Vacuum ultraviolet circular dichroism of keratan sulfate

Biochimica etBiophysicaActa 924 (1987) 99-103 Elsevier

99

BBA22702

V a c u u m u l t r a v i o l e t circular d i c h r o i s m o f k e r a t a n s u l f a t e

E u g e n e S. S t e v e n s

a

a n d B o h a i L i n b,.

~ Department of Chemist~, State University of New York at Binghamton, Binghamton, N Y (U.S.A.) and b Department of Biology, Brookhaven National Laboratoo', Upton, N Y (U.S.A.) (Received 6 October 1986)

Key words: Circular dichroism; Keratan sulfate

The vacuum ultraviolet CD of keratan sulfate reveals an intense negative CD band at 171 nm. Its intensity can be rationalized with a recently proposed quadrant rule in terms of the acetamido group being slightly tilted toward the hexosaminidic linkage oxygen. The same structural feature accounts for the particularly intense negative n-~r* CD band near 210 nm.

Introduction

Keratan sulfate (keratosulfate), a connective tissue glycosaminoglycan, has as its predominant repeating unit a disaccharide structure consisting, of (1 ---, 3)-/3-o-galactopyranosyl-(1 --, 4)-2-acetamido-2-deoxy-/3-o-glucopyranosyl 6-sulfate [1]. It constitutes about half of the glycosaminoglycan content of bovine cornea [2]. The keratan sulfate content of human cartilage increases steadily with age [3], but there is no molecular interpretation of this phenomenon. In corneal preparations of keratan sulfate the extent of sulfation varies, but typically 30% of the galactosyl moieties and approximately 80% of Nacetylglucosamino moieties are sulfated [4]. Other features of sample heterogeneity include variation in residual amino acids and variable contamination with chondroitin sulfates. Corneal and skeletal

* Present address: Institute of Biophysics, Academia Sinica, Beijing, China. Correspondence: E.S. Stevens, Department of Chemistry, State University of New York at Binghamton, Binghamton, NY 13901, U.S.A.

cartilage keratan sulfates are considered to be fragments of two different classes of proteoglycan, since (a) galactosamine is present in cartilage, but not corneal, preparations, (b) aspartate serves as the sole amino acid linking the carbohydrate chain to peptide in corneal preparations, and (c) cartilage keratan sulfate is more highly sulfated [5]. The distinctive structural feature of both classes of keratan sulfate, in comparison with other glycosaminoglycans (hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparin), is the absence of uronic acid residues. This feature, together with the presence of sialic acid, fucose, mannose, and glycosidic linkages to asparagine, give to keratan sulfate some of the appearance of a glycoprotein [6]. On the other hand, the predominant disaccharide repeating unit in keratan sulfate is closely related to that in chrondroitin sulfate, which is reflected in the similarity of the solid state structures they adopt as determined by X-ray diffraction. The sodium salt of keratan sulfate [7] is found in a 2-fold helix with an axial rise per disaccharide of 0.95 nm, similar to the conformation adopted by chondroitin sulfate when the charge of the uronic acid residues is suppressed by lowering the pH. The sodium salt of chondroitin

