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Conformation of keratan sulphate
J. Mol. Biol. (1974) 88, 175-184
Conformation
of Keratan Sdphate
STPJJTHERARNOTT, J. M. Gnss, D. W. L. HUKINS~ Department of Biological Sciences Purdue University West Lafayette, Id 47907, U.S.A. I. C. M. DEA AND D. A. REES Unilever Research Colworthl Welwyn Laboratory Colworth House Sharnbrook, Bedford MK44 IL&, Enjgland (Received 11 April 1974, and in revised form 25 May 1974) X-ray diffraction patterns from stretched films of keratan sulphate, isolated from bovine cornea, indicate that the molecules are twofold helices with an axial rise per disaccharide residue of 0.945 nm. These helices are oriented with their twofold screw axes parallel and about equally spaced, but are not further organized into regular crystalline arrays. Computer methods were used to construct a molecular model with the observed symmetry and axial rise per disaccharide residue, with standard bond lengths, bond’angles and pyranose ring conformations and with a hydrogen bond of length 0.270 nm between 0( 3) of N-acetylglucosamine and O(5) of galactose. This model has no unacceptably short non-bonded interatomic distances. The intensity distribution of the diffraction pattern calculated from the co-ordinates of the model is in reasonable agreement with the observed intensity distribution. This keratan sulphate model is an extended polysaccharide chain fringed with charged sulphate side groups, and is similar to those that have already been reported for chondroitin sulphates and dermatan sulphate, paralleling the similarities in covalent structure and biological occurrence among these substances.
1. Introduction Most of the glycosaminoglycans (mucopolysaccharides) of animal connective tissues share the same alternating fi(l+3), /3(1+4) baokbone. The members differ from one another by the nature and stereochemistry of backbone substitution (Mathews, 1967). They commonly occur as side chains covalently linked to a single polypeptide chain in a proteoglycan complex. Chondroitin 4-sulphate, chondroitin 6-sulphate and dermatan sulphate are not only related closely in structure, but evidently also in their biosynthesis, since some naturally occurring polysaccharides can be regarded as hybrid forms, which incorporate features of two or more of these particular members (Pransson & Malmstrom, 1971). Keratan sulphate and hyaluronic acid are less closely related to this series. Although keratan sulphate may be attached to the same protein backbone as chondroitin sulphates (Brandt & Muir, 1971), it differs from t Present address: Laboratory of Molecular Biophysics, Oxford, South Parks Road, Oxford OX1 3PS, England. 175
Department
of Zoology,
University
of
176
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ET AL.
them in containing no uranic acid residues. Hyaluronic acid is not sulphated, probably is not attached covalently in a proteoglycan, has a molecular weight that is larger by many orders of magnitude than that of the others (Balazs, 1966) and, so far as is known, has a covalent structure that is completely regular and is without any of the variability along the chain that is so characteristic of other glycosaminoglycans. Finally, heparin and heparan sulphate, although sulphated polymers of hexosamine and uranic acid residues, do not share this same basic backbone structure, since they appear to be completely l-+4 linked (Wolfrom et al., 1964; Perlin et al., 1972). To the extent that these polysaccharides are related in covalent structure, they might be expected to show some similarity in their conformations, interactions and biological functions. In this paper we describe an investigation of the conformation of keratan sulphate and attempt to relate the results to our earlier conformational (Rees, 1969) and X-ray (Dea et al., 1973; Arnott et al., 1973ab) studies of chondroitin sulphates, dermatan sulphate, and hyaluronic acid.
FIG. 1. The structural formula of keratan sulphate. the chain conformation are 4AB, $AB, qW and #““.
The 4 conformation
angles used to define
Keratan sulphate (Pig. 1) occurs in cartilage, nucleus pulposus, and cornea (Mathews, 1967; Muir, 1969) and among these sources it shows considerable variation in its detailed chemical composition and mode of linkage to protein (Mathews $ Cifonelli, 1965; Seno et al., 1965; Bhavanandan and Meyer, 1968). Typically, 79% of the N-acetylglucosamine residues and 40% of the galactose residues are sulphated, although the number of sulphate groups and presumably their distribution may vary (Mathews & Cifonelli, 1965; Bhavanandan & Meyer, 1967,196s; Hirano & Meyer, 1971). We have now obtained X-ray diffraction patterns from stretched films of keratan sulphate isolated from bovine cornea, which allow the conformation of the molecules in the films to be determined.
2. Materials and Methods (a) Materials Sodium salts of keratan sulphate extracted from both cornea and cartilage were kindly supplied by Dr M. B. Mathews? and by Dr Mary McCarthy. We could make suitable films only from the bovine cornea1 keratan sulphate supplied by Dr Mathews and not from either of the samples extracted from cartilage. The cornea1 sample, prepared from a papain digest (Cifonelli et al., 1967) of the tissue, had a molar ratio of ester sulphate to hexosamine of 1.17. Its molecular weight, estimated from intrinsic viscosity [y], was 16,000.t t M. B. Mathews, Dept. of Pediatrics,
