Minimalistic supramolecular proteoglycan mimics by co-assembly of aromatic peptide and carbohydrate amphiphiles.

We report the co-assembly of aromatic carbohydrate and dipeptide amphiphiles under physiological conditions as a strategy to generate minimalistic proteoglycan mimics. The resulting nanofibers present a structural, fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) core and a functional carbohydrate (Fmoc-glucosamine-6-sulfate or -phosphate) shell. The size, degree of bundling and mechanical properties of the assembled structures depend on the chemical nature of the carbohydrate amphiphile used. In cell culture medium, these nanofibers can further organize into supramolecular hydrogels. We demonstrate that, similar to proteoglycans, the assembled gels prolong the stability of growth factors and preserve the viability of cultured cells. Our results demonstrate that this approach can be applied to the design of extracellular matrix (ECM) substitutes for future regenerative therapies.

Molecular weight of surface immobilized hyaluronic acid influences CD44-mediated binding of gastric cancer cells.

The physiological importance of the interactions between hyaluronic acid (HA) and its main membrane receptor, CD44, in pathological processes, e.g. cancer, is well recognized. However, these interactions are mainly studied in solution, whereas HA in the extracellular matrix (ECM) is partially immobilized via its interactions with other ECM components. We therefore, developed substrates in which HA is presented in an ECM-relevant manner. We immobilized HA with different molecular weights (Mw) in a Layer-by-Layer (LbL) fashion and studied the interactions of the substrates with CD44 and two human gastric cancer cell lines that overexpress this receptor, namely AGS and MKN45. We demonstrate that MKN45 cells are more sensitive to the LbL substrates as compared with AGS. This difference is due to different CD44 expression: while CD44 is detected mainly in the cytoplasm of AGS, MKN45 express CD44 predominantly at the cell membrane where it is involved in the recognition and binding of HA. The invasiveness of the studied cell lines was also evaluated as a function of HA Mw. Invasive profile characterized by low cell adhesion, high cell motility, high expression of cortactin, formation of invadopodia and cell clusters was observed for MKN45 cells when they are in contact with substrates presenting HA of high Mw.

Interactions between exogenous FGF-2 and sulfonic groups: in situ characterization and impact on the morphology of human adipose-derived stem cells.

FGF-2 is often used as a supplement to stem cells culture medium aiming at preserving their self-renewal capacity and plasticity through the passages. However, little is known on the influence of the underlying substrate in these interactions. In this study, we have used mixed self-assembled monolayers with different ratios of -SO3H and -OH tail groups to investigate the influence of substrate properties (e.g., charge) on the FGF-2 adsorption and activity. QCM-D data demonstrated that, in the presence of -OH groups, the quantity of the adsorbed FGF-2 is proportional to the percentage of surface -SO3H groups. The bioactivity of the adsorbed FGF-2 follows the same tendency as demonstrated by its interactions with anti-FGF-2. Surprisingly, the adlayer of FGF-2 formed on the surface containing only SO3H-tailed SAMs was similar to the surface with 25% of -SO3H groups, demonstrating that FGF-2 adsorption is not solely driven by electrostatic interactions. We related these results with changes in the morphology of adipose-derived stem cells (ASCs) cultured on the same surfaces.

Chlapsin, a chloroplastidial aspartic proteinase from the green algae Chlamydomonas reinhardtii.

Aspartic proteinases have been extensively characterized in land plants but up to now no evidences for their presence in green algae group have yet been reported in literature. Here we report on the identification of the first (and only) typical aspartic proteinase from Chlamydomonas reinhardtii. This enzyme, named chlapsin, was shown to maintain the primary structure organization of typical plant aspartic proteinases but comprising distinct features, such as similar catalytic motifs DTG/DTG resembling those from animal and microbial counterparts, and an unprecedentedly longer plant specific insert domain with an extra segment of 80 amino acids, rich in alanine residues. Our results also demonstrated that chlapsin accumulates in Chlamydomonas chloroplast bringing this new enzyme to a level of uniqueness among typical plant aspartic proteinases. Chlapsin was successfully expressed in Escherichia coli and it displayed the characteristic enzymatic properties of typical aspartic proteinases, like optimum activity at acidic pH and complete inhibition by pepstatin A. Another difference to plant aspartic proteinases emerged as chlapsin was produced in an active form without its putative prosegment domain. Moreover, recombinant chlapsin showed a restricted enzymatic specificity and a proteolytic activity influenced by the presence of redox agents and nucleotides, further differentiating it from typical plant aspartic proteinases and anticipating a more specialized/regulated function for this Chlamydomonas enzyme. Taken together, our results revealed a pattern of complexity for typical plant aspartic proteinases in what concerns sequence features, localization and biochemical properties, raising new questions on the evolution and function of this vast group of plant enzymes.

