(Received for publication, July 13, 1995; and in revised form, October 31, 1995)
From the
Heparin-binding growth-associated molecule (HB-GAM) is a cell surface- and extracellular matrix-associated protein that lines developing axons in vivo and promotes neurite outgrowth in vitro. Because N-syndecan (syndecan-3) was found to function as a receptor in HB-GAM-induced neurite outgrowth, we have now studied whether the heparan sulfate side chains of N-syndecan play a role in HB-GAM-neuron interactions. N-Syndecan from postnatal rat brain was found to inhibit HB-GAM-induced but not laminin-induced neurite outgrowth when added to the assay media. The inhibitory activity was abolished by treating N-syndecan with heparitinase, but it was retained in N-syndecan-derived free glycosaminoglycan chains, suggesting that N-syndecan heparan sulfate at the cell surface is involved in HB-GAM-induced neurite outgrowth. Binding to HB-GAM and inhibition of neurite outgrowth was observed with heparin-related polysaccharides only; galactosaminoglycans were inactive. Significant inhibition of neurite outgrowth was induced by heparin and by N-syndecan heparan sulfate but not by heparan sulfates from other sources. A minimum of 10 monosaccharide residues were required for HB-GAM binding, as well as for inhibition of HB-GAM-induced neurite outgrowth. Experiments with selectively desulfated heparins indicated that 2-O-sulfated iduronic acid units, in particular, are of importance to the interaction with HB-GAM, whereas glucosamine N-sulfate and 6-O-sulfate groups were implicated to a lesser extent. Structural analysis of N-syndecan from 6-day-old rat brain indicated that the heparan sulfate chains contain sequences of contiguous, N-sulfated disaccharide units with an unusually high proportion (82%) of 2-O-sulfated iduronic acid residues. We suggest that this property of N-syndecan heparan sulfate is essential for HB-GAM binding and induction of neurite outgrowth.
Cell surface proteoglycans are suggested to function as
receptors or co-receptors for molecules that mediate
cell-to-extracellular matrix and cell-to-cell interactions and for
growth factors of the extracellular milieu (for reviews see (1, 2, 3, 4, 5, 6, 7) ).
Most of these interactions depend on binding of proteins to the
negatively charged glycosaminoglycan (GAG) ()chains of the
proteoglycans. Binding of proteins to the GAG chains can vary with
regard to affinity and specificity and generally involves electrostatic
interactions with negatively charged GAG
structures(3, 8) . A growing number of heparin-binding
molecules involved in various biological processes have attained
interest during the recent years. Among the best characterized members
of the heparin-binding growth factors are the acidic and basic
fibroblast growth factors. Several reports on the heparin/heparan
sulfate involved in fibroblast growth factor action have
appeared(9, 10, 11, 12, 13) .
HB-GAM (p18) was isolated from rat brain as a neurite outgrowth-promoting protein, the expression of which in brain corresponds to the stage of rapid axonal growth(14) . The full-length cDNA encoding rat HB-GAM has been cloned and sequenced(15) . The same amino acid sequence has been reported for pleiotrophin, a protein isolated from uterus as a mitogen for NIH 3T3 cells(16) . The HB-GAM sequence is more than 90% conserved from man to chicken(15, 16, 17, 18, 19) , and it shares about 50% homology with the mouse midgestation kidney protein (20, 21) and its chicken homolog retinoic acid-inducible heparin-binding protein(22) . The midgestation kidney protein is involved in retinoic acid-induced cell differentiation(20, 21) .
The neurite outgrowth-promoting property of HB-GAM in vitro has been shown in a number of studies(14, 16, 18, 19, 23) . Furthermore, during embryonic and perinatal development, HB-GAM is strongly expressed in axon pathways of the brain (24) and as a component of basement membranes outside the brain(25) . These findings have raised the question as to the occurrence of cell surface receptors for HB-GAM.
A 200-kDa heparan sulfate proteoglycan isolated from cultured brain neurons and from rat brain has been implicated as a receptor or a co-receptor for HB-GAM(26) . Based on partial peptide sequencing and on immunochemical analysis, this component has been identified as N-syndecan (syndecan-3). N-Syndecan cDNA has been previously cloned based on its homology with other syndecans, and N-syndecan has been shown to be strongly expressed in developing nervous tissues, for example in perinatal rat brain(27) . A temporal co-expression during brain development has also been recently demonstrated for HB-GAM and N-syndecan(28) . Furthermore, both proteins are preferentially localized to developing fiber tracts of the rat brain(28) .
