This work was supported in part by an investigator initiated grant from L’Oreal. AD, AFB, GA, JD and LB are employees of L’Oreal Recherche. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: AD JD HLJ LB RV. Performed the experiments: EVS GA RV. Analyzed the data: EVS HLJ RV. Contributed reagents/materials/analysis tools: AD AFB GA JD LB. Wrote the paper: EVS HLJ RV.
Proteoglycans (PGs) are critically involved in major cellular processes. Most PG activities are due to the large interactive properties of their glycosaminoglycan (GAG) polysaccharide chains, whose expression and fine structural features are tightly controlled by a complex and highly regulated biosynthesis machinery. Xylosides are known to bypass PG-associated GAG biosynthesis and prime the assembly of free polysaccharide chains. These are, therefore, attractive molecules to interfere with GAG expression and function. Recently, we have developed a new xyloside derivative, C-Xyloside, that shares classical GAG-inducing xyloside activities while exhibiting improved metabolic stability. We have previously shown that C-Xyloside had beneficial effects on skin homoeostasis/regeneration using a number of models, but its precise effects on GAG expression and fine structure remained to be addressed. In this study, we have therefore investigated this in details, using a reconstructed dermal tissue as model. Our results first confirmed that C-Xyloside strongly enhanced synthesis of GAG chains, but also induced significant changes in their structure. C-Xyloside primed GAGs were exclusively chondroitin/dermatan sulfate (CS/DS) that featured reduced chain size, increased O-sulfation, and changes in iduronate content and distribution. Surprisingly, C-Xyloside also affected PG-borne GAGs, the main difference being observed in CS/DS 4-O/6-O-sulfation ratio. Such changes were found to affect the biological properties of CS/DS, as revealed by the significant reduction in binding to Hepatocyte Growth Factor observed upon C-Xyloside treatment. Overall, this study provides new insights into the effect of C-Xyloside on GAG structure and activities, which opens up perspectives and applications of such compound in skin repair/regeneration. It also provides a new illustration about the use of xylosides as tools for modifying GAG fine structure/function relationships.
Proteoglycans (PGs) are glycoproteins abundantly found in the extracellular matrix (ECM) and at the cell surface, that are critically involved in a large array of cell functions, including cell adhesion, migration, proliferation and differentiation, embryo development, inflammation, pathogen infection or tumour growth and metastasis
The four major types of GAGs borne by PGs are heparan sulfate (HS), chondroitin/dermatan sulfate (CS/DS) and keratan sulfate (KS). They are long, linear polysaccharides characterized by a repeating core disaccharide structure comprising an
GAG structural features, notably extent and patterns of sulfation, largely govern their protein binding and modulating properties. Therefore, the appropriate cell response to signalling proteins is ensured by rapid PG turnover and a highly regulated biosynthesis machinery controlling GAG structure. During PG biogenesis, the synthesis of GAG chains is initiated by the transfer of xylose to specific serine side chains within the protein
GAG biosynthesis can be initiated in the absence of the core protein, using β-D-xylopyranosides
With regards to their multiple activities, the development of strategies designed to selectively tamper with GAG expression is clearly an attractive prospect to decipher the contribution of specific GAGs in biological functions, or for therapeutic applications. The selective inhibition/knock-down/silencing of GAG biosynthesis enzymes has provided valuable insights into the role of both HS and CS/DS during development, tissue repair or tumour progression
Recently, a C-xylopyranoside derivative (C-beta-D-xylopyranoside-2-hydroxy-propane, referred thereafter as C-Xyloside) has been developed to mimic the activity of β-xylosides (that are O-glycosides). This compound showed very similar ability to induce GAG expression as a conventional b-xyloside in cultured dermal fibroblasts
To analyse the effects of C-Xyloside on GAG biosynthesis, we isolated and purified metabolically labelled GAG chains from RD cultured with [3H]glucosamine. Polysaccharides were either recovered from the culture medium (secreted GAGs), or after extraction from RDs with collagenase/Triton/urea (cell-surface and matrix-associated GAGs), yielding four different samples: MedX−/TissueX- and MedX+/TissueX+ corresponding to the culture medium/cell-ECM associated GAGs from untreated or C-Xyloside treated RDs, respectively. These samples were first purified by weak anion-exchange chromatography (DEAE sephacel) followed by analysis of the fractions by scintillation counting.
