Navegando por Palavras-chave "Glycosylation"
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- ItemSomente MetadadadosAnalysis of the Ontogenetic Variation in the Venom Proteome/Peptidome of Bothrops jararaca Reveals Different Strategies to Deal with Prey(Amer Chemical Soc, 2010-05-01) Zelanis, Andre; Tashima, Alexandre Keiji [UNIFESP]; Rocha, Marisa Maria Teixeira; Furtado, Maria F.; Camargo, Antonio Carlos Martins de; Ho, Paulo Lee; Serrano, Solange Maria de Toledo; Inst Butantan; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)Previous studies have demonstrated that the pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Variation in the venom proteome is a well-documented phenomenon; however, variation in the venom peptidome is poorly understood. We report a comparative proteomic and peptidomic analysis of venoms from newborn and adult specimens of B. jararaca and correlate it with the evaluation of important venom features. We demonstrate that newborn and adult venoms have similar hemorrhagic activities, while the adult venom has a slightly higher lethal activity in mice; however, the newborn venom is extremely more potent to kill chicks. the coagulant activity of newborn venom upon human plasma is 10 times higher than that of adult venom. These differences were clearly reflected in their different profiles of SDS-PAGE, gelatin zimography, immunostaining using specific antibodies, glycosylation pattern, and concanavalin A-binding proteins. Furthermore, we report for the first time the analysis of the peptide fraction of newborn and adult venoms by MALDI-TOF mass spectrometry and LC-MS/MS, which revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles and were detected in the venoms showing their canonical sequences and also novel sequences corresponding to BPPs processed from their precursor protein at sites so far not described. As a result of these studies, we demonstrated that the ontogenetic shift in diet, from ectothermic prey in early life to endothermic prey in adulthood, and in animal size are associated with changes in the venom proteome in B. jararaca species.
- ItemAcesso aberto (Open Access)Associação entre Timp1, β1-integrinas e CD63 ao longo da gênese do melanoma(Universidade Federal de São Paulo (UNIFESP), 2010-11-24) Pinto, Mariana Toricelli [UNIFESP]; Jasiulionis, Miriam Galvonas [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Although malignant melanoma is the less frequently diagnosed skin cancer, it shows a poor prognosis due its chemoresistance and metastasis development. One of the adquired abilities of transformed cells is anoikis resistance and this property is closely related to metastasis formation. In our laboratory, we developed a model that allows us to study different steps of melanocyte malignant transformation. Melan-a melanocytes surviving after 1, 2, 3 and 4 deadhesion cycles showed modified morphology and independent PMA growth and have been named, 1C, 2C, 3C and 4C cells, respectively. Different melanoma cell lines were established after submitting 4C spheroids to limiting dilution. Previous results of our group showed increased expression of Timp1 along melanoma genesis and its correlation with anoikis resistance. However, the mechanism involved in this signaling is unknown. Published data demonstrated interaction between CD63, Timp1 and 1-integrins in human breast epithelial cells and its role in apoptosis. Furthermore, aberrant glycosylation in cell adhesion molecules such as integrins provides to cells the ability to survive under anchorage-independent conditions. The aim of this work was analyze the possible interaction among CD63, Timp1 and 1-integrins along melanocyte malignant transformation, possible aberrant N-glycosylation patterns of β1-integrins and their impact in anoikis resistance. Aberrant N-glycosylation patterns were observed in tumorigenic cells. We observed interaction between CD63 and Timp1 and between CD63 and 1-integrins in the melan-a-derived cells 4C, 4C11, and 4C11 +, and interaction between Timp1 and 1-integrins only in melanoma cell lines 4C11 - and 4C11 +. The presence of Timp1 in supernatant from 4C11+ conferred to melan-a cells anoikis resistance. The expression of 1-integrins in our study model is increased in aggressive melanoma lineage, 4C11+, as well as the expression of Mgat-V and aberrant N-glycosylation on cell surface. Moreover, the electrophoretic profile of 1-integrin suggests that melanoma metastatic 4C11+. Lineage present increased aberrant N-glycosylation in this molecule. Treatment of melanoma cells 4C11 + with the N-glycosylation inhibitor, swainsonine, resulted in reduced capacity of these cells to resist to anoikis. This seems to be the first study describing the interaction between Timp1, CD63 and 1-integrin in tumor cells and may contribute to a better understanding of how Timp1 regulates resistance to anoikis during the melanocyte malignant transformation.