0304-4165/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

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4-sulfate occurs as a 3-fold helix [8,9]; even small amounts of calcium ion result in an extended 2-fold structure [10]. In our recent studies of the vacuum ultraviolet circular dichroism (VUCD) of other glycosaminoglycans (chondroitin sulfate [11], heparin [12], dermatan sulfate [13]), we have observed a 175 nm C D band in aqueous solution, and have described a sector rule which relates the sign of that CD band to the geometrical arrangement of oxygen-containing perturbing groups about the acetal chromophores [13]. We report here the resuits of our measurements on corneal keratan sulfate, and compare the results with our earlier findings. Materials and Methods Keratan sulfate (bovine cornea, sodium salt, Sigma) was dissolved directly i n ~ H 2 0 . V U C D measurements were made on a prototype instrument described previously [14] using concentrations of 13.2 m g / m l or 5.3 m g / m l , in a cell of 0.052 m m pathlength. The spectral resolution was 3.2 nm, and the scan rate was 1.0 n m / m i n with a time constant of 100 s. Under these conditions spectra could be measured to 170 nm. In collaboration with Dr. John C. Sutherland a solution spectrum to 170 nm was also obtained at the U.S. National Synchrontron Light Source at Brookhaven National Laboratory. The spectrometer parameters are approximately the same as those described above [15], the significant difference being the use of the high intensity synchrotron light source in place of a conventional deuterium discharge Hinteregger lamp. CD spectra measured on the two instruments were identical except that the signal-to-noise ratio near 170 nm is better with the synchrotron light source. Sulfur analysis (7.33%) and carbon analysis (32.24%) gave a ratio of 1.20 mol sulfur for 14 mol carbon (see Fig. 1), yielding a mean disaccharide molecular weight of 487 for the sodium salt. The ratio of percent carbon found to calculated percent carbon was 0.930, indicating 7.0% water. With these results the calculated percent nitrogen is 2.67, which agreed with the percent nitrogen found, 2.69. Molar ellipticities are reported based on a disaccharide molecular weight of 487.

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Films were cast on calcium fluoride discs by evaporating solutions to dryness in a desiccator. To detect any molecular orientation, successive spectra were obtained as the film was rotated in the light beam by increments of 90 ° Results The C D spectrum to 170 nm of keratan sulfate in 2H20, p2H 7.0, is shown in Fig. 2. A negative C D band appears near 210 nm with molar ellipticity [ 0 ] = - 1 1 . 4 - 1 0 3 d e g . c m 2 . d m o l 1, and a positive band near 188 nm with molar ellipticity [0] = +6.59-103 d e g - c m 2. dmol 1. The vacuum ultraviolet C D instrument allows the measurement of a large, previously unobserved, negative CD band near 171 nm, with [ 0 ] = - 2 4 . 2 . 1 0 3 degcm 2. dmol 1 Film spectra, measured to 155 nm, were qualitatively the same as the solution spectra in the overlapping region. There was an orientation dependence of CD in the films, so that the film spectra are not quantitatively meaningful, but the presence of a large negative band in the vacuum I

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ultraviolet region is nevertheless confirmed by the film spectra. That band appears larger than the 210 nm band in all film orientations. Indeed, the film spectrum obtained by averaging over the several orientations used in the experiment shows the vacuum ultraviolet band to be 3-times larger than the 210 nm band; in solution it is twice as large. Also, in film spectra that negative band appears consistently at 166 nm, which indicates that there is a small blue shift in the solid state spectrum. Discussion

The CD of keratan sulfate above 185 nm was reported by Stone [16]. She observed a negative CD band at 210 nm with [0] = - 8 . 9 . 1 0 3 deg. cm 2. dmol-1, and a positive band at 190 nm with [ 0 ] = +7.9.103 deg-cm2.dmo1-1. With a more highly sulfated keratan sulfate from shark cartilage the negative band was observed at 207.5 nm with [0]= - 9 . 5 . 1 0 3 deg.cm2.dmo1-1 and the positive band at 190 nm with [ 0 ] = - 9 . 5 . 1 0 3 deg. cm ~. dmo1-1 and the positive band at 190 nm with [0] = +5.9.103 deg. cm 2. dmo1-1. Given some expected variation in molar ellipticities arising from sample heterogeneity (see above), our values are in reasonable agreement with hers. In all cases the low energy negative band is stronger than the positive band. Apparent CD intensities can be affected by partial overlap with neighboring bands, and in keratan sulfate, in which all three CD bands have neighboring bands of opposite sign (Fig. 2), the apparent peak intensities can change with even small shifts in band centers. Although the effect of added salt on CD was not measured, previous work by Eyring and Yang [17] on chondroitin sulfate showed virtually no dependence on sodium ion concentration for that structurally similar giycosaminoglycan. n-~r* Transition

The negative band that appears in the CD of all glycosaminoglycans near 210 nm has been assigned to the n-~r* transitions of the acetamido and carboxyl groups [18,19]. In keratan sulfate, which has no uronic acid, the band originates solely from the acetamido group at C-2 of the glucosyl residue. Indeed, the large intensity of the

