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Structural comparison of fibroblast growth factor-specific heparan sulfates derived from a growing or differentiating neuroepithelial cell line
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
Structural comparison of fibroblast growth factor-specific heparan sulfates derived from a growing or differentiating neuroepithelial cell line
Heparan sulfate (HS) glycosaminoglycans are essential modulators of fibroblast growth factor (FGF) activity both in vivo and in vitro, and appear to act by cross-linking particular forms of FGF to appropriate FGF receptors. We have recently isolated and characterized two separate HS pools derived from immortalized embryonic day 10 mouse neuroepithelial 2.3D cells: one from cells in log growth phase, which greatly potentiates the activity of FGF-2, and the other from cells undergoing contact-inhibition and differentiation, which preferentially activates FGF-1. These two pools of HS have very similar functional activities to those species isolated from primary neuroepithelial cells at corresponding stages of active proliferation or differentiation. We present here a structural comparison between these cell line HS species to establish the nature of the changes that occur in the biosynthesis of HS. A combination of chemical and enzymatic cleavage, low pressure chromatography and strong anion-exchange HPLC were used to generate full chain models of each species. Overall, the HS pools synthesized in the dividing cell line pools possessed less complex sulfation than those derived from more differentiated, growth arrested cells.
Introduction
We have previously reported that a particular HSPG is present from embryonic day 10 (E10) through to embryonic day 12 (E12) of murine brain development, when the first morphological signs of the differentiation of precursor cells into a neuronal phenotype become apparent (Nurcombe et al., 1993). The most interesting feature of this proteoglycan was that although the core protein remained constant over the period E10-E12 (Joseph et al., 1996), the affinity of the proteoglycan for FGF-2 and its ability to activate FGF-2 switched to FGF-1. Parallel studies conducted on the 2.3D cells, an immortalized line originally generated from E10 neuroepithelial cells by insertion of the c-myc oncogene (Bernard et al., 1989), showed that a similar transition in proteoglycan affinities occurred, and that this switch in HSPG activating activity was coincident with a similar switch in FGF expression (Nurcombe et al.,1993). In both instances, primary neuroepithelial cell and cell line, it was clear that differences in HS sidechain structure must account for the change in FGF specificity.
Variation in HS species arises from the synthesis of nonrandom, highly sulfated clusters of sugar residues which are separated by sugars which are predominantly N-acetylated. Most modifications, such as ester-O-sulfations and epimerization of glucuronate to iduronate occur in the N-sulfated domains, or directly adjacent to them, so that in the mature chain there are regions of high sulfation separated by domains of low sulfation (Knecht et al., 1967; Winterbourne and Mora, 1981; Gallagher and Walker, 1985; Turnbull and Gallagher, 1991a,b, 1993; Gallagher et al., 1992). This pattern distinguishes HS from heparin, which is essentially highly sulfated along its entire length. Specific binding sites based on the same, simple disaccharide template, are created by superimposition of complex variations in structural modifications as seen in the binding sites for ATIII, FGF-2, and HGF (Saksela et al., 1988; Laurent and Fraser, 1992; Turnbull et al., 1992; Ishihara et al., 1994; Lyon et al., 1994; Zioncheck et al., 1995).
We present here a detailed structural comparison of two HS preparations derived from media conditioned over a murine neuroepithelia derived cell line, the 2.3D cell line (Bernard et al., 1991). One preparation of HS, herein designated GAGB, is derived from the 2.3D cell line in log growth phase that is able to potentiate FGF-2. Another preparation, herein designated GAGA, is from contact inhibited 2.3D cells which in addition, potentiates FGF-1 (Nurcombe et al., 1993). It has now been well established that cells are capable of changing both the GAG moiety attached to a specific core protein, and the sulfation patterns of HSs in culture (Winterbourne and Mora, 1981; Fedarko and Conrad, 1986; Pejler and David, 1987; Pejler et al., 1987; Kato et al., 1994), just as they do over the course of development, injury and disease (Winterbourne and Mora, 1981; David et al., 1992; Jeronimo et al., 1994; Challacombe and Elam, 1995). The main aim of this study was to isolate and purify different HS chains and characterize them as fully as currently possible to determine the conserved structural properties between cell line and primary cell derived-HS that may account for the selectivity for the two FGFs. The conjoint use of heparin lyases and nitrous acid digestion, low pressure chromatography, HPLC, and a unique tetrasaccharide analysis have allowed us to demonstrate that each has a unique structure which subserves their individual function.