- ItemSomente MetadadadosBothrops jararaca venom proteome rearrangement upon neonate to adult transition(Wiley-Blackwell, 2011-11-01) Zelanis, Andre; Tashima, Alexandre Keiji [UNIFESP]; Pinto, Antônio Frederico Michel; Paes Leme, Adriana Franco; Stuginski, Daniel Rodrigues; Furtado, Maria de Fatima Domingues; Sherman, Nicholas E.; Ho, Paulo Lee; Fox, Jay W.; Serrano, Solange Maria de Toledo; Inst Butantan; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP); Ctr Biotecnol UFRGS; LNBio; Univ VirginiaThe pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Similarly, the diet of this species changes from ectothermic prey in early life to endothermic prey in adulthood. in this study we used large and representative newborn and adult venom samples consisting of pools from 694 and 110 specimens, respectively, and demonstrate a significant ontogenetic shift in the venom proteome complexity of B. jararaca. 2-DE coupled to MS protein identification showed a clear rearrangement of the toxin arsenal both in terms of the total proteome, as of the glycoproteome. N-glycosylation seems to play a key role in venom protein variability between newborn and adult specimens. Upon the snake development, the subproteome of metalloproteinases undergoes a shift from a P-III-rich to a P-I-rich profile while the serine proteinase profile does not vary significantly. We also used isobaric tag labeling (iTRAQ) of venom tryptic peptides for the first time to examine the quantitative changes in the venom toxins of B. jararaca upon neonate to adult transition. the iTRAQ analysis showed changes in various toxin classes, especially the proteinases. Our study expands the in-depth understanding of venom complexity variation particularly with regard to toxin families that have been associated with envenomation pathogenesis.
- ItemSomente MetadadadosThe effect of structural motifs on the ectodomain shedding of human angiotensin-converting enzyme(Academic Press Inc Elsevier Science, 2016) Conrad, Nailah; Schwager, Sylva L. U.; Carmona, Adriana Karaoglanovic [UNIFESP]; Sturrock, Edward D.Somatic angiotensin converting enzyme (sACE) is comprised of two homologous domains (N and C domains), whereas the smaller germinal isoform (tACE) is identical to the C domain. Both isozymes share an identical stalk, transmembrane and cytoplasmic domain, and undergo ectodomain shedding by an as yet unknown protease. Here we present evidence for the role of regions distal and proximal to the cleavage site in human ACE shedding. First, because of intrinsic differences between the N and C domains, discrete secondary structures (alpha-helix 7 and 8) on the surface of tACE were replaced with their N domain counterparts. Surprisingly, neither alpha-helix 7 nor alpha-helix 8 proved to be an absolute requirement for shedding. In the proximal ectodomain of tACE residues H-610-L-614 were mutated to alanines and this resulted in a decrease in ACE shedding. An N-terminal extension of this mutation caused a reduction in cellular ACE activity. More importantly, it affected the processing of the protein to the membrane, resulting in expression of an underglycosylated form of ACE. When E-608-H-614 was mutated to the homologous region of the N domain, processing was normal and shedding only moderately decreased suggesting that this region is more crucial for the processing of ACE than it is for regulating shedding. Finally, to determine whether glycosylation of the asparagine proximal to the Pro1199-Leu polymorphism in sACE affected shedding, the equivalent (PL)-L-623 mutation in tACE was investigated. The (PL)-L-623 tACE mutant showed an increase in shedding and MALDI MS analysis of a tryptic digest indicated that (NWT)-W-620 was glycosylated. The absence of an N-linked glycan at N-620, resulted in an even greater increase in shedding. Thus, the conformational flexibility that the leucine confers to the stalk, is increased by the lack of glycosylation reducing access of the sheddase to the cleavage site. (C) 2016 Elsevier Inc. All rights reserved.