210 nm band in keratan sulfate may indicate that the acetamido group is generally the dominant chromophore in the 210 nm region of glycosaminoglycan CD. The intensity of glycosaminogiycan CD near 210 nm varies considerably with chemical composition. We previously have pointed out [13] that in chondroitin sulfates the n-rr * CD is only slightly larger than the sum of molar ellipticities of methyl-2-acetamido-2-deoxy-fl-D-galactopyranoside and methyl-fl-D-glucuronoside, model compounds representing the constituents of chondroitins. This is in contrast to the n-~r* CD in keratan sulfate in which the n-~r* CD band (Fig. 2) is several-times larger than the molar ellipticity of its chromophoric constituent, methyl-2-acetamido-2deoxy-fl-D-glucopyranoside. A theoretical model for the n-~r* rotational strength of acetamido sugars, initially described by Yeh and Bush [20], was recently extended by Cohen and Stevens (unpublished data). The model incorporates the static-field ('one-electron') mechanism of optical activity, whereby the rotational strength arises from the coupling of the n-~r* and •r-~r* electronic transitions of the amide chromophore through the electrostatic field of the permanent partial charges on nearby atoms. In terms of that model, the n-~r* rotational strength of the acetamido group in keratan sulfate is dependent on the orientation of the neighboring C-3 hydroxyl group, the q~, ~p torsional angles of the (1 --, 3) linkage which determines the orientation of the neighboring galactose residue, and the orientation of the acetamido residue. Except for an indirect effect on C-3 hydroxyl group orientation by the galactose residue across the (1 -~ 4) linkage, the C-3 hydroxyl group oriex~tation may not be much different than in the model monomeric compound, methyl-2-acetamido-2-deoxy-glucopyranoside. On the other hand, for q,, ~p torsional angles corresponding to an extended chain, the axial C-4 hydroxyl group of the galactose residue across the (1 ~ 3) linkage is in a position where its oxygen atom makes a negative contribution to the n-rr* CD band, complementing the negative contribution from the (1 --. 3) linkage oxygen atom. Both of these interactions would be strengthened by a rotation of the acetamido group which brings the acetamido

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oxygen atom closer to the (1 --, 3) linkage oxygen atom. In the solid state 2-fold helical conformation of the sodium salt [7], a rotation of 5 ° is observed, relative to an orientation in which the acetamido CO bond eclipses the C - 2 - H bond. In chondroitin 4-sulfate, the 2-acetamido-2-deoxy-/~-D-galactopyranose residue is joined by a (1 -~ 4) linkage to a B-D-glucuronate residue. There is no axial hydroxyl group in a position corresponding to the galactose C-4 hydroxyl group in keratan sulfate. Furthermore, in the 2-fold structure of the calcium salt [10], there is no tilt of the acetamido group toward the neighboring (1-* 4) linkage oxygen atom. Thus, two structural features of keratan sulfate which are potential sources of increased n-vr* CD intensity are absent in chondroitin 4-sulfate. The slight increase in n-vr* CD intensity in chondroitins, relative to its monomers, may, in fact, arise from a slight tilt of the acetamido group toward the//-D-glucuronic acid residue across the (1 --, 4) linkage which would bring the acetamido oxygen atom closer to the hydrogen atom of the C-2 hydroxyl group of the adjoining residue, an interaction which has been attributed as the cause of the slight downfield N H chemical shift in chondroitins, relative to monomeric model compounds [21]. Chondroitin and keratans also differ in sites of ~ulfation, but since chondroitin, chondroitin 4-sulfate, and chondroitin 6-sulfate all have n-~'* CD bands of approximately the same magnitude, the different sites of sulfation between chondroitins and keratans is not a likely source of the large CD nonadditivity in keratan sulfate. The proposal made here that the enhanced n-vr* CD band in keratan sulfate originates in the (1 -* 3) hexosaminidic linkage is similar to that of Cowman et al. [22] that the enhancement in hyaluronate also originates in the hexosaminidic linkage. vr-~r* Transition

Stone [16] originally noted a correlation btween the sign of the CD band in glycosaminoglycans near 190 nm and the linkage site on the amino sugar. Keratan sulfate and heparin have (1 --, 4) linked amino sugars and both display positive CD bands near 190 rim; chondroitins, dermatans, and

hyaluronate have (1-~ 3) amino sugar linkages and display small or negative CD bands near 190 rim.