J. A. Cifonelli and L. Rodbn, acid mucopolysaccharide University of Chicago.
reference
standards,
CONFORMATION
OF KERATAN (b) Film
SULPHATE
177
preparation
Keratan sulphate (10 mg) was dissolved in water (0.5 ml) and dried on a Teflon block. Attempts to stretch strips (about 8 mm long and 2 mm wide) of the resulting tim under constant load were unsuccessful at room temperature. Stretching was achieved, however, to about 2 or 3 times the original length at 65°C with 1.5 g tension in a closed atmosphere, whose relative humidity was kept constant (at about 79%) by a reservoir of saturated potassium bromide solution. X-ray diffraction patterns of unstretched films both before and after this heat treatment were identical, suggesting that the conformation had not been changed by heating. (c) X-ray
diffraction
Specimens were irradiated with focussed (Elliott, 1965) Ni-filtered CuKcr radiation. The relative humidity in the camera was maintained at 75% by sweeping with helium that had been bubbled through a solution of sodium nitrite (75.5% saturated). The diffraction patterns were calibrated by dusting the specimens with calcite, which yields a diffraction ring of spacing 0.3035 nm, and measured with computer-drawn Bernal charts. (d) Molecular
model-building
Computer methods were used to build molecular models under explicitly defined conditions with precise values for bond lengths and bond angles. Galactose and N-acetylglucosamine residues were fixed in the most probable conformation, which is a Reeves Cl chair. The backbone conformation of the helical polydisaccharide is then defined by the values of 4 conformation angles (Fig. 1). If the helix is constrained to have the pitch length and screw symmetry obtained from X-ray diffraction results, only 2 degrees of freedom remain (Arnott & Scott, 1972). The number of degrees of freedom was further reduced to 1 by constraining O(5) of galactose and O(3) of N-acetylglucosamine to be separated by a normal distance (O-270 nm) for hydrogen bond formation across the /3( l-+4) linkage. Similar hydrogen bonds have been proposed for the /3( l-+4) linkages in cellulose (Hermans, 1949) and chitin (Carlstrom, 1962) and have been observed in the crystal structures of the disaccharides cellobiose (Chu & Jeffrey, 1968), lactose (Fries et al., 1971), 1970). Non-bonded interactions between and methyl /3-cellobioside (Ham & Williams, atoms on adjacent pyranose rings severely restrict the values accessible to the 4 variable conformation angles. We have repeated the earlier (Rees, 1969) calculation of “allowed” values for these angles in keratan sulphate using the average pyranose ring co-ordinates of Arnott & Scott (1972). All of this information was combined by a linked-atom least-squares model-building program (Arnott & Scott, 1972), using the centres of the fully allowed regions to define “standard values” for .$AB, $AB, $BA and #BA. of 34’, O’, 31’ and --5”, respectivelyt. This method defines a unique model, in which the precise values of the conformation angles depend on the standard values chosen. Bond lengths and bond angles of the sulphate acetamido side groups were fixed at the stereochemically reasonable values shown in Fig. 2. The bond angle, +?(6), O(6), S), was based on a survey of the limited number of relevant crystal-structure determinations (Truter, 1958; Fitzwater & Rundle, 1959; Cam ,& Marsau, 1970). The acetamido group was fixed in a &ans conformation about the C(Z)-N bond and with the N-H bond trans-antiparallel with respect to the hydrogen atom on C(2) of N~acetylglucosamine. Orientations of the sulphate groups for both galactose and N-acetylglucosamine residues were explored by rotating about the bonds C(5)-C(6) (0 to 360”), C(6)-O(6) (0 to 360”) and 0(6)-S (0 to 120”) in steps of 30”. If any sulphate atom was 0.62 nm closer to any T The torsion angles at a glycosidic &,$** $BA I/C”” The torsion angles are given value 0” frtr bond is rotated clockwise from the 12
linkage
are defined as
= B(H*l > CA1 3 0, CB4), = B(C*l, 0, C*4, H=4) = B(HB1, CB1, 0, CB3): = f3(CBl, 0, CA3 I H*3) . in the eclipsed conformation. They itre positive when the eclipsed conformation (viewed along the central bond).
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S. ARNOTT
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FIG. 2. (a) The atomic l&belling for & disaccharide of kemttan sulphate. 6-sulphe& and residue B is IV-scetylglucosamine 6-sulphate. (b) The bond lengths sulphate.
(in nm) and bond angles (in deg.) resigned
TABLE
Con&wmttin
angles corresponding
Residue A is g&lactose
to the side groups in keratan
1
to the allowed, staggered conformation residues
of the sdphate
groups of galactose and N-metylglucosamine Galactose sulphate Conformation Torsion angle e(O6, ‘25, C6, 06) O(C6, C6, 06, S) B(C6, 06, S, 07)
N-scetylglucosamine sulph&e
(1)
(2)
(1)
(2)
60’ 180” 60”
180° 180” 60”
60’ -160” 60”
- 60” 150° 60”
PLATE I. X-ray diffraction pattern of a stretched film of keratan sulphate. The meridional (vertical) direction corresponds to the parallel axes of the helioes. To obtain this particular pattern the specimen u-as tilted 18” from the perpendicular to the X-ray beam.
CONFORMATION
OF KERATAti
SULPHATE
other atom than the sum of their van der Waals’ radii, the conformation Each sulphate was then represented by a number of conformations represented the centre of the allowed regions. (e) Ca&ulated
inkwity
179 was disallowed. (Table l), which
d&ribzGtion
The distribution of the diffracted intensity was calculated from the co-ordinates for each molecular model by the method described below, and compared with the observed distribution, to further test the acceptability of the model. The intensity distribution was calculated (Franklin & Klug, 1955) in steps of 0.2 nm-1 along each layer line, using Bessel functions of order w in the range - 12 2 n 5 12. Atomic scattering factors were corrected for water in the specimen as described elsewhere (Langridge et al., 1960; Arnott & Hukins, 1973). Because sulphate groups typically occur on 40% of galactose positions and 70% of N-acetylglucosamine positions, their atomic scattering factors were reduced proportionally by an occupancy factor using atomic coordinates corresponding to each of the allowed staggered sulphate orientations. Two additional positions of the 3 equivalent sdphate oxygen atoms were included by adding &30” to the value of the conformation angle B(C(6), O(6), S, 0) in order to allow for thermal motion of the sulphate group, with appropriate reduction of the atomic scattering factors for oxygen. Both these added conformations are allowed. Thermal motion of the entire molecule was allowed for by multiplying the square root of the intensity by exp( - I3 sin20/h2), where B, the isotropic attenuation factor, was given a value of 6 x low2 nm2; 8 is the Bragg angle and h the wavelength of CuKcc X-rays (0.15418 nm).
