Probing the interaction between heparan sulfate proteoglycan with biologically relevant molecules in mimetic models for cell membranes: A Langmuir film study

Investigating the role of proteoglycans associated to cell membranes is fundamental to comprehend biochemical process that occurs at the level of membrane surfaces. in this paper, we exploit syndecan-4, a heparan sulfate proteoglycan obtained from cell cultures, in lipid Langmuir monolayers at the air-water interface. the monolayer served as a model for half a membrane, and the molecular interactions involved could be evaluated with tensiometry and vibrational spectroscopy techniques. Polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) employed in a constant surface pressure regime showed that the main chemical groups for syndecan-4 were present at the air-water interface. Subsequent monolayer decompression and compression showed surface pressure-area isotherms with a large expansion for the lipid monolayers interacting with the cell culture reported to over-express syndecan-4, which was also an indication that the proteoglycan was inserted in the lipid monolayer. the introduction of biological molecules with affinity for syndecam-4, such as growth factors, which present a key role in biochemical process of cell signaling, changed the surface properties of the hybrid film, leading to a model, by which the growth factor binds to the sulfate groups present in the heparan sulfate chains. the polypeptide moiety of syndecan-4 responds to this interaction changing its conformation, which leads to lipid film relaxation and further monolayer condensation. (C) 2012 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Inst Ciencias Ambientais Quim & Farmaceut, Diadema, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Inst Ciencias Ambientais Quim & Farmaceut, Diadema, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, São Paulo, SP, BrazilWeb of Scienc

Heparan sulfate proteoglycans: structure, protein interactions and cell signaling

Heparan sulfate proteoglycans are ubiquitously found at the cell surface and extracellular matrix in all the animal species. This review will focus on the structural characteristics of the heparan sulfate proteoglycans related to protein interactions leading to cell signaling. The heparan sulfate chains due to their vast structural diversity are able to bind and interact with a wide variety of proteins, such as growth factors, chemokines, morphogens, extracellular matrix components, enzymes, among others. There is a specificity directing the interactions of heparan sulfates and target proteins, regarding both the fine structure of the polysaccharide chain as well precise protein motifs. Heparan sulfates play a role in cellular signaling either as receptor or co-receptor for different ligands, and the activation of downstream pathways is related to phosphorylation of different cytosolic proteins either directly or involving cytoskeleton interactions leading to gene regulation. The role of the heparan sulfate proteoglycans in cellular signaling and endocytic uptake pathways is also discussed.Proteoglicanos de heparam sulfato são encontrados tanto superfície celular quanto na matriz extracelular em todas as espécies animais. Esta revisão tem enfoque nas características estruturais dos proteoglicanos de heparam sulfato e nas interações destes proteoglicanos com proteínas que levam à sinalização celular. As cadeias de heparam sulfato, devido a sua variedade estrutural, são capazes de se ligar e interagir com ampla gama de proteínas, como fatores de crescimento, quimiocinas, morfógenos, componentes da matriz extracelular, enzimas, entreoutros. Existe uma especificidade estrutural que direciona as interações dos heparam sulfatos e proteínas alvo. Esta especificidade está relacionada com a estrutura da cadeia do polissacarídeo e os motivos conservados da cadeia polipeptídica das proteínas envolvidas nesta interação. Os heparam sulfatos possuem papel na sinalização celular como receptores ou coreceptores para diferentes ligantes. Esta ligação dispara vias de sinalização celular levam à fosforilação de diversas proteínas citosólicas ou com ou sem interações diretas com o citoesqueleto, culminando na regulação gênica. O papel dos proteoglicanos de heparam sulfato na sinalização celular e vias de captação endocítica também são discutidas nesta revisão.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo (UNIFESP) Departamento de BioquímicaUniversidade Federal de São Paulo (UNIFESP) Departamento de OftalmologiaUNIFESP, Depto. de BioquímicaUNIFESP, Depto. de OftalmologiaSciEL

A novel approach for the characterisation of proteoglycans and biosynthetic enzymes in a snail model

