HYALURONAN
`Volume 1 - Chemical, Biochemical and Biological Aspects
`
`Editors:
`JOHN F. KENNEDY BSc, PhD, DSc, EurChem CChem FRSC, CBiol FIBiol, FCIWEM, FCMI, FIFST
`Director of Birmingham Carbohydrate and Protein Technology Group,
`School of Chemical Sciences, The University of Birmingham, Birmingham B15 21T, England, UK,
`Director of Chembiotech Ltd,
`University of Birmingham Research Park, Birmingham B15 2SQ, England, UK,
`Director of Inovamed Ltd,
`Chembiotech Laboratories, University of Birmingham Research Park, Vincent Drive,
`Birmingham B 15 2SQ, England, UK, and
`Professor of Applied Chemistry,
`The North East Wales Institute of Higher Education, Plas Coch, Mold Road, Wrexham, Clwyd,
`LLl 1 2A W, Wales, UK
`
`GLYN 0. PHILLIPS BSc, PhD, DSc, HonDSc, HonLIB, CChem FRSC
`Chairman of Research Transfer Ltd,
`Newtech Innovation Centre,
`Professorial Fellow,
`The North East Wales Institute of Higher Education, Plas Coch, Mold Road, Wrexham, Clwyd,
`LLl 1 2A W, Wales, UK, and
`Professor of Chemistry,
`The University of Salford, England, UK
`
`PETER A. WILLIAMS BSc, PhD, CChem FRSC
`Director of the Centre for Water Soluble Polymers,
`The North East Wales Institute of Higher Education, Plas Coch, Mold Road, Wrexham, Clwyd,
`LLl 1 2A W, Wales, UK,
`Director of the Centre for Advanced and Renewable Materials at the North East Wales
`Institute and University of Wales, Bangor,
`The North East Wales Institute of Higher Education, Plas Coch, Mold Road, Wrexham, Clwyd,
`LLl 1 2AW, Wales, UK
`Professor of Polymer and Colloid Chemistry,
`The North East Wales Institute of Higher Education, Plas Coch, Mold Road, Wrexham, Clwyd,
`. LLl 1 2A W, Wales, UK
`Guest Editor:
`VINCE C. HASCALL PhD
`Co-Director of the Orthopaedic Surgery Musculoskeletal Research Center,
`Department of Biomedical Engineering ND-20, Lerner Research Institute. Cleveland Clinic
`f(,,undation, Cleveland, Ohio 44195, USA
`Adjunct Professor
`Department of Biochemistry, Case Western Reserve University, Cleveland. Ohio 44106, USA
`Adjunct Professor
`Department of Biochemistry, Rush Presbyterian St. Lukes Medical Center, Chicago, Illinois,
`60612 USA
`
`WOODHEAD PUBLISHING LIMITED
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`PROLLENIUM V. ALLERGAN
`IPR2019-01505 et al.
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`

`

`Published by Woodhead Publishing Ltd, Abington Hall, Abington,
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`www.woodhead-publishing.com
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`First published 2002
`
`© 2002, Woodhead Publishing Ltd
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`

