Interaction of tropocollagen with protein-polysaccharide complexes

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BIOCHIMICAET BIOPHYSICAACTA

BBA 35813 INTERACTION OF TROPOCOLLAGEN WITH PROTEINPOLYSACCHARIDE COMPLEXES AN ANALYSIS OF THE IONIC GROUPS RESPONSIBLE FOR INTERACTION

V. P O D R A Z K Y % F. S. S T E V E N ~, D. S. J A C K S O N ~, J. B. W E I S S b AND S. J. L E I B O V I C H b

•Lapworth Laboratories, Department of Medical Biochemistry; bBiochemistry Section, Rheumatism Research Centre, University of Manchester Connective Tissue Research Group, Manchester, I3 (Great Britain) (Received October 2nd, 197 o)

SUMMARY

Human intervertebral disc protein-polysaccharide complex (PP-L) has been separated by isoelectric focussing analysis into three main subunits and four minor components. PP-L interacted with tropocollagen in the isoelectric-focussing column to yield a single complex zone, containing both uronic acid and hydroxyproline, with an isoionic point intermediate between those of tropocollagen and PP-L. This complex, formed at 4 °, was shown by electron microscopy to be in the form of fibrous long-spacing fibrils, whilst the complex formed at 37 ° consisted of native-type fibrils. Chemical and enzymic modification of the PP-L demonstrated that the sulphate group was largely responsible for interaction with tropocollagen during isoelectric focussing. The initial interaction was independent of the core protein and independent of the length of the glycosaminoglycan chains. The guanidino groups of arginine and the e-amino groups of lysine were found to be the sites of interaction of tropocollagen with PP-L.

INTRODUCTION

Protein-polysaccharide complexes (referred to as PP-L, employing the terminology of GERBER et al. 1) consist of a core of protein with a number of laterally attached chondroitin sulphate and/or keratan sulphate polysaccharide side chains 2. The protein-free sulphated polysaccharides, prepared by enzymic destruction of the core protein, react with tropocollagen at low temperature and acidic pH to form fibrous long spacing fibrils~. At higher temperatures and physiological pH these same sulphated polysaccharides interact with tropocollagen to form native-type fibrils4. Abbreviation" PP-L, protein-polysaccharide complex.

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Using biophysical techniques, intact P P - L preparations were shown to interact ionically with tropocollagen b y MATHEWS5 and MATHEWS AND DECKERs. In a more detailed study TOOLE AND LOWTHER~ reported instantaneous interaction between tropocollagen and P P - L (but not with protein-free sulphated polysaccharides) at 4 ° with the formation of a small proportion of native-type fibrils, whilst at 37 ° the rate of formation of native-type fibrils was slowed although the yield was high. In the present study, chemically modified tropocollagen and modified PP-L have been interacted in an L K B electrofocussing column. The results confirm the previous report s that interaction takes place between the positively charged amino groups of tropocollagen and the negatively charged sulphate groups of PP-L.

MATERIALS AND METHODS

Chemical analyses Uronic acid, sialic acid and total sulphate analyses were performed as described previously 9. Hydroxyproline was estimated by the procedure of WOESSNER10.

Protein polysaccharide complex ( PP-L ) Intervertebral discs were obtained from a 64-year-old normal subject at post mortem who died of myocardial infarction and P P - L was prepared as the potassium salt using the technique of MALAWISTA AND SCHUBERT11.

Neuraminidase-treated PP-L 4 ° mg of P P - L were incubated with neuraminidase (5oo I.U., Koch-Light,

Vibrio cholera) for 24 h at p H 5.4 and 37 °. The digested PP-L was purified by precipitation with 2 vol. ethanol containing IO g/1 potassium acetate ix. The precipitate was redissolved in o.15 M KC1 and reprecipitated a further 3 times with ethanol-potassium acetate as described above. The neuraminidase-treated PP-L contained only 5% of the sialic acid present in the original PP-L preparation.

Papain-treated PP-L PP-L was dissolved in o.15 M KC1 containing 5 mM cysteine and o.75 mM EDTA (tetrasodium salt). The mixture was adjusted to pH 5.6 with 0. 5 M acetic acid; an appropriate quantity of papain (Sigma, twice crystallised) was added to give an enzyme substrate ratio of 1:50. The reaction mixture was incubated at 37 ° for 24 h. The resultant acid polysaccharide fragments were isolated by ethanol-potassium acetate precipitation as described for the neuraminidase-treated PP-L n. This procedure was repeated 3 times.