3. Results Plate I shows a typical X-ray diffraction pattern of a stretched film of kerstan sulphate. A series of patterns from accurately tilted specimens showed no meridional intensity on odd-numbered layer lines. We conclude, therefore, that the molecule has a twofold screw symmetry axis. The spacing between layer lines corresponds to a helix pitch of 1*89 nm. Our diffraction patterns can be most simply explained as arising from twofold single helices with an axial rise per disaccharide residue (h) of 0.945 nm, which results in an extended chain conformation. Other interpretations of the diEraction pattern are multi-chain helices that are stereochemically unreasonable. Figure 3 shows two different views of our two-fold helical model for the ordered conformation of keratan sulphate, with it,s sulphate groups in conformation 1 from t.he allowed forms listed in Table 1. Table 2 lists the co-ordinates of this same model, which was shown by further calculation to have no unreasonable non-bonded interactions. The values of the torsion angles $AB, $AB, $BA and #eA are -1.3”, 5+3”?16.9” and -29-S”, respectively. Although the diffraction pattern is confined to layer lines and, therefore, indicates that the molecules are oriented with their screw axes approximately parallel, the intensity distribution along each layer line is, for the most part, continuous. This shows that the parallel molecules have random translations along and rotations about their axes and are not packed together so that their disaccharide residues fit into a well-developed crystal lattice. However, destructive interference has occurred near the centre of the equator, leaving one broad Bragg reflection of spacing l-48 nm, and the array of molecules therefore has some order when viewed down a molecular screw axis at sufficiently low resolution. This effect is to be expected in an array having equally spaced helical molecules. with their helix axes parallel but with different translations along and rotations about these axes (Arnott, 1973). Apart from this small region of the equator of the diffraction pattern where destructive
180
S..ARNOTT
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I.89 nm
FIG. 3. Two views of the 2-fold helical model for keratan sulpha&e. Both are projections perpendicular to the helix axis, and are related to each other by a 46” rotation about it. Sulphate groups are shown at all their possible sites with an arbitrary allowed conformation.
interference occurs, the calculated intensity distribution is proportional to the square of a cylindrically averaged Fourier transform (Franklin & Klug, 1955). Figure 4 compares this calculated intensity distribution with that observed, and shows that a model of the kind we have proposed can provide a reasonable explanat.ion of the experimental results.
4. Discussion Even within a given tissue, keratan sulphate is not a single molecular species but has a covalent structure that varies between chains and probably within each chain. For example, cornea1 keratan sulphate may be separated into fractions having O-9 to 2-O sulphate groups per disaccharide residue (Cifonelli et al., 1967 ; Fransson & Anseth, 1967) and similar experiments show also that cartilage keratan sulphate is heterogeneous (Cifonelli et al., 1967). The arrangement of the sulphate groups along keratan sulphate chains is not fully understood but some results have been interpreted in terms of sulphate-rich and sulphate-free regions (Bhavanandan & Meyer, 1967,1968). We were able to obtain oriented films using keratan sulphate from cornea
CONFORMATIONOF~%RATANSULPHATE!
181
TABLE 2 Cylindrical
polar (r&Z) and Cartesian (X,Y,Z) w-ordinates of the non-hydrogen in one disaccharide residue of our keratan sulphate model
Atom Cl C2 c’s C4 C5 C6 01 02 03 04 05 06 :7(so,) 0wm) WSO*)
Cl c2 c3 c4 C5 C6 01 03 04 06 N C(amide) O(amide) C(methy1) 06 S 07(5W
W504) Q9W4)
r bm)
Galactose (residue A) 4 (deg.) X (nm)
0.1271 0.1183 0.1780 0.3259 0.3508 0.4924 0.0977 0.0717 0.1739 0.4006 0.2675 0.5184 0.6641 0.7013 0.7654 0.6780
19.98 - 13.31 21.32 21.53 31.26 27.62 - 29.54 -104.63 -2.48 2.13 18.79 33.85 30.94 19.19 35.24 36.13
0.0918 0.0888 0.1026 0.0016 0.1431 0.2455 0.1739 0.2321 0.0977 0.1804 0.1535 0.2638 0.3369 0.3237 0.3781 0.4983 0.5114 0.5002 O-6219
N-acetylglucosamine 20.08 -89.17 -147.38 180.00 53.60 42.87 -2.48 -131.84 150.48 23.54 - 121.95 - 103.79 - 86.89 - 118.31 46.96 35.87 19.70 38.43 40.85
Y (nm)
atoms
2 (nm)
0.1196 0.1152 0.1658 0.3032 0.2999 0.4363 0.0860 -0.0181 0.1737 0.4004 0.2532 0.4305 0.5696 0.6623 0.6251 0.5476
0.0434 --0.0273 0.0647 0.1196 0.1820 0.2283 -0.0481 - 0.0694 -0.0075 0.0149 0.0862 0.2887 0.3414 0.2305 0.4416 0.3998
- -0.0923
(residue B) 0.0862 0.0013 -0.0864 -0.0016 0.0849 0.1799 0.1737 -0.1548 -0.0850 0.1654 -0.0812 - 0.0629 0.0183 -0.1535 0.2581 0.4038 0.4814 0.3918 0.4704
0.0315 -0.0888 -0.0653 0~0000 0.1151 0.1670 -0.0075 -0.1729 0.0481 0.0720 -0.1302 -0.2562 -0.3364 -0.2850 0,2764 0.2920 0.1724 0.3109 0.4067
0.6723 0,7115 0.8312 0.9450 0.8950 1.0008 0.5717 0.8748 1.0502 0.7841 0.5959 0.5426 0.5858 0.4253 0.9525 1.0170 0.9905 1.1603 0.9585
0.2038 0.3386 0.4487 0.4126 0.2734 0.2268 0.1052 0.3653 0.5717 o-4131 0.1772
0.0977 0.0386 0.0270 0.1275
The 2 axis coincides with the helix axis. Co-ordinates of atoms in successive residues may be generated by adding 180” to 4 and 0.945 nm to 2. Snlphate group co-ordinates correspond to the conformation angle values 1 of Table 1.
but not from cartilage. The ordered conformation that occurs in our &lms of cornea1 keratan sulphate has an extended polysaccharide chain that is a single twofold helix with an axial rise per disaccharide residue of 0.945 nm and fringed with charged sulphate groups. It is unlikely that extended conformations of this type can be stable for isolated polysaccharide chains, because there would seem to be no possible mechanism by which favourable intramolecular energy terms can accumulate co-operatively to offset the entropy loss that is associated with conformational ordering. However, keratan sulphate could exist in extended conformations in the
182
8. ARNOTT
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AL.
