596th MEETING. LANCASTER
596th Meeting
Held at the University of Lancaster on 16 and 1 7 September 198 1
Carbohydrate-Protein Interactions in the Extracellular Matrix
Society Colloquium organized and edited by T. E. Hardingham (London) and
I. A. Nieduszynski (Lancaster)
The antithrombin-binding sequence of heparin
ULF LINDAHL, LENNART THUNBERG,
GUDRUN BACKSTROM and JOHAN RIESENFELD
Department of Medical and Physiological Chemistry, Swedish
University of Agricultural Sciences, The Biomedical Center,
P.O. Box.575, S-75123 Uppsala,Sweden
The inhibition of blood clotting by heparin is due to binding of
the polysaccharide to the proteinase inhibitor antithrombin,
resulting in accelerated inactivation of the serine proteinases
involved in the clotting mechanism. Affinity chromatography of
heparin preparations on immobilized antithrombin showed that
only a fraction of the heparin molecules bind with high affinity
to antithrombin; this high-affinity fraction accounts for most of
the anticoagulant activity of the material (for references see
Lindahl et al., 1980). A structural comparison of high-affinity
and low-affinity heparin molecules implicated an iduronosyl -.N-acetylglucosaminyl(-6-O-sulphate)
-.glucuronosyl Nsulphoglucosaminyl(-6-O-sulphate)tetrasaccharide sequence as
the antithrombin-binding structure (Rosenberg & Lam, 1979).
However, the isolation of oligosaccharides with high affinity for
antithrombin enabled a more detailed characterization of the
binding sequence. Such oligosaccharides were isolated by
affinity chromatography on antithrombin-Sepharose of products obtained on partial deaminative cleavage of heparin with
HNO, (Lindahl et al., 1979). The smallest high-affinity
oligosaccharide, an octasaccharide (Thunberg et al., 1980), was
subjected to structural investigation.
-.
Distribution of N-substituents
The octasaccharide was subjected to exhaustive treatment
with HNO, leading to deamination of N-sulphated glucosamine residues and cleavage of the corresponding glucosaminidic linkages. Reduction of the products with NaB3H,
yielded lmol of labelled tetrasaccharide, indicative of a
N-acetylated glucosamine unit (resistant to deamination), for
each 2mol of labelled disaccharide. The location of this
N-acetylated glucosamine residue was established in the following manner (Thunberg et al., 1980). During heparin biosynthesis
D-glucuronic acid units bound at C-4 to N-acetylated glucosamine residues are unable to epimerize into L-iduronic acid (I.
Jacobsson, U. Lindahl, L. Roden & D. S. Feingold, unpublished
work) and therefore remain non-sulphated at C-2. Owing to the
glycol grouping at C-2-C-3, such units are susceptible to
oxidation by periodate. Reduction of the intact octasaccharide
with NaB3H, (introducing a 3H-labelled anhydromannitol unit
at position 8; see the legend to Fig. 1 for explanation) followed
by periodate oxidation and scission by alkali yielded labelled
pentasaccharide (4-13H18) as a major product. The periodatesensitive glucuronic acid residue must therefore be located at
position 3, hence the N-acetylglucosamine unit at position 2.
Positions 4 and 6 would thus be occupied by N-sulphated
glucosamine residues.
VOl.
9
Sequence of hexuronic acid residues
Digestion with purified a-L-iduronidase (for conditions see
Lindahl et al., 1979) converted the octasaccharide into a
heptasaccharide, as shown by gel chromatography on Sephadex
G-50. The heptasaccharide yielded labelled trisaccharide (2[ "14)
or HN0,/NaB3H, treatment, but no tetrasaccharide,
indicating quantitative release of the hexuronic acid at position
1. This unit must therefore be exclusively non-sulphated
L-iduronic acid. The occurrence of D-glucuronic acid at position
3 (see above) was confirmed by digestion of the octasaccharide
with an endo-b-D-glucuronidase isolated from human blood
platelets (U. Lindahl, L. Thunberg, G. Backstrom, J. Riesenfeld,
& A. Wasteson, unpublished work). A pentasaccharide was
released, apparently identical with that ( 4 4 'H18) observed
after treatment with periodate/alkali. In addition to the labelled
pentasaccharide, periodate/alkali treatment of the octasaccharide, 1-I3H18, yielded significant amounts of labelled monosaccharide, but not trisaccharide. The uronic acid residue at
position 5 is thus sulphated, and must therefore be L-iduronic
acid, whereas some of the units in position 7 are non-sulphated.