Results
Determination of chain size
Samples of purified HS chains derived by Pronase treatment were chromatographed through a Sepharose CL-6B column both before and after mild treatment with alkaline borohydride (Table I). Both pools of HSPGs showed a partial resistance to proteolysis by Pronase characteristic of proteoglycans, which are densely substituted with polysaccharide chains. This data indicates that there are at least two HS chains per core protein and that their attachment sites are located close together. Before alkali treatment, GAGB eluted at a Kav 0.31. After treatment it eluted at a Kav of 0.48 (Figure 1A, solid line). Using published calibrations, these correspond to 56 kDa before treatment and 22 kDa after treatment. For GAGA, relative chain sizes before and after this treatment are 72 kDa and 32 kDa, respectively. Assuming an average molecular weight of 400 Da for a disaccharide, the chain sizes are approximately 55 and 80 disaccharides as determined by CL-6B chromatography. Similar chromatographic techniques were employed to determine the size of heparitinase-resistant fragments (Figure 1B). These fragments indicate the distance between highly sulfated domains in the intact chain. A full description of heparitinase, also known as heparinase III, cleavage sites are given at the beginning of the discussion.
Figure 1. Sepharose CL-6B column chromatography to separate HS chains and fragments.The size of full length HS (A) and heparinase-resistant fragments (B) can be calculated from these graphs. The results are summarized in Table I (left ordinate, solid line and GAGB; right ordinate, broken line and GAGA).
Table I
Condition
GAGB
GAGA
Pronase
56,000
72,000
Mild alkali treatment
22,000
32,000
Heparinase
9,500
10,000
Number of heparinase resistant domains
1
2
Structural characterization of the heparan sulfate pools
Chromatography on a Bio-Gel P-10 column of the GAGB and GAGA oligosaccharides after low pH HNO2 treatment gave the elution profiles shown in Figure 2A. Low pH scission releases the N-sulfate groups from heparan sulfate with subsequent cleavage of the adjacent hexosaminidic bond. These profiles show the typical distribution of N-sulfated disaccharides characteristic of heparan sulfate. From these profiles it is possible to calculate the percentage of linkages susceptible to this treatment and thus the percentage of N-sulfated glucosamine residues in the HS chains. GAGB and GAGA were 40% and 44% susceptible, respectively. Heparitinase treatment of the samples yielded elution profiles which were also characteristic of HS (Figure 2B). The majority of the tritium was in the disaccharide peak, unlike the HNO2 profile where the largest peak corresponds to tetrasaccharides. Quantitative analysis of the profiles demonstrated that 74.5% and 74.9% of the linkages in GAGB and GAGA, respectively, were susceptible to this treatment. Results from the separation of heparinase-treated oligosaccharides on a Bio-Gel P-10 column are depicted in Figure 2C. The inset (an expanded scale for fractions 69-100) highlights the differences in the composition of the HS reflected by the spacing of heparinase cleavage sites in the sulfated domains. Quantitative analysis revealed that the GAGB and GAGA chains are 18% and 19.5% susceptible, respectively. The major products of this digestion were not resolved on Bio-Gel P-10 columns (Vo peak) but estimation of their molecular size was possible on a CL-6B column (Figure 1B). The major peaks fractioned on the Sepharose CL-6B column have Kav of 0.65 and 0.63, which correspond to molecular weights of 9.5 and 10 kDa for GAGB and GAGA, respectively. The molecular mass of the heparinase-resistant domains in HS corresponds to the average distance between the centers of highly sulfated regions which contain heparinase-susceptible linkages; the heparinase-resistant oligosaccharides are on average 24 and 25 disaccharide units in length for GAGB and GAGA, respectively. Making the assumption that there are broad similarities in structure within the mixture of chains, heparinase therefore bisects GAGB chains but cuts the GAGA chains into three segments (Table I).
Figure 2. Gel filtration on Bio-Gel P-10 of oligosaccharides produced by various depolymerizing agents. HS from 2.3D cells was isolated as described in the text and was fractionated on a Bio-Gel P-10 column (1 × 200 cm) after the following treatments. (A) low pHHNO2. This profile was used to identify the purity of the HS sample and to calculate the percentage of susceptible linkages. A large fraction of this digest sample was run on a Bio-Gel P-2 column (1 × 120 cm) to isolate the tetrasaccharides and disaccharides for SAX-HPLC analysis. (B) depolymerization by heparitinase. The susceptibility of each species to heparitinase was calculated from this profile. The degree of polymerization (dp) of each peak is represented by the number above that peak and was subsequently used in the calculations. (C) depolymerization byheparinase. Inset: fractions 64-115 of the heparinase scission profile with an expanded scale in order to reveal the proportions of low-Mr products. The nonresolved Vo peak was pooled, freeze dried, and resolved on Sepharose CL-6B (Figure 1B, Table I) (left ordinate, solid line and GAGB; right ordinate, broken line and GAGA).
