- Email: [email protected]
Native collagen fibrils from echinoderms are molecularly bipolar
J. Mol. Biol. (1994) 235, 73-79
Native Collagen Fibrils From Echinoderms are Molecularly Bipolar F. A. Thurmond~f and J. A. Trotter Department of A n a t o m y University of N e w Mexico School of Medicine Albuquerque, N M 87131, U . S . A .
Collagen fibrils are generally assumed to be cylinders with uniform diameters (except possibly at their ends) and to be composed of molecules all of which have the same polarity. These assumptions have been largely untested because of the extreme difficulty associated with isolating entire native fibrils. Intact collagen fibrils are readily extracted from certain echinoderms, however, and we have therefore analyzed the molecular structure of these fibrils. Our electron microscopic analyses show the above assumptions to be false: echinoderm fibrils, which previously have been shown to be symmetrically spindle shaped, are also molecularly bipolar. Their constituent molecules have their N-termini oriented toward the nearest fibril end, and they are antiparallel in the fibril center. The shape and molecular arrangement of these fibrils have implications for fibrillogenesis.
Keywords: Collagen; symmetry; antiparallel; fibril; echinoderm
Fibrillar collagens of echinoderms are similar to those of vertebrates in amino acid composition (Watson & Silvester, 1959; Piez & Gross, 1959; Travis et al., 1967; Katzman et al., 1969; Matsumura, 1973; Pucci-Minafra et al., 1978; Bailey, 1985; Shimizu et al., 1990; Kimura et al., 1993; Trotter & Koob, 1993), triple helix length (Shimizu et al., 1990; Trotter et al., 1993), cross-link chemistry (Bailey et al., 1982; Eyre & Glimcher, 1971, 1973; Van Ness et al., 1988; Trotter & Koob, 1993), gene structure (D'Alessio et al., 1989, 1990; Exposito et al., 1992a,b), and D-periodicity (Marks et al., 1949; Piez & Gross, 1959; Matsumura, 1973, 1974; Pucci-Minafra et al., 1978; Bailey et al., 1982; Bailey, 1985; Trotter & Koob, 1989). Many collagenous tissues of echinoderms differ from those of other phyla, however, in that their mechanical properties are regulated by the nervous system on a time scale of seconds (Wilkie, 1984; Motokawa, 1984). This physiological mutability is undoubtedly related to the capacity of the tissues to be dissociated by gentle procedures into intact, native fibrils (Matsumura, 1973, 1974; Trotter & Koob, 1989). We have utilized the dissoeiability of sea urchin spine ligaments and sea cucumber dermis to analyze the molecular packing of whole native fibrils.
The banding pattern of positively stained native echinoderm fibrils, which reflects the relative density of charged amino acids along the fibril length, is similar but not identical to that of mammalian fibrils (Fig. 1). The major differences are the relative faintness of the a 3 and b 2 bands, and the prominent intensity of the c 3 band in echinoderm fibrils (Trotter et al., 1993). The similarity between echinoderm and vertebrate banding patterns allows the amino-carboxy orientation of the molecules in echinoderm fibrils to be specified, as shown in Figure 1. Isolated echinoderm fibrils between 24 and 436 mm long have been shown to be symmetrically spindle shaped and geometrically similar (Trotter et al., 1993). The ratio of total length to maximum diameter is about 2,000 in sea cucumber fibrils (Trotter et al., 1993) and about 2,500 in sea urchin fibrils (Trotter & Koob, 1989). The symmetrical shape of the fibrils raises questions concerning the orientation and organization of their constituent molecules. If all the molecules in the fibril had the same N-C polarity, each fibril would also have an N-terminus and a C-terminus. Analysis of isolated echinoderm fibrils shows this not to be the case: the molecules in each haft of every fibril are oriented with their C-termini toward the fibril center, and their N-termini toward the nearest end. Echinoderm collagen fibrils are thus not only symmetrically spindle shaped, but are also
tAuthor to whom all correspondence should be addressed. 0022-2836/94/010073-07 $08.00/0
73
© 1994 Academic Press Limited
Communications
74 o
Table
1
Position of centrosymmetric region along fibril
NEG Fibril number
POS
L! ! i N
;
ii
'
!
:
i LJ
•
I L_i._l
dc
I b
c
I I.LLJ a
Figure 1. Comparisoh of the banding patterns observed in negatively (NEG) and positively (POS) stained native echinoderm collagen fibrils with that seen in a positively stained fibril reconstituted from mammalian type I collagen (bottom panel). The many similarities in the positions and intensities of bands in the positively stained fibrils allow the amino/carboxyl polarity of the echinoderm fibril to be aligned with that of the mammalian fibril (labelled N and C in the lower panel). Compared with the positively stained mammalian fibril, the echinoderm fibril shows reduced stain intensity in the a a and b 2 bands, and greatly increased intensity in the c a band (the bands are lettered from right to left, and the sub-bands within them are numbered from right to left, as described in Chapman (1985), but the numbers have not been included in the figure). Intact, native collagen fibrils were isolated from the spine ligaments of the sea urchin Eucidaris tribuloides, and positively and negatively stained with uranyl acetate as described (Trotter & Koob, 1989). The electron micrograph of the reconstituted mammalian type I fibril was kindly provided by Dr. John Chapman.
molecularly bipolar. A n o t h e r s u p e r p o l y m e r with this symmetrical molecular s t r u c t u r e is the native myosin filament of skeletal muscle (Davis, 1988). The bipolar symmetrical arrangement of molecules predicts t h a t in e v e r y fibril there is a region in which the molecular polarity is reversed. Indeed, e v e r y fibril examined (more t h a n 100) contained a single region in which the banding p a t t e r n was c e n t r o s y m m e t r i c (Figs, 2B, 3A). This c e n t r o s y m m e t r i e region, which was in the center of e v e r y fibril (Table 1), was only one to two D-periods long in most fibrils, and was flanked on both sides b y transitional zones four D-periods long (Figs. 2B, 3A, 4B). T h e banding p a t t e r n s observed in the transitional zones and in the c e n t r o s y m m e t r i c central zone can be a c c o u n t e d for b y either of the models shown in Figure 3A, b o t h of which are based on " H o d g e P e t r u s k a " model of the longitudinal a r r a n g e m e n t of collagen molecules (Hodge & P e t r u s k a , 1963). The u p p e r model has the following features: (a) there are equal n u m b e r s of molecules t h r o u g h o u t the entire fibril, including the region of p o l a r i t y reversal; (b) there are equal n u m b e r s of antiparallel molecules in the central (centrosymmetric) D-period, and
I 2 3 4 5 6 7 8 9 l0 Il 12 13
14 15 16 17
18 19 20 21
Species
Length (L) (~m)
SCt SC SU SC SC SC SC SC SC SC SC SU SC SC SC SC SC SU SU SU SU
36 l 13 115 132 139 159 167 175 188 207 212 236 251 258 274 285 290 302 334 456 1218
Distance to Symmetric D-Period (D) (~m)
D/L
17 56 55 65 66 77 81 88 94 102 104 95
0-47 0"50 0'48 0"49 0-47 0-48 0-49 0-50 0"50 0-49 0"49 0-40
120
(}'48
121 136 142
0"47 0.50 0.50 0-49 0-49 0"50 0"49 0"48
142
147 167 225 585
t SC, sea cucumber; SU, sea urchin.
