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Antigenic relatedness of primate procollagens as determined by a competitive radioimmunoassay
Comp. Biochent Physiol., 1978, Vol. 59B, pp. 47 to 49. Pergamon Press. Printed in Great Britain
ANTIGENIC RELATEDNESS OF PRIMATE PROCOLLAGENS AS DETERMINED BY A COMPETITIVE R A D I O I M M U N O A S S A Y MARK B. TAUBMAN AND BURTON GOLDBERG The Department of Pathology, New York University Medical Center, New York, N.Y. 10016, U.S.A.
(Received 14 May 1977) Abstract--l. A radioimmunoassay specific for the nonhelical carboxy terminal portion of human type
I procollagen was used to study the antigenic relatedness of primate procollagens. 2. The assay identified reactive antigen in primate sera and in the media of primate fibroblast cultures. 3. The displacement curves generated in the assay indicated that human and ape type I procollagens have antigenically identical carboxy terminal determinants which are partially cross-reactive with those from Old and New World Monkeys.
Media were dialyzed against H20, lyophilized, and dissolved in the buffer system for the radioimmunoassay.
INTRODUCTION
Procollagen, the precursor of collagen, differs from the collagen molecule by having additional nonhelical sequences, "propeptides", at the amino and carboxy termini. The conversion of procollagen to collagen is effected by enzymatic removal of the propeptides. In the case of type I procollagen, an enzymatic excision at the carboxyterminus (Byers et al., 1975; Fessler et al., 1975; Tanzer et al., 1974) releases the propeptides as a disulfide-linked, three-chain fragment of about 75,000 daltons (Goldberg et al., 1975). We have developed a radioimmunoassay (RIA) specific for the carboxy terminal, disulfide-linked fragment of human type I procollagen. We have reported that human serum contains antigenically indistinguishable material, whereas sera of several other mammalian species contain partially cross-reactive molecules (Taubman et aL, 1974). We have now extended our original studies by testing sera from the three anthropoid superfamilies: Hominoidea (man and apes), Cercopithecoidea (Old World monkeys) and Ceboidea (New World monkeys). It appears that the Hominoidea have antigenically identical carboxyterminal determinants; and additionally, these determinants may be distinguished from those of the Cercopithecoidea and Ceboidea.
RESULTS
Figure 1 illustrates a representative RIA in which purified standard antigen and sera from human, orangutan (Pongo pygmaeus; ape), Guinea baboon (Papio papio; Old World monkey), and night monkey (Aotus trivirgatus; New World monkey) were used as competing antigens. Both the human and orangutan sera displaced more than 959/o of the 125i.antigen and gave curves identical to that generated by the unlabeled standa.rd antigen. These results are consistent with antigenic identity between the standard antigen and the reactive material in human and ape sera (Hunter, 1971). The sera of four other hominoid species (Gor-
illa gorilla, Pan troglodytes, Symphalangus syndactylus, Hylobates lar) were tested and these also gave displacement curves of antigenic identity. Figure 1 also shows that the baboon and Aotus sera were unable to compete for more than 52~o of the 125I-antigen and that they generated curves with slopes different from that of the standard. Sera from 12 other species of Old World monkeys (Erythrocebus patas, Cerco-
pithecus aethiops, Mandrillus sphinx, Theropithecus ng STANDARD ANTIGEN
0.1 MATERIALS AND METHODS
1.0
10
100
100
o~r=~
~EJ
HUMAN
The purification of the carboxyterminal fragment of human type I procollagen and procedures for the generation of rabbit antiserum directed against this fragment have been previously reported (Sherr et al., 1973). Radioimmunoassays were performed as detailed in an earlier report (Taubman et al., 1974): dilutions of competing sera, culture media, or standard antigen were incubated with rabbit antiserum for 2hr at 37°C. 125I-antigen was added and samples were incubated for I hr at 37°C and 36 hr at 4°C. Immune complexes were precipitated with sheep antiserum to rabbit 7S 3,-globulin, and washed precipitates were counted in a 3'-spectrometer. Competing sera were stored at -20°C, and thawed and centrifuged at 2500 g prior to use in the assay. Competing culture media (serum-free) were collected after 24 hr of incubation with confluent layers of primate fibroblasts.