SAX-HPLC of nitrous acid-generated disaccharides
Oligosaccharides derived by HNO2-treatment of HS were reduced, separated on a Bio-Gel P-2 column, pooled and freeze dried. SAX-HPLC separation of disaccharides and tetrasaccharides resulted in the elution profiles in Figures 3 and 4. Nitrous acid cleaves hexosaminidic bonds in HS leading to the production of saccharides with an authentic nonreducing end uronic acid, but the loss of N2 from the reducing end glucosamine and its conversion to a 2,5-anhydromannose residue. For SAX-HPLC, this moiety was reduced to 2,5-anhydromannitol. Each disaccharide peak was identified according to its elution time in comparison with standard HNO2-derived disaccharides. These results are summarized in Table II where the area under each peak has been integrated and shown as a percentage of total disaccharides released by HNO2 digestion. There is a significant increase in 6-O-sulfates between GAGB and GAGA, mainly due to increases in GlcA-AManR(6S) and IdoA-AManR(6S). There is a decrease in 2-O-sulfate because of a marked drop in IdoA(2S)-AManR.
Figure 3. Strong anion exchange-high pressure liquid chromatography of HNO2-generated disaccharides.Disaccharides produced by low pH HNO2 were isolated by Bio-Gel P-2 low pressure chromatography, freeze dried and separated by SAX-HPLC. Disaccharides were eluted as described under the Materials and methods in the text. The elution times of the peaks were compared to whose of authentic standards and labeled accordingly. (A) Represents the elution profile from GAGB disaccharides, and (B) represents the elution profile from GAGA disaccharides. The relative amounts of each of the peaks has been calculated and summarized in Table II. SAX-HPLC of nitrous acid-generated tetrasaccharides
The tetrasaccharide products isolated after an HNO2 depolymerization were separated by SAX-HPLC and the profiles are shown in Figure 4. The positions of non-, mono- and disulfated peaks were established by comparison with dual [35S/3H]-labeled samples run under identical conditions. No trisulfated species were detectable. The percentage of each of the designated peaks as compared to the total population is summarized in Table III. There are some prominent differences in the tetrasaccharide peaks which signify differences in the alternating N-sulfated, N-acetylated regions of the HS chains. The nonsulfated portions (i.e., HNO2-resistant tetrasaccharides of type UA-GlcNAc-GlcA-AManR) show a slight decrease in the transition from GAGB to GAGA whereas there are slight increases in the monosulfated peaks (e.g., 1, 2 and 6). There is an overall increase in the disulfated products.
Table II
Table III
Figure 4. SAX-HPLC of the 2.3D-derived tetrasaccharides produced by HNO2. Tritiated tetrasaccharides produced by low pH HNO2 were isolated from Bio-Gel P-2 low pressure chromatography, freeze dried, and separated by high pressure liquid chromatography and compared to dual 35S/3H labeled standard results (supplied by Dr. G. Jayson, Christie Hospital, Manchester, UK). The numbers correspond to the order of the left column of Table III. (A) represents a complete profile of GAGB, and (B) represents an expanded axis highlighting the fractions which are low in abundance from GAGB. Similar experiments were performed on GAGA, as shown in Tables II-IV.