Elution profiles obtained for both cell-associated PG samples TissueX- and TissueX+ were fairly similar, featuring two very close peaks, eluted at ∼560 and ∼580 mM, respectively (
3H-labeled medium PGs and tissue-associated PGs treated or not with C-Xyloside (7,5 mM, 48 h) were purified using DEAE ion exchange chromatography.
PG-containing fractions were pooled, desalted, then GAG chains were separated from the protein cores by β-elimination and purified again by anion-exchange chromatography. To determine whether C-Xyloside influenced the nature of synthesized GAG chains, we treated the samples with either chondroitinase ABC or a cocktail of heparinases I, II and III and analysed the digestion profiles by gel filtration on a Superdex 75 column. The digestion profile of secreted GAG chains are shown, as an example, in
Purified GAGs chains were digested with chondroitinase ABC (C′ase ABC) or heparinase II/III and analysed on a Superdex 75 column.
The size distributions of purified GAG chains were analysed by size-exclusion chromatography on a Sepharose CL-6B column, using the previously published calibration chart
Molecular size of the HS and CS/DS chains were assessed by gel filtration chromatography on a CL6B column. HS or CS/DS chains were loaded individually on the CL6B column and the fractions collected analyzed by scintillation counting.
Compositional data were obtained by analysing the disaccharide content of purified GAGs. The CS chains were exhaustively digested to disaccharides using the chondroitinase ABC, the completion of the depolymerisation being verified by gel filtration analysis of the degradation products. Disaccharides were then resolved by SAX-HPLC and identified by comparing the peak elution positions with those of commercial disaccharide standards (
Secreted or tissue-associated CS chains from control RDs (A,C) and from C-Xyloside treated RDs (B,D) were exhaustively digested with chondroitinase ABC and the digestion products were analysed by SAX-HPLC, using a 45 min linear gradient of 0–0.75 M NaCl. Elution positions of authentic CD/DS disaccharides standards are indicated by arrows.1, ΔDi-0S; 2, ΔDi-4S; 3, ΔDi-6S; 4, ΔDi-2,4S and ΔDi-4,6S; 5, ΔDi-2,6S.
Tissue-associated HS chains from control RDs (A) and from C-Xyloside treated RDs (B) were exhaustively digested with a combination of heparinases I, II and III and the digestion products were analysed by SAX-HPLC, using a 45 min linear gradient of 0–1 M NaCl. Elution positions of authentic CD/DS disaccharide standards are indicated as follows.
Standard peak N° | Disaccharide structure | Total disaccharides | |||
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MedX− | MedX+ | TissueX− | TissueX+ | ||
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ΔDi-0S | 50.3% | 6.7% | 8.3% | 10.5% |
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ΔDi-4S | 32.6% | 53.7% | 80.2% | 62.0% |
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ΔDi-6S | 15.1% | 35.8% | 8.8% | 24.8% |
|
ΔDi-2.4S |
1.1% | 1.9% | 2.2% | 1.5% |
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ΔDi-2.6S | 0.9% | 2.0% | 0.5% | 0.9% |
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33.7% | 55.6% | 82.4% | 63.5% | |
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16.0% | 37.8% | 9.3% | 25.7% | |
Sulfate/dp2 | 0.5 | 1.0 | 0.9 | 0.9 |
As disaccharides ΔDi-2.4S and ΔDi-4.6S could not be discriminated, corresponding percentage has not been taken into account for the calculation.
Comparison of control and C-Xyloside treated samples revealed major compositional differences. First, CS chains secreted by the C-Xyloside treated RDs featured much higher amounts of mono-sulfated disaccharides (ΔDi-4s + ΔDi-6s = 88%) compared to CS from the MedX- control sample (ΔDi-4s + ΔDi-6s = 47%). Consequently, CS from MedX+ showed a much lower amount of nonsulfated disaccharide ΔDi-0s (7%,
An effect of C-Xyloside treatment was finally observed on tissue-associated HS disaccharide composition (
Standard peak N° | Disaccharide structure | Total disaccharides | |
TissueX− | TissueX+ | ||
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ΔUA-GlcNAc | 46.8% | 58.5% |
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ΔUA-GlcNS | 22.4% | 21.8% |
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ΔUA-GlcNAc.6S | 14.6% | 5.9% |
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ΔUA-GlcNS.6S | 3.0% | 2.0% |
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ΔUA.2S-GlcNS | 11.1% | 9.6% |
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ΔUA.2S-GlcNS.6S | 2.2% | 2.2% |
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36.4% | 33.4% | |
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13.3% | 11.8% | |
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19.7% | 10.1% | |
Sulfate/dp2 | 0.7 | 0.6 |
To analyse the proportion and distribution of iduronic acid (IdoA) and glucuronic acid (GlcA) containing disaccharides, purified CS/DS chains were digested with either chondroitinase ACI or B, and each digest was analysed by gel filtration (
Secreted and tissue-associated CS/DS from control RDs (---) and C-Xyloside treated RDs ( ) were degraded by exhaustive treatment with chondroitinase AC-I (
IdoA/GlcA ratios were deduced from the proportion of chondroitinase ACI or chondroitinase B susceptible linkages of our samples (see table in
Digestion of MedX- CS/DS with chondroitinase B yielded essentially disaccharides, except for a small amount of tetrasaccharides and some large resistant fragments eluting towards
Digestion of cell-associated CS (TissueX- and TissueX+) with chondroitinase ACI or B, resulted in the formation of oligosaccharides corresponding to di-, tetra-, hexa-, octa-, and decasaccharides, suggesting a mixed distribution of the GlcA and IdoA containing units along the polysaccharide chains, both in the control RDs and in the C-Xyloside treated RDs.