The heterologous systems in the study of cardosin B trafficking pathways.

Cardosins are abundant in cardoon pistils and were found to accumulate in different cell compartments: cardosin A was detected in the vacuoles of stigmatic papillae and cardosin B accumulates in the extracellular matrix of the transmitting tissue. Due to the fact that cardoon system imposes some limitations to the study of processing and trafficking events, heterologous species have been employed to study cardosins trafficking pathways. Cardosin B was successfully expressed both in Arabidopsis and Tobacco plants, where it accumulated mainly in the vacuole but it was also detected in the cell wall. The glycosylation pattern of cardosin B was replicated in these systems - high-mannose type glycans. In tobacco leaves, cardosin B is transported through the Golgi in a RAB-D2a-dependent route, and is delivered to the vacuole via the prevacuolar compartment in a RAB-F2b-dependent pathway. 

Dissecting cardosin B trafficking pathways in heterologous systems.

In cardoon pistils, while cardosin A is detected in the vacuoles of stigmatic papillae, cardosin B accumulates in the extracellular matrix of the transmitting tissue. Given cardosins' high homology and yet different cellular localisation, cardosins represent a potentially useful model to understand and study the structural and functional plasticity of plant secretory pathways. The vacuolar targeting of cardosin A was replicated in heterologous species so the targeting of cardosin B was examined in these systems. Inducible expression in transgenic Arabidopsis and transient expression in tobacco epidermal cells were used in parallel to study cardosin B intracellular trafficking and localisation. Cardosin B was successfully expressed in both systems where it accumulated mainly in the vacuole but it was also detected in the cell wall. The glycosylation pattern of cardosin B in these systems was in accordance with that observed in cardoon high-mannose-type glycans, suggesting that either the glycans are inaccessible to the Golgi processing enzymes due to cardosin B conformation or the protein leaves the Golgi in an early step before Golgi-modifying enzymes are able to modify the glycans. Concerning cardosin B trafficking pathway, it is transported through the Golgi in a RAB-D2a-dependent route, and is delivered to the vacuole via the prevacuolar compartment in a RAB-F2b-dependent pathway. Since cardosin B is secreted in cardoon pistils, its localisation in the vacuoles in cardoon ovary and in heterologous systems, suggests that the differential targeting of cardosins A and B in cardoon pistils results principally from differences in the cells in which these two proteins are expressed.

Cardosins in postembryonic development of cardoon: towards an elucidation of the biological function of plant aspartic proteinases.

Following on from previous work, the temporal and spatial accumulation of the aspartic proteinases (EC 3.4.23) cardosin A and cardosin B during postembryonic seed development of cardoon (Cynara cardunculus) was studied, mRNA and protein analyses of both cardosins suggested that the proteins accumulate during seed maturation, and that cardosin A is later synthesised de novo at the time of radicle emergence. Immunocytochemistry revealed that the precursor form of cardosin A accumulates in protein bodies and cell walls. This localisation in seeds is different from that previously described for cardoon flowers, suggesting a tissue-dependent targeting of the protein. It is known that procardosins are active and may have a role in proteolysis and processing of storage proteins. However, the presence of procardosin A in seeds could be related to the proposed role of the plant-specific insert in membrane lipid conversion during water uptake and solute leakage in actively growing tissues. This is in accordance with the recently proposed bifunctional role of aspartic proteinase precursor molecules that possess a membrane-destabilising domain in addition to a protease domain. Mature cardosin B, but not its mRNA, was detected in the first hours after seed imbibition and disappeared at the time of radicle emergence. This extracellular aspartic protease has already been implicated in cell wall loosening and remodelling, and its role in seed germination could be related to loosening tissue constraints for radicle protusion. The described pattern of cardosin A and B expression suggests a finely tuned developmental regulation and prompts an analysis of their possible roles in the physiology of postembryonic development.