palCB 0.1 (No. 728, a) or against pal(I) chain (No. 709, b) were reacted with ‘251-labeled pal-CB 0.1. Inhibitors were peptides pctl-CB 0.1 (c+-o), monomeric pc+CB 0.1 (M), pal-CB 0.1 Co1 1 (x---x), and pal(II1) Co1 l-3 from bovine type III procollagen (Cl---O). All inhibitors except the last were from sheep.
observed with antisera to pctl-CB0.1 Co1 1. The binding capacity of the antisera except the latter one was in the range l-10 ng pal--CB 0.1 Co1 2/ml which on a molar basis is about lOOO-foldless binding activity than observed with the large procollagen peptides (see Table 1).
chains were strongly inhibitory but differed by a factor of 2-5 in inhibiting potency when compared with pal-CB0.1 Co1 1. No inhibitory activity could be detected on pa2 chains even when tested in 5&100-fold higher concentration than pal(I) chains. The possible occurrence of minor populations of antibody directed against precursor specific regions in pal(I) other than the globular domain was examined by direct binding studies. The collagen-like part has yet not been isolated as a distinct fragment and could therefore not be used in this study. However, another small fragment pul
Lack of antibody response to pu2 chain
Since pu2 chains did not react with antibody to the aminoterminal segment in pal(I) chain (Fig. 3) it was of interest to determine the response of rabbits or rats after injection with pa2 chains. Incubation of undiluted antisera to pu2 chain with 1251-labeled pa2 showed that less than 10% of the peptide-bound radioactivity was bound. However, some of the anti-pa2 chain antisera showed a distinct although weak reaction with labeled peptides from pal(I) chain. Presumably, the pa2 preparation used for immunization contained sufficient amounts of contaminating
two tyrosine residues and could therefore be labeled with lz51. Binding studies with labeled pc&CB 0.1 Co1 2 showed a distinct reaction with antisera to procollagen or pal(I) chain and also with antisera to type I collagen (Fig. 4). Essentially no binding was
bl
al
I
I
to-’
____
I
10’
I
103
I
I
10-Z
I
I
100
I
I
102
Inhibitor [ pMole]
Fig. 3. Radioimmune inhibition assay with antisera to the globular region in procollagen (peptide pal-CB 0 1 Co1 1) and ‘251-labeled pal
971
Antigenicity of Procollagen
-60
\
Antiserum
dilution
Fig. 4. Binding of ‘ZSI-labeled peptide pctlCB 0.1 Co1 2 by antisera to procollagen ( x ~ x ), collagen (M), pal(I) chain (Cl---U) and peptide pctl-CB 0.1 Co1 1 (A-A). Immune complexes were precipitated by goat antiserum to rabbit IgG. pal(I) to provoke these antibodies. These contaminants were probably cross-linked p/l components or disulfide-linked procollagen both of which chromatograph near the region of pa2 chains on CM cellulose. No binding of pz2 chains was also observed with antisera to procollagen, pal(I) chain and the peptides pal-CB 0.1 and pa-CB0.1 Co1 1. Identification of antigenic determinants shared by the natioe and unfolded globular region of pal(I) chain The effect of reducing all disulfide bonds in the globular region of pal(I) chain on inhibiting activity is shown in Fig. 5. Reduced and alkylated peptides did not inhibit the reaction of most of the antisera with native peptides except when used at 5000-10,000-fold higher concentrations than native peptides (Fig. 5a). A few antisera have been found which show a weak reaction with the labeled. un-
folded peptide (Table 1). These particular antisera could be distinctly inhibited by low concentrations of unfolded peptide but the extent of inhibition did not exceed 10% (Fig. 5b). The data may indicate that native and unfolded peptides have antigenic determinants in common but the antibody levels directed toward these determinants are far lower than those reacting with conformational determinants. Alternatively, sufficient levels of unfolded peptide may occur in the supposedly native preparation to elicit antibodies. To distinguish between these possibilities we examined the binding of antibodies to procollagen or pal-CB 0.1 Co1 1 with labeled reduced pal-CB 0.1 Co1 1 by inhibition assays. In the case of shared determinants we should observe a similar inhibition with both the native and unfolded peptide. On the other hand, an antibody reaction specific for the unfolded peptide alone should be inhibited much better by this peptide (Young & Leung, 1970). The examples shown in Fig. 6 support the first interpretation. Both the native and unfolded peptide completely inhibit the reaction even though there were distinct differences in the amount of antigen required to achieve 50% inhibition as well as in the slopes of the inhibition curves. Most likely these differences may reflect the relative proportions of different antibodies in the antisera. While unfolded peptide only reacts with a minor population of antibody, native peptide is able to react with all antibodies and so higher amounts of peptides are required to reach equivalent levels in inhibition. Antibodies against determinants shared by native and unfolded peptide could also be efficiently inhibited by a tryptic digest prepared from reduced palCB 0.1 Co1 1 (Fig. 6). Therefore, it should be possible to locate some of these determinants by using individual tryptic peptides. Antibodies to the shared determinants could also be inhibited by the reduced and alkylated precursorspecific peptide from type III procollagen (Fig. 6). About lOO-fold greater amounts were required for a significant reaction when compared with the unfolded peptide from type I procollagen. Considerably less
./’
b)
/ .