changing ratios of antiparallel molecules in the transitional zones; and (c) the C-telopeptides of colinear antiparallel molecules a b u t t one another. In the lower model, in contrast, the n u m b e r of molecules increases from 1X in the " n o r m a l " asymmetric regions to 2X in the c e n t r o s y m m e t r i c D-period. The C-telopeptides of antiparallel molecules are in register, b u t do not a b u t t one another. This model predicts t h a t the central zone should be thicker t h a n the regions adjacent to it and, indeed, some of the fibrils examined had a bulge in the e x p e c t e d place (see Fig. 2B). D i a m e t e r m e a s u r e m e n t s were inconsistent, however, and a true test of this model will require mass determinations of the sort described by Holmes et al. (1992). In the central s y m m e t r i c D-period the c a bands of antiparallel molecules are in register, and the c2 bands of molecules of one polarity are aligned with the d bands of oppositely polarized ones (Fig. 4A). In addition, the a s band of one molecular polarity is in register with the a 4 band of the opposite polarity. This banding p a t t e r n has two planes of mirror s y m m e t r y , one passing through the c a band, and the other passing t h r o u g h the aa/a 4 region. The same a n t i p a r a l l e l packing, designated DPSt (for D-periodic symmetric) or DPS III, has been noted previously in fibrils reconstituted u n d e r certain conditions (e.g. elevated phosphate) from mammalian collagens (Doyle et al., 1974, 1975; Williams et al., 1978; Bruns, 1976; Piez, 1984), b u t it has not until recently been t h o u g h t to exist in native fibrils or to be functionally significant. However, the v e r y tAbbreviation used: DPS, D-periodic symmetric.
75
Communications .a.
l~'~,:q~-
• .,-.:."-I : .),~-."'.~' .:.... . ".. ::-~ ',,~ ~ ........
,,.
i:::!.
.
.
.
•
".:
- . ~ ~ ~
7
:
-..--.~.~;,,,, ~,'_:.-:
i :,.i: :,!. :
". : :;
i:i!:.:,:
I~:::"..: ."-...,....-..i" ..."'.":..-.,.,. k:~: :-":/~:/--:B
$~Ir
:
Figure 2. Electron micrographs of an entire fibril and of the central regions of 9 fibrils from sea urchin ligaments (SU) and from sea cucumber dermis (SC). A. An entire sea urchin fibril, 170/lm long is seen in the electron micrograph in the top panel. Its ends are marked (arrowheads). B. Positively and negatively stained central portions of fibrils similar to that in the boxed zone in A. Starting at the top left, the fibrils were from SU, SC, SU, SC. Similarly for the right hand side the fibrils were from SU, SU, SC, SU, SC. Note that the banding pattern is symmetrical in the regions marked by the vertical lines. In contrast, the banding pattern on both sides of the central region becomes polarized, with the amino termini of the constituent molecules oriented away from the center (compare these images with Fig. 1). There is a characteristic bulge in the central portion of some fibrils, where the symmetrical banding pattern is seen. Intact collagen fibrils were isolated from sea urchin ligaments and from sea cucumber dermis as described (Trotter & Koob, 1989). Fibrils were positively stained with uranyl acetate (A and the right side of B) or negatively stained with phosphotungstic acid (left side of B).
recent findings of H o l m e s et al. (1993), reported in the p a p e r following this one, show t h a t some verteb r a t e fibrils are also bipolar, and contain a " s w i t c h " region where the b a n d i n g p a t t e r n is D P S - I I I . The molecular model presented in the p a p e r b y H o l m e s et al. is identical to the u p p e r one shown in Figure 3A, except t h a t the antiparallel molecules are verti-
cally displaced so t h a t the C-telopeptides are axially aligned b u t do not a b u t t one another. The ability of the molecular models shown in Figure 3A to a c c o u n t for the banding p a t t e r n s observed b o t h in the c e n t r o s y m m e t r i c and in the transitional D-periods is shown in the c o m p u t e r g e n e r a t e d images of Figure 4. I n Figure 4A, the t o p
Communications
76 A
4 i
]D
1 I
•
,~ M
N
ID
,
•
4
i
B N
N "~,
C
o
N
mo,~ ~~_____~ C N
Figure 3. A. Molecular models that can account for the banding patterns seen in the middle of the negatively stained fibril by the antiparallel packing of oppositely polarized molecules. The carboxyl (c) termini (arrowheads at the ends of schematized molecules) are oriented toward the middle of the fibril. The C-telopeptides of individual molecules are located at the carboxyl end of the overlap zones. The N-termini of individual molecules are located at the amino end of the overlap zones. In the central, symmetrically banded portion of the fibril the D-period remains the same and the C-telopeptides (arrowheads) of oppositely polarized molecules are in register. This gives rise to the prominent, thin, stainexcluding (white) lines in this region. On either side of the region of complete symmetry (marked by the vertical dashed line), which is 1 D-period long, is a transitional region, 4 D-periods long, produced by the changing ratio of oppositely polarized molecules. B. Possible crosslinking configuration where the C-telopeptides of oppositely polarized molecules are in register. The left panel shows the 3-hydroxypyridinium crosslinks that occur in the normally polarized regions of vertebrate collagen fibrils (redrawn after Eyre, 1987). The right panel shows the hypothetical trivalent crosslink that could stabilize antiparallel molecules in the central zones of echinoderm fibrils. It was produced by rotating one molecule through an angle of 180° about an axis through the C-telopeptide. image is an electron mierograph of a parallel (asymmetric) region of a positively stained fibril with the amino ends of the molecules pointing toward the left; the next micrograph below it is the same image inverted left to right, and positioned so as to bring the c 3 bands into alignment. The arrows between these two images show the position of two selected molecules in each image. The third image down is a computer-generated addition of the first two, and the fourth image down is an electron micrograph of the centrosymmetric region of a native fibril. I t is apparent t h a t the third and fourth images correspond precisely. The centrosymmetric pattern is thus produced by equal numbers of antiparallel molecules arranged as shown.
In Figure 4B, the middle image was generated by combining two oppositely oriented fibrils aligned with their c 3 bands in register, as just described, except t h a t each fibril extended for ten D-periods. The density of each image was linearly attenuated so t h a t the last D-period on the left contained only molecules with amino ends toward the left, the last D-period on the right contained only those with amino ends to the right while the middle D-period (bracket) contained equal numbers of each. The middle D-period has the centrosymmetric banding pattern seen in Figure 4A, whereas the D-periods on either side of the central one gradually change to the usual asymmetric pattern. One way to follow the transition is to look at the width of the
Communications
77
N
C
f N
C:
I
I
11
a3a4
C3
a3a4
B
!
