(Received for publication, December 11, 1995; and in revised form, February 6, 1996)
From the
Porcine intestinal heparin was extensively digested with Flavobacterium heparinase and size-fractionated by gel
chromatography. Subfractionation of the hexasaccharide fraction by
anion exchange high pressure liquid chromatography yielded 10
fractions. Six contained oligosaccharides derived from the repeating
disaccharide region, whereas four contained glycoserines from the
glycosaminoglycan-protein linkage region. The latter structures were
reported recently (Sugahara, K., Tsuda, H., Yoshida, K., Yamada, S., de
Beer, T., and Vliegenthart, J. F. G.(1995) J. Biol. Chem. 270,
22914-22923). In this study, the structures of one tetra- and
five hexasaccharides from the repeat region were determined by chemical
and enzymatic analyses as well as 500-MHz H NMR
spectroscopy. The tetrasaccharide has the hexasulfated structure
typical of heparin. The five hexa- or heptasulfated hexasaccharides
share the common core pentasulfated structure
HexA(2S)
1-4GlcN(NS,6S)
1-4IdoA
/GlcA
1-4GlcN(6S)
1-4GlcA
1-
4GlcN(NS) with one or two additional sulfate groups
(
HexA, GlcN, IdoA, and GlcA represent
4-deoxy-
-L-threo-hex-4-enepyranosyluronic acid, D-glucosamine, L-iduronic acid, and D-glucuronic acid, whereas 2S, 6S, and NS stand for
2-O-, 6-O-, and 2-N-sulfate, respectively).
Three components have the following hitherto unreported structures:
HexA(2S)
1-4GlcN(NS,6S)
1-4GlcA
1-4GlcN(NS,6S)
1-4GlcA
1-4GlcN(NS,6S),
HexA(2S)
1-4GlcN(NS,6S)
1-4IdoA
1-4GlcNAc(6S)
1-4GlcA
1-4GlcN(NS,3S),
and
HexA(2S)
1-4GlcN(NS,6S)
1-4IdoA(2S)
1-4GlcNAc(6S)
1-4GlcA
1-
4GlcN(NS,6S). Two of the five hexasaccharides are structural
variants derived from the antithrombin III-binding sites containing
3-O-sulfated GlcN at the reducing termini with or without a
6-O-sulfate group on the reducing N,3-disulfated GlcN
residue. Another contains the structure identical to that of the above
heptasulfated antithrombin III-binding site fragment but lacks the
3-O-sulfate group and therefore is a pro-form for the binding
site. Another has an extra sulfate group on the internal IdoA residue
of this pro-form and therefore can be considered to have diverged from
the binding site in the biosynthetic pathway. Thus, the isolated
hexasaccharides in this study include the three overlapping pairs of
structural variants with an apparent biosynthetic precursor-product
relationship, which may reflect biosynthetic regulatory mechanisms of
the binding site.
Heparin is a highly sulfated, linear polysaccharide that has
various biological activities such as inhibition of blood coagulation
(Marcum and Rosenberg, 1989), modulation of cellular proliferation
(Clowes and Karnovsky, 1977; Thornton et al., 1983),
potentiation of angiogenesis (Folkman and Ingber, 1989), and
interactions with various growth factors (Maciag et al., 1984;
Shing et al., 1984; Klagsbrun and Shing, 1985; Nakamura et
al., 1986). The basic polymeric structure of heparin is an
alternating repeat sequence of the disaccharide units
4IdoA
/GlcA
1
4GlcN
1
, which can be
variably sulfated (for reviews see Rodén(1980),
Gallagher and Lyon(1989), and Lindahl(1989)). It is synthesized on a
serine residue of a protein core named ``serglycin'' through
a specific structure, the so-called carbohydrate-protein linkage
region:
GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser
(Lindahl and Rodén, 1965). Although the principal
structure of the repeating disaccharide region, known as the regular
region (Casu, 1985), is composed of the major trisulfated disaccharide
unit,
4IdoA(2-sulfate)
1
4GlcN(N,6-disulfate)
1
,
undersulfation and substantial structural variability are observed in
the irregular region, which is distributed along the chain flanked by
the regular region and accounts for approximately one quarter of the
heparin polysaccharide chain. The structural variability is often the
basis of a wide variety of domain structures with a number of
biological activities ascribed to heparin.