Because biochemical and cell biological studies have implicated N-syndecan as an HB-GAM receptor(26) , we have carried out further studies on HB-GAM-N-syndecan interactions. The carbohydrate moiety of N-syndecan consists mostly or exclusively of heparan sulfate chains(26, 27) , and we have therefore focused our studies on heparin-type structures that are likely to function as HB-GAM-binding sites. Because N-syndecan carbohydrates are available in a very limited amount, we have taken the strategy to define the structural requirements of active carbohydrates using heparin and other glycosaminoglycans as model components and attempted to correlate these findings to studies on N-syndecan. We suggest that the binding of HB-GAM to carbohydrates is specific for heparin-type structures and that N-syndecan heparan sulfate fulfills the requirements for such interaction.
N-Syndecan glycopeptides were prepared
by digestion with sequencing grade trypsin (Boehringer Mannheim).
Heparan sulfate-containing fractions were concentrated by HB-GAM
affinity chromatography, and the 100-kDa glycopeptides were ethanol
precipitated for 30 min at -70 °C and centrifuged at 20,000
g for 30 min at 4 °C.
Glycosaminoglycan chains were released from intact N-syndecan by treatment with alkaline borohydride(31) . The released saccharides were purified by HB-GAM affinity, desalted by passage through Sephadex G-25, and lyophilized. The carbohydrate contents of intact N-syndecan or its released GAG chains were measured by the carbazole reaction for hexuronic acid(32) .
Figure 1: Neurite outgrowth from cerebral neurons on surfaces coated with recombinant HB-GAM or laminin and effects of exogenous heparin-related compounds. Neuron-enriched cell suspensions were prepared from cerebral hemispheres of 16-day-old embryonic rats and cultured for 20 h on substrates coated with 4 µg/ml recombinant HB-GAM (A-D) or 50 µg/ml laminin (E and F). The cells were fixed with 2% paraformaldehyde and stained with 0.05% toluidine blue. A, control cells on HB-GAM-coated substrate. B, 2-O-desulfated heparin added to culture medium at 6 µg/ml. C, N-syndecan added to the culture medium at GAG concentration of 2.6 µg/ml. D, heparitinase-digested N-syndecan (Heparitinase III, Sigma; 1 unit/ml; phosphate-buffered saline overnight at 37 °C) added to the culture medium at 17 µg/ml. E, control cells grown on laminin-coated substrate. F, cells grown on laminin with 10 µg/ml of N-syndecan added to the assay medium. Scale bar, 50 µm.
Figure 2:
HB-GAM-induced neurite outgrowth in the
presence of heparin, its modified forms, and other glycosaminoglycans.
Cells were prepared and cultured on HB-GAM-coated substrates as in Fig. 1with different concentrations of glycosaminoglycan
preparations added to the culture medium. Cells were fixed with 2%
paraformaldehyde and stained with 0.05% toluidine blue. The number of
cells with neurites/number of cells on a field was counted on five
random fields for each concentration; the results given are averages.
The standard error for each concentration was < 10%. A,
heparin (), 6-O-desulfated heparin (
),
2-O-desulfated heparin (x), and N-desulfated heparin
(
). B, heparin (
), N-syndecan (
), N-syndecan-derived glycosaminoglycans (
), chondroitin
sulfate (
), aorta HS (
), and kidney HS
(
).
To study whether the inhibitory effect of N-syndecan is specific with respect to the substrate used, the assays were also carried out on laminin-coated culture wells. No effect on neurite outgrowth or other aspects of cell morphology could be observed on laminin-coated substrates at the concentrations of N-syndecan that inhibited HB-GAM-induced neurite outgrowth (Fig. 1, E and F). An N-syndecan fraction that completely inhibited HB-GAM-induced neurite outgrowth at 5 µg/ml was tested up to 41 µg/ml on laminin-coated substrate, but no inhibition was observed (33 ± 3.3% of cells with neurites as compared with 33 ± 5.7% in the control).