Because keratan sulfate has no uronic acid constituent, the 188 nm CD band originates entirely in the ~r-vr* acetamido transition. The intensity of the band in keratan sulfate (Fig. 2) is somewhat greater than the positive band of methyl-2acetamido-2-deoxy-[/-D-glucopyranoside [23] in which [ 0 ] = + 3 . 5 . 1 0 -~ d e g . c m 2 - d m o l 1. The origin of this nonadditivity is not known. Vacuum ultraviolet CD

The negative sign of the 171 nm CD band is consistent with the quadrant rule recently proposed for that band in saccharides [13]. According to the quadrant rule, the negative dichroism at 171 nm in keratan sulfate arises from the combined effect of three negative contributions: (1) perturbation of the /~-D-galactose ring oxygen atom by the galactose axial 0-4 atom; (2) perturbation of the (1 - , 4) linkage oxygen atom by the B-Dgalactose 0-2 atom; and (3) perturbation of the (1-~ 3) linkage oxygen atom by the acetamido group. The first of these contributions is fixed by the stereochemistry of the sugar ring, but the glycosidic oxygen contributions depend on chain geometry. For application of the sector rule we use a projection based on a locally extended chain conformation, in which C-2 lies approximately in the C-1-O-1-CH_~ plane. Variation in the linkage geometry will affect the magnitude of the CD band but, given the steric restrictions across the glycosidic linkage, it is unlikely to affect its sign. Chondroitins also display negative CD near 175 nm [11] as is expected from application of the quadrant rule to that closely related structure. The CD in keratan sulfate is, however, several-times more intense than in chondroitins. In chondroitins, the quadrant rule indicates that the negative CD arises from contributions analogous to those in keratan sulfate: (1) perturbation of the 2-acetamido-2-deoxy-/~-D-galactopyranose ring oxygen atom by the axial 0-4 atom; (2) perturbation of the (1---, 3) linkage oxygen atom by the /~-D-glucuronate 0-2 atom; and (3) perturbation of the (1 ~ 4) linkage oxygen atom by the acetamido group. The first of these is fixed by the stereochemistry of the sugar ring, and

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would thereby have approximately the same magnitude as in keratan sulfate. The second contribution, for the locally extended chain geometries of chondroitins and keratan sulfates, would also be similar in magnitude. The perturbation of the amino sugar linkage oxygen atom by the acetamido group increases as that group is rotated toward the linkage oxygen, relative to an orientation in which the acetamido CO bond eclipses the C-2-H bond. The increased intensity of the vacuum ultraviolet CD band in keratan sulfate, relative to chondroitin sulfates, can therefore be rationalized by the same structural feature that also rationalizes its increased n-~r* CD band intensity; i.e., a rotation of the acetamido group toward the neighboring linkage oxygen atom. In the solid state, such a rotation is observed in the 2-fold helical conformation of keratan sulfate [7], but not in the 2-fold helical conformation of chondroitin sulfate [10]. The factors which determine the precise orientation of acetamido groups in glycosaminoglycans are not known, but the present work indicates that circular dichroism may be sensitive to subtle differences in that conformational feature. Since chondroitin and chondroitin 6-sulfate display similar vacuum ultraviolet CD [11], sulfation alone and the differing sites of sulfation in keratans and chondroitins do not provide a likely alternative explanation of the increased vacuum ultraviolet CD intensity in keratan sulfate.

Acknowledgments This work was partially supported by NIH Grant GM24862. We are grateful to Dr. John C. Sutherland for providing access to the Port U9B vacuum CD spectrometer at the National Synchrotron Light Source at Brookhaven National Laboratory. Port U9B is supported by the Office of Health and Environmental Research, U.S. Department of Energy; the NSLS is supported by

the Office of Energy Research, U.S. Department of Energy.

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