Recent structural studies of the binding domains to ATIII (For review see Lindahl(1989)) and basic fibroblast growth factor (Maccarana et al., 1993) are the best known examples showing the relationships between the complicated fine structures and biological functions. The ATIII-binding site requires a minimal pentasaccharide sequence uniquely 3-O-sulfated on the central GlcN residue. This specific pentasaccharide has been shown to be primarily responsible for the anticoagulant activity of heparin (Lindahl et al., 1983). It has also been demonstrated that the binding domain to basic fibroblast growth factor requires a 2-O-sulfated IdoA residue and N-sulfated GlcN residue(s) for its specific interaction with the growth factor. Some structural variability has been observed within both binding sequences (Lindahl et al., 1984; Yamada et al., 1993; Maccarana et al., 1993).
We have been studying the basic primary structure of heparin to clarify the structural basis of its various biological activities. Previously, we demonstrated its structural variability by isolating six glycoserines from the carbohydrate-protein linkage region (Sugahara et al., 1992, 1995) and a number of tetrasaccharides from the repeating disaccharide region of porcine intestinal heparin after extensive digestion with bacterial heparin lyases (Yamada et al., 1993, 1994, 1995). In this study, we isolated and characterized five hexasaccharide structures from the repeating disaccharide region of the same heparin preparation after extensive enzymatic digestion to investigate the structure beyond the above tetrasaccharide sequences. These included three hitherto unreported hexasaccharide structures and structural variants with an apparent biosynthetic precursor-product relationship for the ATIII-binding site.
Materials-Stage 14 heparin was purchased from
American Diagnostica (New York, NY) and purified by DEAE-cellulose
chromatography as reported previously (Sugahara et al., 1992).
Heparinase (EC 4.2.2.7) and purified heparitinases I (EC 4.2.2.8) and
II (no EC number) were obtained from Seikagaku Corp. (Tokyo, Japan).
-Glycuronate-2-sulfatase (EC 3.1.6.-), abbreviated
as 2-sulfatase, and heparitinase V (no EC number) were purified from Flavobacterium heparinum (McLean et al., 1984) and Flavobacterium sp. Hp206 (Yoshida et al.,
1989), respectively. Sephadex gels were from Pharmacia Biotech Inc.,
and Bio-Gel resins were from Bio-Rad. NaB
H
(15
Ci/mmol) was supplied by American Radiolabeled Chemicals, Inc. (St.
Louis, MO). 4-Methylumbelliferyl-
-L-iduronide was from
Sigma, and p-nitrophenyl-
-D-glucuronide was from
Nacalai Tesque (Kyoto, Japan). Seven standard unsaturated disaccharides
were prepared from heparin as reported previously (Yamada et
al., 1992). Standard heparin disaccharides prepared by deaminative
cleavage (Shively and Conrad, 1976a, 1976b) were gifts from Dr. H. E.
Conrad, University of Illinois.
-Glucuronidase (EC 3.2.1.31)
purified to homogeneity from Ampullaria (freshwater apple
shell) hepatopancreas (Tsukada and Yoshino, 1987) was obtained from
Tokyo Zouki Chemical Co., Tokyo. Human liver
-iduronidase (EC
3.2.1.76) was purified as reported previously (Freeman and Hopwood,
1992).
Figure 1: Capillary electrophoresis of the isolated hexasaccharide fractions. The isolated hexasaccharide fractions (1.0 nmol each) were subjected to electrophoresis as described under ``Experimental Procedures.'' A, Fr. b-15; B, Fr. b-19; C, Fr. b-20; D, Fr. b-22; E, Fr. b-24.
Figure 2:
HPLC
analysis of the enzyme digests of Fr. b-15. Fr. b-15 (0.5 nmol) was
digested with heparitinase I (A) and successively with
2-sulfatase and then heparitinase I (B) or with heparitinase V (C) as described under ``Experimental Procedures.''