We then assessed the consequences of C-Xyloside induced structural changes on the ability of CS to bind to HGF. HGF binding properties of RD GAGs were analysed using a filter binding assay. To do so, the growth factor (1 µg) was incubated with 10 000 cpm of each GAG sample, then drawn through a nitrocellulose membrane. Free GAG chains were recovered in the wash-through (and buffer rinses), while HGF bound material was step-eluted from the membrane with increasing NaCl concentrations. Secreted CS/DS did not significantly bind to HGF, regardless of C-Xyloside treatment (
Secreted (
GAGs are polysaccharides that are critically involved in many biological processes. However, study of these molecules remains extremely difficult because of their inherent heterogeneity and structural complexity. This is clearly exemplified in skin, where GAGs are believed to play important, yet not fully defined roles. GAGs are indeed major components of dermal ECM and participate in tissue cohesiveness and hydration. Through their ability to bind to and modulate the activity of a number of growth factors, GAGs are also involved in cell adhesion and migration, as well as skin organogenesis and wound healing. The structure and integrity of GAGs is therefore essential for skin homeostasis and regeneration, leaving open the question of a potential role of GAGs in the different skin stem cell compartments (including epidermal and dermal stem cells) known to be involved in skin homeostasis. The consequences of skin ageing on GAGs remain poorly understood. However, increasing evidence suggest that age-related alteration of the dermal connective tissue may involve a remodeling of GAG expression and structure
In this context, xylosides could provide an important orthogonal method to selectively alter the expression and structure of the GAGs present at cell surfaces, although their effects on the polysaccharide fine structural features remains poorly documented. On this basis, we have recently developed a C-Xyloside, that presents the same GAG-priming activity as classical β-xylosides, but exhibits improved chemical stability and therefore shows greater potential for future
Our results first confirmed the previously observed induction of GAG synthesis by C-Xyloside
Features | Effects of C-Xyloside treatment | |
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|
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Total GAG amount | 1 → 15 | No change |
Ratio HS : CS/DS | 1∶5 → no more HS | 1∶4 → 1∶7 |
GAG chain size | CS/DS: 45 → 15 kDa | CS/DS : 45 → 30 kDa HS : 45 → 15 and 45 kDa |
CS/DS global charge (sulfate/dp2) | ∼0.5→ ∼1 | No change (∼0.9) |
Ratio CS/DS 4-O−/6-O-sulfation | ∼2∶1→ ∼2∶1 | ∼7∶1→ ∼2.5∶1 |
HS N−/O-sulfation | – | N–S : no change; O–S: −11% |
CS/DS GlcA : IdoA distribution | Segregated → more evenly distributed | No change |
HGF binding (CS/DS) | No change | −26% of binding |
Altogether, our data indicate that C-Xyloside has numerous and complex effects on GAGs. C-Xyloside does not simply induce an upregulation of CS/DS production, but also affects GAG structure, including subtle features such as altered sulfation profiles. Interestingly, these modifications differ, depending on whether the polysaccharide chains are primed by the xyloside or attached to a protein core. This suggests that C-Xyloside may have some regulatory function on GAG biosynthesis machinery. Effects of C-Xyloside on proteoglycans have been previously reported. Syndecan-1, Syndecan-4 and Perlecan depleted expression in an atrophic skin model was restored to normal level by treatment with C-Xyloside
As GAG structural and functional properties are closely intertwined, we foresaw that C-Xyloside induced structural modifications of the polysaccharide would have consequences on its activity. We therefore tested the ability of GAGs from untreated and C-Xyloside treated RDs to bind HGF, a growth factor that can interact with both HS and CS/DS. HGF is a multifunctional growth factor promoting motility and proliferation of many different cell types, mainly of mesenchymal origin