FIG. 4. Calculated cylindrically averaged Fourier transform squared (oontinuous line) superimposed on the observed diffracted intensity (rectangular boxes are peaks in the continuous diffraction and the circle is a Bragg reflection). Closed, hatched end open boxes are intense, medium and weak diffraction. The widths of the diffraction maxima are indicated by the horizontal dimensions of the boxes. The observed intensity was measured with, a, Joyce-Lo&b1 scanning microdensitometer. The layer line is I, the reciprocal space radial co-ordinate is 6 and the cralcuWed intensity, from zero on each layer line, is given in arbitrary units (where the line is dotted the value haa been divided by 10).
extracellular. matrix, where co-operative stabilization might be derived from intermolecular contacts. On the other hand, this binding behaviour need not necessarily involve conformational ordering. The variable charge density that exists within chains or between them would lead to variable binding and interaction behaviour Ovith water, ions and other connective tissue components, and hence to the possibility of different conformational behaviour by different chains or chain segments. In any densely sulphsted part of a chain, the sulphates on either side of a’ I+3 linkage would be close enough to repel each other strongly unless they qrere bound to a positively charged ion or group in the vicinity; such chain segments might then be particularly likely to exist in a locally ordered conformation. Our keratan sulphate conformation and the other known conformations of glycosaminoglycans can be considered to be related to each other as states bf the same
CONFORMATION
OF KERATAN
SlJLPHATE
183
wire spring that are interconvertible by rather slight twisting or extension. Hyaluronic acid (Atkins et al., 1972a), chondroitin 6-sulphate (Atkins et al., 19726) and chondroitin 4-sulphate (Isaac & Atkins, 1973) when in the presence of acid, which suppresses the ionization of their uranic acid residues, will adopt conformations similar to that which we now report for keratan sulphate. This suppression can be considered to make the uronate residue electrostatically equivalent to a neutral hexose and, therefore, to emphasize the chemical similarity with uranic acid-free keratan sulphate. The twofold conformation, which represents a fully extended and fully twisted state of the glycosaminoglycan helix that we have likened to a wire spring, appears then to be most characteristic of those members of the family that are without uronate residues. That the various glycosaminoglycans have biological functions that are related and yet distinct, is further indicated by the variations that occur with development and with ageing. For example, the keratan sulphate content of human cartilage has been found to rise steadily with age between 1 year and 40 years, from about 5% to 60% of the total glycosaminoglycan, while the chondroitin 4-sulphate content falls from about 45% to loo/, (Mathews & Glagov, 1966). The content of chondroitin 6-sulphate changes little over the period. Unfortunately, the new understanding of glycosaminoglycan conformations has not yet led to any new principles by which we can give a molecular interpretation of such phenomena. We regard the delineation of the range of conformational behaviour of glycosaminoglycans as a stage towards physical studies on systems that are closer to the full complexity of biological situations and which we hope will eventually show, in molecular detail, how they function in living tissues. The work at Purdue University was supported by National Science Foundation grant no. GB35965X. One author (J. M. G.) was a Fellow of the Arthritis Foundation and another (D. W. L. H.) was a Fellow of the Jane Coffin Childs Memorial Fund for Medical Research.
Arnott, Arnott, Arnott, Arnott, Arnott,
REFERENCES Assoc. 9, 31-56. S. (1973). Trans. Amer. Crystallogr. S. & Hukins, D. W. L. (1973). J. Mol. Biol. 81, 93-105. S. & Scott, W. E. (1972). J. Chem. Sot. Perlcin Trans. 2, 3241335. S., Guss, J. M., Hukins, D. W. L. & Mathews, M. B. (19730). Science, 180, 743-745. S., Guss, J. M., Hukins, D. W. L. & Mathews, M. B. (1973b). Biochem. Biophys.
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Atkins, E. D. T., Phelps, C. F. & Sheehan, J. K. (1972a). Biochem. J. 128, 1255-1263. Atkins, E. D. T., Gaussen, R., Isaac, D. H., Nandanwar, V. & Sheehan, J. K. (19725). J. Polymer Sci. (sect. B), 10, 863-865. Balazs, E. A. (1966). .E’ed. Proc. 25, 1817-1822. Bhavanandan, V. P. & Meyer, K. (1967). J. Biol. Chem. 242, 4352-4359. Bhavanandan, V. P. & Meyer, K. (1968). J. Biol. Chem. 243, 1052-1059. Brandt, K. D. & Muir, H. (1971). Biochem. J. 123, 747-755. Cam, Y. & Marsau, P. (1970). G.R. Hebd. Seances Acad. Sci. (ser. G), Sci. Chim. (Paris) 270, 309-312. Carlstrom, D. (1962). Biochim. Riophys. Acta, 59, 361-364. Chu, S. S. C. & Jeffrey, G. A. (1968). Acta Crystullogr. ser. B, 24, 830-838. Cifonelli, J. A., Saunders, A. & Gross, J. I. (1967). Carbohydr. Res. 3, 478-485. Dea, I. C. M., Moorhouse, R., Rees, D. A., Arnott, S., Guss, J. M. & Balazs, E. A. (1973). Science, 179, 560-562. Elliott, A. (1965). J. Sci. In&rum. 42, 312-316.