I
102
10-l
Inhibitor [ pMok]
Fig. 5. Comparison of native and reduced and alkylated procollagen peptides by radioimmune assay. Antisera against native peptides pal-CB 0.1 (a) and pal-CB 0.1 Co1 1 (b) were reacted against 1251-labeled native peptides pal-CBO.l and pal-CB 0.1 Co1 1, respectively. Inhibitors of these reactions were native pal-CB 0.1 (M), reduced and alkylated pal-CB 0.1 (A---A), native palCB 0.1 Co1 1 (M) and reduced and alkylated palCB 0.1 Co1 1 (A---A).
912
H. ROHDE, U. BECKER, H. NOWACK and R. TIMPL
10-l
10'
Inhibitor [pMole]
Fig. 6. Demonstration of antibodies to sequential antigenic determinants in the globular peptide palCB 0.1 Co1 1. Antisera No. 670 (a) and 667 (b) to peptide pal-CB 0.1 Co1 1, and to procollagen (c) were reacted against iz51-labeled, reduced and alkylated pal-CB 0.1 Co1 1. Inhibitors were native peptide palCB 0.1 Co1 1 (M), reduced and alkylated pal-CB 0.1 Co1 1 (x--x ), a tryptic digest of reduced pal-CBO.1 Co1 1 (O---O), and reduced and alkylated peptide pal(II1) Co1 l-3 from type III procollagen (A---A). cross-reactivity has been observed between the native peptides (Fig. 2). The data resemble therefore previous observations (Arnon & Maron, 1971) that sequential determinants in proteins show higher cross-reactivity than conformational determinants. Specificity of the antibody response to the unfolded globular region Antisera prepared against reduced and alkylated p&CB 0.1 Co1 1 bound to the unfolded but not to the native peptide (Table 1). The specificity of the antibodies was further studied by inhibition assay (Fig. 7). While reduced p&CB 0.1 Co1 1 was an effeceven concentrations, low at tive inhibitor 5000-lO,OO@fold greater amounts of native peptide did not completely inhibit the reaction. DISCUS!SION
Previous studies have shown that the precursorspecific segment of procollagen from dermatosparac'OQr
tic animals is a globular peptide whose conformation is stabilized by intra-chain disulfide bridges (Furthmayr et al., 1972). More recent studies demonstrated an additional segment with a typical collagen sequence (Becker et al., 1976b) and the presence of a short non-helical segment in this region (Fietzek & Kuhn, 1976). It was therefore of interest to compare the contribution of each domain to the immunological properties of procollagen. The present study confirms and extends previous observations (Timpl et al., 1973) that for antigenic activity the globular domain of pal(I) chain is the most important region in this preparation of procollagen. The collagenous domain is apparently only weakly immunogenic but may modulate to some extent antigenic expression of the determinants in the globular part (Fig. 2). Similar differences between the cyanogen bromide and collagenase-derived procollagen peptides could also be detected by hemagglutination-inhibition (Becker et al., 1976b) and have also been reported in a study on chick procollagen (von
x
al
T8o 2 5 :E 60 .g
/ x
C 40-
/
1.1 -Lo4o I
16'
10'
I
I
103
I
I
10-2
100
102
Inhibitor [pMole]
Fig. 7. Radioimmune inhibit& assay with antisera Nos. 713 (a) and 707 (b) against reduced and alkylated peptide palCB 0.1 Co1 1. The ‘251-labeled antigen was reduced and alkylated pal-CB 0.1. Co1 1. Inhibitors were native peptide palCB 0.1 Co1 1 (o----O) and reduced and alkylated pal-CB 0.1 Co1 1 ( x -x ).
