HS2ST1‐dependent signaling pathways determine breast cancer cell viability, matrix interactions, and invasive behavior

Heparan sulfate proteoglycans (HSPGs) act as signaling co‐receptors by interaction of their sulfated glycosaminoglycan chains with numerous signaling molecules. In breast cancer, the function of heparan sulfate 2‐O‐sulfotransferase (HS2ST1), the enzyme mediating 2‐O‐sulfation of HS, is largely unknown. Hence, a comparative study on the functional consequences of HS2ST1 overexpression and siRNA knockdown was performed in the breast cancer cell lines MCF‐7 and MDA‐MB‐231. HS2ST1 overexpression inhibited Matrigel invasion, while its knockdown reversed the phenotype. Likewise, cell motility and adhesion to fibronectin and laminin were affected by altered HS2ST1 expression. Phosphokinase array screening revealed a general decrease in signaling via multiple pathways. Fluorescent ligand binding studies revealed altered binding of fibroblast growth factor 2 (FGF‐2) to HS2ST1‐expressing cells compared with control cells. HS2ST1‐overexpressing cells showed reduced MAPK signaling responses to FGF‐2, and altered expression of epidermal growth factor receptor (EGFR), E‐cadherin, Wnt‐7a, and Tcf4. The increased viability of HS2ST1‐depleted cells was reduced to control levels by pharmacological MAPK pathway inhibition. Moreover, MAPK inhibitors generated a phenocopy of the HS2ST1‐dependent delay in scratch wound repair. In conclusion, HS2ST1 modulation of breast cancer cell invasiveness is a compound effect of altered E‐cadherin and EGFR expression, leading to altered signaling via MAPK and additional pathways

Methodologies to generate, extract, purify and fractionate yeast ECM for analytical use in proteomics and glycomics

In a multicellular organism, the extracellular matrix (ECM) provides a cell-supporting scaffold and helps maintaining the biophysical integrity of tissues and organs. At the same time it plays crucial roles in cellular communication and signalling, with implications in spatial organisation, motility and differentiation. Similarly, the presence of an ECM-like extracellular polymeric substance is known to support and protect bacterial and fungal multicellular aggregates, such as biofilms or colonies. However, the roles and composition of this microbial ECM are still poorly understood.Authors would like to acknowledge Joana Tulha for assistance on yeasts overlay photographs, and to Rui Armada for C. albicans ECM SDS-PAGE experiment. The proteomic analysis was carried out at the proteomics facility UCM-PCM, a member of the ProteoRed network. The polysaccharide analysis was performed at the Laboratory of Glycoconjugates Biochemistry and Cellular Biology, UFRJ, Brazil. Fabio Faria-Oliveira was supported by a PhD scholarship SFRH/BD/45368/2008 from FCT (Fundacao para a Ciencia e a Tecnologia). This work was funded by Marie Curie Initial Training Network GLYCOPHARM (PITN-GA-2012-317297), and by FCT/MEC through Portuguese funds (PIDDAC) - PEst-OE/BIA/UI4050/2014. The authors would also like to acknowledge Hugh S. Johnson for critical reading of the manuscript regarding English usage

The specificity of interactions between proteins and sulfated polysaccharides

Sulfated polysaccharides are capable of binding with proteins at several levels of specificity. As highly acidic macromolecules, they can bind non-specifically to any basic patch on a protein surface at low ionic strength, and such interactions are not likely to be physiologically significant. On the other hand, several systems have been identified in which very specific substructures of sulfated polysaccharides confer high affinity for particular proteins; the best-known example of this is the pentasaccharide in heparin with high affinity for antithrombin, but other examples may be taken from the study of marine invertebrates: the importance of the fine structure of dermatan sulfate (DS) to its interaction with heparin cofactor II (HCII), and the involvement of sea urchin egg-jelly fucans in species specific fertilization. A third, intermediate, kind of specific interaction is described for the cell-surface glycosaminoglycan heparan sulfate (HS), in which patterns of sulfate substitution can show differential affinities for cytokines, growth factors, and morphogens at cell surfaces and in the intracellular matrix. This complex interplay of proteins and glycans is capable of influencing the diffusion of such proteins through tissue, as well as modulating cellular responses to them.<br>Os polissacarídeos sulfatados são capazes de se ligar às proteínas com diferentes níveis de especificidade. São macromoléculas altamente ácidas que podem se ligar de forma inespecífica a qualquer domínio básico da superfície de uma proteína em soluções com baixa força iônica, contudo tais interações não parecem ser fisiologicamente significativas. Por outro lado, foram identificados vários sistemas nos quais componentes estruturais muito específicos dos polissacarídeos sulfatados conferem alta afinidade para algumas proteínas. O exemplo mais conhecido é o pentassacarídeo da heparina com alta afinidade pela antitrombina. Outros exemplos podem ser observados no estudo de invertebrados marinhos, tais como a importância da estrutura fina do dermatam sulfato para sua interação com o cofator II da heparina e o envolvimento defucanas sulfatadas encontradas no gel que envolve osóvulos dos ouriços-do-mar na espécie especificidade da fertilização. Um terceiro exemplo de interação específica é aquele descrito para o glicosaminoglicano heparam sulfato encontrado na superfície celular. Neste caso, o padrão de sulfatação pode determinar diferentes afinidades do carboidrato por citoquinas, fatores de crescimento e outras proteínas encontradas na superfície celular e na matriz extracelular. Estas interações complexas entre proteínas e carboidratos são capazes de influenciar a difusão das proteínas através dos tecidos, assim como modelar a resposta celular a estas moléculas