Experiments outlined below showed that this non-sulphated
uronic acid may be either glucuronic or iduronic acid.
Location of 0-sulphate groups
The disaccharides formed on HN0,/NaB3H, treatment of the
octasaccharide contained a mixture of di-0- and mono-0sulphated 3H-labelled species, as shown by paper electrophoresis. Reduction of the octasaccharide with unlabelled borohydride before deamination eliminated some of the di-0sulphated and practically all of the mono-0-sulphated labelled
disaccharides, which would thus correspond to positions 7 and 8
in Fig. 1. Identification of the disaccharides by ion-exchange
chromatography indicated that position 7 may be occupied by
sulphated as well as by non-sulphated iduronic acid or by
glucuronic acid, and furthermore that the anhydromannose unit
in position 8 may be either sulphated or non-sulphated. Positions
5 and 6, represented by di-0-sulphated disaccharide, each
display a sulphate group, at C-2 of the iduronic acid (in accord
with the periodate oxidation pattern) and at C-6 of the
glucosamine unit respectively.
The 0-sulphate substitution of the glucosamine residue in
position 4 is variable. Recent findings indicate that this unit
carries a unique 3-0-sulphate group, previously unrecognized in
glycosaminoglycans (Lindahl et al., 1980). Incubation of the
pentasaccharide 4-13H18 with a newly discovered urinary
3-0-sulphatase (Leder, 1980) thus resulted in loss of a sulphate
group from a fraction of the molecules. The susceptible
glucosamine residues (Corresponding to position 4) apparently
lacked a sulphate substituent at C-6. However, most of the
glucosamine residues in position 4 are 3,6-di-O-sulphated, as
demonstrated by
the formation of di-0-sulphated
499
BIOCHEMICAL SOCIETY TRANSACTIONS
500
anhydroI’H1mannitol on periodate/alkali cleavage of the tetrasaccharide l-I’H14 (isolated after HNO,/NaB’H, treatment of
the octasaccharide). The occurrence of 3-0-sulphated
glucosamine residues in the antithrombin-binding sequence was
recently confirmed by ”C n.m.r. spectroscopy (Meyer et al.,
1981).
Analysis of tetrasaccharide 1-[’H14 by paper electrophoresis
revealed two major components, containing two and three
(0-)sulphate groups per molecule respectively. This variability
would seem to be accounted for by the presence or absence of
the 6-sulphate group at position 4. Given the 3-0-sulphate group
at the same position, and the lack of sulphate substituents at
positions 1 and 3, the remaining sulphate group must be located
at C-6 of the N-acetylglucosamine residue in position 2.
Structurefunction relationship
The functional role of the various components of the
antithrombin-binding octasaccharide may be evaluated by a
number of criteria.
(a) Identification of structural variants. Those components of
the octasaccharide that are essential to antithrombin binding
must be present in all high-affinity species and should therefore
display structural constancy. Inversely, it is unlikely that sugar
residues or substituents expressing structural variability should
be directly involved in binding to antithrombin. The disaccharide
unit represented by positions 7 and 8 would thus fall outside the
actual binding sequence. By the same criterion the 6-0-sulphate
group at position 4 appears to be non-essential to the
interaction.