Disaccharide
GAGB (%)
GAGA (%)
IdoA/GlcA-AManR
13.4
24.2
IdoA(2S)-AManR
50.6
27.6
GlcA-AManR(6S)
5.9
9.7
IdoA-AManR(6S)
4.1
5.4
IdoA(2S)-AManR(6S)
23.8
23
GlcA(2S)-AManR
2.1
1.9
GlcA-AManR(3S)
nd
nd
GlcA- AManR(3,6S)
nd
nd
UNKNOWN
0.0
8.0
Sulfation number : peak number
GAGB (%)
GAGA (%)
Nonsulfated : 1
64.9
58.6
Monosulfated : 1
0.84
1.14
Monosulfated : 2
0.8
0.9
Monosulfated : 3
0.7
0.6
Monosulfated : 4
0.7
1.1
Monosulfated : 5
8.3
5.8
Monosulfated : 6
13.0
19.7
Monosulfated : 7
5.2
5.5
Monosulfated : 8
nd
nd
Monosulfated : 9
nd
nd
Disulfated : 1
2.4
1.5
Disulfated : 2
0.78
1.2
Disulfated : 3
1.5
2.2
Disulfated : 4
0.9
1.7
Trisulfated
nd
nd
Total
100
100
Analysis of the total disaccharide composition of the HS pools
In order to fully characterize the composition of the samples of HS, the chains were subjected to complete lyase depolymerization using heparitinases I, II, III, and IV. The products of this digestion were separated on a Bio-Gel P-2 column with over 95% of the radioactivity accounted for in the disaccharide peaks (data not shown), indicating sufficiently complete digestion to provide a representative compositional analysis. For analysis on SAX-HPLC the disaccharides were pooled and freeze dried. The differences in total composition of the HS chains in GAGB and GAGA were quite subtle. Analysis of elution profiles such as the representative profile in Figure 5 (data summarized in Table IV) revealed that, in general, the amounts of the different disaccharides was typical of HS, and both samples were broadly similar. However, in GAGA, the number of HexUA(2S)-GlcNAc units (peak 7) increased by 50%, HexUA-GlcNAc(6S) increased by 35%, and there was a slight increase in the trisulfated species HexUA(2S)-GlcNSO3(6S) (peak 6). The significance of these structural differences in relation to the possible functions of these molecules is unknown. Overall, in the 2.3D cell line, there was a relatively low level of O-sulfation at 0.27 and 0.31 sulfates per disaccharide for the GAGB and GAGA, respectively.
Figure 5. SAX-HPLC of the disaccharides produced by complete lyase depolymerization. Disaccharides produced by lyase depolymerization were separated on a Bio-Gel P-2 column, freeze dried, and separated by SAX-HPLC as described under Materials and methods in the text. Each of the peaks is labeled, and a summary of the proportions of each peak is in Table IV. (A) is a representative elution profile from GAGB, and (B) is a representative elution profile from GAGA.
Table IV
| Peak number | Disaccharide | GAGB (%) | GAGA (%) |
| 1 | [Delta]HexUA-GlcNAc | 55.4 | 50.7 |
| 3 | [Delta]HexUA-GlcNSO3 | 22.2 | 19.1 |
| 2 | [Delta]HexUA-GlcNAc(6S) | 3.2 | 4.7 |
| 7 | [Delta]HexUA(2S)-GlcNAc | 1.8 | 2.6 |
| 4 | [Delta]HexUA-GlcNSO3(6S) | 2.5 | 2.8 |
| 5 | [Delta]HexUA(2S)-GlcNSO3 | 9.0 | 9.1 |
| 8 | [Delta]HexUA(2S)-GlcNAc(6S) | 0 | 0 |
| 6 | [Delta]HexUA(2S)-GlcNSO3(6S) | 5.1 | 5.8 |
| 9 | Unknown | 0.7 | 5.1 |
Discussion
A mixture of structurally complex chain sequences are generated during the biosynthesis of HS and the data described above represent the average for the pool of HS chains isolated. The analysis makes clear that chain organization differs systematically between the developmental stages. Although direct sequencing of whole HS chains is not yet feasible, sufficient advances in analytical techniques in the past 10 years have made it possible to identify unique elements within chains derived from a single source. These include enzymatic cleavage with different heparin lyases and chemical cleavage with low pH HNO2. Heparinase (also known as heparitinase III) cleaves at linkages of the type GlcNSO3(±6) [alpha]1-4 IdoA(2S) (Linhardt et al., 1990; Desai et al., 1993) creating resistant sequences of structure IdoA(2S) [alpha]1-4 [GlcNR(±6S) [alpha]1-4 UA] [beta]1-4 GlcNSO3(±6S) (R = N-acetyl or N-sulfate moiety) (Turnbull and Gallagher, 1991; Jandik et al., 1994; Pye and Kumar, 1995). Heparitinase II (also known as heparinase II) has a wide spectrum of activity cleaving linkages of the type GlcNSO3(±6S) [alpha]1-4 IdoA leading primarily to the generation of disaccharides from HS. Heparitinase I (also known as heparinase III) cleaves mainly at GlcNR(±6S) [alpha]1-4 GlcA linkages (R = N-acetyl or N-sulfate moiety) (Linhardt et al., 1990; Pye and Kumar, 1995) creating resistant products with the structural motif GlcA [alpha]1-4 [GlcNSO3(±6S) [alpha]1-4 IdoA(+2S)]n [alpha]1-4 GlcNR. Linkages susceptible to low pH HNO2 are those containing N-sulfates of the type GlcNSO3(±6S) [alpha]1-4 UA(±2S) (Shively and Conrad, 1976a), whereas resistant linkages are UA [alpha]1-4 [GlcNAc(±6S) [alpha]1-4 GlcA]n-GlcNSO3). After a pool of HS chains is subjected to any of these treatments, gel filtration, SAX-HPLC, and PAGE can be used to elucidate subtle structural or compositional differences between HS samples.