Oligosaccharides released from PP-L by hyaluronidase digestion 21 mg PP-L was treated with 700 I.U. hyaluronidase (Koch-Light, ex ovine testes) at p H 5.0 and 37 ° for 17 h. The reaction was terminated by heating to 60 ° for IO min, and the low-molecular-weight oligosaccharides were then collected b y dialysis against distilled water for 24 h. The diffusable fragments were freeze-dried, dissolved in IOO ml water and centrifuged at 96 ooo × g for I h at 4 °. The uronic acid content of the supernatant was found to be 30/,g/ml.

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Desulphated PP-L P P - L was treated with 0.06 M HC1 in methanol as described by KANTOR AND SCHUBERTTM. Analysis indicated that 63% of the initial ester sulphate had been removed during desulphation. The uronic acid content remained unaltered during the desulphation process.

Chromatographic subfractionation of PP-L Four major and two minor subfractions of PP-L were prepared as previously described 9 using DEAE-Sephadex chromatography.

Tropocollagen Acetic acid-soluble tropocoUagen from calf skin, and neutral salt-soluble collagen from the skins of weanling rats, were prepared by previously described methods13,14.

Aeetoacetylated tropoeollagen Acetic acid-soluble calf skin tropocollagen (70 rag) dissolved in o.I M acetic acid was adjusted to p H 8.5 with 0.5 M N a O H and reacted with IOO molar excess of aqueous diketene 15 at 18 ° for 18 h. The p H of reaction mixture was maintained at 8.5 by addition of 0.5 M N a O H when necessary. The acetoacetylated tropocollagen was dialysed and freeze-dried before use.

Guanidinated tropocollagen Tropocollagen was guanidinated according to the method described by BARET

et al. a6. Pepsin-treated tropocollagen Tropocollagen was digested with pepsin (Armour) for 18 h employing an enzyme substrate ratio of 1:50, at p H 3.5 and 18 °. The resultant tropocollagen, from which the telopeptides had been removed by pepsin, was dialysed and freeze-dried.

Ultracentrifugal analysis of PP-L preparations The original PP-L, desulphated PP-L and papain-treated P P - L samples, were dissolved in 0.05 M acetate buffer (pH 7.6) and centrifuged at 2Ol ooo × g for 48 rain at 25 ° . Sedimentation coefficients were obtained by plotting the sedimentation rates obtained for concentrations of 1.o%, 0.5% and o.25% and extrapolating to zero concentration.

LKB isoelectricfocussing analysis Each of the modified P P - L preparations was subjected to isoelectricfocussing analysis 9 alone and also when mixed with tropocollagen at 4 °. In a similar manner, each of the modified tropocollagen preparations was examined in the presence and absence of intact PP-L. The proportions of tropocollagen:PP-L were varied over a wide range, i.e. from I :io to IO :I on a weight to weight basis. When complex formation took place, (indicated by an opaque zone with an intermediate isoelectric point (pI) between those of the two starting materials), this complex was collected. The isoelectric Bioch,m. Biophys. Acta, 229 (1971) 690-697

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focussing column was emptied by means of a peristaltic pump at the rate of i ml/min and I-ml fractions collected9. The pH of a fraction containing a zone of precipitated protein defined the p I of that zone. Part of the precipitate formed during the interaction of PP-L and tropocollagen was immediately transferred to previously prepared grids for electron microscopy and the excess liquid drained from the grid. The remainder of this precipitate was collected by centrifugation, dispersed in distilled water and dialysed against 2 1 distilled water. The non-diffusable complex was then subiected to uronic acid and hydroxyproline analysis 1°. The influence of temperature on this interaction was studied by maintaining the isoelectric focussing column at 4 °, 20 ° and 37 ° in successive experiments. The ampholine and sucrose solutions were pre-equilibrated to the correct running temperature before the PP-L and tropocollagen were mixed.

Electron microscopy Complexes formed during L K B electrofocussing were drained from the apparatus and immediately placed on carbon-coated collodion films supported on copper grids, at the temperature at which the LKB column had been run. The unfixed samples were negatively stained with 1% sodium phosphotungstate (pH 7.0) and examined in an A.E.I.-EM 6B electron microscope. RESULTS

Ultracentrifugal analysis o/PP-L preparations The initial PP-L exhibited a bimodal distribution with s o values of I I and 2.3 S, respectively whilst the desulphated and papain-treated PP-L each contained a single peak with s o values of 2.2 and 2.1 S, respectively.