The digest was subjected to HPLC on an amine-bound silica column using
a linear gradient of NaHPO
from 16 to 800
mM over 90 min. Elution positions of the standard
disaccharides isolated from heparin/heparan sulfate are indicated in A: 1,
DiHS-0S; 2,
DiHS-6S; 3,
DiHS-NS; 4,
DiHS-diS
; 5,
DiHS-diS
; 6,
DiHS-diS
; 7,
DiHS-triS. The
peaks marked by asterisks are often observed between 35 and 40
min upon high sensitivity analysis and are due to an unknown substance
eluted from the column resin. The broad peaks observed at around 5 and
10 min were derived from the incubation buffer or the enzyme
preparation.
Figure 3: HPLC analysis of the enzyme digests of Fr. b-24. Fr. b-24 (0.25 nmol) was digested with a mixture of heparitinase I and heparinase (A), heparinase (B), heparitinase I (C), or 2-sulfatase and then heparitinase I successively (D) as described under ``Experimental Procedures.'' For the HPLC conditions, see the legend to Fig. 2.
The major component in Fr. b-24 accounted for 75% of the
UV-absorbing materials in this fraction as judged by capillary
electrophoresis (Fig. 1E). Exhaustive digestion of this
fraction with a mixture of heparinase and heparitinase I yielded
DiHS-triS,
DiHS-diS
, and
DiHS-diS
with recoveries of 158, 96, and 125%, respectively (Fig. 3A). Upon incubation with heparinase only, Fr.
b-24 was degraded into two unsaturated components,
DiHS-triS and a
component that eluted near the elution position of a tetrasulfated
tetrasaccharide, with recoveries of 171 and 110%, respectively (Fig. 3B), whereas it was degraded by heparitinase I
into two unsaturated components,
DiHS-diS
and a
component that eluted near the elution position of a pentasulfated
tetrasaccharide, with recoveries of 100 and 106%, respectively (Fig. 3C). These results together indicate that the
major component in Fr. b-24 is a pentasulfated hexasaccharide composed
of equimolar amounts of three disaccharide units corresponding to
DiHS-triS,
DiHS-diS
, and
DiHS-diS
, and that the excess recovery of
DiHS-triS upon heparinase or heparinase/heparitinase I digestion
was probably due to degradation of contaminating oligosaccharide(s).
The sequential arrangement of the three disaccharide units in the major
hexasaccharide was determined based upon the sensitivity to
2-sulfatase. When digested successively with 2-sulfatase and then
heparitinase I, the presumably pentasulfated tetrasaccharide peak
shifted to a position corresponding to the loss of one sulfate group on
HPLC, indicating that
DiHS-triS had been located on the
nonreducing terminal side of the major compound in Fr. b-24. Therefore,
the structure of the major compound in this fraction is proposed as
HexA(2S)-GlcN(NS,6S)-HexA(2S)-GlcNAc(6S)-HexA-GlcN(NS,6S).
Fr. b-19 was resolved into several subcomponents by capillary
electrophoresis, the major component accounting for only 54% of the
UV-absorbing materials in this fraction (Fig. 1B), but
it was not possible to fractionate it preparatively into its
subcomponents. Therefore, it was first digested with 2-sulfatase and
then the digest was analyzed by HPLC. The 2-sulfatase treatment
resulted in a peak shift of 8 min of 61% of the parent compound on
HPLC, indicating that the major product lost one sulfate group (data
not shown). The major product, designated as Fr. b-19S, was isolated
and subjected to structural analysis. The yield was 167 nmol/100 mg of
the starting heparin. It was degraded by heparitinase I, yielding
almost exclusively DiHS-diS
(Table 1).
Therefore, the major component in Fr. b-19S was deduced to be a
hexasulfated hexasaccharide composed of 3 mol of the disulfated
disaccharide unit corresponding to
DiHS-diS
, i.e.