!
Figure 4. A computer-reconstruction that accounts for the symmetrical and transitional banding patterns in the central regions of positively stained fibrils. A shows in the top panel a portion of a fibril with its N-terminus toward the left. The next micrograph down shows the same fibril, but with its N-terminus towards the right and aligned such that the c 3 bands are in register. The arrows between these two micrographs represent two selected molecules from each orientation, with the arrowheads representing the C-termini. The 3rd panel down is a computer-generated image created by superimposing the 2 images above it. The 4th panel down shows the central region of a native fibril, positively stained with uranyl acetate. Note that the native and computer-generated banding patterns are identical. The C-telopeptides of the antiparallel molecules (arrowheads) are in register at the a3/a 4 bands, while the N-telopeptides of antiparallel molecules (short vertical lines) are separated by the length of the central triplet of the symmetrical pattern. B shows the I 1 central D-periods in a native fibril (lower panel), and above it a computer-generated image created by superimposing the images of left and right oriented fibril regions in which the density of the region with its N-terminus toward the left was decreased linearly from 1 to 0 over 9 D-periods, beginning at the second a2 band from the left, and the region with its N-terminus toward the right was treated similarly, but with the density decreasing toward the left. This is the density equivalent of the molecular model at the bottom of Fig. 3, which is represented here by the schematic in A. The superimposition of these two images produces uniform image intensity, but the ratio of the two banding patterns is 1:1 only in the central period (bracket), and changes linearly over the 4 D-periods toward the right and the left. This transition pattern closely matches the natural one.
78
Communications
unstained zone on either side of the ,central triplet (the middle band of which is the c a band). In the centrosymmetric D-period these two zones are equal in width. Moving toward the left, the zone to the left of the triplet becomes narrower as the zone to its right becomes wider. The opposite occurs moving to the right along the fibril. The bottom image in Figure 4B is a positively stained native fibril, the banding pattern of which corresponds closely to the computer generated image just described. The arrange'ment of antiparallel molecules t h a t produces the images seen in the centrosymmetric and transitional regions of native fibrils brings the C-telopeptides of antiparallel molecules into axial alignment, since they are located at the gap/overlap interface, in the region of the a3/a 4 bands. The bright (stain-excluding) band in the negatively stained central region (Figs 2B, 3A) probably represents the aligned antiparailel C-telopeptides (Doyle el al., 1974). In contrast, the N-termini of antiparallel molecules are not aligned. Strength and stability in the centrosymmetric and transitional zones must be at least as great as in the rest of the fibril. These qualities are thought to be dependent on the presence of intermolecular covalent crosslinks (Eyre, 1987). The existing evidence indicates t h a t most or all of the crosslinks in echinoderm fibrils include residues in the telopeptides (Bailey et ai., 1982; Bailey, 1985; Pucci-Minafra el at., 1978; Shimizu et al., 1990; Trotter & Koob, 1993). Therefore the molecular arrangement described above, by bringing the C-telopeptides of antiparallel molecules into alignment, m a y make it possible for crosslinking residues in either or both of them to form crosslinks with exactly the same helical residues in molecules having either polarity (Fig. 3B). Figure 3B shows on the left a sketch of the trivalent hydroxypyridinium crosslink of mammalian collagen fibrils, and on the right the same sketch, modified by rotating one of the collagen molecules through 180 ° about an axis through its C-telopeptide. This arrangement allows antiparallel molecules to be covalently crosslinked into a stable, three-dimensional structure. On the other" hand, the N-teiopeptides of antiparallel molecules are not aligned, so t h a t crosslinks between them and antiparallel molecules would require the participation of one or more additional lysines in the helical regions (Fig. 4A). In the absence of direct evidence these suggestions must remain speculative. Assuming t h a t large fibrils develop by the addition of molecules or molecular aggregates to smaller fibrils, the features of native fibrils described above suggest t h a t fibril formation operates under certain constraints, which also must be incorporated into any model of fibrillogenesis: at all stages of growth every fibril must (1) have a geometrically similar spindle shape; (2) contain a central region with equal numbers of antiparallel molecules; and (3) be formed of molecules which, except in the centrosymmetric region, are oriented with their N-termini toward the nearest end. Recent experiments on
fibriliogenesis in vitro indicate that similar constraints may apply to mammalian type I collagen. Fibrils formed from pCcollagen by controlled cleavage of the C-propeptide were observed to be spindle shaped and bipolar, and were formed by polymerization toward the N-termini, which were located at the tapered ends of the fibrils (Kadler et at., 1990; Holmes et al., 1992). Maintenance of geometric similarity requires that fibrils of all lengths must have a constant ratio between total length and diameter in the fibril center, and it has been shown t h a t this ratio is about 2,000 to 2,500 (Trotter & Koob, 1989; Trotter et al., 1993). Hence the growth of fibrils entails the addition of new antiparallel collagen molecules in the fibril center as well as parallel ones throughout the rest of the fibril. This observation suggests that, if the formation of an antiparailel nucleus is the initial event in fibrillogenesis, the nucleus may also remain active during subsequent growth of the fibril. This model of collagen fibrillogenesis differs from t h a t thought to operate during myosin filament formation, in which the antiparallel nucleus functions only during the initiation of aggregate formation, and in which the mature filaments have both constant diameter and length (Davis, 1988). The finding t h a t all of the echinoderm fibrils we observed and most of the embryonic chick tendon fibrils observed by Holmes et al. (1993) are molecularly bipolar raises the distinct possibility that this structural feature may be widespread among animal phyla. Because this feature can only be discerned by ultrastructural analyses of intact fibrils, how widespread it is won't be known until methods have been developed for isolating intact fibrils from other animals. The authors thank T. Koob and P. Purslow for helpful discussions, R. Stump for help with image processing, and J. Chapman for providing an electron micrograph of a mammalian type I collagen fibril. We are also grateful to J. Chapman for sharing with us prior to publication the paper that follows this one, and for helpful suggestions concerning our manuscript. This research was supported by grants from the National Science Foundation and the Office of Naval Research. References Bailey, A. J. (1985). The collagen of the echinodermata. In Biology of Invertebrate and Lower Vertebrate Collagens (Bairati, A. & Garonne, R., eds), pp 369388, Plenum Press, New York. Bailey, A. J., Gathercole, L. J.. Dlugosz, J., Keller, A. & Voyle, C. A. (1982). Proposed resolution of the paradox of extensive crosslinking and low tensile strength of cuvierian tubule collagen from the sea cucumber Holothuria forskali. Int. J. Biol. Macromal. 4, 329-334. Bruns, R. R. (1976). Supramolecular structure of polymorphic collagen fibrils. J. Cell Biol. 68, 521-538. Chapman, J. A. (1985). Electron microscopy of tile collagen l~bril. In Biology of Invertebrate and Lower Vertebrate Collagens (Bairati, A. & Garone, R., eds), pp. 515-537. Plenum Press, New York.
