O-fucosylation stabilizes the TSR3 motif in thrombospondin-1 by interacting with nearby amino acids and protecting a disulfide bond; 35597280

Thrombospondin type-1 repeats (TSRs) are small protein motifs containing six conserved cysteines forming three disulfide bonds that can be modified with an O-linked fucose. Protein O-fucosyltransferase 2 (POFUT2) catalyzes the addition of O-fucose to TSRs containing the appropriate consensus sequence, and the O-fucose modification can be elongated to a Glucose-Fucose disaccharide with the addition of glucose by ß3-glucosyltransferase (B3GLCT). Elimination of Pofut2 in mice results in embryonic lethality in mice, highlighting the biological significance of O-fucose modification on TSRs. Knockout of POFUT2 in HEK293T cells has been shown to cause complete or partial loss of secretion of many proteins containing O-fucosylated TSRs. In addition, POFUT2 is localized to the endoplasmic reticulum (ER) and only modifies folded TSRs, stabilizing their structures. These observations suggest that POFUT2 is involved in an ER quality control mechanism for TSR folding and that B3GLCT also participates in quality control by providing additional stabilization to TSRs. However, the mechanisms by which addition of these sugars result in stabilization are poorly understood. Here, we conducted molecular dynamics (MD) simulations and provide crystallographic and NMR evidence that the Glucose-Fucose disaccharide interacts with specific amino acids in the TSR3 domain in thrombospondin-1 that are within proximity to the O-fucosylation modification site resulting in protection of a nearby disulfide bond. We also show that mutation of these amino acids reduces the stabilizing effect of the sugars in vitro. These data provide mechanistic details regarding the importance of O-fucosylation and how it participates in quality control mechanisms inside the ER

Dietas com nitrogênio não-proteico para fêmeas bovinas superovuladas sem prévia adaptação durante curto tempo e em diferentes fases do ciclo estral Effect of short term non-protein nitrogen feeding for superovulated beef cows without previous adaptation and at different periods of the oestrus cycle

Objetivou-se neste estudo avaliar o efeito do fornecimento, sem prévia adaptação, durante curto tempo e em diferentes fases do ciclo estral, de dietas contendo nitrogênio não-proteico (NNP) na produção, na qualidade e no grau de desenvolvimento de embriões recuperados em fêmeas bovinas superovuladas. Sessenta e oito vacas Nelore foram distribuídas em três grupos: um controle e dois com fornecimento de ureia antes (UA = do dia -5 ao dia 0) e após (UD = dia 0 ao dia 5) a inseminação artificial. As vacas foram mantidas em pastagem e receberam concentrado (3,0 kg/animal/dia) durante 16 dias. Foram formulados dois concentrados, e as dietas totais (concentrado e consumo estimado de pastagem) apresentaram 12,0% (dieta controle) e 14,6% (dieta NNP) de proteína bruta (PB). As vacas foram sincronizadas, superovuladas e inseminadas. Sete dias (dia 7) após a inseminação (dia 0), realizou-se a colheita e análise dos embriões. Amostras de sangue foram coletadas nos dias -5, 0 e 5 para determinação da concentração de nitrogênio ureico plasmático (NUP), glicose, insulina e progesterona. A época de fornecimento de ureia influenciou as concentrações médias de NUP nos dias -5, 0 e 5, mas não tiveram efeito nas concentrações de glicose, insulina e progesterona. O período de fornecimento da ureia teve efeito na porcentagem de mórulas compactas em relação ao total de estruturas (UA = 51,4 vs. UD = 15,3%), em relação ao total de oócitos fecundados (UA = 62,5 vs. UD = 30,6%) e em relação ao total de embriões viáveis (UA = 68,8 vs. UD = 38,6%). No grupo que recebeu ureia depois da inseminação artificial, houve redução de 70,2% da proporção de mórulas compactas por total de estruturas em relação ao grupo que recebeu ureia antes da inseminação. Dietas com nitrogênio não-proteico fornecidas imediatamente após a inseminação promovem aceleração do desenvolvimento embrionário inicial.<br>The aim of this study was to evaluate the effects of short term non-protein nitrogen feeding at different periods of the oestrus cycle in superovulated cows, without previous adaptation, on yield, quality and development degree of recovered embryos. A total of sixty-eight Nelore cows were distributed in three groups: the control group (C) and two groups with urea supply before (UB; urea supply from day -5 to day 0) and after (UA; supply from day 0 to day 5) artificial insemination. Animals were kept grazing and received 3.0 kg/animal/day of concentrate during 16 days. Two concentrates were formulated and the total diets (concentrate and estimate forage intake) showed 12.0% (control diet) and 14.6% (non-protein diet) of crude protein. Animals were synchronized, superovulated and inseminated. The embryos were collected and analysed seven days (day 7) after insemination (day 0). Blood samples were collected on days -5, 0 and 5 to determine concentration of plasmatic urea nitrogen, glucose, insulin and progesterone. The time of urea supply affected average plasmatic urea nitrogen concentration on days -5, 0 5 but it did not affect concetrations of glucose, insulin and progesterone. The moment of urea inclusion had effect on compact morula percentage in relation to the total number of structures (UB = 51.4 vs. UA = 15.3%), to the total number of fertilized oocytes (UB = 62.5 vs. UA = 30.6%) and to the total number of viable embryos (UB = 68.8 vs. UA = 38.6%). In the after insemination group of urea supply there was 70.2% of reduction on compact morula proportion over the total structures in relation to the group that received urea before insemination. Non-protein nitrogen feeding immediately after insemination promotes faster rates of embryo development