(b) Chemical or enzymic modification of the octasaccharide:
effects on antithrombin binding. The results of such experiments
are summarized in Table 1. The N-sulphate groups at position 6
and, in particular, at position 4 are both required for
high-affinity binding (Riesenfeld et al., 198 1). [The importance
of the N-sulphate group at position 6 is further emphasized by
the fact that no hexasaccharide with high affinity for antithrombin was detected after partial depolymerization of heparin with
nitrous acid (Thunberg et al., 1980). Formation of a hexasaccharide by deaminative cleavage of the glucosaminidic
linkage at position 6 entails loss of the corresponding N-sulphate
group.] In contrast, neither enzymic removal of the nonsulphated Giduronic acid unit at position 1 nor N-deacetylation
(by hydrazinolysis) at position 2 had any apparent effect on the
interaction. However, removal of the disaccharide corresponding to positions 1 and 2 by selective deamination of the
Ndeacetylated octasaccharide yielded a low-affinity hexasaccharide, suggesting that the 6-0-sulphate group at position 2
may be involved in binding to antithrombin. Accordingly, the
pentasaccharide 4-I3H18 showed low atlinity for antithrombin.
(c) Identification of components that are unique to the
antithrombin-binding sequence of the heparin molecule. Such
components may be tentatively assumed to promote the
interaction between the two macromolecules. The 3-0-sulphate
group in position 4 is the only constituent of the antithrombin-binding sequence that has not previously been
identified in heparin-like polysaccharides. A method to detect
3-0- and 3,6di-O-sulphated glucosamine residues in such
polymers has been developed, with the use of high-pressure
ion-exchange
chromatography
of the corresponding
anhydro1’Hlmannitol derivatives. Preliminary results suggest
that 3-0-sulphate groups are absent not only from heparin
molecules with low affinity for antithrombin, but also from those
regions of the high-affinity molecules that lie outside the
antithrombin-binding sequence.
In conclusion, our results suggest that the actual antithrombin-binding region of the heparin molecule is contained
within the pentasaccharide sequence 2-6 (Fig. 1). The two
N-sulphate groups at positions 4 and 6, the 6-Osulphate group
at position 2 and the 3-0-sulphate group at position 4 all appear
to be involved in the binding to antithrombin. The roles of the
0
X
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X
v1
1981
50 1
596th MEETING, LANCASTER
Table 1. Afinity of heparinfragments for antithrombin
For designation of the fragments, see the legend to Fig. 1. Affinity properties are expressed by the range of salt concentrations
required to elute the various fragments from antithrombin-Sepharose. Each affinity class corresponds to a distinct peak in the
chromatograms (see Fig. 3 in Riesenfeld et al., 1981). For additional information, see the text.
Affinity for antithrombin
Fragment
LOW
0.05-0.5 M-NaCI
Medium
0.5-0.7 M-NaCI
+
+
1-1 ’H 18
1-[’H18, N-deacetylated at position 2
I-[’Hl8, N-desulphated at position 4
1-13H]8, N-desulphated at position 6
2-1 ’H I8
3-[’HI8
4-[’H18
+
High
0.7-1.2~-NaCI
+
+
i
+
Remarks and references
Thunberg et al. (1980)
Hydrazinolysis of octasaccharide
Riesenfeld et a/. (1 98 1)
Riesenfeld et al. (1981)
Digestion with a-L-iduronidase
Deamination of N-deacetylated
octasaccharide
Cleavage of octasaccharide by treatment
with periodate/alkali or with endo-b-Dglucuronidase
0-sulphate groups in positions 5 and 6 remain to be evaluated. Leder, I. G. (1980) Biochem. Biophys. Res. Commun. 94,1183-1 I89
Finally, it may be noted that, although the antithrombin-binding Lmdahl, U., Backstrom, G., Hook, M., Thunberg, L., Fransson, L.-A.
& Linker, A. (1979) Proc. Natl. Acad. Sci.U S A . 76,3198-3202
region is crucial to anticoagulant activity, the expression of such
activity also depends on other regions of the heparin molecule Lindahl, U., Backstrom, G . Thunberg, L. & Leder, I. G. (1980) Proc.
Natl. Acad. Sci. U S A . 77,655 1-6555
(Laurent et al., 1978; Holmer et al., 1979; Oosta et al., 1981).
This work was supported by grants from the Swedish Medical
Research Council (2309), KabiVitrum AB, Stockholm, and the
National Swedish Board for Technical Development.
