Insights into the N-Sulfation Mechanism: Molecular Dynamics Simulations of the N-Sulfotransferase Domain of Ndst1 and Mutants

Sulfation patterns along glycosaminoglycan (GAG) chains dictate their functional role. the N-deacetylase N-sulfotransferase family (NDST) catalyzes the initial downstream modification of heparan sulfate and heparin chains by removing acetyl groups from subsets of N-acetylglucosamine units and, subsequently, sulfating the residual free amino groups. These enzymes transfer the sulfuryl group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS), yielding sulfated sugar chains and 3'-phosphoadenosine-5'-phosphate (PAP). for the N-sulfotransferase domain of NDST1, Lys833 has been implicated to play a role in holding the substrate glycan moiety close to the PAPS cofactor. Additionally, Lys833 together with His716 interact with the sulfonate group, stabilizing the transition state. Such a role seems to be shared by Lys614 through donation of a proton to the bridging oxygen of the cofactor, thereby acting as a catalytic acid. However, the relevance of these boundary residues at the hydrophobic cleft is still unclear. Moreover, whether Lys833, His716 and Lys614 play a role in both glycan recognition and glycan sulfation remains elusive. in this study we evaluate the contribution of NDST mutants (Lys833, His716 and Lys614) to dynamical effects during sulfate transfer using comprehensive combined docking and essential dynamics. in addition, the binding location of the glycan moiety, PAPS and PAP within the active site of NDST1 throughout the sulfate transfer were determined by intermediate state analysis. Furthermore, NDST1 mutants unveiled Lys833 as vital for both the glycan binding and subsequent N-sulfotransferase activity of NDST1.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional do Desenvolvimento Cientifico e TecnologicoCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilUniv Fed Rio Grande do Sul, Ctr Biotecnol, Porto Alegre, RS, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilFAPESP: 2010/52426-3Web of Scienc

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

Characterization of urinary glycosaminoglycans from mucopolysaccharidosis IVA patients

A Mucopolissacaridose do tipo IVA (MPS IVA) é uma doença genética do tecido conjuntivo, que envolve mutações no gene N-acetil galactosamina-6-sulfato sulfatase. Esta enzima participa da degradação seqüencial do condroitim sulfato (CS) e queratam sulfato (KS), removendo sulfato da N-acetilgalactosamina-6-sulfato e galactose–6-sulfato do terminal não redutor destes GAGs. Quando esta enzima está ausente, os órgãos acumulam produtos de digestão incompleta destes GAGs, levando a um aumento na excreção de tais compostos na urina. Neste estudo nós analisamos KS e CS purificados de urina doada por pacientes portadores de MPS IVA e de indivíduos normais. GAGs totais foram extraídos da urina de indivíduos normais e pacientes utilizando uma resina de troca iônica . O KS foi isolado, incubando GAGs totais com extrato bruto de enzimas da F. heparinum, que digere todos os GAGs, com exceção do KS. Os produtos menores remanescentes foram separados do composto intacto por cromatografia de gel filtração sequenciais, Sephadex G-25 (PD10) e Sephadex G-10. KS foi detectado através de eletroforese em poliacrilamida e confirmado com queratanases específicas. Na tentativa de separar KS e CS, mantendo a integridade de ambos, os GAGs totais foram submetidos a cromatografia em Sephadex G-50 para fracionamento de acordo com a massa molecular. O perfil de eluição de CS foi detectado com reagente de carbazol que reage com ácido urônico, presente no CS e não no KS. Este foi detectado nas frações através de blotting com o anticorpo monoclonal específico para queratam sulfato. A maior parte do KS foi detectado na membrana de nitrocelulose, indicando um possível KS-ligado a peptídeo na urina. O mesmo procedimento de blotting foi realizado, e a membrana de nitrocelulose corado com reagente Ponceau, revelando a presença de proteína comigrando com o KS previamente detectado. Para confirmar o complexo KS-peptídeo GAGs totais foram submetidos a cromatografia hidrofóbica em coluna de Octyl-Sepharose, e eluídos com um gradiente linear de sulfato de amônio 2M e uma lavagem final com isopropanol50%. Uma população de KS foi retida na coluna, sendo eluído apenas com isopropanol 50%, devido à interação hidrofóbica do peptídeo com a resina. KS, CS e proteína foram detectados nessa população retida. Amostras de GAGs totais foram submetidos a uma reação química de β-eliminação, que remove açucares O-ligados a proteínas, e então aplicados a HPLC, em colunas de fase reversa, C4 Ultrasphere rotein and C18 Sephasil. Vários fragmentos protéicos ou peptídicos foram purificados nas colunas, que estão sendo sequenciados para análise estrutural..BV UNIFESP: Teses e dissertaçõe