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D. R. & Rundle, R. E. (1959). 2. Krystdogr. 112, 362-374. R. E. & Klug, A. (1955). Acta Cryetallogr. 8, 777-780. L. A. & Anseth, A. (1967). Eqn. Bye Res. 6, 107-119. L. A. & Malstrom, A. (1971). Eur. J. Biochem. 18, 422-430. C., Rao, S. T. & Sundaralingam, M. (1971). Acta Crystallogr.
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Ham, J. T. & Williams, D. G. (1970). Aota CrystaZEogr. ser. B, 26, 1373-1383. Hermans, P. H. (1949). Physics and Chemistry of Cellu.$ose I%bres, Elsevier, New York. Hirano, S. & Meyer, K. (1971). Biochem. Biophys. Res. Commun. 44, 1371-1375. Isaac, D. H. & Atkins, E. D. T. (1973). Nature New Biol. 244, 252-253. Langridge, R., Wilson, H. R., Hooper, C. W., Wilkins, M. H. F. & Hamilton, L. D. (1960). J. Mol. Biol. 2, 19-37. Mathews, M. B. (1967). In The Connective Tti.vue (Wagner, B. M. and Smith, D. E., eds), pp. 304-329, Williams and Wilkins, Baltimore. Mathews, M. B. & Cifonelli, J. A. (1965). J. Biol. Chem. 240, 4140-4145. Mathews, M. B. & Glagov, 8. (1966). J. Clin. Invest. 45, 1103-1111. Muir, H. (1969). Amer. J. Med. 47, 673-690. Perlin, A. S., Ng Ying Kin, N. M. K., Bhattacharjee, S. S. & Johnson, L. F. (1972). Camzd. J. Chem. 50, 2437-2441. Rees, D. A. (1969). J. Chem. Sot. (ser. B), 217-226. Seno, N., Meyer, K., Anderson, B. & Hoffman, P. (1965). J. Biol. Chem. 240, 1005-1010. Truter, M. R. (1958). Acta CryetaZZogr. 11, 680-685. Wolfram, M. L., Vercellotti, J. R., Tomomatsu, H. & Horton, D. (1964). J. Org. Chem. 29, 540-547.
Conformation
of Keratan Sdphate
STPJJTHERARNOTT, J. M. Gnss, D. W. L. HUKINS~ Department of Biological Sciences Purdue University West Lafayette, Id 47907, U.S.A. I. C. M. DEA AND D. A. REES Unilever Research Colworthl Welwyn Laboratory Colworth House Sharnbrook, Bedford MK44 IL&, Enjgland (Received 11 April 1974, and in revised form 25 May 1974) X-ray diffraction patterns from stretched films of keratan sulphate, isolated from bovine cornea, indicate that the molecules are twofold helices with an axial rise per disaccharide residue of 0.945 nm. These helices are oriented with their twofold screw axes parallel and about equally spaced, but are not further organized into regular crystalline arrays. Computer methods were used to construct a molecular model with the observed symmetry and axial rise per disaccharide residue, with standard bond lengths, bond’angles and pyranose ring conformations and with a hydrogen bond of length 0.270 nm between 0( 3) of N-acetylglucosamine and O(5) of galactose. This model has no unacceptably short non-bonded interatomic distances. The intensity distribution of the diffraction pattern calculated from the co-ordinates of the model is in reasonable agreement with the observed intensity distribution. This keratan sulphate model is an extended polysaccharide chain fringed with charged sulphate side groups, and is similar to those that have already been reported for chondroitin sulphates and dermatan sulphate, paralleling the similarities in covalent structure and biological occurrence among these substances.
1. Introduction Most of the glycosaminoglycans (mucopolysaccharides) of animal connective tissues share the same alternating fi(l+3), /3(1+4) baokbone. The members differ from one another by the nature and stereochemistry of backbone substitution (Mathews, 1967). They commonly occur as side chains covalently linked to a single polypeptide chain in a proteoglycan complex. Chondroitin 4-sulphate, chondroitin 6-sulphate and dermatan sulphate are not only related closely in structure, but evidently also in their biosynthesis, since some naturally occurring polysaccharides can be regarded as hybrid forms, which incorporate features of two or more of these particular members (Pransson & Malmstrom, 1971). Keratan sulphate and hyaluronic acid are less closely related to this series. Although keratan sulphate may be attached to the same protein backbone as chondroitin sulphates (Brandt & Muir, 1971), it differs from t Present address: Laboratory of Molecular Biophysics, Oxford, South Parks Road, Oxford OX1 3PS, England. 175
Department
of Zoology,
University
of
176
S. ARNOTT
ET AL.
them in containing no uranic acid residues. Hyaluronic acid is not sulphated, probably is not attached covalently in a proteoglycan, has a molecular weight that is larger by many orders of magnitude than that of the others (Balazs, 1966) and, so far as is known, has a covalent structure that is completely regular and is without any of the variability along the chain that is so characteristic of other glycosaminoglycans. Finally, heparin and heparan sulphate, although sulphated polymers of hexosamine and uranic acid residues, do not share this same basic backbone structure, since they appear to be completely l-+4 linked (Wolfrom et al., 1964; Perlin et al., 1972). To the extent that these polysaccharides are related in covalent structure, they might be expected to show some similarity in their conformations, interactions and biological functions. In this paper we describe an investigation of the conformation of keratan sulphate and attempt to relate the results to our earlier conformational (Rees, 1969) and X-ray (Dea et al., 1973; Arnott et al., 1973ab) studies of chondroitin sulphates, dermatan sulphate, and hyaluronic acid.
FIG. 1. The structural formula of keratan sulphate. the chain conformation are 4AB, $AB, qW and #““.