HexA-GlcN(NS,6S)-HexA-GlcN(NS,6S)-HexA-GlcN(NS,6S).
Consequently, the structure of the major component in the parent
fraction b-19 was
HexA(2S)-GlcN(NS,6S)-HexA-GlcN(NS,6S)-HexA-GlcN(NS,6S).
Heparitinase I digestion of both Fr. b-20 and -22 resulted in two
unsaturated components, the trisulfated disaccharide DiHS-triS and
a component that eluted near the elution position of the tri- or
tetrasulfated tetrasaccharide (data not shown). Recoveries of the di-
and tetrasaccharide components from Fr. b-20 or -22 were 109 and 62% or
122 and 106%, respectively (Table 1). The lower recoveries of the
presumable tetrasaccharide components of these fractions compared with
those of their counterpart disaccharides suggested that the excess
disaccharides were derived from minor components in these fractions
consistent with the results of capillary electrophoresis. The
presumable tetrasaccharide components from both Fr. b-20 and -22 were
resistant to heparinase and heparitinases I and II (data not shown),
probably due to the 3-O-sulfation of the reducing GlcN as
reported previously for the ATIII-binding site-derived tetrasaccharides
(Yamada et al., 1993). The presumable tetrasaccharides were
co-chromatographed on HPLC with the authentic tetrasaccharides
containing 3-O-sulfated GlcN residue (Yamada et al.,
1993), demonstrating that the tri- and tetrasulfated tetrasaccharides
derived from Fr. b-20 and -22 were identical to
HexA-GlcNAc(6S)-GlcA-GlcN(NS, 3S) and
HexA-GlcNAc(6S)-GlcA-GlcN(NS, 3S,6S), respectively.
Sensitivities of the compounds in Fr. b-20 and -22 to 2-sulfatase
were examined to characterize the sequential arrangement of the
constituent di- and tetrasaccharide units. After 2-sulfatase digestion,
Fr. b-20 and -22 gave a single peak on HPLC, which eluted approximately
10 min earlier than the corresponding parent compound, indicating that
the major compound in each fraction had a sulfate group on the C-2
position of the HexA residue at the nonreducing terminus.
Therefore, the structures of the major compounds in Fr. b-20 and -22
were
HexA(2S)-GlcN(NS,6S)-HexA-GlcNAc(6S)-GlcA-GlcN(NS,3S)
and
HexA(2S)-GlcN(NS,6S)-HexA-GlcNAc(6S)-GlcA-GlcN(NS,3S,6S),
respectively.
The
resultant di- and/or tetrasaccharide(s) from each hexasaccharide
fraction were isolated by gel filtration on Bio-Gel P-2 and were
analyzed by HPLC. These fractions obtained from Fr. b-15, -20, or -22
were confirmed to contain a disaccharide and a tetrasaccharide
component as judged from their elution positions on HPLC (data not
shown) (Bienkowski and Conrad, 1985). The tetrasaccharide component
presumably derived from the reducing side of each of these original
hexasaccharides was subjected to digestion with human liver
-iduronidase (Freeman and Hopwood, 1992) and Ampullaria
-glucuronidase (Tsukada and Yoshino, 1987) to identify the
uronic acid residues at the nonreducing termini. After
-iduronidase digestion, a part (40, 57, or 60%) of the
tetrasaccharide peak derived from Fr. b-15, -20, or -22 eluted
7-14 min earlier than the corresponding parent compound on HPLC.
As representative chromatograms, those obtained with the
tetrasaccharide from Fr. b-22 are shown in Fig. 4. Upon
-glucuronidase digestion, however, the tetrasaccharide fraction
obtained from each of the three fractions was totally resistant to the
action of the enzyme (data not shown). These results suggest that the
nonreducing terminal uronic acid of the major tetrasaccharide obtained
from Fr. b-15, -20, or -22 by nitrous acid treatment is not GlcA but
rather IdoA. The reason for the partial insensitivity to the action of
-iduronidase is unclear at present but may have been due to side
product(s) of the deamination reactions. It has been reported that the
so-called ring contraction tetrasaccharides can be formed during
deamination reactions (Bienkowski and Conrad, 1985). Based upon the
above results, the structures of the major components in these
fractions were deduced as follows: Fr. b-15,
HexA(2S)-GlcN(NS,6S)-IdoA-GlcNAc(6S)-HexA-GlcN(NS,6S);
Fr. b-20,
HexA(2S)-GlcN(NS,6S)-IdoA-GlcNAc(6S)-GlcA-GlcN(NS,
3S); Fr. b-22,
HexA(2S)-GlcN(NS,6S)-IdoA-GlcNAc(6S)-GlcA-GlcN(NS,
3S,6S).