Sepharose CL-6B chromatography was used to estimate the size of full length GAGB and GAGA HS chains and the heparinase resistant fragments derived from them (Figure 1, Table I). The data showed that GAGB was bisected by heparinase whereas GAGA was trisected. The disaccharides released by nitrous acid cleavage are summarized in Table II and the interesting pattern of released tetrasaccharides in Table III. The disaccharide composition established by complete depolymerization for the two GAG samples is summarized in Table IV.
Analysis of the disaccharides from HNO2-degraded HS from the 2.3D cells medium corresponds with the relative amounts of HS-derived disaccharides found in the conditioned medium of a hepatocyte cell line. There is a high proportion of IdoA(2S)-AManR, followed by IdoA(2S)- AManR(6S) and IdoA/GlcA- AManR (Bienkowski and Conrad, 1984). There is a significant decrease in IdoA(2S)-AManR and an increase in GlcA-AManR(6S) in the transition from GAGB to GAGA. In addition, E12-derived HS is significantly different from E10-derived HS in that there is approximately twice as much IdoA(2S)-AManR(6S) (unpublished observations). Both of these results may reflect the crucial role suggested for 6-O-sulfates in binding FGF-1 and potentiating its activity in vivo (Gallagher and Walker, 1985). The total sulfations in the 2.3D samples were 0.65 and 0.68 sulfates/disaccharide for GAGB and GAGA respectively (Table V), which is at the top end of the levels identified by Gallagher and Walker (1985). These percentages are consistently lower than the levels identified for the primary neuroepithelial-cell derived HS, which are similar to those identified in bovine kidney and human skin fibroblasts and [sim]50% higher than identified in human endothelial cells (Lyon et al., 1994a). This may reflect a loss of sulfation in tissue culture. It is also interesting that there is a lower level N-sulfation in primary cells, although it is difficult to determine whether the lower N-sulfation ratio is important as there is an overall increase in total sulfation in the primary cell-derived HS.
Table V
| Sulfation | GAGB (%) | GAGA (%) |
| Total sulfation/100 disaccharides | 65.5 | 67.6 |
| 6-O-Sulfate | 10.8 | 13.3 |
| 2-O-Sulfate | 15.9 | 17.5 |
| N-Sulfate | 38.8 | 36.8 |
| O-Sulfate | 26.7 | 30.8 |
| Ratios of sulfations | ||
| 2-O-Sulfate/6-O-sulfate | 1.47 | 1.31 |
| N-Sulfate/O-sulfate | 1.45 | 1.19 |
| N-Sulfate/2-O-sulfate | 2.44 | 2.10 |
| N-Sulfate/6-sulfate | 3.59 | 2.77 |
We are currently attempting to model these neuroepithelial HSs by integrating the data on the size of the whole chains, the size of heparinase-resistant fragments, the proportions of oligosaccharide fragments of different sizes generated from heparinase and HNO2 cleavage, the composition of the highly sulfated domains and the total disaccharide composition. Turnbull and Gallagher characterized skin fibroblast HS (Turnbull and Gallagher, 1990, 1991b) and proposed a model which posited the now widely accepted domain structure model of HS. They also concluded that heparinase-sensitive disaccharides are located in short domains consisting of GlcNSO3(±6S)-IdoA(±2S) repeats. These are separated by regions of polysaccharide that are heparinase resistant, enriched with N-acetylated disaccharides and low in both iduronic acid and sulfate moieties. Following this discovery, many other HS pools have been characterized which are similar in their overall organization (Lyon et al., 1994a,b; Sanderson et al., 1994; Hiscock et al., 1995; Pye and Kumar, 1995); this study also demonstrates that 2.3D-derived HS share this structural theme. The models reveal that overall chain organization differs dramatically between samples, as well as the sequences within individual sulfated subdomains. Similar to primary cell HS, the major difference apparent is the size and complexity of the sulfated domains. In comparing the tissue culture samples with the primary cell-derived HS, the 2.3D HS appear to be much less complicated, with reduced sulfation, implying fewer sulfated domains than their primary cell counterpart.