`

`BIOLOGICAL PROPERTIES OF HY ALURONAN ARE
`CONTROLLED AND SEQUESTERED BY TERTIARY
`STRUCTURES
`
`John E. Scottl and Frank Heatley2
`
`lchemical MoT,hology, Manchester University Medical School, Oxford Rd, Manchester Ml3 9PT
`Chemistry Dept. Manchester University, Manchester Ml3 9PL
`
`ABSTRACT
`
`Hyaluronan (HA) is the characteristic polysaccharide component of vitreous
`humor (from which it was first purifiedl ), Wharton's jelly and synovial fluid. The
`unusual mechanical properties of these gels and viscoeleastic fluids were attributed
`non-specifically to interactions between HA molecules, which are stiff in solution
`partly because they prefer to take up 2-fold helical secondary structures, stabilised
`by H-bonds2 (Fig lA). HA has since been found in many tissues from many species3.
`Probably all animals produce it. Unexpectedly, a new class of specific and potent
`biological activities shown by HA fragments (in angiogenesis, inflammation etc)
`emerged4-6. Highly polymerised HA does not share these properties. HA is vitally
`important during development7. It is a pluripotent material with a simple strucure
`(FiglA).
`that
`this diversity? We suggest
`there a unifying concept behind
`Is
`physiological properties of HA are controlled and sequestered by reversible tertiary
`structures8. We introduce an NMR approach which can monitor their formation and
`behaviour. Biological properties may thereby be linked to specific chemical aspects of
`HA and HA supramolecular organisation.
`
`KEYWORDS
`
`{3 Sheets, twofold helices, angiogenesis, hyaluronidase, erythrocyte lysis.
`
`INTRODUCTION
`
`13c NMR studies on HA in aqueous solution proved that acetamido and
`carboxylate groups were involved in NH-coo- H-bonds, probably in stacked
`antiparallel agregates formally similar to {3 sheets as seen in proteins8 (Fig IB), but
`uncommon if not unique in the polysaccharide field. The 13c acetamido C=O signal
`is much broadened (unlike all the other 13c resonances) because rotation of the amide
`group is restricted by participation in an H-bond. This broadening can be used as a
`reporter of H-bond formation, the first specific spectrometric test so far in this field.
`
`DISCUSSION
`
`It is a necessary consequence of the tertiary structure model that the acetamido
`NH is oriented trans-to the C2H of the glucosamine ring, permitting the NH-COO H-
`
`

`

`118
`
`Characterisation and solution properties of hyaluronan
`A
`
`~ ,CH l
`~ O"'c
`\
`
`9
`·--····o ... c""o
`
`
`1·
`
`~NH
`OH
`OH
`
`.. .... •···~ ··· HO O
`CH,OH
`- - -~······ ·HO~~O--·····
`... HO
`o····
`3
`1
`
`o 4
`
`O
`
`CH,OH
`
`OH
`
`OH---.......... ___
`,NH·----·---···O="·C.'\-
`e b
`·· .. o =-c
`Ill ~ 'cH,
`
`B
`
`~--
`
`Figure l (A) secondary structure of HA (a tetrasaccharide fragment) as a two-fold
`helix with hydrogen bonds (dotted lines).
`(B) tertiary structure of HA viewed from the side, showing three HA
`molecules in antiparallel array stacked above each other. Arrows to the left
`and right point to the reducing ends; cross hatched areas are the hydrophobic
`patches; vertical dotted lines delineate sugar units. Square symbols (C) denote
`acetamido groups, circles (0) carboxylate groups. Filled symbols are on the
`distal side and open symbols on the proximal side. Vertical arrows indicating
`H-bonds from donor NH to acceptor -Coo- are in pairs alternately pointing
`up and down, linking each HA molecule with both its neighbours in a {3 sheet(cid:173)
`like structure8. Overlapping hydrophobic patches provide additional
`stabilisation by hydrophobic bonding.
`Four different environments in which the amide group can exist in HA are
`labelled I, II, Ill and IV. I and II are not H-bonded to carboxylates, I because
`there is no suitably placed carboxylate, and II because the relevant carboxylate
`has been converted into a poor receptor for H-bonding e.g.as a methyl ester8.
`III and IV are H-bonded either in secondary (twofold helices) or tertiary (/3
`sheets) structures. Characteristic NMR signatures are associated with each
`environment (see refs 8 and 12).
`
`