S~NOAR
G~
.~__.,&__-A---cz B I ~ O N (ow;
4o ~
-_:-L
20 0.1
1.0
10
100
pl SERUM Fig. l. Radioimmunoassay: displacement curves generated by primate sera. (NW): New World monkey; (OW): Old World monkey; Standard: purified antigen derived from the carboxy terminus of human type I procollagen.
47
48
MARK B. TAUBMANAND BURTONGOLDBERG Table 1. Antigenic relatedness of procollagens in primate sera and culture media
A. Serum Hominoids: Human Apes (5) Old World Monkeys (13) New World Monkeys (4) B. Culture medium Hominoids: Human Apes (2) Old World "Monkeys (2)
Ratio of slopes
S.D.
N
0.99 0.97 0.60 0.52
+ 0.05 + 0.08 _ 0.12 _+ 0.10
20 15 34 14
1.01 0.93 0.49
_+ 0.06 + 0.09 _ 0.05
5 2 4
Slopes were calculated from the linear portion (20-80% displacement range) of curves plotted as in Figs 1 and 2. Slopes from sera and culture media were divided by the slope of the standard antigen to give ratios. S.D.--standard deviation; N: total number of samples tested. Numbers in parentheses represent the number of different species tested.
gelada, Papio cynoeephalus, Cercocebus atys, Cynopithecus niger, Macaca arctoides, Macaca nemestrina, Macaca fascicularis, Macaca silenus, Macaca mulatta) and 3 additional species of New World monkeys
(Cebus capucinus, Lagothrix laoothricha, Ateles sp) generated curves similar to those shown for baboon and Aotus. Such displacement curves indicate that Old and New World monkey sera contain molecules which share only some of the antigenic determinants of the earboxyterminal propeptides (Hunter, 1971). To quantify the antigenic cross-reactions detected in the RIA, we computed the slopes of the linear portions (20-80% displacement range) of the various displacement curves. The slopes of curves generated by sera were then divided by the slope generated by the standard antigen (Table 1A). The average ratios thus obtained show that the slopes from human and ape sera did not differ significantly from the standard slope or from each other (P > 0.3), whereas slopes from Old and New World monkey sera differed significantly from the slope of the standard antigen (P < 0.01). Culture media from primate fibroblast cultures were also tested as sources of competing antigens in the RIA. Figure 2 shows that media from strains of
ng S T A N D A R D
0.1
ANTIGEN
1,0
10
I
I
100 _J
100 I
HUMAN CL
.O....O . . . . . . . . . . . .
,.0
8o; STANO,~RD
,
~ 7, 6O
O"
BABOON (OW) .~
RHESUS(ow)
0/
~
20 0.001
0.01
0.1
1.0
10.0
pg MEDIUM
Fig. 2. Radioimmunoassay: displacement curves generated by primate fibroblast culture media.
human, gorilla (Gorilla gorilla; ape), and chimpanzee (Pan troglodytes; ape) fibroblasts generated curves consistent with antigenic identity to the standard antigen, whereas culture media from baboon (Papio cynocephalus; Old World) and rhesus (Macaca mulatta; Old World) gave curves indicative of incomplete cross-reactivity. Quantitative data from such experiments are given in Table 1B and the agreement with the respective data from sera is evident. It is unlikely that homologous displacement curves for each primate group would have been artefactually generated when two independent sources of antigen were used. Accordingly, we conclude that we have demonstrated valid antigenic identities or cross-reactivities between the various primate procollagens. We have also tested several non-primate mammalian sera. These generated a variety of displacement curves, ranging from no detectable displacement in the case of rodents (outbred and inbred mice, rats, guinea pig, hamster) to curves of incomplete crossreactivity (sheep, horse, calf, mule) similar to those observed for Old and New World monkeys. The failure to detect antigen in rodent sera may be explained by the fact that our antisera were raised in rabbits, a rodent species. When rodent serum was mixed with human serum in the assay, a normal displacement curve was generated, indicating.that rodent serum does not interfere non-specifically in the RIA. Antiserum drawn at various times from the same rabbit or taken from different rabbits may detect different antigenic determinants and therefore generate different displacement curves (Hanke et al., 1973). We performed several experiments using early (4-6 weeks after primary injection) and late (5-7 months after primary injection) bleeds from several rabbits. Use of the different antisera apparently caused small changes in the slopes of the competition curves generated by each primate group; however, no antiserum was able to distinguish between the propeptide antigens of human and apes, and each antiserum preserved the differences between this class of antigens and those in sera of Old and New World monkeys. In addition, a few antisera yielded greater differences between the slopes generated by Old and New World
Antigenic relatedness of primate procollagens monkey sera, suggesting that the propeptides of these two groups may be antigenically distinguishable.