Isoelectricfocussing analysis (a) Interaction of tropocollagen with PP-L Neutral salt-soluble tropocollagen and acetic acid-soluble tropocollagen be-

6.75

6.76 6.60

m

5.02

4.52 •

4,22 C 4.10 3.70

!

(Q)

(b)

(c)

Fig. I. Isoelectricfocussing analysis of P P - L and tropocollagen. ( a ) I n t a c t PP-L. ( b ) T r o p o collagen. (c) P P - L plus tropocollagen in ratio 1:2 (w/w). (d) P P - L plus tropocollagen in ratio I : I (w/w). The n u m b e r s refer to t h e p I values o f each zone.

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haved in an identical manner alone and when mixed with PP-L. Tropocollagen yielded a single zone with a p I of 6.75. Intact PP-L yielded three major subunits with p I ' s of 3.39, 3.52 and 3.7 ° (Fig. I). Mixtures of tropocollagen and PP-L yielded a single component which had a p I between those of tropocollagen and PP-L. The ratio of tropocollagen:PP-L was varied from I :IO to IO:I ; and in each case a single zone was formed showing a characteristic shift in p I towards lower values with increasing proportions of PP-L (Fig. I). Uronic acid and hydroxyproline analyses confirmed the presence of both PP-L and tropocollagen in the isolated complexes. The small quantities of the complexes prevented accurate quantitation of the ratios of PP-L and tropocollagen in each complex.

(b) Effect of temperature on the interaction of tropocollagen with PP-L During isoelectricfocussing at 4 °, the interaction product was fibrous longspacing fibrils s. When the temperature was subsequently raised to 20 ° and 37 ° the fibrous long-spacing structure remained and no native-type fibrils were observed. When tropocollagen and PP-L were initially incubated at 2o °, a mixture of nativetype and fibrous long-spacing fibrils was observed. Initial incubation of tropocollagen and PP-L at 37 ° produced only native-type fibrils.

(c) Interaction of tropo6ollagen with PP-L subfractions The DEAE-Sephadex subfractions 9 all combined with tropocollagen in a manner similar to intact PP-L. Subfraction 2 of PP-L was previously shown to contain no uronic acid 9 but was still capable of interacting with tropocollagen and resulted in a changed pI. In each case, the p I of the complex was lowered by increasing the quantity of tropocollagen in the original mixture. The two minor components of PP-L 9, with isoelectric points near 6. 7, did not interact with tropocollagen to form a stable complex. I t was possible to observe the lack of interaction in the following manner. Tropocollagen alone had virtually the same p I as the two minor components of PP-L; however, when intact PP-L was mixed with tropocollagen, all the tropocollagen combined with the three major PP-L bands resulting in a complex with a p I close to 4. In the zone normally occupied by tropocollagen alone, two faint bands were observed with p I ' s identical to the two minor components of intact PP-L.

(d) Interaction of tropocollagen with modified PP-L Neuraminidase-treated PP-L contained only 5% of the initial sialic acid content but still combined with tropocollagen in a manner similar to intact PP-L (Fig. 2). Desulphated PP-L contained 37% of its original sulphate groups and separated into five distinct subffactions (Fig. 2), none of which was capable of interaction with tropocollagen. Papain-digested PP-L contained five main subfractions with low p I values and a minor fraction with a p I of 6.8 (Fig. 3). All the major subfractions combined with tropocollagen although the minor fraction with a p I of 6.8 remained as a zone with p I 6.65-6.70. The oligosaccharides released from hyaluronidase-treated PP-L interacted with tropocollagen to give a single zone with a p I value dependent on the ratio of tropocollagen and oligosaccharide used (Fig. 3).