A number of significant structural differences were observed over the change from active growth to quiescence (i.e., GAGB to GAGA). Since HS must serve a variety of different functions in vivo, the structural differences dictating function appear to be subtle. It is highly likely that these differences are the key to the individual and specific function of HS in the extracellular environment. Methods for direct sequencing of HS saccharides will be required to substantiate this view. The fundamental hypothesis underlying the present study was that the changes in the FGF-activating activities of secreted HS from first dividing and then differentiating cells were due to systematic changes in the disaccharide composition of the HS chains. A combination of chemical and enzymatic cleavages has been used to elucidate the structural characteristics of two HS species isolated from different stages of growth of a neuroepithelial-derived cell line: one from proliferating cells that selectively activates FGF-2, and one from differentiating cells that selectively activates FGF-1. There are differences in the disaccharide compositions of the two HS species, compatible with the idea that distinct HS sequences are expressed in order to selectively activate different FGFs. Taken together with similar results from the structural analyses of other HS-binding molecules such as antithrombin III, hepatocyte growth factor and interferon-[chi], we predict that every molecule that requires HS for activation will have a distinct and specific disaccharide sequence dedicated to it, which will result in distinct HS chains. Currently such heparin-binding molecules encompass members of the transforming growth factor-[beta] family (TGF-[beta]), the platelet derived growth factor (PDGF) family, all of the FGFs, the pleiotropin-like family, and structural molecules such as the laminins, the fibronectins, the amyloid precursor protein family, and the collagens, among many others. We further suggest that such heparin-binding molecules will have configurations of basic amino acids which are spatially distinctive for the binding of such HS sequences.
One of the most interesting observations in this study is that the HS from undifferentiated, dividing cells is both smaller and simpler in structure than the HS from more differentiated, contact-inhibited cells. This change is paralleled in primary neuroepithelial cells, which secrete shorter and simpler chains when interacting with FGF-2, and longer and more complex chains when interacting with FGF-1 (Brickman et al., 1998). We know that GAGB contains a subdomain which promotes the interaction of FGF-2 with FGFR 1 IIIc (Brickman et al., 1995). Partial degradation of these HS chains demonstrated that a single, highly sulfated fragment, 18 saccharides in length, contains both a subdomain specific for the receptor and a subdomain specific for FGF-2 (Walz et al., 1997). Similar specific binding relationships presumably apply for the GAGA sequence, as its cellular effects are quite distinct. The exact composition of the sulfated domains which interact with the specific receptors remains to be determined. We conclude that the differences between chains that generate such significant differential specificity between ligands is quite subtle. This conclusion has been confirmed by gradient-PAGE of depolymerized HS (results not shown). Clearly only a combination of many sensitive techniques can elucidate the detail of the differences of these structures.
The spacing between the receptor-binding and the growth factor-binding regions of an HS molecule is critical to its recruitment into a ternary complex and thus its bioactivity (Kan et al., 1993; Wang et al., 1995; Pantoliano et al., 1994). We favor the idea that a single sulfated subdomain contains both a region specific for the FGF receptor and a region specific for the FGF. Our results imply that the use of heparin or heparin fragments in tissue culture experiments to augment the activity of agents such as FGF, heparin binding-growth associated molecule (HB-GAM) or midkine (MK) are likely to mask crucial and specific control mechanisms.
The other important question raised by the analysis is how a single cell type regulates the switching of the production of one class of HS chain for another, closely related chain. We have as yet no data which informs us whether the sudden production of FGF-1 in contact-inhibited, neurofilament-expressing cells induces a change in HS synthesis, or whether the new HS is made in readiness for the growth factor switch. Recent evidence from our laboratory has tended to confirm our original hypothesis that the sudden synthesis of FGF-1 is a key event in the subsequent emergence of the neuronal phenotype; if this is true, the change in HS specificity toward promotion of FGF-1 bioactivity becomes a seminal event in the appearance of neurons. This hypothesis can now be tested by specifically interfering with the enzymatic processes which lead to the subtle differences in HS structure at the appropriate phenotypic stage, and monitoring neuronal differentiation should be disrupted if HS structure is altered.