Communications D'Alessio, M. D., Ramirez, F., Suzuki, H. R., Solursh, M. & Gambino, R. (1989). Structure and developmental expression of a sea urchin fibrillar collagen gene. Proc. Nat. Acad. Sci., U.S.A. 86, 9303-9307. D'Alessio, M. D., Ramirez, F., Suzuki, H. R., Solursh, M. & Gambino, R. (1990). Cloning of a fibrillar collagen gene expressed in the mesenchymal cells of the developing sea urchin embryo. J. Biol. Chem. 265, 7050-7054. Davis, J. S. (1988). Assembly processes in vertebrate skeletal thick filament formation. Annu. Rev. Biophys. Biophys. Chem. 17, 217-239. Doyle, B. B., Hulmes, D. J. S., Miller, A., Parry, D. A. D., Piez, K. A. & Woodhead-Galloway, J. (1974). Axially projected collagen structures. Proc. Roy. Soc. sect. B, 187, 37-46. Doyle, B. B., Hukins, D. W. L., Hulmes, D. J. S., Miller, A. & Woodhead-Galloway, J. (1975). Collagen polymorphism: its origins in the amino acid sequence. J. Mol. Biol. 91, 79-99. Exposito, J.-Y., D'Alessio, M. & Ramirez, F. (1992a). Novel amino-terminal propeptide configuration in a fibrillar procollagen undergoing alternative splicing. d. Biol. Chem. 267, 1704-1708. Exposito. J.-Y., D'Alessio, M., Solursh, M. & Ramirez, F. (1992b). Sea urchin collagen evolutionarily homologous to vertebrate pro-a2 (I) collagen. J. Biol. Chem. 267, 15559-15562. Eyre, D. R. (1987). Collagen stability through covalent crosslinking. In Advances in Meat Research: Collagen a~ a Food (Pearson, A. M., Dotson, T. R. & Bailey. A. J., eds), vol. 4, pp. 69-85, Van Nostrand Reinhold, New York. Eyre, D. R. & Glimcher, M. J. (1971). Comparative biochemistry of collagen crosslinks. Biochim. Biophys. Acta, 243, 525-529. Eyre, D. R. & Glimcher, M. J. (1973). Evidence for glycosylated crosslinks in the collagen of the body wall of the sea cucumber. Proc. Roy. Soc. Biol. Med. 144, 400~03. Hodge, A. J. & Petruska, J. A. (1963). Recent studies with the electron microscope on ordered aggregates of the tropocollagen molecule. In Aspects of Protein Structure (Ramachandran, G. N., ed.), pp. 289-300, Academic Press, New York. Holmes, D. F., Chapman, J. A., Prockop, D. J. & Kadler, K. E. (1992). Growing tips of type I collagen fibrils formed in vitro are near-paraboloidal in shape, implying a reciprocal relationship between accretion and diameter. Proc. Nat. Acad. Sci., U.S.A. 89, 9855-9859. Holmes, D. F., Lowe, M. P. & Chapman, J. A. (1993). Vertebrate (chick) collagen fibrils formed in vitro can exhibit a reversal in molecular polarity. J. Mol. Biol. 235, 80-83. Kadler, K. E., Hojima, Y. & Prockop, D. J. (1990). Collagen fibrils in vitro grow from pointed tips in the C- and N-terminal direction. Biochem. d. 268, 339-343. Katzman, R. L., Bhattacharyya, A. K. & Jeanloz, R. W. (1969). The amino acid and carbohydrate composition of the collagen from Thyone Briareus. Biochim. Biophys. Acta, 184, 523-528.
79
Kimura, S., Omura, Y., Ishida, M. & Shirai, H. (1993). Molecular characterization of fibrillar collagen from the body wall of starfish Asterias amurensis. Comp. Biochem. Physiol. sect. B, 104, 663-668. Marks, M. H., Bear, R. S. & Blake, C. H. (1949). X-ray diffraction evidence of collagen-type protein fibers in the echinodermata, coelenterata and porifera. J. Expl. Zool. 111, 55-78. Matsumura, T. (1973). Shape, size and amino acid composition of collagen fibril of the starfish Asterias amurensis. Comp. Biochem. Physiol. sect. B, 44, 1197-1205. Matsumura, T. (1974). Collagen fibrils of the sea cucumber, Stichopus japonicus: purification and morphological study. Connect. Tiss. Res. 2, 117-125. Motokawa, T. (1984). Connective tissue catch in echinoderms. Biol. Rev. 59, 255-270. Piez, K. A. (1984). Molecular and aggregate structures of the collagens. In Extracellular Matrix Biochemistry (Piez, K. A. & Reddi, A. H. eds), pp. 1-39, Elsevier, New York. Piez, K. A. & Gross, J. (1959). The amino acid composition and morphology of some invertebrate and vertebrate collagens. Biochim. Biophys. Acta, 34, 24-39. Pucci-Minafra, I., Galante, R. & Minafra, S. (1978). Identification of collagen in the Aristotle's lantern of Paracentrotus lividus. J. Submicr. Cytol. 10, 53-63. Shimizu, K., Amemiya, S. & Yoshizato, K. 0990). Biochemical and immunological characterization of collagen molecules from echinothurioid sea urchin Asthenosoma ijinmi. Biochim. Biophys. Acta, 1038, 39-46. Travis. D. F., Francois, C. J., Bonar, L. C. & Glimcher, M. J. (1967). Comparative studies of the organic matrices of invertebrate mineralized tissues. J. UItrastruct. Res. 18, 519-550. Trotter, J. A. & Koob, T. J. (1989). Collagen and proteoglycan in a sea urchin ligament with mutable mechanical properties. Cell Tiss. Res. 258, 527-539. Trotter, J. A. & Koob, T. J. (1993). Biochemical characterization of fibrillar collagen from the mutable spine ligament of the sea urchin Eucidaris tribuloides. Camp. Biochem. Physiol. In the press. Trotter, J. A., Thurmond, F. A. & Koob, T. J. (1993). Molecular structure and functional morphology of echinoderm collagen fibrils. Cell Tiss. Res. In the press. Van Ness, K. P., Koob, T. J. & Eyre, D. R. (1988). Collagen cross-linking: distribution of hydroxypyridinium cross-links among invertebrate phyla and tissues. Comp. Biochem. Physiol. sect. B, 91,531-534. Watson, M. R. & Silvester, N. R. (1959). Studies of invertebrate collagen preparations. Biochem. J. 71, 578-584. Wilkie, I. C. (1984). Variable tensility in echinoderm collagenous tissues: a review. Mar. Behav. Physiol. 11, 1-34. Williams, B. R., Gelman, R. A. & Poppke, D. C. (1978). Collagen fibril formation. J. Biol. Chem. 253, 6578-6585.