DISCUSSION
Our data indicate that human and ape procollagens have antigenically' identical carboxyterminal determinants which may be distinguished from those of Old and New World monkeys. Similar immunologic comparisons have been made for other primate proteins and the similarities and differences have been related to the phylogenetic relationships among the primates (Prager & Wilson, 1971; Sarich & Wilson, 1967). For example, divergence in antigenicity between like proteins of different anthropoid species has been compared with the time elapsed since these species shared a common ancestor. Our demonstration of cross-reactivity among procollagens of widely divergent species indicates some conservation of structure. The amino and carboxy terminal sequences of the procollagens are assumed to function in molecular assembly, secretion, and fibrillogenesis (Speakman, 1971). Each function could represent a selective pressure for conservation of structure. It is not known if the propeptides serve other functions once they are enzymatically cleaved from the precursor molecule. However, there are many precedents for the participation of such split products in physiologic reactions (Neurath & Walsh, 1976). We have evidence (unpublished work) that the procollagen antigen in normal serum is the cleaved carboxy terminal fragment rather than the intact procollagen molecule. The persistence of the fragment in serum raises the possibility that it might have Some biological activity, as yet undefined. Acknowledgements--We thank Dr G. Kuhn, III, of Yerkes Regional Primate Research Center, Atlanta, GA; Dr L. Miller, N. I. H., Laboratory of Parasitic Diseases, Bethesda, MD; Dr W. Socha, Laboratory for Experimental Medicine and Surgery in Primates, Tuxedo, N.Y.,; and Dr C. J. Sherr, Viral Leukemia and Lymphoma Branch, National Cancer Institute, Bethesda, MD for generously supplying primate sera and culture media.
C.B.P.59/I B--D
49
This study was supported by N. I. H. Grant No. 1 RO1 HL 1755-02. M. B. Taubman is the recipient of N. I. H. Medical Scientist Fellowship No. 5-T05-GM-01668-13.
REFERENCES
BYERS P. H., CLICK E. M., HARPER E. & BORNSTEIN P. (1975) Interchain disulfide bonds in procollagen are located in a large nontriple-helical COOH-terminal domain. Proc. natn. Acad. Sci. U.S.A. 72, 3009-3013. FESSLERL. I., MORRIS N. P. & FESSLER J. H. (1975) Procollagen: Biological scission of amino and carboxyl extension peptides. Proc. natn. Aead. S c i . U.S.A. 72, 4905-4909. GOLDBERGB., TAUBMANM. B. & RADINA. (1975) Procollagen peptidase: its mode of action on the native substrate. Cell 4, 45-50. HANKEN., PRAGERE. M. & WILSONA. C. (1973) Quantitative immunological and electrophoretic comparison of primate lysozymes. J. biol. Chem. 248, 2824-2828. HUNTER W. M. (1971) The preparation of radioiodinated proteins of high activity, their reaction with antibody in vitro: the radioimmunoassay. Handbook of Experimental Immunology (Edited by WEIR D. M.), Chap. 17, F. A. Davis Co., Philadelphia. NEURATH H. & WALSH K. A. (1976) Role of proteolytic enzymes in biological regulation. Proc. natn. Acad. Sci. U.S.A, 73, 3825-3832. PRAGER E. M. & WILSON A. C. (1971) The dependence of immunological cross-rcactivity upon sequence resemblance among lysozymes. J. biol. Chem. 246, 5978-5989. SARICH V. M. & WILSON A. C. (1967) Rates of albumin evolution in primates. Proc. natn. Acad. Sci. U.S.A. 58, 142-148. SHERRC. J., T^UaMANM. B. & GOLDBERGB. (1973) Isolation of a disulfide-stabilized, three-chain polypeptide fragment unique to the precursor of human collagen. J. biol. Chem. 248, 7033-7038. SPEAKMAt
D. E. & PARt E. D. (1974) Procollagen: intermediate forms containing several types of peptide chains and non-collagen peptide extensions at NH 2 and COOH ends. Proc. natn. Acad. Sci. U.S.A. 71, 3009-3013. TAUBMAN M. B., GOLDBERG B. & SHERR C. J. (1974) Radioimmunoassay for human procollagen. Science 186, 1115-1117.