The 4 conformation
angles used to define
Keratan sulphate (Pig. 1) occurs in cartilage, nucleus pulposus, and cornea (Mathews, 1967; Muir, 1969) and among these sources it shows considerable variation in its detailed chemical composition and mode of linkage to protein (Mathews $ Cifonelli, 1965; Seno et al., 1965; Bhavanandan and Meyer, 1968). Typically, 79% of the N-acetylglucosamine residues and 40% of the galactose residues are sulphated, although the number of sulphate groups and presumably their distribution may vary (Mathews & Cifonelli, 1965; Bhavanandan & Meyer, 1967,196s; Hirano & Meyer, 1971). We have now obtained X-ray diffraction patterns from stretched films of keratan sulphate isolated from bovine cornea, which allow the conformation of the molecules in the films to be determined.
2. Materials and Methods (a) Materials Sodium salts of keratan sulphate extracted from both cornea and cartilage were kindly supplied by Dr M. B. Mathews? and by Dr Mary McCarthy. We could make suitable films only from the bovine cornea1 keratan sulphate supplied by Dr Mathews and not from either of the samples extracted from cartilage. The cornea1 sample, prepared from a papain digest (Cifonelli et al., 1967) of the tissue, had a molar ratio of ester sulphate to hexosamine of 1.17. Its molecular weight, estimated from intrinsic viscosity [y], was 16,000.t t M. B. Mathews, Dept. of Pediatrics,
J. A. Cifonelli and L. Rodbn, acid mucopolysaccharide University of Chicago.
reference
standards,
CONFORMATION
OF KERATAN (b) Film
SULPHATE
177
preparation
Keratan sulphate (10 mg) was dissolved in water (0.5 ml) and dried on a Teflon block. Attempts to stretch strips (about 8 mm long and 2 mm wide) of the resulting tim under constant load were unsuccessful at room temperature. Stretching was achieved, however, to about 2 or 3 times the original length at 65°C with 1.5 g tension in a closed atmosphere, whose relative humidity was kept constant (at about 79%) by a reservoir of saturated potassium bromide solution. X-ray diffraction patterns of unstretched films both before and after this heat treatment were identical, suggesting that the conformation had not been changed by heating. (c) X-ray
diffraction
Specimens were irradiated with focussed (Elliott, 1965) Ni-filtered CuKcr radiation. The relative humidity in the camera was maintained at 75% by sweeping with helium that had been bubbled through a solution of sodium nitrite (75.5% saturated). The diffraction patterns were calibrated by dusting the specimens with calcite, which yields a diffraction ring of spacing 0.3035 nm, and measured with computer-drawn Bernal charts. (d) Molecular
model-building
Computer methods were used to build molecular models under explicitly defined conditions with precise values for bond lengths and bond angles. Galactose and N-acetylglucosamine residues were fixed in the most probable conformation, which is a Reeves Cl chair. The backbone conformation of the helical polydisaccharide is then defined by the values of 4 conformation angles (Fig. 1). If the helix is constrained to have the pitch length and screw symmetry obtained from X-ray diffraction results, only 2 degrees of freedom remain (Arnott & Scott, 1972). The number of degrees of freedom was further reduced to 1 by constraining O(5) of galactose and O(3) of N-acetylglucosamine to be separated by a normal distance (O-270 nm) for hydrogen bond formation across the /3( l-+4) linkage. Similar hydrogen bonds have been proposed for the /3( l-+4) linkages in cellulose (Hermans, 1949) and chitin (Carlstrom, 1962) and have been observed in the crystal structures of the disaccharides cellobiose (Chu & Jeffrey, 1968), lactose (Fries et al., 1971), 1970). Non-bonded interactions between and methyl /3-cellobioside (Ham & Williams, atoms on adjacent pyranose rings severely restrict the values accessible to the 4 variable conformation angles. We have repeated the earlier (Rees, 1969) calculation of “allowed” values for these angles in keratan sulphate using the average pyranose ring co-ordinates of Arnott & Scott (1972). All of this information was combined by a linked-atom least-squares model-building program (Arnott & Scott, 1972), using the centres of the fully allowed regions to define “standard values” for .$AB, $AB, $BA and #BA. of 34’, O’, 31’ and --5”, respectivelyt. This method defines a unique model, in which the precise values of the conformation angles depend on the standard values chosen. Bond lengths and bond angles of the sulphate acetamido side groups were fixed at the stereochemically reasonable values shown in Fig. 2. The bond angle, +?(6), O(6), S), was based on a survey of the limited number of relevant crystal-structure determinations (Truter, 1958; Fitzwater & Rundle, 1959; Cam ,& Marsau, 1970). The acetamido group was fixed in a &ans conformation about the C(Z)-N bond and with the N-H bond trans-antiparallel with respect to the hydrogen atom on C(2) of N~acetylglucosamine. Orientations of the sulphate groups for both galactose and N-acetylglucosamine residues were explored by rotating about the bonds C(5)-C(6) (0 to 360”), C(6)-O(6) (0 to 360”) and 0(6)-S (0 to 120”) in steps of 30”. If any sulphate atom was 0.62 nm closer to any T The torsion angles at a glycosidic &,$** $BA I/C”” The torsion angles are given value 0” frtr bond is rotated clockwise from the 12
linkage
are defined as
= B(H*l > CA1 3 0, CB4), = B(C*l, 0, C*4, H=4) = B(HB1, CB1, 0, CB3): = f3(CBl, 0, CA3 I H*3) . in the eclipsed conformation. They itre positive when the eclipsed conformation (viewed along the central bond).
178
S. ARNOTT
ET
AL.
FIG. 2. (a) The atomic l&belling for & disaccharide of kemttan sulphate. 6-sulphe& and residue B is IV-scetylglucosamine 6-sulphate. (b) The bond lengths sulphate.
(in nm) and bond angles (in deg.) resigned
TABLE
Con&wmttin
angles corresponding
Residue A is g&lactose
to the side groups in keratan
1
to the allowed, staggered conformation residues
of the sdphate
groups of galactose and N-metylglucosamine Galactose sulphate Conformation Torsion angle e(O6, ‘25, C6, 06) O(C6, C6, 06, S) B(C6, 06, S, 07)
N-scetylglucosamine sulph&e
(1)
(2)
(1)
(2)
60’ 180” 60”
180° 180” 60”
60’ -160” 60”
- 60” 150° 60”
PLATE I. X-ray diffraction pattern of a stretched film of keratan sulphate. The meridional (vertical) direction corresponds to the parallel axes of the helioes. To obtain this particular pattern the specimen u-as tilted 18” from the perpendicular to the X-ray beam.