Edited by D. DeRosier ( R e c e i v e d 6 M a y 1993; accepted 19 A u g u s t 1 9 9 3 )
Native Collagen Fibrils From Echinoderms are Molecularly Bipolar F. A. Thurmond~f and J. A. Trotter Department of A n a t o m y University of N e w Mexico School of Medicine Albuquerque, N M 87131, U . S . A .
Collagen fibrils are generally assumed to be cylinders with uniform diameters (except possibly at their ends) and to be composed of molecules all of which have the same polarity. These assumptions have been largely untested because of the extreme difficulty associated with isolating entire native fibrils. Intact collagen fibrils are readily extracted from certain echinoderms, however, and we have therefore analyzed the molecular structure of these fibrils. Our electron microscopic analyses show the above assumptions to be false: echinoderm fibrils, which previously have been shown to be symmetrically spindle shaped, are also molecularly bipolar. Their constituent molecules have their N-termini oriented toward the nearest fibril end, and they are antiparallel in the fibril center. The shape and molecular arrangement of these fibrils have implications for fibrillogenesis.
Keywords: Collagen; symmetry; antiparallel; fibril; echinoderm
Fibrillar collagens of echinoderms are similar to those of vertebrates in amino acid composition (Watson & Silvester, 1959; Piez & Gross, 1959; Travis et al., 1967; Katzman et al., 1969; Matsumura, 1973; Pucci-Minafra et al., 1978; Bailey, 1985; Shimizu et al., 1990; Kimura et al., 1993; Trotter & Koob, 1993), triple helix length (Shimizu et al., 1990; Trotter et al., 1993), cross-link chemistry (Bailey et al., 1982; Eyre & Glimcher, 1971, 1973; Van Ness et al., 1988; Trotter & Koob, 1993), gene structure (D'Alessio et al., 1989, 1990; Exposito et al., 1992a,b), and D-periodicity (Marks et al., 1949; Piez & Gross, 1959; Matsumura, 1973, 1974; Pucci-Minafra et al., 1978; Bailey et al., 1982; Bailey, 1985; Trotter & Koob, 1989). Many collagenous tissues of echinoderms differ from those of other phyla, however, in that their mechanical properties are regulated by the nervous system on a time scale of seconds (Wilkie, 1984; Motokawa, 1984). This physiological mutability is undoubtedly related to the capacity of the tissues to be dissociated by gentle procedures into intact, native fibrils (Matsumura, 1973, 1974; Trotter & Koob, 1989). We have utilized the dissoeiability of sea urchin spine ligaments and sea cucumber dermis to analyze the molecular packing of whole native fibrils.
The banding pattern of positively stained native echinoderm fibrils, which reflects the relative density of charged amino acids along the fibril length, is similar but not identical to that of mammalian fibrils (Fig. 1). The major differences are the relative faintness of the a 3 and b 2 bands, and the prominent intensity of the c 3 band in echinoderm fibrils (Trotter et al., 1993). The similarity between echinoderm and vertebrate banding patterns allows the amino-carboxy orientation of the molecules in echinoderm fibrils to be specified, as shown in Figure 1. Isolated echinoderm fibrils between 24 and 436 mm long have been shown to be symmetrically spindle shaped and geometrically similar (Trotter et al., 1993). The ratio of total length to maximum diameter is about 2,000 in sea cucumber fibrils (Trotter et al., 1993) and about 2,500 in sea urchin fibrils (Trotter & Koob, 1989). The symmetrical shape of the fibrils raises questions concerning the orientation and organization of their constituent molecules. If all the molecules in the fibril had the same N-C polarity, each fibril would also have an N-terminus and a C-terminus. Analysis of isolated echinoderm fibrils shows this not to be the case: the molecules in each haft of every fibril are oriented with their C-termini toward the fibril center, and their N-termini toward the nearest end. Echinoderm collagen fibrils are thus not only symmetrically spindle shaped, but are also
tAuthor to whom all correspondence should be addressed. 0022-2836/94/010073-07 $08.00/0
73
© 1994 Academic Press Limited
Communications
74 o
Table
1
Position of centrosymmetric region along fibril
NEG Fibril number
POS
L! ! i N
;
ii
'
!
:
i LJ
•
I L_i._l
dc
I b
c
I I.LLJ a
Figure 1. Comparisoh of the banding patterns observed in negatively (NEG) and positively (POS) stained native echinoderm collagen fibrils with that seen in a positively stained fibril reconstituted from mammalian type I collagen (bottom panel). The many similarities in the positions and intensities of bands in the positively stained fibrils allow the amino/carboxyl polarity of the echinoderm fibril to be aligned with that of the mammalian fibril (labelled N and C in the lower panel). Compared with the positively stained mammalian fibril, the echinoderm fibril shows reduced stain intensity in the a a and b 2 bands, and greatly increased intensity in the c a band (the bands are lettered from right to left, and the sub-bands within them are numbered from right to left, as described in Chapman (1985), but the numbers have not been included in the figure). Intact, native collagen fibrils were isolated from the spine ligaments of the sea urchin Eucidaris tribuloides, and positively and negatively stained with uranyl acetate as described (Trotter & Koob, 1989). The electron micrograph of the reconstituted mammalian type I fibril was kindly provided by Dr. John Chapman.
molecularly bipolar. A n o t h e r s u p e r p o l y m e r with this symmetrical molecular s t r u c t u r e is the native myosin filament of skeletal muscle (Davis, 1988). The bipolar symmetrical arrangement of molecules predicts t h a t in e v e r y fibril there is a region in which the molecular polarity is reversed. Indeed, e v e r y fibril examined (more t h a n 100) contained a single region in which the banding p a t t e r n was c e n t r o s y m m e t r i c (Figs, 2B, 3A). This c e n t r o s y m m e t r i e region, which was in the center of e v e r y fibril (Table 1), was only one to two D-periods long in most fibrils, and was flanked on both sides b y transitional zones four D-periods long (Figs. 2B, 3A, 4B). T h e banding p a t t e r n s observed in the transitional zones and in the c e n t r o s y m m e t r i c central zone can be a c c o u n t e d for b y either of the models shown in Figure 3A, b o t h of which are based on " H o d g e P e t r u s k a " model of the longitudinal a r r a n g e m e n t of collagen molecules (Hodge & P e t r u s k a , 1963). The u p p e r model has the following features: (a) there are equal n u m b e r s of molecules t h r o u g h o u t the entire fibril, including the region of p o l a r i t y reversal; (b) there are equal n u m b e r s of antiparallel molecules in the central (centrosymmetric) D-period, and
I 2 3 4 5 6 7 8 9 l0 Il 12 13
14 15 16 17
18 19 20 21
Species
Length (L) (~m)
SCt SC SU SC SC SC SC SC SC SC SC SU SC SC SC SC SC SU SU SU SU
36 l 13 115 132 139 159 167 175 188 207 212 236 251 258 274 285 290 302 334 456 1218
Distance to Symmetric D-Period (D) (~m)
D/L
17 56 55 65 66 77 81 88 94 102 104 95
0-47 0"50 0'48 0"49 0-47 0-48 0-49 0-50 0"50 0-49 0"49 0-40
120
(}'48
121 136 142
0"47 0.50 0.50 0-49 0-49 0"50 0"49 0"48
142
147 167 225 585
t SC, sea cucumber; SU, sea urchin.