The inhibitory activity of heparin was essentially lost upon
selective 2-O-desulfation (Fig. 2A), which
resulted in an IC 10
-fold higher than that of
heparin. Also selective 6-O-desulfation and N-desulfation followed by N-acetylation resulted in
an appreciable, albeit less dramatic, loss of inhibitory capacity,
shifting the dose-response curves to about 10
-fold higher
concentrations. The inhibition of HB-GAM-induced neurite outgrowth by
heparin thus appears to depend on the concerted effect of different
sulfate substituents, the 2-O-sulfate groups on iduronic acid
units being of particular importance.
In contrast to heparin, chondroitin sulfate, aorta heparan sulfate, kidney heparan sulfate (Fig. 2B), lung heparan sulfate, and dermatan sulfate (data not shown) were all ineffective or displayed little inhibitory activity. Notably, detergent-free N-syndecan and its heparan sulfate chains were clearly more active than any of the other heparan sulfate preparations tested (Fig. 2B).
Dose-response
curves similar to those shown in Fig. 2were also generated for
different heparin-derived oligosaccharides. The IC concentrations, compiled in Table 1, show that heparin
oligosaccharides from disaccharide to octasaccharide were not
inhibitory, whereas some inhibition was observed for the
decasaccharide. For larger oligosaccharides the inhibitory activity was
clearly increased with increasing polymer length.
Figure 3:
Competitive binding of H-labeled heparin and unlabeled polysaccharides to HB-GAM.
Unlabeled polysaccharide competitors were incubated at various
concentrations with 10 µg of recombinant HB-GAM (1.85
µM) in the presence of 334 Bq of
H-labeled
heparin (120 nM;
20-24 saccharides).
Protein-saccharide complexes were recovered on a nitrocellulose filter
and protein-bound saccharides were released by incubation in 2 M NaCl. Released radioactivity was counted. The mean values of
duplicates are given in the figure. A, unlabeled heparin
(
), 6-O-desulfated heparin (
),
2-O-desulfated heparin (
), N-desulfated
heparin (
), and totally desulfated re-N-acetylated
heparin (
). B, unlabeled heparin (
), aorta HS
(
), chondroitin sulfate (
), lung HS (
), and kidney
HS (
).
Of other polysaccharides tested, only
aorta heparan sulfate displayed significant binding activity, although
10-10
-fold higher concentrations were
required as compared with heparin (Fig. 3B).
Chondroitin sulfate, dermatan sulfate, lung heparan sulfate, or kidney
heparan sulfate did not bind to HB-GAM (Fig. 3B).
The requirement for polymer size in HB-GAM binding was studied using H-labeled heparin oligosaccharides. The smallest
oligosaccharide showing appreciable binding (approximately 10% of added
oligosaccharide) was the decasaccharide (Fig. 4).
Figure 4:
Binding of heparin oligosaccharides to
HB-GAM. Recombinant HB-GAM (10 µg) was incubated with H-labeled heparin oligosaccharides (167 Bq,
60
nM) ranging from tetrasaccharide to a fragment containing
>18 monosaccharides. HB-GAM-bound oligosaccharides were captured on
nitrocellulose filters, and bound oligosaccharides were determined as
in Fig. 3.
Figure 5:
Nitrous acid degradation of N-syndecan HS and compositional analysis of nitrous acid
cleaved disaccharides. N-Syndecan HS was degraded by
exhaustive treatment with nitrous acid at pH 1.5 and labeled with
NaBH
reduction. The resulting saccharides were
separated by gel chromatography on Biogel P-10 (A). Nitrous
acid-cleaved disaccharides were separated by anion exchange HPLC on a
Partisil-10 SAX column (B). The numbers correspond to
the elution positions of known disaccharide standards as follows: Peak 1, nonsulfated HexA-aMan
; Peak 2,
GlcA-aMan
(6-OSO
); Peak 3,
IdoA-aMan
(6-OSO
); Peak 4,
IdoA(2-OSO
)-aMan
; Peak 5,
IdoA(2-OSO
)-aMan
(6-OSO
).