913
Antigenicity of Procollagen der Mark et al., 1973). Since the major antigenic determinants in the globular region are conformation dependent it is likely that the influence of the collagenous domain is related to conformational alterations. However, antisera produced against the globular procollagen peptide did not distinguish between the peptide and procollagen. Apparently, the conformational changes produced by removing the collagenous from ‘the globular domain are very small and are not recognized regularly in the antibody response. A weak reaction was detected against the non-helical segment (peptide pctl-CB 0.1 Co1 2) which joins the precursor-specific portion of pal(I) with the aminoterminal end of ml(I) chain. An antigenic determinant of collagen has been previously located in this region (Rauterberg et al., 1972) which explains why antibodies to collagen react with pcz_CB 0.1 Co1 2. The presence in this peptide of determinants unique to procollagen remains to be determined. There was at least no cross-reaction with antibodies to the globular procollagen peptide pc&CB 0.1 Co1 1. The pa2 chain did not react with antibody prepared against pal(I) and in addition showed essentially no immunogenic activity. The precursor-specific, aminoterminal segment of pa2 mainly consists of a collagenlike sequence (Becker et al., 1976; Becker, Fietzek and Timpl, unpublished). It is however, still possible that an intact pct2 chain containing the carboxyl segment possesses different immunogenic properties. Antibodies to determinants of pal(I) chain showed also no or only little reaction with a peptide unique to the aminoterminal region in pctl(II1) chain of type III procollagen. A strong antibody response can be produced against type III procollagen and these antibodies also distinguish between type I and type III procollagen (Nowack et al., 1976a). The precursorspecific part of type III procollagen consists, like in pal(I) chain, of a large globular, a collagenous and a non-helical domain but showed significant differences in its amino acid composition (Nowack et al., 1976~).Thus, globular determinants are unique to distinct types of procollagen and determine their strong immunogenic activity. Two types of antigenic determinants could be demonstrated in the globular region. One type is unique for the native, disulfide-bonded structure, the other is shared by the native and by the reduced and alkylated peptide. Both sites differ in their immunogenic capacity and usually less than 10% of the total antibodies produced are directed toward unfolded determinants. The immunological findings agree with recent circular dichroism studies on the globular procollagen peptide (Bruckner & Engel, unpublished). The data suggest that the native peptide possesses regions which assume a distinct structure while other regions have an extended, linear conformation. Reduction and alkylation of all disulfide bonds destroys completely the conformation. The two kinds of determinants in the globular region of procollagen correspond to the two categories of conformational and sequential determinants to which Sela et al. (1967) have attributed the antigenicity of proteins. Whereas there is no doubt on the existence of conformational determinants in globular proteins, serious objections have been raised against evidence for sequential determinants (Crump-
ton, 1974; Sachs et al., 1972). Young and Leung (1970), for example, compared antibodies against native lysozyme with those against the unfolded protein and found no difference in their reaction patterns with unfolded lysozyme. Antigenicity detected in unfolded globular proteins may therefore be altematively explained by antibodies against denatured antigen which was present in the native preparation used for immunization. This explanation appears unlikely for procollagen since considerable differences were observed by inhibition assays between antibodies to sequential determinants in the native peptide and antibodies to the unfolded procollagen peptide. Rather the data indicate that denaturation unmasks determinants which are ordinarily buried in the region of the disulfide bridges. Trypsin treatment did not significantly change antigenie activity of the sequential determinants in the procollagen peptide. More recent studies with isolated tryptic fragments showed antigenicity in regions as small as a hexapeptide sequence (Rohde & Timpl, unpublished). It is unlikely that such small fragments exhibit a distinct conformation but it cannot be excluded that they become folded when bound to the antibody combining site (Crumpton, 1974; Sachs et a/., 1972). Since the precise localization of these determinants is feasible, information on the three-dimensional structure of the procollagen peptide should clarify conformation of those sites which are operationally defined here as sequential determinants. Acknowledgements-These studies were supported by Grants Ti 95/l and Ti 95/2 from the Deutsche Forschungs-
gemeinschaft.
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Arnon R. & Maron E. (1971) J. molec. Biol. 60, 225. Becker U., Nowack H., Gay S. & Timpl R. (1976a) Immunology 31, 51. Becker U., Timpl R., Helle 0. & Prockop D. J. (1976b) Biochemistry 15, 2853. Bornstein P. (1974) Ann. Rev. Biochem. 43, 567.
Crumpton M. (1974) The Antigens, (Edited by Sela M.), Vol. 2, p. 1. Academic Press, NY. Fietzek P. P. & Kiihn K. (1976) Int. Rev. Connective Tissue Res. I, 1. Fjelstad M. & Helle 0. (1974) J. Pathol. 112, 183. Furthmayr H. & Timpl R. (1971) Analyt. Biochem. 41, 510. Furthmayr H., Timpl R., Stark M., Lapiere C. M. & Kiihn K. (1972) FEBS lett. 28, 247. Hahn E., Nowack H., GGtze d. & Timpl R. (1975) Eur. J. Immun. 5, 288.
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