(e) Interaction of modified tropocollagen with PP-L Acetoacetylated tropocoUagen contained one major component (pI 4.1o) and two minor components (Fig. 4). None of these combined with intact PP-L, since if

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INTERACTION OF TROPOCOLLAGEN WITH COMPLEXES

6.60

6,65

1

5.15

~--,,,~3.68

° ~3.65

(0)

(c)

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Fig. 2. Isoelectric focussing analysis of modified PP-L and tropocollagen. (a) Neuraminidasetreated PP-L. (b) Neuraminidase-treated PP-L plus tropocollagen. (c) Desulphated PP-L. (d) Desulphated PP-L plus tropocollagen. The numbers refer to the p I values of each zone. Fig. 3. Isoelectric focussing analysis of modified PP-L and tropocollagen. (a) Papain-digested PP-L. (b)Papain-digested PP-L plus tropocollagen in ratio i : i (w/w). (c)Papain-digested PP-L plus tropocollagen in ratio I : 2 (w/w). (d) Diffusable oligosaccharides from hyalurinodasetreated PP-L (IOO/*g) plus tropocollagen. (e) Diffusable oligosaccharides from hyaluronidasetreated PP-L (15o/,g) plus tropocollagen.

such interaction had taken place the three major PP-L subfractions with pI 3.4-3.8 would have been replaced by a single complex zone of higher pI. Guanidinated tropocollagen interacted with PP-L in the same way as untreated tropocollagen (Fig. 4). Pepsin-treated tropocollagen interacted with PP-L in the same manner as untreated tropocollagen.

I

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6.72

6.60

m~l

4.10

3.50

(o)

~

4.52

4,28 3.89 3.62 3.45

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(d)

Fig. 4. Isoelectric focussing analysis of modified tropocollagen and PP-L. (a) Acetoacetylated tropocollagen. (b)Acetoacetylated tropocollagen plus PP-L. (c) Guanidinated tropocollagen. (d) Guanidinated tropocollagen plus PP-L. The numbers refer to the p I values of each zone.

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DISCUSSION

Ultracentrifugal analysis The initial PP-L preparation contained two components as observed by SAJDERA AND HASCALL17. During desulphation and papain digestion of PP-L, depolymerisation took place with the result that a single component was observed in ultracentrifugal analysis, having s = 2.1-2.2 S, similar to the lower-molecular-weight fragment present in the initial PP-L. This component probably corresponds to single polysaccharide chains. Isoelectric focussing analysis Intact PP-L, the main subfractions of PP-L, papain-treated PP-L, oligosaccharides from hyaluronidase-treated PP-L, and neuraminidase-treated PP-L, all interacted with untreated tropocollagen, whilst partially desulphated PP-L was incapable of forming a stable complex. One essential requirement for tropocollagenPP-L interaction was clearly the presence of the sulphate groups. The fact that desulphation only removed two-thirds of the total sulphate groups, leaving the uronic acid content unchanged, and that the partially desulphated product was incapable of interaction with tropocollagen, suggested that the remaining sulphated groups were located in the interior of the PP-L and were not exposed to contact with the tropocollagen in solution. It would seem that the COO- ... NH8 + interaction plays a relatively minor role in stabilising the tropocollagen-PP-L complex under these conditions. Neither the presence of sialic acid, the intact core protein, nor the size of the sulphated polysaccharide chains (e.g. diffusable oligosaccharides), are essential for this primary interaction, although they could play a subsequent role in fibril formation in vivo. The ability of both guanidinated tropocollagen and pepsin-treated tropocollagen to interact normally with PP-L whilst acetoacetylated tropocollagen failed to interact, can only be interpreted by defining the positively charged amino groups of lysine and arginine as the site of interaction on tropocollagen with the negatively charged sulphate groups of the PP-L. The fact that tropocollagen, from which telopeptides had been removed, reacted with PP-L indicates that the interaction takes place along the length of the tropocollagen molecule and is not confined to the telopeptide regions as was suggested by LOWTHER et al. TM. Earlier fingerprinting studies s with collagenase digests clearly showed that the peptides from the helical part of tropocollagen and polymeric collagen were linked through guanidino and e-NH3+ to S04- of PP-L in preparations obtained from human intervertebral disc4. Even in the presence of excess PP-L, only one zone of interaction was observed with tropocollagen (Fig. I). This could be explained if the excess PP-L interacted with the PP-L bound to tropocollagen to form a matrix in which the collagen was embedded; a situation analagous to that existing in cartilagenous tissues. In the intact intervertebral disc and cartilage, the PP-L is associated with polymeric collagen fibrilsS,19,2°. Previous studies ~1,22 have demonstrated that most of the e-NHz groups of lysine are chemically masked in polymeric collagen. It is therefore suggested that the main ionic groups which stabilise the intact disc and cartilage complex of polymeric collagen and PP-L are arginine guanidino groups of collagen Biochim. Biophys. Acta, 229 (1971) 69o-697

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fibrils and the sulphate groups of PP-L. It is worth recording that this interaction can be completely inhibited by premixing PP-L with either arginine or salmine, so that interaction takes place between the sulphate groups of PP-L and guanidino groups of arginine or salmine prior to adding polymerised collagen. Similarly, a water-soluble sulphonated acrylate polymer~8, prepared as a model for PP-L, interacted immediately and irreversibly with polymerised collagen, preventing its interaction with additional PP-L, which was added subsequently.