Separation of the disaccharides by
anion-exchange HPLC showed a predominant peak of the
mono-O-sulfated species,
IdoA(2-OSO)-aMan
, along with smaller amounts of
nonsulfated, other mono-O-sulfated, and di-O-sulfated
components (Fig. 5B; Table 2). As much as 82% of
the disaccharides contained a 2-O-sulfated IdoA unit, whereas
only 30% were 6-O-sulfated. The total (N- and O-) sulfate content of the heparan sulfate chain was
calculated to 0.92 residue/disaccharide unit. Interestingly, only about
one-third of the tetrasaccharides carried an O-sulfate
substituent, as indicated by high voltage paper electrophoresis (data
not shown).
HB-GAM was initially isolated as a neurite
outgrowth-promoting protein that was eluted from heparin-Sepharose by
1 M NaCl(14) . The salt concentration needed to
reverse the HB-GAM-heparin interaction thus approaches that required to
impede high affinity binding to heparin of other proteins such as
antithrombin and fibroblast growth factors(38, 39) .
The strong binding of HB-GAM to heparin suggests that heparin-type
carbohydrates might be involved in the biological function(s) of
HB-GAM. This assumption was strengthened by the recent findings that N-syndecan serves as a receptor or a co-receptor for HB-GAM (26) and that addition of exogenous heparin, as well as
heparitinase treatment of neurons, both inhibit HB-GAM-induced neurite
outgrowth(24) .
The purpose of the present study was to define the glycosaminoglycan structure(s) responsible for HB-GAM binding and implicated in the functional properties of the protein. To this end we employed a neurite outgrowth bioassay as well as a protein binding assay, comparing the effects on these assays of various exogenous carbohydrates. In the bioassay we used rat forebrain neurons that were freshly prepared for each assay, thus attempting to retain the normal cell surface carbohydrate structures that tend to be rapidly changed in vitro.
Heparin at very low concentrations (10-30 ng/ml) was found to clearly inhibit neurite outgrowth induced by substrate-bound HB-GAM. By contrast, heparin did not affect neurite outgrowth on laminin-coated substrates at concentrations clearly exceeding those active with HB-GAM. Interestingly, preparations of detergent-free N-syndecan also inhibited HB-GAM-induced but not laminin-induced neurite outgrowth. This finding is in agreement with observations that brain N-syndecan does not bind to commonly occurring matrix proteins, such as laminin or fibronectin(40) .
Although the inhibitory effect of N-syndecan was clearly attributed to the heparan sulfate side chains, the potency was significantly lower than that displayed by heparin, expressed on a carbohydrate basis (Fig. 2B). Heparan sulfate is generally more heterogeneous in structure than is heparin, and it thus seems reasonable to assume that a particular saccharide sequence implicated in the biological activity would be less abundant in heparan sulfate. Release of the polysaccharide chains from the core protein resulted in a further modest decrease in inhibitory capacity (Fig. 2B), suggesting that the biological activity is facilitated by the intact proteoglycan assembly.
Compared with N-syndecan and N-syndecan-derived
glycosaminoglycans, other glycosaminoglycan preparations were clearly
less active (Fig. 2B). A preparation of heparan sulfate
from human aorta thus was required in 10-fold higher concentration
than the N-syndecan polysaccharide to similarly inhibit
HB-GAM-induced neurite outgrowth; other polysaccharides tested,
including lung and kidney heparan sulfates, chondroitin sulfate, and
dermatan sulfate, showed even lower activity.
The results of
competitive binding of unlabeled polysaccharides, along with
[H]heparin, to HB-GAM in solution essentially
reflected those obtained in the neurite outgrowth assay. Native heparin
thus showed the most efficient binding, followed by aorta heparan
sulfate, which was required at 10
-10
-fold
higher concentration to achieve similar displacement of the labeled
heparin. Again, other polysaccharides, including heparan sulfate
preparations, were essentially inactive. Interestingly, the aorta
heparan sulfate is relatively low sulfated, with an overall sulfate
content of
0.6 sulfate groups/disaccharide unit(13) ,
suggesting the occurrence of a specific HB-GAM-binding region.
Unfortunately, the amounts of N-syndecan-derived
polysaccharide were insufficient for this assay.