CONFORMATION
OF KERATAti
SULPHATE
other atom than the sum of their van der Waals’ radii, the conformation Each sulphate was then represented by a number of conformations represented the centre of the allowed regions. (e) Ca&ulated
inkwity
179 was disallowed. (Table l), which
d&ribzGtion
The distribution of the diffracted intensity was calculated from the co-ordinates for each molecular model by the method described below, and compared with the observed distribution, to further test the acceptability of the model. The intensity distribution was calculated (Franklin & Klug, 1955) in steps of 0.2 nm-1 along each layer line, using Bessel functions of order w in the range - 12 2 n 5 12. Atomic scattering factors were corrected for water in the specimen as described elsewhere (Langridge et al., 1960; Arnott & Hukins, 1973). Because sulphate groups typically occur on 40% of galactose positions and 70% of N-acetylglucosamine positions, their atomic scattering factors were reduced proportionally by an occupancy factor using atomic coordinates corresponding to each of the allowed staggered sulphate orientations. Two additional positions of the 3 equivalent sdphate oxygen atoms were included by adding &30” to the value of the conformation angle B(C(6), O(6), S, 0) in order to allow for thermal motion of the sulphate group, with appropriate reduction of the atomic scattering factors for oxygen. Both these added conformations are allowed. Thermal motion of the entire molecule was allowed for by multiplying the square root of the intensity by exp( - I3 sin20/h2), where B, the isotropic attenuation factor, was given a value of 6 x low2 nm2; 8 is the Bragg angle and h the wavelength of CuKcc X-rays (0.15418 nm).
3. Results Plate I shows a typical X-ray diffraction pattern of a stretched film of kerstan sulphate. A series of patterns from accurately tilted specimens showed no meridional intensity on odd-numbered layer lines. We conclude, therefore, that the molecule has a twofold screw symmetry axis. The spacing between layer lines corresponds to a helix pitch of 1*89 nm. Our diffraction patterns can be most simply explained as arising from twofold single helices with an axial rise per disaccharide residue (h) of 0.945 nm, which results in an extended chain conformation. Other interpretations of the diEraction pattern are multi-chain helices that are stereochemically unreasonable. Figure 3 shows two different views of our two-fold helical model for the ordered conformation of keratan sulphate, with it,s sulphate groups in conformation 1 from t.he allowed forms listed in Table 1. Table 2 lists the co-ordinates of this same model, which was shown by further calculation to have no unreasonable non-bonded interactions. The values of the torsion angles $AB, $AB, $BA and #eA are -1.3”, 5+3”?16.9” and -29-S”, respectively. Although the diffraction pattern is confined to layer lines and, therefore, indicates that the molecules are oriented with their screw axes approximately parallel, the intensity distribution along each layer line is, for the most part, continuous. This shows that the parallel molecules have random translations along and rotations about their axes and are not packed together so that their disaccharide residues fit into a well-developed crystal lattice. However, destructive interference has occurred near the centre of the equator, leaving one broad Bragg reflection of spacing l-48 nm, and the array of molecules therefore has some order when viewed down a molecular screw axis at sufficiently low resolution. This effect is to be expected in an array having equally spaced helical molecules. with their helix axes parallel but with different translations along and rotations about these axes (Arnott, 1973). Apart from this small region of the equator of the diffraction pattern where destructive
180
S..ARNOTT
El’
AL.
I.89 nm
FIG. 3. Two views of the 2-fold helical model for keratan sulpha&e. Both are projections perpendicular to the helix axis, and are related to each other by a 46” rotation about it. Sulphate groups are shown at all their possible sites with an arbitrary allowed conformation.
interference occurs, the calculated intensity distribution is proportional to the square of a cylindrically averaged Fourier transform (Franklin & Klug, 1955). Figure 4 compares this calculated intensity distribution with that observed, and shows that a model of the kind we have proposed can provide a reasonable explanat.ion of the experimental results.
4. Discussion Even within a given tissue, keratan sulphate is not a single molecular species but has a covalent structure that varies between chains and probably within each chain. For example, cornea1 keratan sulphate may be separated into fractions having O-9 to 2-O sulphate groups per disaccharide residue (Cifonelli et al., 1967 ; Fransson & Anseth, 1967) and similar experiments show also that cartilage keratan sulphate is heterogeneous (Cifonelli et al., 1967). The arrangement of the sulphate groups along keratan sulphate chains is not fully understood but some results have been interpreted in terms of sulphate-rich and sulphate-free regions (Bhavanandan & Meyer, 1967,1968). We were able to obtain oriented films using keratan sulphate from cornea
CONFORMATIONOF~%RATANSULPHATE!