Figure 4:
-Iduronidase digestion of the
tetrasaccharide prepared from Fr. b-22 by
HNO
/NaB
H
treatment. Fr. b-22 was
subjected to nitrous acid depolymerization at pH 1.5, and the resultant
H-labeled tetrasaccharides were isolated by gel filtration
chromatography on a Bio-Gel P-2 column and analyzed by HPLC on an
amine-bound silica column using a stepwise salt gradient as indicated
by the dashed line. Fractions were collected at 1-min
intervals at a flow rate of 1 ml/min, and their radioactivity was
determined by liquid scintillation counting. A, the presumable
tetrasaccharide (7500 cpm) derived from Fr. b-22; B, the
-iduronidase digest of the tetrasaccharide (3900
cpm).
Deaminative cleavage of Fr. b-19S yielded no appreciable
tetrasaccharides but two kinds of disaccharide components, which eluted
at around the positions of HexA-anMan
(6S) and
GlcA-anMan
(6S), respectively, in a molar ratio of 1.0:1.7.
These results indicate that both internal uronic acid residues of the
major hexasaccharide component in Fr. b-19S and its parent fraction
b-19 are GlcA. Thus, the structure of the major hexasaccharide in Fr.
b-19S and -19 was deduced respectively as follows: Fr. b-19S,
HexA-GlcN(NS,6S)-GlcA-GlcN(NS,6S)-GlcA-GlcN(NS,6S);
Fr. b-19,
HexA(2S)-GlcN(NS,6S)-GlcA-GlcN(NS,6S)-GlcA-GlcN(NS,6S).
Fr. b-24 was also degraded into a disaccharide and a tetrasaccharide
component (data not shown) as judged from the gel filtration profiles
of the deaminative cleavage products and the elution positions on the
subsequent HPLC of the isolated di- and tetrasaccharides. The
disaccharide eluted at around the position of
HexA(2S)-anMan
(6S) on HPLC. Deaminative cleavage
products of this fraction were not further analyzed because the
internal uronic acid residues were easily identified as IdoA(2S) and
nonsulfated GlcA by 500-MHz
H NMR analysis as described
below.
The internal uronic acid residue of
each isolated hexasaccharide was unambiguously identified by 500-MHz H NMR spectroscopy based upon the chemical shifts of the
anomeric proton signals and the coupling constants J
. Anomeric proton signals of an
IdoA and a
GlcA residue in heparin/heparan sulfate oligosaccharides are
observed at around
5.2-5.0 and 4.7-4.5, respectively
(Merchant et al., 1985; Yamada et al., 1995). The
coupling constants J
of
IdoA and
GlcA
in heparin/heparan sulfate oligosaccharides are approximately 3.0 and
8.0 Hz, respectively (Horne and Gettins, 1992; Yamada et al.,
1995). In the spectrum of Fr. b-24, two internal uronic acid residues
were identified as IdoA and GlcA based on the chemical shifts of the
anomeric proton signals, at
5.184 and 4.576 (Fig. 5), and
the coupling constants J
, 3.0 and 7.5 Hz,
respectively. The chemical shifts of H-1 and H-2 of the IdoA residue
were shifted downfield by approximately 0.2 and 0.6 ppm, respectively,
when compared with those of the nonsulfated IdoA residue of the
tetrasaccharides isolated from bovine kidney heparan sulfate (Sugahara et al., 1994), supporting the 2-sulfation of IdoA-4 of the
compound in Fr. b-24 (Yamada et al., 1994). Based upon these
NMR data and the sequential arrangement of the disaccharide units
determined by enzymatic analysis, the following structure is proposed
for the major compound in this fraction: Fr. b-24,
HexA(2S)-GlcN(NS,6S)-IdoA(2S)-GlcNAc(6S)-GlcA-GlcN(NS,6S).
