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

Clinical and laboratorial study of 19 cases of mucopolysaccharidoses

The mucopolysaccharidoses (MPS) are a heterogeneous group of inborn errors of lysosomal glycosaminoglycan (GAG) metabolism. The importance of this group of disorders among the inborn errors of metabolism led us to report 19 cases. METHOD: We performed clinical, radiological, and biochemical evaluations of the suspected patients, which allowed us to establish a definite diagnosis in 19 cases. RESULTS: Not all patients showed increased GAG levels in urine; enzyme assays should be performed in all cases with strong clinical suspicion. The diagnosis was made on average at the age of 48 months, and the 19 MPS cases, after a full clinical, radiological, and biochemical study, were classified as follows: Hurler -- MPS I (1 case); Hunter -- MPS II (2 cases); Sanfilippo -- MPS III (2 cases); Morquio -- MPS IV (4 cases); Maroteaux-Lamy -- MPS VI (9 cases); and Sly -- MPS VII (1 case). DISCUSSION: The high relative frequency of Maroteaux-Lamy disease contrasts with most reports in the literature and could express a population variability.As mucopolissacaridoses (MPS) constituem um grupo de erros inatos do metabolismo lisossomal dos glicosaminoglicanos (GAG) bastante heterogêneo. A importância das MPS levou-nos a relatar as características de 19 casos. MÉTODO: Realizamos uma avaliação clínica, radiológica e bioquímica, incluindo estudos enzimáticos, que nos permitiram estabelecer o diagnóstico definitivo em 19 casos. RESULTADOS: Nem todos os pacientes apresentaram níveis elevados de GAG na urina, devendo os ensaios enzimáticos serem realizados em todos os pacientes com forte suspeita clínica. O diagnóstico foi estabelecido em média aos 48 meses de idade e os casos, após amplo estudo clínico, radiológico e bioquímico, foram classificados como: Hurler -- MPS I (1 caso); Hunter -- MPS II (2 casos); Sanfilippo -- MPS III (2 casos); Morquio -- MPS IV (4 casos); Maroteaux-Lamy -- MPS VI (9 casos); e Sly -- MPS VI (1 caso). DISCUSSÃO: A proporção relativamente alta de MPS VI (Maroteaux-Lamy) contrasta com a maioria dos dados da literatura e pode expressar uma variabilidade populacional

Participation of heparin binding proteins from the surface of Leishmania (Viannia) braziliensis promastigotes in the adhesion of parasites to Lutzomyia longipalpis cells (Lulo) in vitro

Background: Leishmania (V.) braziliensis is a causative agent of cutaneous leishmaniasis in Brazil. During the parasite life cycle, the promastigotes adhere to the gut of sandflies, to avoid being eliminated with the dejection. the Lulo cell line, derived from Lutzomyia longipalpis (Diptera: Psychodidae), is a suitable in vitro study model to understand the features of parasite adhesion. Here, we analyze the role of glycosaminoglycans (GAGs) from Lulo cells and proteins from the parasites in this event.Methods: Flagellar (F-f) and membrane (M-f) fractions from promastigotes were obtained by differential centrifugation and the purity of fractions confirmed by western blot assays, using specific antibodies for cellular compartments. Heparin-binding proteins (HBP) were isolated from both fractions using a HiTrap-Heparin column. in addition, binding of promastigotes to Lulo cells or to a heparin-coated surface was assessed by inhibition assays or surface plasmon resonance (SPR) analysis.Results: the success of promastigotes subcellular fractionation led to the obtainment of F-f and M-f proteins, both of which presented two main protein bands (65.0 and 55.0kDa) with affinity to heparin. the contribution of HBPs in the adherence of promastigotes to Lulo cells was assessed through competition assays, using HS or the purified HBPs fractions. All tested samples presented a measurable inhibition rate when compared to control adhesion rate (17 +/- 2.0% of culture cells with adhered parasites): 30% (for HS 20 mu g/ml) and 16% (for HS 10 mu g/ml); HBP M-f (35.2% for 10 mu g/ml and 25.4% for 20 mu g/ml) and HBP F-f (10.0% for 10 mu g/ml and 31.4% for 20 mu g/ml). Additionally, to verify the presence of sulfated GAGs in Lulo cells surface and intracellular compartment, metabolic labeling with radioactive sulfate was performed, indicating the presence of an HS and chondroitin sulfate in both cell sections. the SPR analysis performed further confirmed the presence of GAGs ligands on L. (V.) braziliensis promastigote surfaces.Conclusions: the data presented here point to evidences that HBPs present on the surface of L. (V.) braziliensis promastigotes participate in adhesion of these parasites to Lulo cells through HS participation.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Lab Biol Mol & Doencas Endem, BR-21040360 Rio de Janeiro, BrazilLab Ultraestrutura Celular, BR-21040360 Rio de Janeiro, BrazilFiocruz MS, IOC, Lab Bioquim & Fisiol Insetos, BR-21040360 Rio de Janeiro, BrazilFiocruz MS, IPEC, Lab Vigilancia Leishmanioses, BR-21040360 Rio de Janeiro, BrazilUniversidade Federal de São Paulo, UNIFESP, Dept Bioquim, São Paulo, BrazilUniv Rosario, Escuela Med, Bogota, DC, ColombiaUniversidade Federal de São Paulo, UNIFESP, Dept Bioquim, São Paulo, BrazilCNPq: 300731/2010-8CNPq: 509737/2010-2CAPES: EDITAL - 11/2009FAPERJ: E-26/103.060/2008FAPERJ: E-26/110.257/2010Web of Scienc

Heparan sulfates and heparins: similar compounds performing the same functions in vertebrates and invertebrates?

The distribution and structure of heparan sulfate and heparin are briefly reviewed. Heparan sulfate is a ubiquitous compound of animal cells whose structure has been maintained throughout evolution, showing an enormous variability regarding the relative amounts of its disaccharide units. Heparin, on the other hand, is present only in a few tissues and species of the animal kingdom and in the form of granules inside organelles in the cytoplasm of special cells. Thus, the distribution as well as the main structural features of the molecule, including its main disaccharide unit, have been maintained through evolution. These and other studies led to the proposal that heparan sulfate may be involved in the cell-cell recognition phenomena and control of cell growth, whereas heparin may be involved in defense mechanisms against bacteria and other foreign materials. All indications obtained thus far suggest that these molecules perform the same functions in vertebrates and invertebrates.Universidade Federal de São Paulo (UNIFESP)A02Universidade Federal do Rio Grande do NorteUniversidade de Mogi das CruzesUNIFESPSciEL

Mucopolissacaridoses: aspectos bioquímicos, tentativas terapêuticas e caracterização de uma nova síndrome

BV UNIFESP: Teses e dissertaçõe

Análise estrutural de heparam sulfato urinário no diagnóstico diferencial das mucopolissacaridoses

BV UNIFESP: Teses e dissertaçõe