ANTIGENIC RELATEDNESS OF PRIMATE PROCOLLAGENS AS DETERMINED BY A COMPETITIVE R A D I O I M M U N O A S S A Y MARK B. TAUBMAN AND BURTON GOLDBERG The Department of Pathology, New York University Medical Center, New York, N.Y. 10016, U.S.A.
(Received 14 May 1977) Abstract--l. A radioimmunoassay specific for the nonhelical carboxy terminal portion of human type
I procollagen was used to study the antigenic relatedness of primate procollagens. 2. The assay identified reactive antigen in primate sera and in the media of primate fibroblast cultures. 3. The displacement curves generated in the assay indicated that human and ape type I procollagens have antigenically identical carboxy terminal determinants which are partially cross-reactive with those from Old and New World Monkeys.
Media were dialyzed against H20, lyophilized, and dissolved in the buffer system for the radioimmunoassay.
INTRODUCTION
Procollagen, the precursor of collagen, differs from the collagen molecule by having additional nonhelical sequences, "propeptides", at the amino and carboxy termini. The conversion of procollagen to collagen is effected by enzymatic removal of the propeptides. In the case of type I procollagen, an enzymatic excision at the carboxyterminus (Byers et al., 1975; Fessler et al., 1975; Tanzer et al., 1974) releases the propeptides as a disulfide-linked, three-chain fragment of about 75,000 daltons (Goldberg et al., 1975). We have developed a radioimmunoassay (RIA) specific for the carboxy terminal, disulfide-linked fragment of human type I procollagen. We have reported that human serum contains antigenically indistinguishable material, whereas sera of several other mammalian species contain partially cross-reactive molecules (Taubman et aL, 1974). We have now extended our original studies by testing sera from the three anthropoid superfamilies: Hominoidea (man and apes), Cercopithecoidea (Old World monkeys) and Ceboidea (New World monkeys). It appears that the Hominoidea have antigenically identical carboxyterminal determinants; and additionally, these determinants may be distinguished from those of the Cercopithecoidea and Ceboidea.
RESULTS
Figure 1 illustrates a representative RIA in which purified standard antigen and sera from human, orangutan (Pongo pygmaeus; ape), Guinea baboon (Papio papio; Old World monkey), and night monkey (Aotus trivirgatus; New World monkey) were used as competing antigens. Both the human and orangutan sera displaced more than 959/o of the 125i.antigen and gave curves identical to that generated by the unlabeled standa.rd antigen. These results are consistent with antigenic identity between the standard antigen and the reactive material in human and ape sera (Hunter, 1971). The sera of four other hominoid species (Gor-
illa gorilla, Pan troglodytes, Symphalangus syndactylus, Hylobates lar) were tested and these also gave displacement curves of antigenic identity. Figure 1 also shows that the baboon and Aotus sera were unable to compete for more than 52~o of the 125I-antigen and that they generated curves with slopes different from that of the standard. Sera from 12 other species of Old World monkeys (Erythrocebus patas, Cerco-
pithecus aethiops, Mandrillus sphinx, Theropithecus ng STANDARD ANTIGEN
0.1 MATERIALS AND METHODS
1.0
10
100
100
o~r=~
~EJ
HUMAN
The purification of the carboxyterminal fragment of human type I procollagen and procedures for the generation of rabbit antiserum directed against this fragment have been previously reported (Sherr et al., 1973). Radioimmunoassays were performed as detailed in an earlier report (Taubman et al., 1974): dilutions of competing sera, culture media, or standard antigen were incubated with rabbit antiserum for 2hr at 37°C. 125I-antigen was added and samples were incubated for I hr at 37°C and 36 hr at 4°C. Immune complexes were precipitated with sheep antiserum to rabbit 7S 3,-globulin, and washed precipitates were counted in a 3'-spectrometer. Competing sera were stored at -20°C, and thawed and centrifuged at 2500 g prior to use in the assay. Competing culture media (serum-free) were collected after 24 hr of incubation with confluent layers of primate fibroblasts.
S~NOAR
G~
.~__.,&__-A---cz B I ~ O N (ow;
4o ~
-_:-L
20 0.1
1.0
10
100
pl SERUM Fig. l. Radioimmunoassay: displacement curves generated by primate sera. (NW): New World monkey; (OW): Old World monkey; Standard: purified antigen derived from the carboxy terminus of human type I procollagen.