Figure 5:
One-dimensional 500-MHz H NMR
spectrum of the structure in Fr. b-24 recorded in
H
O. The numbers and letters in the spectrum refer to the corresponding residues in the
structure.
The two internal uronic acid residues of the hexasaccharide in Fr.
b-15 were identified as IdoA and GlcA, based on the chemical shifts
( 5.024 and 4.566) of the anomeric proton signals, respectively.
Analysis of the deamination products showed that the IdoA residue was
located at position 4, suggesting in turn that the GlcA residue is
located at position 2. Thus, the following structure is proposed for
the major compound in this fraction: Fr. b-15,
HexA(2S)-GlcN(NS,6S)-IdoA-GlcNAc(6S)-GlcA-GlcN(NS,6S).
Likewise, the structures of the major compounds in three other fractions were determined and summarized in Table 3. Some of the chemical shifts of Fr. b-20 were not assigned due to the low quality of the spectra.
The five sulfated hexasaccharide structures isolated in this
study share the pentasulfated hexasaccharide backbone
HexA(2S)
1-4GlcN(NS,6S)
1-4IdoA
/GlcA
1-4GlcN(6S)
1-4GlcA
1-4GlcN(NS)
with one or two additional sulfate groups on GlcN-1, GlcN-3, and/or
IdoA-4. Two of these, Fr. b-15 and -22, have been isolated previously
(Linhardt et al., 1992), whereas the other three (Fr. b-19,
-20, and -24) were isolated for the first time as discrete structures
in this study. All the hexasaccharides contain the common trisulfated
disaccharide unit on their nonreducing sides and the GlcN(NS)
residue at their reducing termini, reflecting the substrate specificity
of heparinase used for digestion of the starting heparin. These
structural features are in good agreement with the established
specificity of heparinase that cleaves the glucosaminidic linkage in
the GlcN(NS)
1-4IdoA(2S) sequence in a polymer
(Linker and Hovingh, 1984; Merchant et al., 1985) and that in
the
GlcN(NS)
1-4IdoA(2S)
1-4GlcN(NS,6S)
sequence in small oligosaccharides (Yamada et al., 1994,
1995).
Heparin and heparan sulfate have been demonstrated to exhibit
various biological activities (Kjellén and
Lindahl, 1991). Especially their specific interactions with various
growth factors have recently attracted much attention. However, the
functional domain structures elucidated to date are limited to only a
few examples, including the minimum pentasaccharide sequences for ATIII
binding and basic fibroblast growth factor binding, which have been
demonstrated as
GlcN(6S)1-4GlcA
1-4GlcN(NS,3S)
1-4IdoA(2S)
1-4GlcN(NS,6S)
(Lindahl et al., 1983) and
GlcA
1-4GlcN(NS)
1-4HexA1-4GlcN(NS)
1-4IdoA(2S)
(Maccarana et al., 1993), respectively. The oligosaccharide
sequences for high affinity binding to acidic fibroblast growth factor,
fibroblast growth factor 4, and hepatocyte growth factor have been
partially characterized (Bârzu et al.,
1989; Ishihara, 1994; Guimond et al., 1993; Lyon et
al., 1994), but the essential sulfate groups in these sequences
have not yet been identified.