Materials and methods
Materials
Trypsin was supplied by Calbiochem and DNase from Boehringer Mannheim. d-[6-3H]Glucosamine (sp. 21Ci/mmol) was obtained from Amersham Life Science. Heparitinases I (EC 4.2.2.8), II (no EC number assigned) and III (EC 4.2.2.7) and chondroitin ABC lyase (EC 4.2.2.4) were obtained from Seikagaku Kogyo Co., Tokyo, Japan. Heparitinase IV was from Sigma (Sydney, Australia). Cell-culture media was supplied by Gibco. Bio-Gel P-2 and P-10 and the Trans-blot tank were from Bio-Rad Laboratories. CL-6B gel, DEAE-Sephacel, columns, peristaltic pumps, fraction collectors, and tubing were from Pharmacia Biotech Inc. (Sydney, Australia). ProPac PA1 analytical columns for the HPLC were from Dionex (Surrey, United Kingdom). Centriflo CF25 Membrane Cones were supplied by Amicon (Sydney, Australia). Scintillant (Ultima Gold) was from Packard (Melbourne, Australia) as were the scintillation vials. Biotrace RP nylon membrane was supplied by Gelman Sciences. En3Hance spray surface autoradiography enhancer was obtained from NEN Research Products, DuPont (UK) Ltd. Autoradiography cassettes were supplied by Genetic Research Ltd. X-Omat AR x-ray film, and development chemicals were supplied by Kodak.
Cell culture and radiolabeling
2.3D cells were grown in 250 ml tissue culture flasks in 5% FCS/DMEM in a 10% CO2/air-humidified incubator as previously described (Brickman et al., 1995). When isolating logarithmic growth HS, radiolabel was added 24 h post-passaging and the cells allowed to grow unhindered for 3 days; the HS in this preparation was designated GAGB. To isolate HS from contact-inhibited 2.3D cells, media on the cells was changed to 0.5% FCS/DMEM post-confluence and radiolabeled (20 µCi/ml) 24 h after the media was changed. The HS in this preparation was designated GAGA. Cells were maintained at confluence for 3 days and then the media collected and frozen at -20°C until required.
Preparation of intact heparan sulfate chains
The conditioned media was subjected to ion-exchange chromatography on a DEAE-Sephacel column (3 ml) equilibrated in 150 mM NaCl with phosphate-buffered saline (PBS), pH 7.2. The media was manually loaded onto the column and eluted under gravity. The column was washed and the bound material eluted with 1 M NaCl in 50 mM PBS, and 2 ml fractions were collected. Fractions containing the 3H-glucosamine labeled GAGs were pooled, concentrated, and desalted on Centriflo Cones (as per manufacturer's instructions), freeze dried, and resuspended in a minimal volume (100-500 µl) of neuraminidase buffer (25 mM Na-acetate pH 5.0). Samples were treated with neuraminidase (0.25 U/sample) for 4 h. Five volumes of 100 mM Tris-acetate (pH 8.0) were then added to the sample, which was then digested with chondroitin ABC lyase (0.25 U/sample) for 4 h at 37°C and further digested overnight with an equal amount of fresh enzyme. Finally, the core protein and the lyases were digested away with Pronase (1/5 total volume of 10 mg/ml Pronase in 500 mM Tris-acetate, 50 mM calcium acetate, pH 8.0) at 37°C for 24 h. The entire mixture was then diluted 1:10 with deionized water, passed through a 2 ml DEAE-Sephacel column, and eluted as previously described, and 1 ml fractions were collected. The sample was finally desalted on a 1 ×35 cm Bio-Gel P2 column, the Vo fraction collected and freeze dried.Samples were then eluted in [sim]200 µl of 500 mM NaOH/1 M NaBH4,incubated for 16 h at 4°C, and then neutralized to pH 7 with glacial acetic acid. A small amount of saturated ammonium bicarbonate was added and samples run on a CL-6B column (1 ×120 cm) for size determination of the released HS chains (Wasteson, 1971).
