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 addition, a recent publication reports that senescence of dermal stem cells occurs during the aging process and affects their functions 
, highlighting the need to investigate the possible connection between GAGs, stem cells and aging in the near future. In that context, it would be interesting to focus on the role of growth factors and certain chemokines/cytokines (such as IL7, Fractalkine, G-CSF) that bind to GAGs and are known to be secreted by keratinocytes during the wound healing process 
. These factors appear like potential candidates to understand the mechanisms involved in the (mis)communication between the different stem cell compartments along human lifetime. GAGs may therefore constitute attractive targets in cosmetology, wound healing, or for the development of new therapeutic agents against skin disease.
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 in vivo
/therapeutic applications 
. During our previous studies, GAG-inducing activity of C-xyloside was confirmed and was shown to be very similar as that of a control β-xyloside in cultured human dermal fibroblasts 
. In addition, we showed using a number of models that C-Xyloside restored compromised proteoglycan expression in atrophic dermis 
, improved dermal-epidermal junction 
and promoted epidermal keratinocyte migration 
. However, understanding further the underlying mechanisms would require first the precise characterisation of GAG structural alterations induced by such compound. Here, we have investigated these aspects in the line of our previous study on monolayer-cultured dermal fibroblasts 
. However, to improve further the relevance of our data, we have used here a reconstituted human skin dermis model, to provide an extracellular environment as close as possible to the physiological one, while limiting the study to this single cell type.
Our results first confirmed the previously observed induction of GAG synthesis by C-Xyloside 
. We found that C-Xyloside treatment of RDs resulted in a 15 fold increase of GAGs found in the extracellular medium, these GAGs being exclusively CS/DS chains (). Such results are in agreement with a number of studies, which showed that most xylosides specifically primed assembly of free CS/DS chains, produced in high amounts and exported outside the cell 
. Likewise, we also found that C-xyloside primed CS/DS chains were of significantly shorter size, likely as a result of the GAG biosynthesis burden. However, and unlike some findings with other xylosides, C-Xyloside did not lead to a significant reduction in tritium incorporation within tissue-associated GAGs, suggesting no effect on the expression of GAGs borne by proteoglycans. Disaccharide analysis revealed effects of C-Xyloside on both xyloside-primed and proteoglycan-associated GAG chains. C-Xyloside primed CS chains found in the extracellular medium showed an increased level of overall sulfation, mainly due to higher amounts of monosulfated species to the detriment of the unsulfated one. However, the nature of sulfation remained unchanged. Interestingly, the polysaccharide organisation in CS or DS domains was also affected. Chains from untreated RDs were mainly of the CS type, with IdoA-containing disaccharides well segregated into distant domains within the polysaccharide. In contrast, C-Xyloside treatment resulted in a more regular distribution of these disaccharides along the chain (). Polysaccharides borne by proteoglycans present at the cell surface and in the surrounding ECM were also structurally affected by C-Xyloside, although in very different ways. C-Xyloside treatment did not affect the polysaccharide overall charge, and IdoA distribution. However, it modified the nature of sulfation on these chains, the main difference being a reduction of ~25% of the 4-O-sulfated disaccharides compensated by an equivalent increase of 6-O-sulfated disaccharides. Although unable to induce HS synthesis, C-Xyloside also affected the structure of PG-associated HS. Treatment with C-Xyloside resulted in a substantial reduction in O-sulfation, mostly 6-O-sulfation (). Interestingly, the most significant effect was a reduction of (ΔUA-GlcNAc,6S) disaccharide and the consequent increase in (ΔUA-GlcNAc) disaccharide. Such disaccharide is predominantly found in HS S-domain flanking regions. C-Xyloside may therefore induce directed alterations of 6-O-sulfation patterns within these areas, which could lead to changes in defined biological functions.
Summary of C-Xyloside effects on GAG expression and structure.
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 
. However, our study provides the first evidence of a regulatory effect on the activity of GAG biosynthesis sulfotransferases. Further work will be needed to determine whether this effect is due to a direct modulation of biosynthesis enzyme expression, or to the burdened biosynthesis machinery, leading to enzyme titration and assembly on PGs of distinct saccharide motifs.
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 
. In skin, HGF is mainly produced by dermal fibroblasts and has a paracrine action on epidermal keratinocytes, thus contributing to important dermis/epidermis crosstalk 
. Our results showed that the structural modifications of cell-associated CS/DS induced by C-Xyloside greatly reduced their ability to interact with HGF. Although the functional consequences of this loss of binding activity have not been examined yet, one attractive hypothesis is that C-Xyloside may modify these dermis/epidermis crosstalks by facilitating diffusion of HGF towards keratinocytes. However, much work will be needed to completely decipher the mechanisms involved, in a biological system as complex as the skin, and with other cytokines/growth factors likely to be affected by these alterations.
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 
. Accordingly, our results show that the C-Xyloside-induced structural changes in HS, which are restricted to the relatively low sulfated S-domain flanking regions, do not affect HGF binding. For DS, a comparative analysis of mammalian DS and highly sulfated DS isolated from marine tunicates Ascidia nigra
showed no specific O-sulfation requirements for binding to HGF but, unlike HS/heparin, no significant correlation between overall sulfation and binding affinity 
. This study also emphasized the critical role played by DS IdoA units, which could compensate for a relatively low level of overall sulfation by increasing the polysaccharide chain flexibility and facilitating protein-saccharide contacts 
. Our data is in partial agreement with these conclusions, since secreted GAG chains which feature lower IdoA content than their tissue-associated counterparts () failed to promote binding to HGF (), and the significant increase of overall sulfation induced by C-Xyloside did not suffice to restore binding. However, our results also show a requirement for 4-O-sulfation, as C-Xyloside 4-O-sulfation reduction on tissue-associated CS/DS resulted in impaired HGF binding, despite increased 6-O-sulfation (). This is in apparent contradiction with the results obtained on Ascidian DS, which binds to HGF but lacks 4-O-sulfates. One likely explanation would be that this DS species is particularly enriched in IdoA (nearly 100% of uronates, compared to ~50% in our dermal CS/DS preparations), which may compensate for the absence of 4-O-sulfates. Our data thus support further the existence of a critical interplay between DS sulfation and iduronate content 
. Moreover, they highlight the importance of 4-O-sulfates for the interaction, which may become a prerequisite for the binding of HGF to GAGs with low levels of IdoA.
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.