Holmer, E., Soderstrom, G. & Andersson, L.-0. (1979) Eur. J.
Biochem. 93,l-5
Laurent, T. C., Tengblad, A., Thunberg, L., Hook, M. & Lindahl, U.
(1978)Biochem.J. 175,691-701
Meyer, B., Thunberg, L., Lmdahl, U., Larm, 0. & Leder, I. G. (1981)
Carbohydr. Res. 88, C1-C4
Oosta, G. M., Gardner, W. T., Beeler, D. L. & Rosenberg, R. D. (1981)
Proc. Natl. Acad. Sci. U S A A78,829-833
.
Riesenfeld, J., Thunberg, L., Hook, M. & Lindahl, U. (1981) J. Biol.
Chem. 256,2389-2394
Rosenberg, R. D. & Lam, L. (1979) Proc. Natl. Acad. Sci. U S A . 76,
1218-1222
Thunberg, L., Backstrom, G., Grunberg, H., Riesenfeld, J. & Lindahl,
U. (1980) FEBS Lett. 117,203-206
Multivalent proteins and multidentate polysaccharides: Interaction of platelet factor 4 and
heparin
MOLLIE LUSCOMBE,* SUSAN E. MARSHALL,*
DUNCAN S. PEPPER? and J. JOHN HOLBROOK*
*Department of Biochemistry, University of Bristol Medical
Schoof,Bristof BS8 I TD, U.K., and ?Scottish National Blood
Transfusion Service, 2 Forrest Road, Edinburgh, Scotland,
UX.
It is useful to distinguish two classes of complexes formed from
globular proteins and acidic polysaccharides. One class results
from the interaction of a specific sequence of monosaccharide
units with a specific site on the surface of a protein (typified by
the reaction of high-aflinity heparin with antithrombin III). Such
specific interactions will be sensitive to point mutations that alter
the protein site.
A second class of protein-polysaccharide complexes reflects
the interaction of a more or less regular repeat of acidic sugars
along the polysaccharide chain with repeated basic sites on the
protein surface. For globular proteins repeated basic sites may
arise from the subunit structure of the protein molecule (as they
do in the case of the tetrameric protein platelet factor 4) or from
multiple (but different) basic patches on a protein with no
subunit structure. In spite of the non-specific nature of these
ionic bonds, it is the purpose of the present communication to
show that such interactions can lead to the formation of
recognizably ordered protein-polysaccharide complexes. These
interactions are unlikely to be sensitive to single point mutations.
As an example of this second kind of interaction, we (Bock et
VOl. 9
al., 1980) have studied by the technique of polarization of
fluorescence the interaction of heparin with two closely
homologous (60% identical residues; Begg et al., 1978) platelet
proteins (factor 4 and D-thromboglobulin). Platelet factor 4 is a
constituent of platelets that neutralizes the anticoagulant effects
of heparin (Kaplan et al., 1979; Rucinski et al., 1979). The
factor 4 is a stable tetramer of identical subunits (each of M , =
7800; Kurachi, 1978), whereas p-thromboglobulin exists in a
concentration-dependent equilibrium of tetramer and subunits.
In the presence of excess of protein the platelet factor
4-heparin system assembles to give polysaccharide chains
‘peppered’ with protein molecules. Addition of further heparin
results in a 1:l complex of tetrameric protein and polysaccharide in which all the basic sites on the protein are satisfied
by one polysaccharide chain. The stability of this complex can
be very high if the polysaccharide is long enough for a single
chain to be able to wrap around the protein. In experiments
where we introduce a fluorescent label at the end of the heparin
chain it is possible to show that the protein acts as a ‘molecular
ruler in that polysaccharides with molecular weight 8600 bind
very differently to those with molecular weight 10 500. Even on
the addition of a very large excess of large heparin we do not
&serve a complex in which four heparin chains bind to the
tetrameric protein. The preferential stability of 1:1 complexes
with heparins longer than the critical length arises from entropic
considerations. The critical length is as might be predicted from
the crystallographic results on both the protein (Kurachi, 1978)