`

`Biological properties
`
`119
`
`bonds to form between stacked anti~arallel polysaccharide chains (Fig. lB). It was
`known, using 1 H NMR to measure JcH-:¥1 coupling, that the trans- arrangement
`was present in monomers and oligomers of up to 27 disaccharidesl2 which,
`however, do not aggregate efficiently to tertiary structures8. We tried to extend these
`data to high mo!. mass HA, but all signals were broadened, as is usual in high
`polymers, and although the NH signal was broader than other peaks in the spectrum
`(Fig. 2), as expected if it was intrinsically two peaks with a large value of 3JcH-NH
`coupling, a precise value of the coupling constant could not be obtained. The
`broadness of this peak is probably also due to lack of rotation of the amide group, as
`already shown by 13c NMR8. If the sharpening of the 13c acetamido C=O
`resonance on warming is secondary to simple rupture of NH---+COO H-bonds, the
`trans- CH-NH orientation should persist at high temperatures and this is compatible
`with the persistent broadening of the NH signal up to -80 °c (Fig 2).
`
`il,ljiiiij!llljillllllllllli,1\1,11111,1111tjiil,1
`
`176.5
`
`175.0
`
`173.5
`
`ppm
`
`111111111111,T1111!1ll[iiil\iiiijiltllllll(illi(l1iq111111111p111111111
`7.5
`ppm
`7.8
`
`8.1
`
`8.4
`
`Figure 2. Left, 13c NMR resonances of high mo!. mass HA acetamido C=O (174.5
`- 175 ppm, amide IV) at 23 °c (lower trace) and 80 °c (upper trace). The
`sharp signal at 173.5 -174ppm is the carboxylate C=O resonances. HA
`concentration lOmg/ml in D2O containing 0.14M NaCl. Note the marked
`sharpening of the acetamido C=O resonance on warming.
`Right, 1 H NMR resonance of high mol. mass HA NH proton. HA
`concentration 2mg/ml in 4: l H2OID2O solution containing 0.15M NaCl. The
`broad resonance is of similar width at both 23 °c (lower trace) and 80 °c
`( upper trace).
`
`

`

`120
`
`Characterisation and solution properties of hyaluronan
`
`It would be predicted that any process that caused disaggregation of high mol.
`mass HA tertiary structure would bring about sharpening of the 13c acetamido C=O
`signal. Simple warming of HA solutions is one such process (Fig 2). The predicted
`spectral change was completely reversible on cooling. The cycle was repeated several
`times with identical results. HA aggregation is therefore specific and reversible.
`Marked decreases in viscosity of HA solutions on warming9 parallel the temperature
`dependant NMR changes. We suggest that rupture of intermolecular H-bonds in the /3
`sheet is an important part of this phenomenon.
`
`O•l
`
`O ·Z
`
`.:
`=-
`
`O·O
`
`-0· 1
`
`B
`
`/
`
`-O· l~l--;;0-~l-~1 --,l,':-z:------:J'-:,J,----='3.--,.4--,3--'-.5-'
`
`Trmporaturc cio~ncfoneo nf hyalumnRto 11nlutinn vi~o:'lity.
`eel pH 7. (0 ) pH ~-5; M,. 4-0 x 10'; 0· 15 ,c . N~CI ; con«nlra~ion, 0·06 g rl l • ' ·
`
`C
`
`3 . .:.
`
`3.6
`
`0----'-----------'-----'---
`
`28
`
`JO
`
`l .:?
`t O! / T (K - 1)
`
`Arrhtnius p/01 [In ( k, X I0' )againsr 1o>;n of rtactio11
`of periodatt with rhe g/ycol-conraining
`compounds
`methyl 4-O-merhyl-,-o-glucop_vranoside ( A ). chondroirin
`6-su/phate ((cid:127)) and hyoluronatt ( • )
`The venical lines show the spread of values of Ink,
`at each temperature point. The plotted point is the
`arithmetical average. The plots are drawn by
`ins pection. All solutions coniained 0.2><•NaCJO • .
`The glucoside concentrations were all 0.4m>< in
`0.4m.'4-Na!O • . Hya luronate concentrations were
`either 6.53m>< or 8.7mM in either 8.0mM• or I0mM•
`NalO,. exccp1 for the readings at 364 a nd 3S8 K.
`which were l.9mM in polymer and !mM in Na!O,.
`Chondroitin 6-;ulphatc conccntradons were 7. 7! mM
`in S.Om><•PCriodate.
`
`10 ~ ................................. .......,u...i ...... ...u. ................... r.....u
`0.0028
`0.003
`0.0032
`0.0034
`1/T/K' '
`Figure 3. Arrhenius plots (In. [variable] against l/°K) of (A) second order rate
`constants of periodate oxidation of HA, chondroitin 6 sulphate and methyl 4-
`0-methyl-a-D-glucopyranosidel 1, (B) viscosity of HA solutions9, and (C) the
`widths of the high mo!. mass HA acetarnido C=O resonance (Scott and
`Heatley, submitted). All show a change in slope at -40 °c (see text).
`
`