47
48
MARK B. TAUBMANAND BURTONGOLDBERG Table 1. Antigenic relatedness of procollagens in primate sera and culture media
A. Serum Hominoids: Human Apes (5) Old World Monkeys (13) New World Monkeys (4) B. Culture medium Hominoids: Human Apes (2) Old World "Monkeys (2)
Ratio of slopes
S.D.
N
0.99 0.97 0.60 0.52
+ 0.05 + 0.08 _ 0.12 _+ 0.10
20 15 34 14
1.01 0.93 0.49
_+ 0.06 + 0.09 _ 0.05
5 2 4
Slopes were calculated from the linear portion (20-80% displacement range) of curves plotted as in Figs 1 and 2. Slopes from sera and culture media were divided by the slope of the standard antigen to give ratios. S.D.--standard deviation; N: total number of samples tested. Numbers in parentheses represent the number of different species tested.
gelada, Papio cynoeephalus, Cercocebus atys, Cynopithecus niger, Macaca arctoides, Macaca nemestrina, Macaca fascicularis, Macaca silenus, Macaca mulatta) and 3 additional species of New World monkeys
(Cebus capucinus, Lagothrix laoothricha, Ateles sp) generated curves similar to those shown for baboon and Aotus. Such displacement curves indicate that Old and New World monkey sera contain molecules which share only some of the antigenic determinants of the earboxyterminal propeptides (Hunter, 1971). To quantify the antigenic cross-reactions detected in the RIA, we computed the slopes of the linear portions (20-80% displacement range) of the various displacement curves. The slopes of curves generated by sera were then divided by the slope generated by the standard antigen (Table 1A). The average ratios thus obtained show that the slopes from human and ape sera did not differ significantly from the standard slope or from each other (P > 0.3), whereas slopes from Old and New World monkey sera differed significantly from the slope of the standard antigen (P < 0.01). Culture media from primate fibroblast cultures were also tested as sources of competing antigens in the RIA. Figure 2 shows that media from strains of
ng S T A N D A R D
0.1
ANTIGEN
1,0
10
I
I
100 _J
100 I
HUMAN CL
.O....O . . . . . . . . . . . .
,.0
8o; STANO,~RD
,
~ 7, 6O
O"
BABOON (OW) .~
RHESUS(ow)
0/
~
20 0.001
0.01
0.1
1.0
10.0
pg MEDIUM
Fig. 2. Radioimmunoassay: displacement curves generated by primate fibroblast culture media.
human, gorilla (Gorilla gorilla; ape), and chimpanzee (Pan troglodytes; ape) fibroblasts generated curves consistent with antigenic identity to the standard antigen, whereas culture media from baboon (Papio cynocephalus; Old World) and rhesus (Macaca mulatta; Old World) gave curves indicative of incomplete cross-reactivity. Quantitative data from such experiments are given in Table 1B and the agreement with the respective data from sera is evident. It is unlikely that homologous displacement curves for each primate group would have been artefactually generated when two independent sources of antigen were used. Accordingly, we conclude that we have demonstrated valid antigenic identities or cross-reactivities between the various primate procollagens. We have also tested several non-primate mammalian sera. These generated a variety of displacement curves, ranging from no detectable displacement in the case of rodents (outbred and inbred mice, rats, guinea pig, hamster) to curves of incomplete crossreactivity (sheep, horse, calf, mule) similar to those observed for Old and New World monkeys. The failure to detect antigen in rodent sera may be explained by the fact that our antisera were raised in rabbits, a rodent species. When rodent serum was mixed with human serum in the assay, a normal displacement curve was generated, indicating.that rodent serum does not interfere non-specifically in the RIA. Antiserum drawn at various times from the same rabbit or taken from different rabbits may detect different antigenic determinants and therefore generate different displacement curves (Hanke et al., 1973). We performed several experiments using early (4-6 weeks after primary injection) and late (5-7 months after primary injection) bleeds from several rabbits. Use of the different antisera apparently caused small changes in the slopes of the competition curves generated by each primate group; however, no antiserum was able to distinguish between the propeptide antigens of human and apes, and each antiserum preserved the differences between this class of antigens and those in sera of Old and New World monkeys. In addition, a few antisera yielded greater differences between the slopes generated by Old and New World
Antigenic relatedness of primate procollagens monkey sera, suggesting that the propeptides of these two groups may be antigenically distinguishable.