- ItemSomente MetadadadosThe mucin-like glycoprotein super-family of Trypanosoma cruzi: structure and biological roles(Elsevier B.V., 2001-05-01) Serrano, Alvaro Acosta; Almeida, Igor Correia de [UNIFESP]; Freitas Junior, Lucio Holanda Gondim de [UNIFESP]; Yoshida, Nobuko [UNIFESP]; Schenkman, Sergio [UNIFESP]; Johns Hopkins Univ; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)Trypanosoma cruzi expresses at its surface large amounts of mucin-like glycoproteins. the T. cruzi mucins (TcMUC), a group of highly glycosylated GPI-anchored proteins rich in Thr, Ser, and Pro residues, are expressed in high copy numbers in both insect and mammalian stages of the parasite. These molecules are encoded by a multigene family and contain a unique type of glycosylation consisting of several sialylated O-glycans linked to the protein backbone via Nw-acetylglucosamine residues. the TcMUC are important because of their role in host cell invasion and the ability to induce secretion of proinflammatory cytokines and nitric oxide in activated macrophages. the TcMUC are also significant in being the major substrate for the cell surface trans-sialidase. in this review, we summarize the recent knowledge on the molecular structure and function of this family of T. cruzi glycoproteins. (C) 2001 Elsevier Science B.V. All rights reserved.
- ItemSomente MetadadadosPadrão de glicosilação da glicoproteína do vírus da raiva produzida em células drosophila melanogaster S2: diferentes sistemas de expressão e condições de cultivo tese(Universidade Federal de São Paulo (UNIFESP), 2019-11-13) Silva, Livia Pilatti Mendes Da [UNIFESP]; Augusto, Elisabeth De Fatima Pires [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Rabies virus glycoprotein (RVGP) produced in animal cells had become an attractive alternative for development of a new vaccine against rabies with lower biosecurity risk and lower costs. In recombinant proteins production host cell lineage and cultivation parameters and conditions are important factors for glycoform profile of final protein, which impacts directly in its function. Objectives: Determine and compare the glycosylation pattern of rabies virus glycoprotein (RVGP) expressed in insect Drosophila melanogaster Schneider 2 (S2), in different expression systems and culture conditions. Methods: S2 cells expressing RVGP in cell membrane using inducible (S2MtRVGP-imm) or constitutive (S2AcRVGP-2) promoter, and expressing the soluble RVGP ectodomain, using an inducible promoter (S2BipMt_RVGP-Ecto), stablished in previous studies, were used in the present work. S2BipMt_RVGP-Ecto cells were cultivated in schott flasks and S2MtRVGP-imm cells were cultivated in schott and spinner flasks, in both cases with different induction parameters and cultivation time. S2AcRVGP-2 cells grown in bioreactor, in batch or fed-batch with glutamine supplementation. An immunoaffinity chromatography methodology for membrane rRVGP (rRVGP-S2Mt and rRVGP-S2Ac) were stablished using a commercial monoclonal antibody attached to a HiTrap NHSactivated HP (GE). Soluble ectodomain (rRVGP-S2Ecto) were purified by Immobilized Metal Affinity Chromatography (IMAC-Ni+2). Glycosylation pattern were analyzed by hydrophobic interaction chromatography (HILIC) or by HILIC accoupled with a mass spectrometry (UPLC-FLR-MS/MS). Results: For S2BipMt_RVGP-Ecto cells, the variation in inductor concentration presented biological effect that reflected on cell growth and rRVGP production. Inductor concentration and cell concentration at induction resulted in an increase in rRVGP production speed by S2MtRVGP-imm cells. Cultivation on spinner flasks results in a higher production of rRVGP-S2Mt comparing with schott flasks cultivation. The strategy of low initial glutamine concentration with supplementation during the S2AcRVGP-2 cell cultivation decreased the ammonium formation after the end of exponential phase, with an impact on growth and rRVGP production. Immunoaffinity chromatography methodology purification generated samples with high level of purity and efficiency up to 99 % of trimeric rRVGP-S2Mt. For rRVGP-S2Ac the same methodology worked with less efficiency and low level of purity. IMAC-Ni+2 used for rRVGP-S2Ecto presented low level of purity due to the low amount of expressed glycoprotein. In glycan pattern analyzes, samples of rRVGP-S2Ecto presented in higher proportion paucimannosidic structures: F(6)M2, M3 and F(6)M3. In rRVGP-S2Mt samples, the structures F(6)M2 e M3 were detected in higher proportion. rRVGP-S2Ac samples presented higher proportion of structures M2, F(6)M2 e M3. Conclusions: Increase of inductor concentration and of cell concentration at induction time results in a biological effect, by increasing expression speed of rRVGP. Specific production speed of rRVGP has a direct influence on decrease of N-glycans processing. Furthermore, different cultivation modes resulted in variation in glycan pattern, and a drastic reduction in initial glutamine concentration resulted in an increase on glycoforms with five or more mannose residues.