Nitrous acid treatment of HS chains
HS was chemically depolymerized by low pH-HNO2 (pH < 1.5) as described by Shively and Conrad (1976a,b) and modified by Bienkowski and Conrad (1985). A small portion of the mixture was run on a Bio-Gel P10 column (1 × 200 cm) to obtain a profile of the fragments released by this treatment, and the rest of the mixture was separated on a Bio-Gel P-2 column (1 × 120 cm) to isolate disaccharides and tetrasaccharides for strong anion exchange-high pressure liquid chromatography (SAX-HPLC).
Lyase depolymerization of HSPGs
Heparitinase (heparitinase I), heparitinase II and heparitinase IV were used at a concentration of 25 mU/ml in 100 mM-sodium acetate, 0.2 mM-calcium acetate, pH 7.0. Heparinase was used at a concentration of 50 mU/ml in the same buffer. Samples were digested in the presence of 100 µg of carrier HS. Each sample was separately incubated at 37°C for 16 h and then a second aliquot of enzyme added and incubated for a further 4 h. Sequential digests for recovery of disaccharides for SAX-HPLC analysis were performed at 37°C as follows: heparinase for 2 h, heparitinase for 1 h, heparitinase II for 18 h, and finally an aliquot of each lyase and heparitinase IV for 6 h. Sample volumes were decreased to less than 100 µl by desiccation and run on a Bio-Gel P-2 column to isolate disaccharides.
Gel chromatography
Gel chromatography of intact chains or scission products was performed on Sepharose CL-6B (1 × 120 cm) or Bio-Gel P-2 (1 × 120 cm) or Bio-Gel P-10 (1 × 200 cm) columns. The running buffer for the CL-6B and the Bio-Gel P-10 columns was 0.5M NH4HCO3 and for the Bio-Gel P-2 column was 0.25 M NH4HCO3. Samples were eluted at 4 ml/h with 1 ml fractions collected. Estimates of the size of fragments resolved on Sepharose CL-6B were based on published calibrations (Wasteson, 1971; Laurent et al., 1978).
SAX-HPLC analysis of disaccharides and tetrasaccharides
Disaccharide composition of the HS was analyzed on SAX-HPLC after either complete depolymerization with a mixture of lyases as described above or HNO2 treatment. Disaccharides and/or tetrasaccharides were recovered by Bio-Gel P-2 chromatography, and fractions corresponding to disaccharides or tetrasaccharides were pooled, freeze-dried, and stored at -20°C. The lyase-derived disaccharides were subjected to SAX-HPLC on a ProPac PA1 analytical column (4 × 250 mm) as follows. After equilibration in the mobile phase (double-distilled water adjusted to pH 3.5 with HCl) at 1 ml/min, samples were injected and disaccharides eluted with a linear gradient of NaCl from 0-1 M over 45 min in the same mobile phase. The eluant was collected in 0.5 ml fractions and the radioactivity measured by scintillation counting for comparison with lyase-derived disaccharides standards. Nitrous acid-derived tetrasaccharides were subjected to the same conditions (with smaller fractions collected) and compared to double labeled standard results which were supplied by Dr. Gordon Jayson (Christie Hospital, Manchester, UK). Alternatively, HNO2-derived disaccharides were separated using two ProPac PA1 columns in the same mobile phase. A shallow, noncontinuous gradient was used over the course of 97 min. From 0-51 min a gradient from 0-150 mM NaCl was employed and from 52-121 min a gradient of 150-500 mM NaCl was used. The eluant was collected as described above and compared to standards.
Acknowledgments
We thank the National Health and Medical Research Council of Australia and the UK Cancer Research Campaign for helping to support this work. Y.G.B. was supported by an Overseas Postgraduate Research Award and a Melbourne University Postgraduate Scholarship.
Abbreviations
FGF, fibroblast growth factor; GAG, glycosaminoglycan; HS, heparan sulfate; HSPGs, heparan sulfate proteoglycans; E10, embryonic day 10; GAGB, nonconfluent, 2.3D-derived HS; GAGA, confluent 2.3D-derived HS; SAX-HPLC, strong anion exchange-high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; GlcA, glucuronic acid; AManR, reduced anhydromannose; IdoA, iduronic acid; GlcNAc, N-acetylated glucosamine; GlcNSO3, N-sulfated glucosamine; HexUA, hexuronic acid; dp, degree of polymerization; GlcUA, glucosamine.
References
3To whom correspondence should be addressed at: Van Cleef/Roet Centre for Nervous Diseases, Department of Medicine (Dept. of Neuroscience), Monash University, Alfred Hospital, Commercial Road, Prahran, Victoria 3181, Australia
4Present address: School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, England, UK
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