changing ratios of antiparallel molecules in the transitional zones; and (c) the C-telopeptides of colinear antiparallel molecules a b u t t one another. In the lower model, in contrast, the n u m b e r of molecules increases from 1X in the " n o r m a l " asymmetric regions to 2X in the c e n t r o s y m m e t r i c D-period. The C-telopeptides of antiparallel molecules are in register, b u t do not a b u t t one another. This model predicts t h a t the central zone should be thicker t h a n the regions adjacent to it and, indeed, some of the fibrils examined had a bulge in the e x p e c t e d place (see Fig. 2B). D i a m e t e r m e a s u r e m e n t s were inconsistent, however, and a true test of this model will require mass determinations of the sort described by Holmes et al. (1992). In the central s y m m e t r i c D-period the c a bands of antiparallel molecules are in register, and the c2 bands of molecules of one polarity are aligned with the d bands of oppositely polarized ones (Fig. 4A). In addition, the a s band of one molecular polarity is in register with the a 4 band of the opposite polarity. This banding p a t t e r n has two planes of mirror s y m m e t r y , one passing through the c a band, and the other passing t h r o u g h the aa/a 4 region. The same a n t i p a r a l l e l packing, designated DPSt (for D-periodic symmetric) or DPS III, has been noted previously in fibrils reconstituted u n d e r certain conditions (e.g. elevated phosphate) from mammalian collagens (Doyle et al., 1974, 1975; Williams et al., 1978; Bruns, 1976; Piez, 1984), b u t it has not until recently been t h o u g h t to exist in native fibrils or to be functionally significant. However, the v e r y tAbbreviation used: DPS, D-periodic symmetric.
75
Communications .a.
l~'~,:q~-
• .,-.:."-I : .),~-."'.~' .:.... . ".. ::-~ ',,~ ~ ........
,,.
i:::!.
.
.
.
•
".:
- . ~ ~ ~
7
:
-..--.~.~;,,,, ~,'_:.-:
i :,.i: :,!. :
". : :;
i:i!:.:,:
I~:::"..: ."-...,....-..i" ..."'.":..-.,.,. k:~: :-":/~:/--:B
$~Ir
:
Figure 2. Electron micrographs of an entire fibril and of the central regions of 9 fibrils from sea urchin ligaments (SU) and from sea cucumber dermis (SC). A. An entire sea urchin fibril, 170/lm long is seen in the electron micrograph in the top panel. Its ends are marked (arrowheads). B. Positively and negatively stained central portions of fibrils similar to that in the boxed zone in A. Starting at the top left, the fibrils were from SU, SC, SU, SC. Similarly for the right hand side the fibrils were from SU, SU, SC, SU, SC. Note that the banding pattern is symmetrical in the regions marked by the vertical lines. In contrast, the banding pattern on both sides of the central region becomes polarized, with the amino termini of the constituent molecules oriented away from the center (compare these images with Fig. 1). There is a characteristic bulge in the central portion of some fibrils, where the symmetrical banding pattern is seen. Intact collagen fibrils were isolated from sea urchin ligaments and from sea cucumber dermis as described (Trotter & Koob, 1989). Fibrils were positively stained with uranyl acetate (A and the right side of B) or negatively stained with phosphotungstic acid (left side of B).
recent findings of H o l m e s et al. (1993), reported in the p a p e r following this one, show t h a t some verteb r a t e fibrils are also bipolar, and contain a " s w i t c h " region where the b a n d i n g p a t t e r n is D P S - I I I . The molecular model presented in the p a p e r b y H o l m e s et al. is identical to the u p p e r one shown in Figure 3A, except t h a t the antiparallel molecules are verti-
cally displaced so t h a t the C-telopeptides are axially aligned b u t do not a b u t t one another. The ability of the molecular models shown in Figure 3A to a c c o u n t for the banding p a t t e r n s observed b o t h in the c e n t r o s y m m e t r i c and in the transitional D-periods is shown in the c o m p u t e r g e n e r a t e d images of Figure 4. I n Figure 4A, the t o p
Communications
76 A
4 i
]D
1 I
•
,~ M
N
ID
,
•
4
i
B N
N "~,
C
o
N
mo,~ ~~_____~ C N
Figure 3. A. Molecular models that can account for the banding patterns seen in the middle of the negatively stained fibril by the antiparallel packing of oppositely polarized molecules. The carboxyl (c) termini (arrowheads at the ends of schematized molecules) are oriented toward the middle of the fibril. The C-telopeptides of individual molecules are located at the carboxyl end of the overlap zones. The N-termini of individual molecules are located at the amino end of the overlap zones. In the central, symmetrically banded portion of the fibril the D-period remains the same and the C-telopeptides (arrowheads) of oppositely polarized molecules are in register. This gives rise to the prominent, thin, stainexcluding (white) lines in this region. On either side of the region of complete symmetry (marked by the vertical dashed line), which is 1 D-period long, is a transitional region, 4 D-periods long, produced by the changing ratio of oppositely polarized molecules. B. Possible crosslinking configuration where the C-telopeptides of oppositely polarized molecules are in register. The left panel shows the 3-hydroxypyridinium crosslinks that occur in the normally polarized regions of vertebrate collagen fibrils (redrawn after Eyre, 1987). The right panel shows the hypothetical trivalent crosslink that could stabilize antiparallel molecules in the central zones of echinoderm fibrils. It was produced by rotating one molecule through an angle of 180° about an axis through the C-telopeptide. image is an electron mierograph of a parallel (asymmetric) region of a positively stained fibril with the amino ends of the molecules pointing toward the left; the next micrograph below it is the same image inverted left to right, and positioned so as to bring the c 3 bands into alignment. The arrows between these two images show the position of two selected molecules in each image. The third image down is a computer-generated addition of the first two, and the fourth image down is an electron micrograph of the centrosymmetric region of a native fibril. I t is apparent t h a t the third and fourth images correspond precisely. The centrosymmetric pattern is thus produced by equal numbers of antiparallel molecules arranged as shown.
In Figure 4B, the middle image was generated by combining two oppositely oriented fibrils aligned with their c 3 bands in register, as just described, except t h a t each fibril extended for ten D-periods. The density of each image was linearly attenuated so t h a t the last D-period on the left contained only molecules with amino ends toward the left, the last D-period on the right contained only those with amino ends to the right while the middle D-period (bracket) contained equal numbers of each. The middle D-period has the centrosymmetric banding pattern seen in Figure 4A, whereas the D-periods on either side of the central one gradually change to the usual asymmetric pattern. One way to follow the transition is to look at the width of the
Communications
77
N
C
f N
C:
I
I
11
a3a4
C3
a3a4
B
!