From a structural point of view, our data provide new information on GAG/HGF interaction. GAG structural requirements for binding to HGF remains unclear. Heparin and HS display the highest affinity for the growth factor, with a direct correlation between binding and charge content but no definite requirements regarding sulfation positions
In conclusion, this study supplies missing structural information that should help understanding the exact mechanisms underlying the beneficial effects of C-Xyloside on dermis/epidermis homeostasis and regeneration. In a more general perspective, this study delivers a detailed and unprecedented survey of the effect of a xyloside on GAG expression and fine structure and provides new insights into the activity mechanisms of xylosides. Much work would be needed to determine the whole repertoire of C-Xyloside activities and applications. C-xyloside will most likely affect differently GAG binding properties for its various ligands. C-Xyloside effects on GAG structure may also vary from one cell type to another, and may be highly dependent on the amount of GAGs, or the proportion of CS/DS versus HS, naturally expressed by these cells. Finally, although C-xyloside overall effect on GAG expression was comparable to that of a conventional β-xyloside, we cannot exclude differences in the way these compounds alter GAG fine structure. Likewise, variations in xyloside structure (particularly of their aglycone moiety) may trigger different activities. However, this study shows for the first time that the GAG modifying activities of these molecules are not restricted to xyloside-primed GAG chains, and demonstrates that these modifications lead to a fine tuning of GAG structure. Finally, as exemplified with the analysis of HGF binding activity, we bring out the use of xylosides as an efficient strategy to induce GAG structural modifications and decipher fine structure/activity relationships involved in biological functions of these polysaccharides, as well as for potential corrective applications.
Skin fibroblasts were recovered from anonymous breast surgical waste. Samples were obtained for research purposes and donors gave their written informed consent as is required by French Bioethics Law of 2004 (article L1245-2). This law exempts this research from requiring formal ethical approval.
D-[1-3H] Glucosamine (sp. radioactivity 20–45 Ci/mmol) was obtained from Perkin Elmer. Scintillation mixture (Optiphase HiSafe 3) was obtained from PerkinElmer Life Sciences. C-β-D-xylopyranoside-2-hydroxy-propane (C-Xyloside, Pro-xylane™) was obtained from L’Oréal Research Laboratories (Clichy, France), as previously described
4,5-unsaturated CS/DS disaccharide standards (ΔUA-GalNAc (ΔDi-0S), ΔUA-GalNAc4S (ΔDi-4S), ΔUA-GalNAc6S (ΔDi-6S), ΔUA2S-GalNAc (ΔDi-2S), ΔUA2S-GalNAc4S (ΔDi-2,4S), ΔUA2S-GalNAc6S (ΔDi-2,6S), ΔUA-GalNAc4S6S (ΔDi-4,6S), ΔUA2SGalNAc4S6S (ΔDi-2,4,6S)) were from Iduron (Manchester, UK). All other chemicals were from Sigma or of equivalent commercial grade.
Normal human breast skin was obtained after written informed consent from healthy subjects following plastic mammary reduction and according to the principles expressed in the Declaration of Helsinki. Dermal fibroblasts were then isolated from skin samples as previously described
Following metabolic labelling, the medium was removed and RDs were rinsed twice with PBS. Medium and washes were pooled and stored at −20°C. RDs were incubated with collagenase (2 mg/ml in PBS, 5 mM CaCl2) at 37°C for 2 h and centrifuged for 5 minutes at 3000 rpm. Supernatants were recovered and cells and ECM were incubated with a Triton X-100 extracting solution (20 mM phosphate pH 6.5, 0.25 M NaCl, supplemented with 1% Triton X-100 (v/v), 10 mg/ml bovine serum albumin and Complete™ protease inhibitor cocktail) for 30 minutes at 4°C, under stirring. Extracts were centrifuged for 10 minutes at 15 000 rpm, supernatants were retrieved and pellets were incubated overnight at 4°C with urea-extract buffer (20 mM phosphate pH 6.5, 0.25 M NaCl, 6 M urea, 1% Triton X-100 (v/v), 10 mg/ml bovine serum albumin, Complete™ protease inhibitor cocktail). The resulting extracts were centrifuged for 10 minutes at 15 000 rpm, supernatants were recovered and pooled with the two previous supernatant fractions.