181
TABLE 2 Cylindrical
polar (r&Z) and Cartesian (X,Y,Z) w-ordinates of the non-hydrogen in one disaccharide residue of our keratan sulphate model
Atom Cl C2 c’s C4 C5 C6 01 02 03 04 05 06 :7(so,) 0wm) WSO*)
Cl c2 c3 c4 C5 C6 01 03 04 06 N C(amide) O(amide) C(methy1) 06 S 07(5W
W504) Q9W4)
r bm)
Galactose (residue A) 4 (deg.) X (nm)
0.1271 0.1183 0.1780 0.3259 0.3508 0.4924 0.0977 0.0717 0.1739 0.4006 0.2675 0.5184 0.6641 0.7013 0.7654 0.6780
19.98 - 13.31 21.32 21.53 31.26 27.62 - 29.54 -104.63 -2.48 2.13 18.79 33.85 30.94 19.19 35.24 36.13
0.0918 0.0888 0.1026 0.0016 0.1431 0.2455 0.1739 0.2321 0.0977 0.1804 0.1535 0.2638 0.3369 0.3237 0.3781 0.4983 0.5114 0.5002 O-6219
N-acetylglucosamine 20.08 -89.17 -147.38 180.00 53.60 42.87 -2.48 -131.84 150.48 23.54 - 121.95 - 103.79 - 86.89 - 118.31 46.96 35.87 19.70 38.43 40.85
Y (nm)
atoms
2 (nm)
0.1196 0.1152 0.1658 0.3032 0.2999 0.4363 0.0860 -0.0181 0.1737 0.4004 0.2532 0.4305 0.5696 0.6623 0.6251 0.5476
0.0434 --0.0273 0.0647 0.1196 0.1820 0.2283 -0.0481 - 0.0694 -0.0075 0.0149 0.0862 0.2887 0.3414 0.2305 0.4416 0.3998
- -0.0923
(residue B) 0.0862 0.0013 -0.0864 -0.0016 0.0849 0.1799 0.1737 -0.1548 -0.0850 0.1654 -0.0812 - 0.0629 0.0183 -0.1535 0.2581 0.4038 0.4814 0.3918 0.4704
0.0315 -0.0888 -0.0653 0~0000 0.1151 0.1670 -0.0075 -0.1729 0.0481 0.0720 -0.1302 -0.2562 -0.3364 -0.2850 0,2764 0.2920 0.1724 0.3109 0.4067
0.6723 0,7115 0.8312 0.9450 0.8950 1.0008 0.5717 0.8748 1.0502 0.7841 0.5959 0.5426 0.5858 0.4253 0.9525 1.0170 0.9905 1.1603 0.9585
0.2038 0.3386 0.4487 0.4126 0.2734 0.2268 0.1052 0.3653 0.5717 o-4131 0.1772
0.0977 0.0386 0.0270 0.1275
The 2 axis coincides with the helix axis. Co-ordinates of atoms in successive residues may be generated by adding 180” to 4 and 0.945 nm to 2. Snlphate group co-ordinates correspond to the conformation angle values 1 of Table 1.
but not from cartilage. The ordered conformation that occurs in our &lms of cornea1 keratan sulphate has an extended polysaccharide chain that is a single twofold helix with an axial rise per disaccharide residue of 0.945 nm and fringed with charged sulphate groups. It is unlikely that extended conformations of this type can be stable for isolated polysaccharide chains, because there would seem to be no possible mechanism by which favourable intramolecular energy terms can accumulate co-operatively to offset the entropy loss that is associated with conformational ordering. However, keratan sulphate could exist in extended conformations in the
182
8. ARNOTT
ET
AL.
FIG. 4. Calculated cylindrically averaged Fourier transform squared (oontinuous line) superimposed on the observed diffracted intensity (rectangular boxes are peaks in the continuous diffraction and the circle is a Bragg reflection). Closed, hatched end open boxes are intense, medium and weak diffraction. The widths of the diffraction maxima are indicated by the horizontal dimensions of the boxes. The observed intensity was measured with, a, Joyce-Lo&b1 scanning microdensitometer. The layer line is I, the reciprocal space radial co-ordinate is 6 and the cralcuWed intensity, from zero on each layer line, is given in arbitrary units (where the line is dotted the value haa been divided by 10).
extracellular. matrix, where co-operative stabilization might be derived from intermolecular contacts. On the other hand, this binding behaviour need not necessarily involve conformational ordering. The variable charge density that exists within chains or between them would lead to variable binding and interaction behaviour Ovith water, ions and other connective tissue components, and hence to the possibility of different conformational behaviour by different chains or chain segments. In any densely sulphsted part of a chain, the sulphates on either side of a’ I+3 linkage would be close enough to repel each other strongly unless they qrere bound to a positively charged ion or group in the vicinity; such chain segments might then be particularly likely to exist in a locally ordered conformation. Our keratan sulphate conformation and the other known conformations of glycosaminoglycans can be considered to be related to each other as states bf the same
CONFORMATION
OF KERATAN
SlJLPHATE
183
wire spring that are interconvertible by rather slight twisting or extension. Hyaluronic acid (Atkins et al., 1972a), chondroitin 6-sulphate (Atkins et al., 19726) and chondroitin 4-sulphate (Isaac & Atkins, 1973) when in the presence of acid, which suppresses the ionization of their uranic acid residues, will adopt conformations similar to that which we now report for keratan sulphate. This suppression can be considered to make the uronate residue electrostatically equivalent to a neutral hexose and, therefore, to emphasize the chemical similarity with uranic acid-free keratan sulphate. The twofold conformation, which represents a fully extended and fully twisted state of the glycosaminoglycan helix that we have likened to a wire spring, appears then to be most characteristic of those members of the family that are without uronate residues. That the various glycosaminoglycans have biological functions that are related and yet distinct, is further indicated by the variations that occur with development and with ageing. For example, the keratan sulphate content of human cartilage has been found to rise steadily with age between 1 year and 40 years, from about 5% to 60% of the total glycosaminoglycan, while the chondroitin 4-sulphate content falls from about 45% to loo/, (Mathews & Glagov, 1966). The content of chondroitin 6-sulphate changes little over the period. Unfortunately, the new understanding of glycosaminoglycan conformations has not yet led to any new principles by which we can give a molecular interpretation of such phenomena. We regard the delineation of the range of conformational behaviour of glycosaminoglycans as a stage towards physical studies on systems that are closer to the full complexity of biological situations and which we hope will eventually show, in molecular detail, how they function in living tissues. The work at Purdue University was supported by National Science Foundation grant no. GB35965X. One author (J. M. G.) was a Fellow of the Arthritis Foundation and another (D. W. L. H.) was a Fellow of the Jane Coffin Childs Memorial Fund for Medical Research.
Arnott, Arnott, Arnott, Arnott, Arnott,
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