`

`Biological properties
`
`121
`
`Arrhenius plots of the NMR signal width showed a break (a melting) at 40-50
`0 c (Fig 3). Similar plots of viscosity also showed a break at about 40 oc9, as did
`those of periodate oxidation kinetics of HAlO(Fig. 3). Taken together these data
`suggest that at physiological temperatures HA is on the edge of a critical change in
`supramolecular organisation. On the upper side of the break glycol groups react more
`easiiy with periodate, amido groups rotate more freely and viscosity becomes a
`property of single chains which can entangle randomly with other long chains.
`Disaggregation of the tertiary structure on warming parallels a marked increase
`in a biological activity, viz.
`inhibition of complement-mediated
`iysis of
`erythrocytes I 1. This effect, too, is reversible on cooling. Disaggregated HA chains
`interact with complement, thereby inactivating it. On reaggregation the HA active
`sites are masked by inter-HA liaisons. The aggregated form sequesters biological
`activity that is an intrinsic property of HA primary structure.
`This hypothesis implies that certain biological activities of HA would be
`expressed if aggregation was prevented. Indeed, angiogenesis (the formation of new
`blood vessels) is inhibited by HA oligosaccharides although highly polymerised HA is
`without effect6. Angiogenesis is fundamental to the facilitation of tumor growth. The
`13c NMR method showed that HA oligosaccharides do not aggregate8, since they
`cannot form enough collaborative intermolecular bonds to produce stable aggregates
`at ambient temperatures. They act freely and independantly, deploying sites that
`would be masked or otherwise engaged in the tertiary structure.
`in solution produced
`Similarly, ultrasonication of high moi. mass HA
`fragments which activated an autoregulatory loop in murine macrophages, or induced
`chemik:ine expression in alveolar macrophages - activities which were not shown by
`undegraded HA4,5. Probably some fragments were too small to participate in stable
`tertiary structures and could hence exert an activity that was hidden in the high mol.
`mass HA tertiary structures. It was suggested that HA degradation products could
`porentiate inflammatory reactions in tissues4.
`The NMR evidence provides proof of a tertiary HA structure, probably a (3.
`sheet-like array, which is stabilised by H-bonds and hydrophobic bonds at ambient
`temperatures, provided the HA chains are long enough for sufficient cooperative
`intermolecular bonds to form. Disaggregation of the tertiary structure exposes the
`hydrophobic patches in particular, opening up biological activities and potential
`control mechanisms that were hidden in the tertiary structure.
`Easy transitions between secondary and tertiary structures in physiological
`conditions, which the Arrhenius plots suggest is probabie, offer convenient and
`economic mechanisms for switching between functions dependant on these structures
`(Fig. I).
`Conversely, the tertiary structure may present possibilities that are not present
`in the single stranded secondary structure. An intriguing finding by Kreil's group13
`is that a hyaluronidase with specific tissue localisation patterns is nor able to break
`down HA of a size less than about 50 disaccharide units. This is around the size at
`which stable tertiary structures would be expected to form8. We suggest that the
`enzyme, termed HY AL 2, is specific for the aggregated form oi HA but not able to
`hydrolyse the singie stranded form. The more familiar hyaluronidases, e.g. from
`testicles, are able to work on single stranded HA. Serendipitously, one was termed
`HYAL I i3_
`
`