!
Figure 4. A computer-reconstruction that accounts for the symmetrical and transitional banding patterns in the central regions of positively stained fibrils. A shows in the top panel a portion of a fibril with its N-terminus toward the left. The next micrograph down shows the same fibril, but with its N-terminus towards the right and aligned such that the c 3 bands are in register. The arrows between these two micrographs represent two selected molecules from each orientation, with the arrowheads representing the C-termini. The 3rd panel down is a computer-generated image created by superimposing the 2 images above it. The 4th panel down shows the central region of a native fibril, positively stained with uranyl acetate. Note that the native and computer-generated banding patterns are identical. The C-telopeptides of the antiparallel molecules (arrowheads) are in register at the a3/a 4 bands, while the N-telopeptides of antiparallel molecules (short vertical lines) are separated by the length of the central triplet of the symmetrical pattern. B shows the I 1 central D-periods in a native fibril (lower panel), and above it a computer-generated image created by superimposing the images of left and right oriented fibril regions in which the density of the region with its N-terminus toward the left was decreased linearly from 1 to 0 over 9 D-periods, beginning at the second a2 band from the left, and the region with its N-terminus toward the right was treated similarly, but with the density decreasing toward the left. This is the density equivalent of the molecular model at the bottom of Fig. 3, which is represented here by the schematic in A. The superimposition of these two images produces uniform image intensity, but the ratio of the two banding patterns is 1:1 only in the central period (bracket), and changes linearly over the 4 D-periods toward the right and the left. This transition pattern closely matches the natural one.
78
Communications
unstained zone on either side of the ,central triplet (the middle band of which is the c a band). In the centrosymmetric D-period these two zones are equal in width. Moving toward the left, the zone to the left of the triplet becomes narrower as the zone to its right becomes wider. The opposite occurs moving to the right along the fibril. The bottom image in Figure 4B is a positively stained native fibril, the banding pattern of which corresponds closely to the computer generated image just described. The arrange'ment of antiparallel molecules t h a t produces the images seen in the centrosymmetric and transitional regions of native fibrils brings the C-telopeptides of antiparallel molecules into axial alignment, since they are located at the gap/overlap interface, in the region of the a3/a 4 bands. The bright (stain-excluding) band in the negatively stained central region (Figs 2B, 3A) probably represents the aligned antiparailel C-telopeptides (Doyle el al., 1974). In contrast, the N-termini of antiparallel molecules are not aligned. Strength and stability in the centrosymmetric and transitional zones must be at least as great as in the rest of the fibril. These qualities are thought to be dependent on the presence of intermolecular covalent crosslinks (Eyre, 1987). The existing evidence indicates t h a t most or all of the crosslinks in echinoderm fibrils include residues in the telopeptides (Bailey et ai., 1982; Bailey, 1985; Pucci-Minafra el at., 1978; Shimizu et al., 1990; Trotter & Koob, 1993). Therefore the molecular arrangement described above, by bringing the C-telopeptides of antiparallel molecules into alignment, m a y make it possible for crosslinking residues in either or both of them to form crosslinks with exactly the same helical residues in molecules having either polarity (Fig. 3B). Figure 3B shows on the left a sketch of the trivalent hydroxypyridinium crosslink of mammalian collagen fibrils, and on the right the same sketch, modified by rotating one of the collagen molecules through 180 ° about an axis through its C-telopeptide. This arrangement allows antiparallel molecules to be covalently crosslinked into a stable, three-dimensional structure. On the other" hand, the N-teiopeptides of antiparallel molecules are not aligned, so t h a t crosslinks between them and antiparallel molecules would require the participation of one or more additional lysines in the helical regions (Fig. 4A). In the absence of direct evidence these suggestions must remain speculative. Assuming t h a t large fibrils develop by the addition of molecules or molecular aggregates to smaller fibrils, the features of native fibrils described above suggest t h a t fibril formation operates under certain constraints, which also must be incorporated into any model of fibrillogenesis: at all stages of growth every fibril must (1) have a geometrically similar spindle shape; (2) contain a central region with equal numbers of antiparallel molecules; and (3) be formed of molecules which, except in the centrosymmetric region, are oriented with their N-termini toward the nearest end. Recent experiments on
fibriliogenesis in vitro indicate that similar constraints may apply to mammalian type I collagen. Fibrils formed from pCcollagen by controlled cleavage of the C-propeptide were observed to be spindle shaped and bipolar, and were formed by polymerization toward the N-termini, which were located at the tapered ends of the fibrils (Kadler et at., 1990; Holmes et al., 1992). Maintenance of geometric similarity requires that fibrils of all lengths must have a constant ratio between total length and diameter in the fibril center, and it has been shown t h a t this ratio is about 2,000 to 2,500 (Trotter & Koob, 1989; Trotter et al., 1993). Hence the growth of fibrils entails the addition of new antiparallel collagen molecules in the fibril center as well as parallel ones throughout the rest of the fibril. This observation suggests that, if the formation of an antiparailel nucleus is the initial event in fibrillogenesis, the nucleus may also remain active during subsequent growth of the fibril. This model of collagen fibrillogenesis differs from t h a t thought to operate during myosin filament formation, in which the antiparallel nucleus functions only during the initiation of aggregate formation, and in which the mature filaments have both constant diameter and length (Davis, 1988). The finding t h a t all of the echinoderm fibrils we observed and most of the embryonic chick tendon fibrils observed by Holmes et al. (1993) are molecularly bipolar raises the distinct possibility that this structural feature may be widespread among animal phyla. Because this feature can only be discerned by ultrastructural analyses of intact fibrils, how widespread it is won't be known until methods have been developed for isolating intact fibrils from other animals. The authors thank T. Koob and P. Purslow for helpful discussions, R. Stump for help with image processing, and J. Chapman for providing an electron micrograph of a mammalian type I collagen fibril. We are also grateful to J. Chapman for sharing with us prior to publication the paper that follows this one, and for helpful suggestions concerning our manuscript. This research was supported by grants from the National Science Foundation and the Office of Naval Research. References Bailey, A. J. (1985). The collagen of the echinodermata. In Biology of Invertebrate and Lower Vertebrate Collagens (Bairati, A. & Garonne, R., eds), pp 369388, Plenum Press, New York. Bailey, A. J., Gathercole, L. J.. Dlugosz, J., Keller, A. & Voyle, C. A. (1982). Proposed resolution of the paradox of extensive crosslinking and low tensile strength of cuvierian tubule collagen from the sea cucumber Holothuria forskali. Int. J. Biol. Macromal. 4, 329-334. Bruns, R. R. (1976). Supramolecular structure of polymorphic collagen fibrils. J. Cell Biol. 68, 521-538. Chapman, J. A. (1985). Electron microscopy of tile collagen l~bril. In Biology of Invertebrate and Lower Vertebrate Collagens (Bairati, A. & Garone, R., eds), pp. 515-537. Plenum Press, New York.