The medium or tissue samples were applied to an ion-exchange DEAE-Sephacel column (1×10 cm). The column was first washed with 20 mM Phosphate pH6.5, 0.3 M NaCl, to remove contaminating proteins and hyaluronic acid, then PGs were resolved on a linear 330 min gradient of 0.35–0.75 M NaCl in 20 mM Phosphate pH 6.5, at a flow rate of 0.250 ml/min. Fractions of 1 ml were collected and aliquots of 50 µl were removed for scintillation counting (Packard Tri-Carb 2100 TR β-counter). Fractions corresponding to detected radioactive peaks were pooled and freeze-dried. Samples were solubilised in distilled water and desalted on a PD-10 column (GE Healthcare), equilibrated in distilled water.
GAG chains were then released from the core protein by β-elimination, as previously described
Purified GAG chains were analysed by size-exclusion chromatography, using a Sepharose CL-6B column (1 cm diameter×120 cm length) equilibrated in PBS, at a flow rate of 4 ml/h. Fractions (1 ml) were collected and 3H content was monitored by scintillation counting. Fraction numbers were normalised to
Purified [3H]-CS/DS chains (∼20,000 cpm) were exhaustively digested to disaccharides by adding 500 mU of chondroitinase ABC in 50 mM Tris-HCl pH 7,5, 50 mM NaCl, 2 mM CaCl2, 0.01% (w/v) bovine serum albumin, at 37°C for 24 h. The enzyme was denatured by boiling the samples for 5 min and precipitated by centrifugation at 13 000 rpm. The completion of the reaction was confirmed by gel chromatography analysis, using two Superdex Peptide 10/300GL columns in series equilibrated in 1 mM KH2PO4, 3 mM Na2HPO4,2H2O, 350 mM NaCl pH 7,4, at a flow rate of 0.5 ml/min. Generated disaccharides were subsequently reduced by incubation in 0.1 M NaBH4, 10 mM NaOH for 2 h at room temperature. Remaining NaBH4 was hydrolysed by acidification with 2 M acetic acid and pH was neutralised with NaOH. Samples were then applied to a Propac PA1 strong-anion exchange column (4×250 mm; Dionex) equilibrated in water pH 3.5. After a wash with water pH 3.5, disaccharides were resolved over a linear gradient of 0–0.75 M NaCl, pH 3.5, at a flow rate of 1 ml/min. Fractions (0.5 ml) were collected and analysed by scintillation counting. Disaccharides peaks were identified by comparison with the elution positions of CD/DS disaccharides standards.
Purified [3H]-HS chains (∼50 000 cpm) were exhaustively digested to disaccharides after successive addition of 10 mU of heparinase I in 100 mM sodium acetate pH 7.1, 0.5 mM calcium acetate at 30°C for 24 h, then heparinase II and heparinase III (10 mU of each) at 37°C for 24 h. Digests were boiled and centrifuged to inactivate and remove enzymes. Like CS/DS chains, the completion of the reaction was confirmed by gel chromatography using the twinned Superdex Peptide columns. Samples were applied to the Propac PA1 equilibrated in water pH 3.5 and resolved on a linear NaCl gradient, 0–1 M NaCl, pH 3.5 over 45 min at a flow rate of 1 ml/min. Fractions (0.6 ml) were collected and analysed by scintillation counting. Disaccharides peaks were identified by comparing the elution positions of HS disaccharides standards.
[3H] purified GAG chains (50 000 cpm) were exhaustively digested with either chondroitinase ACI (0.25 units/ml in 33 mM Tris-HCl, 33 mM sodium acetate, 0.008% (w/v) bovine serum albumin, pH 7.3) or chondroitinase B (12.5 units/ml in 20 mM Tris-HCl, 50 mM NaCl, 4 mM CaCl2, 0.01% (w/v) bovine serum albumine pH 7.4) at 37°C for 24 h. Digests were then boiled and centrifuged. Supernatants were applied to two Superdex Peptide 10/300GL columns in series equilibrated with 0.35 M NaCl and run at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected and counted for radioactivity. The percentage of galactosaminyl bonds cleaved by each enzyme was calculated from the distribution of 3H radiolabeled peaks, relative to the total eluted 3H radiolabel, using the standard algorithm. GlcA content was therefore calculated from chondroitinase ACI digestion profile as follow: %GlcA = Σ (
Filter binding analysis was performed as previously described
The authors would like to thank Prof. M. Nitz for critical reading of the manuscript.