Taken together, the
results suggest that HB-GAM binds to ``heparin-like'' (i.e. highly sulfated) regions of heparan sulfate chains and
that this interaction is essential to the neurite outgrowth-promoting
effect of the protein. This assumption was supported by experiments
involving selective chemical desulfation of heparin. In particular,
elimination of IdoA 2-O-sulfate groups yielded a product with
dramatically impaired ability to bind HB-GAM and to abolish
HB-GAM-induced neurite outgrowth. N-Sulfate groups were
similarly implicated, although the effects of N-desulfation on
neurite outgrowth were somewhat less marked (Fig. 2A).
The results of GlcN 6-O-desulfation are less straightforward,
because this modification was accompanied by a loss of 30% of the
2-O-sulfate groups(13) . However, it seems justified
to also implicate 6-O-sulfate groups in HB-GAM binding,
although they appear to be less essential than the 2-O-sulfate
substituents. All three sulfate residues of the major disaccharide unit
of heparin, IdoA(2-O-SO
)-GlcNSO
that
is frequently 6-O-sulfated in the glucosamine residue, thus
contribute to the interaction with HB-GAM. The binding epitope
apparently differs from those involved in interactions with basic
fibroblast growth factor, which requires 2-O- but no
6-O-sulfate groups(11, 13) , and with
hepatocyte growth factor, which seems to depend more on 6-O-
than on 2-O-sulfate groups(41) . The occurrence of a
distinct binding site for HB-GAM is emphasized by the demonstration of
a minimal molecular size, corresponding to a decasaccharide, for a
functionally active heparin oligosaccharide (Fig. 4).
The
finding that HB-GAM binds to heparin-type saccharide sequences raises
the question as to the occurrence of such structures in N-syndecan. In particular, it was anticipated that analysis of
the heparan sulfate constituents of N-syndecan, isolated from
postnatal rat brain, would elucidate the structural basis for the more
efficient interaction of this polysaccharide with HB-GAM, as compared
with other heparan sulfates. Indeed, such analysis revealed
characteristics of relevance to HB-GAM binding. The degree of N-sulfation, corresponding to 60% of the total
glucosamine units, is intermediary to the values reported for typical
heparan sulfates (40-50%) and for heparin (>80%)(42) .
A similarly high N-sulfate content was recently described for
a heparan sulfate from rat liver(43) . However, a more
conspicuous feature was noted in the high proportion of
2-O-sulfated IdoA units within the N-sulfated block
regions, which amounted to 82% of the total disaccharide units. This
value should be compared with the corresponding narrow range
54-58%, which was recently found in a survey of five heparan
sulfate preparations from different bovine organs. (
)Apparently, the heparan sulfate chains of brain N-syndecan contain regions with unusually high contents of the
constituent, 2-O-sulfated IdoA units, which is primarily
implicated in HB-GAM binding. It seems likely that this property is
reflected by the relatively high activity of N-syndecan in the
neurite outgrowth bioassay. Further work is needed to define the
minimal binding structure and the distribution of critical N-,
2-O-, and 6-O-sulfate groups within the 10-saccharide
sequence. It is recalled, as clearly demonstrated in connection with
the basic fibroblast growth factor-heparin/heparan sulfate interaction,
that only a limited number of the sulfate groups in a defined heparin
sequence are in fact essential to binding; others are redundant but do
not interfere with the interaction(8, 13) . A heparan
sulfate sequence might be specifically tailored during biosynthesis of
the corresponding proteoglycan to express only those substituents that
are functionally involved in a particular interaction.
In
conclusion, a specific transmembrane protein, N-syndecan, is
substituted with heparan sulfate chains that contain regions with a
higher density of IdoA(2-OSO) units than are commonly found
in heparan sulfates. This feature presumably explains the propensity of N-syndecan chains for interaction with HB-GAM, which are of
importance to the development of axonal processes. Because HB-GAM and N-syndecan are in addition spatiotemporally co-expressed in
developing brain(28) , it seems likely that HB-GAM indeed binds
to N-syndecan heparan sulfate in developing fiber tracts in
tissue. HB-GAM was also recently shown to co-localize with heparan
sulfate in the developing neuromuscular synapse(44) ; however,
the synaptic protein carrying the heparan sulfate chains has not yet
been identified.