Communications D'Alessio, M. D., Ramirez, F., Suzuki, H. R., Solursh, M. & Gambino, R. (1989). Structure and developmental expression of a sea urchin fibrillar collagen gene. Proc. Nat. Acad. Sci., U.S.A. 86, 9303-9307. D'Alessio, M. D., Ramirez, F., Suzuki, H. R., Solursh, M. & Gambino, R. (1990). Cloning of a fibrillar collagen gene expressed in the mesenchymal cells of the developing sea urchin embryo. J. Biol. Chem. 265, 7050-7054. Davis, J. S. (1988). Assembly processes in vertebrate skeletal thick filament formation. Annu. Rev. Biophys. Biophys. Chem. 17, 217-239. Doyle, B. B., Hulmes, D. J. S., Miller, A., Parry, D. A. D., Piez, K. A. & Woodhead-Galloway, J. (1974). Axially projected collagen structures. Proc. Roy. Soc. sect. B, 187, 37-46. Doyle, B. B., Hukins, D. W. L., Hulmes, D. J. S., Miller, A. & Woodhead-Galloway, J. (1975). Collagen polymorphism: its origins in the amino acid sequence. J. Mol. Biol. 91, 79-99. Exposito, J.-Y., D'Alessio, M. & Ramirez, F. (1992a). Novel amino-terminal propeptide configuration in a fibrillar procollagen undergoing alternative splicing. d. Biol. Chem. 267, 1704-1708. Exposito. J.-Y., D'Alessio, M., Solursh, M. & Ramirez, F. (1992b). Sea urchin collagen evolutionarily homologous to vertebrate pro-a2 (I) collagen. J. Biol. Chem. 267, 15559-15562. Eyre, D. R. (1987). Collagen stability through covalent crosslinking. In Advances in Meat Research: Collagen a~ a Food (Pearson, A. M., Dotson, T. R. & Bailey. A. J., eds), vol. 4, pp. 69-85, Van Nostrand Reinhold, New York. Eyre, D. R. & Glimcher, M. J. (1971). Comparative biochemistry of collagen crosslinks. Biochim. Biophys. Acta, 243, 525-529. Eyre, D. R. & Glimcher, M. J. (1973). Evidence for glycosylated crosslinks in the collagen of the body wall of the sea cucumber. Proc. Roy. Soc. Biol. Med. 144, 400~03. Hodge, A. J. & Petruska, J. A. (1963). Recent studies with the electron microscope on ordered aggregates of the tropocollagen molecule. In Aspects of Protein Structure (Ramachandran, G. N., ed.), pp. 289-300, Academic Press, New York. Holmes, D. F., Chapman, J. A., Prockop, D. J. & Kadler, K. E. (1992). Growing tips of type I collagen fibrils formed in vitro are near-paraboloidal in shape, implying a reciprocal relationship between accretion and diameter. Proc. Nat. Acad. Sci., U.S.A. 89, 9855-9859. Holmes, D. F., Lowe, M. P. & Chapman, J. A. (1993). Vertebrate (chick) collagen fibrils formed in vitro can exhibit a reversal in molecular polarity. J. Mol. Biol. 235, 80-83. Kadler, K. E., Hojima, Y. & Prockop, D. J. (1990). Collagen fibrils in vitro grow from pointed tips in the C- and N-terminal direction. Biochem. d. 268, 339-343. Katzman, R. L., Bhattacharyya, A. K. & Jeanloz, R. W. (1969). The amino acid and carbohydrate composition of the collagen from Thyone Briareus. Biochim. Biophys. Acta, 184, 523-528.
79
Kimura, S., Omura, Y., Ishida, M. & Shirai, H. (1993). Molecular characterization of fibrillar collagen from the body wall of starfish Asterias amurensis. Comp. Biochem. Physiol. sect. B, 104, 663-668. Marks, M. H., Bear, R. S. & Blake, C. H. (1949). X-ray diffraction evidence of collagen-type protein fibers in the echinodermata, coelenterata and porifera. J. Expl. Zool. 111, 55-78. Matsumura, T. (1973). Shape, size and amino acid composition of collagen fibril of the starfish Asterias amurensis. Comp. Biochem. Physiol. sect. B, 44, 1197-1205. Matsumura, T. (1974). Collagen fibrils of the sea cucumber, Stichopus japonicus: purification and morphological study. Connect. Tiss. Res. 2, 117-125. Motokawa, T. (1984). Connective tissue catch in echinoderms. Biol. Rev. 59, 255-270. Piez, K. A. (1984). Molecular and aggregate structures of the collagens. In Extracellular Matrix Biochemistry (Piez, K. A. & Reddi, A. H. eds), pp. 1-39, Elsevier, New York. Piez, K. A. & Gross, J. (1959). The amino acid composition and morphology of some invertebrate and vertebrate collagens. Biochim. Biophys. Acta, 34, 24-39. Pucci-Minafra, I., Galante, R. & Minafra, S. (1978). Identification of collagen in the Aristotle's lantern of Paracentrotus lividus. J. Submicr. Cytol. 10, 53-63. Shimizu, K., Amemiya, S. & Yoshizato, K. 0990). Biochemical and immunological characterization of collagen molecules from echinothurioid sea urchin Asthenosoma ijinmi. Biochim. Biophys. Acta, 1038, 39-46. Travis. D. F., Francois, C. J., Bonar, L. C. & Glimcher, M. J. (1967). Comparative studies of the organic matrices of invertebrate mineralized tissues. J. UItrastruct. Res. 18, 519-550. Trotter, J. A. & Koob, T. J. (1989). Collagen and proteoglycan in a sea urchin ligament with mutable mechanical properties. Cell Tiss. Res. 258, 527-539. Trotter, J. A. & Koob, T. J. (1993). Biochemical characterization of fibrillar collagen from the mutable spine ligament of the sea urchin Eucidaris tribuloides. Camp. Biochem. Physiol. In the press. Trotter, J. A., Thurmond, F. A. & Koob, T. J. (1993). Molecular structure and functional morphology of echinoderm collagen fibrils. Cell Tiss. Res. In the press. Van Ness, K. P., Koob, T. J. & Eyre, D. R. (1988). Collagen cross-linking: distribution of hydroxypyridinium cross-links among invertebrate phyla and tissues. Comp. Biochem. Physiol. sect. B, 91,531-534. Watson, M. R. & Silvester, N. R. (1959). Studies of invertebrate collagen preparations. Biochem. J. 71, 578-584. Wilkie, I. C. (1984). Variable tensility in echinoderm collagenous tissues: a review. Mar. Behav. Physiol. 11, 1-34. Williams, B. R., Gelman, R. A. & Poppke, D. C. (1978). Collagen fibril formation. J. Biol. Chem. 253, 6578-6585.
Edited by D. DeRosier ( R e c e i v e d 6 M a y 1993; accepted 19 A u g u s t 1 9 9 3 )