The hexasaccharides isolated in this
study appear to be large enough to potentially exhibit binding
activities toward growth factors or other functional proteins. None of
them, however, showed ATIII-mediated inhibition of factor Xa as
examined according to Morita et al.(1977). Although the
hexasaccharides in Fr. b-20 and -22 contain a 3-sulfated
GlcN(NS) residue at their reducing termini, they lack a part
of the minimum pentasaccharide sequence on their reducing sides, i.e. IdoA(2S)1-4GlcN(NS,6S). The isolated
hexasaccharides are not expected to contain the functional domains for
binding to basic fibroblast growth factor. The hexasaccharides, except
for that in Fr. b-24, do not have an IdoA(2S) residue essential for
binding to basic fibroblast growth factor. Although the hexasaccharide
in Fr. b-24 contains an IdoA(2S) residue in its sequence, it lacks the
disaccharide extension GlcA
1-4GlcN(NS) of the basic
fibroblast growth factor binding sequence on the nonreducing side. It
remains to be determined whether the binding domains to the other
growth factors or biologically active proteins are embedded in the
isolated hexasaccharides.
The most interesting structural feature of the isolated hexasaccharides is that they include three overlapping pairs of structural variants with an apparent biosynthetic precursor-product relationship for the ATIII-binding site. Structurally, the hexasaccharide in Fr. b-15 is a pro-form of that in Fr. b-22. The former lacks the 3-sulfate group on GlcN-1 of the latter. Likewise, the hexasaccharide in Fr. b-20 is a pro-form of that in Fr. b-22, the former lacking the 6-sulfate of GlcN-1 of the latter. The hexasaccharide in Fr. b-15 can be considered as a pro-form of that in Fr. b-24, where the former lacks the 2-sulfate on IdoA-4 of the latter. Among the above hexasaccharides, the previously isolated structures found in Fr. b-15 and -22 (Linhardt et al., 1992) have been subjects of much discussion as biosynthetic precursors and product, respectively, of the ATIII-binding site as described below.
Only about one-third of chains of commercial porcine intestinal heparin contain an ATIII-binding site and have a high affinity for ATIII. The critical 3-O-sulfation of GlcN required for the ATIII-high affinity concludes the biosynthesis of the ATIII-binding site (Kusche et al., 1988). Based upon the isolation of a precursor tetrasaccharide sequence, which lacked the 3-O-sulfate group from heparin chains with ATIII-low affinity, Kusche et al. (1990) proposed that essentially each low affinity chain would contain a potential but not utilized 3-O-sulfation site. Linhardt et al.(1992) challenged this hypothesis, proposing that the existence of a low affinity heparin may not simply be the result of the incomplete action of 3-O-sulfotransferase in the final step, but rather some earlier step involved in the formation of the precursor sites may be primarily responsible for high and low ATIII affinity heparins. Recently, Razi and Lindahl(1995) proposed an intriguing hypothesis that the 3-O-sulfotransferase may be inhibited by a sulfated saccharide sequence outside the 3-O-sulfate acceptor region based upon the observation that an octasaccharide fraction isolated from ATIII-low affinity heparin, unlike low affinity heparin polysaccharide (Kusche et al., 1990), yielded high affinity components following incubation with a GlcN 3-O-sulfotransferase preparation.
The multiple pro-form structures demonstrated in this study probably do not represent precursors and products but rather reflect structural variants that have diverged during the modification reactions in the as yet unresolved but precisely programmed biosynthetic scheme. Practically, they will be valuable acceptor substrates for sulfotransferases to investigate such biosynthetic mechanisms by which the production of specific functional carbohydrate sequences is regulated. For example, it would be of interest to examine whether the hexasaccharide structures in Fr. b-15 and -20 serve as acceptor substrates for IdoA 2-O-sulfotransferase and GlcN 6-O-sulfotransferase (Habuchi et al., 1995) to produce the hexasaccharide structures found in Fr. b-24 and -22, respectively. It will be intriguing to evaluate these hexasaccharides as regulatory elements as well because they may control biosynthetic modifying enzymes.
They will also be useful for characterizing heparin/heparan sulfate-degrading enzymes including both the bacterial heparinase/heparitinases and mammalian heparanases. The former enzymes are essential tools for structural studies (Yoshida et al., 1989), whereas the latter have been implicated in tumor metastasis (for review see Nakajima et al.(1988)), T cell adhesion (Gilat et al., 1995), and chemoattractant functions (Hoogewerf et al., 1995).