Proteoglycans encompass a heterogeneous group of glycoconjugates where proteins are substituted with linear, highly negatively charged glycosaminoglycan chains. Sulphated glycosaminoglycans are ubiquitous to the animal kingdom of the Eukarya domain. Information on the distribution and characterisation of proteoglycans in invertebrate tissues is limited and restricted to a few species. By the use of multidimensional protein identification technology and immunohistochemistry, this study shows for the first time the presence and tissue localisation of different proteoglycans, such as perlecan, aggrecan, and heparan sulphate proteoglycan, amongst others, in organs of the gastropoda Achatina fulica. Through a proteomic analysis of Golgi proteins and immunohistochemistry of tissue sections, we detected the machinery involved in glycosaminoglycan biosynthesis, related to polymer formation (polymerases), as well as secondary modifications (sulphation and uronic acid epimerization). Therefore, this work not only identifies both the proteoglycan core proteins and glycosaminoglycan biosynthetic enzymes in invertebrates but also provides a novel method for the study of glycosaminoglycan and proteoglycan evolution. (C) 2011 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)NIHUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniv Texas El Paso, Dept Biol Sci, Border Biomed Res Ctr, El Paso, TX 79912 USAUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilNIH: 2G12RR008124-16A1NIH: 2G12RR008124-16A1S1Web of Scienc

Activation of the Low Molecular Weight Protein Tyrosine Phosphatase in Keratinocytes Exposed to Hyperosmotic Stress

Herein, we provide new contribution to the mechanisms involved in keratinocytes response to hyperosmotic shock showing, for the first time, the participation of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) activity in this event. We reported that sorbitol-induced osmotic stress mediates alterations in the phosphorylation of pivotal cytoskeletal proteins, particularly Src and cofilin. Furthermore, an increase in the expression of the phosphorylated form of LMWPTP, which was followed by an augment in its catalytic activity, was observed. of particular importance, these responses occurred in an intracellular milieu characterized by elevated levels of reduced glutathione (GSH) and increased expression of the antioxidant enzymes glutathione peroxidase and glutathione reductase. Altogether, our results suggest that hyperosmostic stress provides a favorable cellular environment to the activation of LMWPTP, which is associated with increased expression of antioxidant enzymes, high levels of GSH and inhibition of Src kinase. Finally, the real contribution of LMWPTP in the hyperosmotic stress response of keratinocytes was demonstrated through analysis of the effects of ACP1 gene knockdown in stressed and non-stressed cells. LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells, mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These results describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ Estadual Campinas, Dept Bioquim, Inst Biol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, SP, BrazilUniv Estadual Paulista, Dept Quim & Bioquim, IBB, São Paulo, BrazilUniv São Paulo, Dept Genet & Biol Evolut, São Paulo, SP, BrazilUniv Fed ABC, Ctr Ciencias Nat & Humanas, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, São Paulo, SP, BrazilFAPESP: 2006/07315-3CNPq: PQ-2Web of Scienc

Crosstalk between tumor cells and lymphocytes modulates heparanase expression

Abstract Background Heparanase (HPSE) is an endo-beta-glucuronidase that degrades heparan sulfate (HS) chains on proteoglycans. The oligosaccharides generated by HPSE promote angiogenesis, tumor growth and metastasis. Heparanase-2 (HPSE2), a close homolog of HPSE, does not exhibit catalytic activity. Previous studies have demonstrated that serum or plasma from breast cancer patients showed increased expression of both heparanases in circulating lymphocytes. The aim of this study was to better understand the mechanisms involved in the upregulation of heparanases in circulating lymphocytes. Methods Lymphocytes collected from healthy women were incubated in the presence of MCF-7 breast cancer cells (co-culture) to stimulate HPSE and HPSE2 overexpression. The protein level of heparanases was evaluated by immunocytochemistry, while mRNA expression was determined by quantitative RT-PCR. Results The medium obtained from co-culture of MCF-7 cells and circulating lymphocytes stimulated the expression of HPSE and HPSE2. Previous treatment of the co-culture medium with an anti-heparan sulfate proteoglycan antibody or heparitinase II inhibited the upregulation of heparanases in circulating lymphocytes. The addition of exogenous heparan sulfate (HS) enhanced the expression of both heparanases. Moreover, the co-cultured cells, as well as MCF-7 cells, secreted a higher number of exosomes expressing an increased level of HS compared to that of the exosomes secreted by circulating lymphocytes from women who were not affected by cancer. Conclusions The results revealed that HS is likely responsible for mediating the expression of heparanases in circulating lymphocytes. HS secreted by tumor cells might be carried by exosome particles, confirming the key role of tumor cells, as well as secreted HS, in upregulating the expression of heparanases, suggesting a possible mechanism of crosstalk between tumor cells and circulating lymphocytes