Stromal reaction and low molecular weight proteoglycans leucine-rich

During cancer cell growth many tumors exhibit various grades of desmoplasia, unorganized production of fibrous or connective tissue, composed mainly of collagen fibers and myofibroblasts. The accumulation of extracellular matrix (ECM) surrounding tumors directly affects cancer cell proliferation, migration and spread, therefore the study of desmoplasia is of vital importance. Myofibroblasts synthesize an amalgam of products including collagens and other ECM proteins, such as proteoglycans and are activated during a desmoplastic reaction. Small leucine rich proteoglycans have been characterized surrounding breast and pancreatic tumors and have the ability to suppress cell proliferation. In this study we have analyzed desmoplasia co-cultivating colorectal cancer cells (Caco-2 and HCT116) and myofibroblasts using various co-culture systems. Our findings demonstrate that direct cell-cell contact between myofibroblasts and colorectal cancer cells evokes an upregulation of the expression of ECM components (collagen I, collagen III, collagen IV, collagen V, biglycan and fibromodulin) by myofibroblasts. The ECM accumulation produced when myofibroblasts are co-cultivated with colorectal cancer cells appears unorganized and in bundles. This ECM accumulation slowed the migration and invasion of the colorectal tumor cells in both monolayer and 3-D co-culture systems. The participation of the ECM components analyzed in this study in desmoplasia is also demonstrated in vivo in human colorectal carcinoma tissue, validating our in vitro system.A progressão do câncer e metástase invariavelmente envolve a interação com fibroblastos e com o ambiente circundante. Os fibroblastos estromais auxiliam a proliferação de células tumorais, invasão e matástase pela produção de fatores de crescimento e citocinas e pela modificação do ambiente circundante do tumor, pela modulação da matriz extracelular (MEC). Os proteoglicanos pequenos ricos em leucina (SRLPs) são componentes ativos da MEC, podendo estar alterados no estroma circundante a tumores. O efeito das células estromais em células de câncer é bem descrito; entretanto, pouco é sabido sobre o efeito das células de câncer na biologia e comportamento dos fibroblastos, incluindo a expressão de proteoglicanos, colágenos e MMPs. Nossos resultados revelam uma redução significativa na expressão dos componentes da MEC como colágenos I, II, III e IV, e os SLRPs decorim, biglicam, lumicam e fibromodulina em co-cultura de fibroblastos e duas linhagens de células de câncer de próstata; PC3 (derivada de metástase óssea) e DU145 (derivada de metástase cerebral). Interessantemente, foi observada uma diminuição da expressão global de TGFβ quando co-cultivados com as células de câncer de próstata, bem como despolimerização dos filamentos de actina e aumento da expressão de vimentina e integrina 51. A distribuição da vimentina se alterou de padrão alterado para fibrilar, característico de células invasivas. A expressão de MT1-MMP foi aumentada, sendo localizada em protusões de invadopodia estendidas pela MEC. Nossos dados demonstram como células de câncer de próstata alteram drasticamente o fenótipo dos fibroblastos, possuindo provavelmente papel importante na migração dessas células pelo estroma.TEDEBV UNIFESP: Teses e dissertaçõe

Transplantation of human umbilical mesenchymal stem cells cures the corneal defects of Mucopolysaccharidosis VII mice