Electron microscopy The fact that the complex formed between PP-L and tropocollagen at 37 ° resulted in the formation of native-type fibrils with both neutral salt-soluble and acetic acid-soluble tropocollagen, suggests that the ionic interactions studied in this report are similar to those taking place during fibrogenesis in vivo. ACKNOWLEDGEMENTS

V.P. wishes to thank the Nuffield Foundation and the Wellcome Trust for their generous provision of a research fellowship during 1958-59 and 1959-7o, respectively. S.J.L. wishes to thank the Medical Research Council for a research studentship. We wish to thank Professor J. H. Kellgren for his continued interest and practical help in providing intervertebral discs from which the PP-L was prepared for these studies. The technical assistance of Miss S. Eltherington is gratefully acknowledged. REFERENCES i B. R. GERBER, E. C. FRANKLIN AND M. SCHUBERT, J. Biol. Chem., 235 (196o) 2870. 2 N. SENO, K. MEYER, ]3. ANDERSON AND P. HOFFMAN, J. Biol. Chem., 240 (1965) lOO5. 3 F. O. SCltMITT, J. GROSS AND J. H. HIGHBERGER, in J. F. DANIELLI AND R. ]3ROWN, SOC. Expa. Biol. Syrup. No. 9, Fibrous Proteins and Their Biological Sigmficance, C a m b r i d g e U n i v e r s i t y Press, 1955, P. 148. 4 G. C. WOOD, Biochem. ]., 75 (196o) 605. 5 M. ]3. MATHEWS, Biochem. J., 96 (1965) 71o. 6 M. B. MATHEWS AND L. DECKER, Biochem. J., IO9 (1968) 517 . 7 ]3. P. TOOLE AND A. G. LOWrHER, Bwchem. J., lO9 (1968) 857. 8 F. S. STEVEN, J. KNOT~, D. S. JACKSON AND V. PODRAZKY, Biochim. Biophys. Acla, 188 (1969) 307. 9 V. PODRAZKY, F. S. STEVEN, M. E. GRANT AND D. S. JACKSON, Biochim. Biophys. Acta, 221 (197 ° ) 549. IO J. F. WOESSNER, Arch. Biochem. Biophys., 93 (1961) 44 o. I I I. MALAWlSTA AND M. SCHUBER'r, J. Biol. Chem., 23o (1958) 230. 12 T. G. KANrOR AND M. SCHUBERT, J. Am. Chem. Soc., 79 (1957) 152. 13 F. S. STEVEN AND D. S. JACKSON, Biochem. J., lO 4 (1967) 534. 14 D. S. JACKSON AND E. G. CLEAR',', in D. GLICK, Methods in Biochemical Analysis, Vol. 15, Wiley, N e w York, 1967, p. 25. 15 A. MARZOTTO, P. PAJETTA, L. GALZlGNA AND E. SCOFFONE, Bwchim. Biophys. Acta, 154 (1968) 450. 16 R. BARET, J. RENAl AND M. MOURGUE, Compt. Rend. Soc. Bzol., 159 (1965) 587. 17 S. W, SAJDERA AND V. C. HASCALL, J. Biol. Chem., 244 (1969) 77. 18 D. A. LOWTHER, ]3. P. TOOLE AND A . C . HARRINGTON, in E . A . BALAZS, Chemistry and Molecular Biology of the Intercellular Matrix, Vol. 2, Ed. A c a d e m i c Press, N e w York, 197 o, p. 1135. 19 F. S. STEVEN, D. S. JACKSON AND K. BROADY, Biochim. Biophys. Acta, I6o ti968) 435. 20 F. S. STEVEN, ~4~.BROADY AND D. S. JACKSON, Biochim. Biophys. Acta, 175 (1969) 225. 21 F. S. SrEVEN, D. S. JACKSON AND K. BROADY, Biochim. Biophys. Acta, 188 (1969) 334. 22 R. C. PAGE AND E. P. ]3ENDITT, F E B S Letters, 9 (197 o) 49. 23 N. TUCKER AND F. S. STEVEN, in p r e p a r a t i o n .

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