`

`122
`
`Characterisation and solution properties ofhyaluronan
`
`The viscoelastic properties of high mol. mass HA are very probably
`fundamentally determined by the ability to form tertiary structures. A new NMR
`technique (rheo-NMR) has been used in preliminary experiments to show that the
`effect of shear on HA solutions can be observed by monitoring for ~ sheets using the
`13c carbonyl signal (Fischar, Callaghan, Scott & Heatley, submitted).
`Elasticity in connective tissue extracellular matrices, in which analogous
`tertiary structures
`involving glycosaminoglycans similar
`to HA (chondroitins,
`keratans) may exist 14, probably depends on similar reversible interactions (Scott,
`unpublished).
`
`REFERENCES
`
`1. K. Meyer, The biological significance of hyaluronic acid and hyaluronidase.
`Physiol. Rev. 1947, 27, 335-359
`2. J.E.Scott, F. Heatley, & W.E Hull,. Secondary structure of hyaluronate in
`solution: A lH n.m.r. investigation at 300 and 500 MHz in dimethyl sulphoxide d6
`solution. Biochem.J. 1984, 220, 197-205
`(ed.) The Chemistry, Biology and Medical Applications of
`3.T.C. Laurent,
`Hyaluronan and its Derivatives. 1998, Portland Press Ltd, London
`4. C.M.McKee, M.B. Penno, M.K. Cowman, M.D. Burdick, R.M. Strieter, C. Bao,
`P.W. Noble, Hyaluronan (HA) fragments induce gene expression in alveolar
`macrophages J. Clin. lnvest.1996, 98, 2403-2413
`5. P.W.Noble, C.M. McKee, M.K. Cowman, H.S. Shin, Hyaluronan fragments
`activate an NF-KBII-K Ba autoregulatory loop in murine macrophages. I.Exp.
`Med.1996, 183, 2373-2378
`6. D.C West & Kumar S. Hyaluronan and angiogenesis. In Hyaluronan. Ciba
`Foundation Symposium No.143, 1989, 187-207
`7. B. P. Toole, Hyaluronan-cell interactions in morphogenesis. In The Chemistry,
`Biology and Medical Applications of Hyaluronan and its Derivatives. (Laurent, T.C.
`ed.) 1998, Portland Press Ltd, London
`8. J.E. Scott, & F. Heatley, Hyaluronan forms specific stable cooperative tertiary
`structures in solution. A 13c n.m.r. study. PNAS1999, 96, 4850-4855
`9. E.D.Morris, D.A. Rees, E.J Welsh,. Conformation and dynamic interactions in
`hyaluronate solutions J. Mol. Biol.1980, 138, 383-400
`10.N.S. Chang, R.J. Boackle, & G. Armand, Hyaluronic acid-complement
`interactions.I. Reversible heat-induced anticomplementary activity. Molecular
`Immunology,1985 22, 391-397
`11.J.E. Scott & M.J. Tigwell, Periodate oxidation and
`glycosaminoglycuronans in solution Biochem. J.1978, 173, 103-114.
`12.M.K. Cowman, D.M. Hittner & J. Feder-Davis,13c NMR studies of hyaluronan.
`Conformational sensitivity to varied environments. Afacromolecules1996, 29, 2893-
`2902
`13. G. Lepperdinger, B. Strobl, & G. Kreil, 'HYAL2, a human gene expressed in
`many cells which encodes a lysosomal hyaluronidase with a novel type of specificity'
`I.Biol. Chem.1998, 273, 22466-22470
`14. J.E. Scott, Supramolecular organisation and the 'shape module' concept'
`animal matrix biology. J.Biochem. Mol. Biol & Biophys.1999, 2, 155-167
`
`in
`
`the
`
`shapes of
`
`