Mucopolysaccharidosis (MPS) are a family of related disorders caused by a mutation in one of the lysosomal exoglycosidases which leads to the accumulation of glycosaminoglycans (GAGs). MPS VII, caused by a mutation in β-glucuronidase, manifests hepatomegaly, skeletal dysplasia, short stature, corneal clouding and developmental delay. Current treatment regimens for MPS are not effective for treating corneal clouding and impaired mental development. We hypothesized that human umbilical mesenchymal stem cells (UMSC) transplanted into the corneal stroma could participate in the catabolism of GAGs providing a means of cell therapy for MPS. For such treatment, human UMSC were intrastromally transplanted into corneas of MPS VII mice. UMSC transplantation restored the dendritic and hexagonal morphology of host keratocytes and endothelial cells, respectively, and in vivo confocal microscopy (HRTII) revealed reduced corneal haze. Immunohistochemistry using antibodies against HS and CS chains as well as LAMP2 revealed a decrease in GAG content and both lysosomal number and size in the treated corneas. Labeling UMSC intracellular compartments prior to transplantation revealed the distribution of UMSC vesicles throughout the corneal stroma and endothelium. An in vitro co-culture assay between skin fibroblasts isolated from MPSVII mice and UMSC demonstrated that neutral vesicles released by the UMSC are taken up by the fibroblasts and proceed to fuse with the acidic lysosomes. Therefore, transplanted UMSC participate both in extracellular GAG turnover and enable host keratocytes to catabolize accumulated GAG products, suggesting that UMSC could be a novel alternative for treating corneal defects associated with MPS and other congenital metabolic disorders

Heparan sulfate regulates hair follicle and sebaceous gland morphogenesis and homeostasis.

Hair follicle (HF) morphogenesis and cycling are a result of intricate autonomous epithelial-mesenchymal interactions. Once the first HF cycle is complete it repeatedly undergoes cyclic transformations. Heparan sulfate (HS) proteoglycans are found on the cell surface and in the extracellular matrix where they influence a variety of biological processes by interacting with physiologically important proteins, such as growth factors. Inhibition of heparanase (an HS endoglycosidase) in in vitro cultured HFs has been shown to induce a catagen-like process. Therefore, this study aimed to elucidate the precise role of HS in HF morphogenesis and cycling. An inducible tetratransgenic mouse model was generated to excise exostosin glycosyltransferase 1 (Ext1) in keratin 14-positive cells from P21. Interestingly, EXT1(StEpiΔ/StEpiΔ) mice presented solely anagen HFs. Moreover, waxing the fur to synchronize the HFs revealed accelerated hair regrowth in the EXT1(StEpiΔ/StEpiΔ) mice and hindered cycling into catagen. The ablation of HS in the interfollicular epidermal cells of mature skin led to the spontaneous formation of new HFs and an increase in Sonic Hedgehog expression resembling wild-type mice at P0, thereby indicating that the HS/Sonic Hedgehog signaling pathway regulates HF formation during embryogenesis and prevents HF formation in mature skin. Finally, the knock-out of HS also led to the morphogenesis and hyperplasia of sebaceous glands and sweat glands in mature mice, leading to exacerbated sebum production and accumulation on the skin surface. Therefore, our findings clearly show that an intricate control of HS levels is required for HF, sebaceous gland, and sweat gland morphogenesis and HF cycling

Heparan sulfate regulates hair follicle and sebaceous gland morphogenesis and homeostasis.

Hair follicle (HF) morphogenesis and cycling are a result of intricate autonomous epithelial-mesenchymal interactions. Once the first HF cycle is complete it repeatedly undergoes cyclic transformations. Heparan sulfate (HS) proteoglycans are found on the cell surface and in the extracellular matrix where they influence a variety of biological processes by interacting with physiologically important proteins, such as growth factors. Inhibition of heparanase (an HS endoglycosidase) in in vitro cultured HFs has been shown to induce a catagen-like process. Therefore, this study aimed to elucidate the precise role of HS in HF morphogenesis and cycling. An inducible tetratransgenic mouse model was generated to excise exostosin glycosyltransferase 1 (Ext1) in keratin 14-positive cells from P21. Interestingly, EXT1(StEpiΔ/StEpiΔ) mice presented solely anagen HFs. Moreover, waxing the fur to synchronize the HFs revealed accelerated hair regrowth in the EXT1(StEpiΔ/StEpiΔ) mice and hindered cycling into catagen. The ablation of HS in the interfollicular epidermal cells of mature skin led to the spontaneous formation of new HFs and an increase in Sonic Hedgehog expression resembling wild-type mice at P0, thereby indicating that the HS/Sonic Hedgehog signaling pathway regulates HF formation during embryogenesis and prevents HF formation in mature skin. Finally, the knock-out of HS also led to the morphogenesis and hyperplasia of sebaceous glands and sweat glands in mature mice, leading to exacerbated sebum production and accumulation on the skin surface. Therefore, our findings clearly show that an intricate control of HS levels is required for HF, sebaceous gland, and sweat gland morphogenesis and HF cycling