DISCUSSION
Our data indicate that human and ape procollagens have antigenically' identical carboxyterminal determinants which may be distinguished from those of Old and New World monkeys. Similar immunologic comparisons have been made for other primate proteins and the similarities and differences have been related to the phylogenetic relationships among the primates (Prager & Wilson, 1971; Sarich & Wilson, 1967). For example, divergence in antigenicity between like proteins of different anthropoid species has been compared with the time elapsed since these species shared a common ancestor. Our demonstration of cross-reactivity among procollagens of widely divergent species indicates some conservation of structure. The amino and carboxy terminal sequences of the procollagens are assumed to function in molecular assembly, secretion, and fibrillogenesis (Speakman, 1971). Each function could represent a selective pressure for conservation of structure. It is not known if the propeptides serve other functions once they are enzymatically cleaved from the precursor molecule. However, there are many precedents for the participation of such split products in physiologic reactions (Neurath & Walsh, 1976). We have evidence (unpublished work) that the procollagen antigen in normal serum is the cleaved carboxy terminal fragment rather than the intact procollagen molecule. The persistence of the fragment in serum raises the possibility that it might have Some biological activity, as yet undefined. Acknowledgements--We thank Dr G. Kuhn, III, of Yerkes Regional Primate Research Center, Atlanta, GA; Dr L. Miller, N. I. H., Laboratory of Parasitic Diseases, Bethesda, MD; Dr W. Socha, Laboratory for Experimental Medicine and Surgery in Primates, Tuxedo, N.Y.,; and Dr C. J. Sherr, Viral Leukemia and Lymphoma Branch, National Cancer Institute, Bethesda, MD for generously supplying primate sera and culture media.
C.B.P.59/I B--D
49
This study was supported by N. I. H. Grant No. 1 RO1 HL 1755-02. M. B. Taubman is the recipient of N. I. H. Medical Scientist Fellowship No. 5-T05-GM-01668-13.
REFERENCES
BYERS P. H., CLICK E. M., HARPER E. & BORNSTEIN P. (1975) Interchain disulfide bonds in procollagen are located in a large nontriple-helical COOH-terminal domain. Proc. natn. Acad. Sci. U.S.A. 72, 3009-3013. FESSLERL. I., MORRIS N. P. & FESSLER J. H. (1975) Procollagen: Biological scission of amino and carboxyl extension peptides. Proc. natn. Aead. S c i . U.S.A. 72, 4905-4909. GOLDBERGB., TAUBMANM. B. & RADINA. (1975) Procollagen peptidase: its mode of action on the native substrate. Cell 4, 45-50. HANKEN., PRAGERE. M. & WILSONA. C. (1973) Quantitative immunological and electrophoretic comparison of primate lysozymes. J. biol. Chem. 248, 2824-2828. HUNTER W. M. (1971) The preparation of radioiodinated proteins of high activity, their reaction with antibody in vitro: the radioimmunoassay. Handbook of Experimental Immunology (Edited by WEIR D. M.), Chap. 17, F. A. Davis Co., Philadelphia. NEURATH H. & WALSH K. A. (1976) Role of proteolytic enzymes in biological regulation. Proc. natn. Acad. Sci. U.S.A, 73, 3825-3832. PRAGER E. M. & WILSON A. C. (1971) The dependence of immunological cross-rcactivity upon sequence resemblance among lysozymes. J. biol. Chem. 246, 5978-5989. SARICH V. M. & WILSON A. C. (1967) Rates of albumin evolution in primates. Proc. natn. Acad. Sci. U.S.A. 58, 142-148. SHERRC. J., T^UaMANM. B. & GOLDBERGB. (1973) Isolation of a disulfide-stabilized, three-chain polypeptide fragment unique to the precursor of human collagen. J. biol. Chem. 248, 7033-7038. SPEAKMAt
D. E. & PARt E. D. (1974) Procollagen: intermediate forms containing several types of peptide chains and non-collagen peptide extensions at NH 2 and COOH ends. Proc. natn. Acad. Sci. U.S.A. 71, 3009-3013. TAUBMAN M. B., GOLDBERG B. & SHERR C. J. (1974) Radioimmunoassay for human procollagen. Science 186, 1115-1117.