Surface (S)-layer proteins are model systems for studying protein glycosylation in bacteria and simultaneously hold promises for the design of novel, glyco-functionalized modules for nanobiotechnology due to their 2D ...Surface (S)-layer proteins are model systems for studying protein glycosylation in bacteria and simultaneously hold promises for the design of novel, glyco-functionalized modules for nanobiotechnology due to their 2D self-assembly capability. Understanding the mechanism governing S-layer glycan biosynthesis in the Gram-positive bacterium Paenibacillus alvei CCM 2051T is necessary for the tailored glyco-functionalization of its S-layer. Here, the putative oligosaccharyl:S-layer protein transferase WsfB from the P. alvei S-layer glycosylation gene locus is characterized. The enzyme is proposed to catalyze the final step of the glycosylation pathway, transferring the elongated S-layer glycan onto distinct tyrosine O-glycosylation sites. Genetic knock-out of WsfB is shown to abolish glycosylation of the S-layer protein SpaA but not that of other glycoproteins present in P. alvei CCM 2051T, confining its role to the S-layer glycosylation pathway. A transmembrane topology model of the 781-amino acid WsfB protein is inferred from activity measurements of green fluorescent protein and phosphatase A fused to defined truncations of WsfB. This model shows an overall number of 13 membrane spanning helices with the Wzy_C domain characteristic of O-oligosaccharyl:protein transferases (O-OTases) located in a central extra-cytoplasmic loop, which both compares well to the topology of OTases from Gram-negative bacteria. Mutations in the Wzy C motif resulted in loss of WsfB function evidenced in reconstitution experiments in P. alvei ΔWsfB cells. Attempts to use WsfB for transferring heterologous oligosaccharides to its native S-layer target protein in Escherichia coli CWG702 and Salmonella enterica SL3749, which should provide lipid-linked oligosaccharide substrates mimicking to some extent those of the natural host, were not successful, possibly due to the stringent function of WsfB. Concluding, WsfB has all features of a bacterial O-OTase, making it the most probable candidate for the oligosaccharyl:S-layer protein transferase of P. alvei, and a promising candidate for the first O-OTase reported in Gram-positives.展开更多
Surface layer (S-layer) proteins are one of the most commonly observed cell envelope components in both Archaea and Bacteria. It has versatile functions and holds considerable application potential in biotechnology. B...Surface layer (S-layer) proteins are one of the most commonly observed cell envelope components in both Archaea and Bacteria. It has versatile functions and holds considerable application potential in biotechnology. Bifidobacteria are representative probiotics conferring health promoting properties. However, there is little study of S-layer in bifidobacteria yet. The distribution and characteristics of S-layer in bifidobacteria are unknown. In this study, search for S-layer protein in the identical protein groups in NCBI yielded 49 hits belonging to bifidobacteria. These proteins were annotated as either “S-layer (domain) protein” or “putative S-layer (y) domain protein” that distributed among 26 species of Bifidobacterium genus. Multiple alignments suggest S-layer proteins are relatively conservative. Phylogenetic analysis of 24 S-layer (domain) protein sequences groups them into three distinct clusters, with the majority species in Cluster-2. S-layer (domain) protein has a universe motif DUF4381, though its function is unknown. Meanwhile, two other motifs CARDB and EphA2_TM involved in cell adhesion and cell signaling respectively, presented in most S-layer (domain) protein in bifidobacteria. All S-layer proteins have a typical N-terminal Sec-dependent signal peptide and a C-terminal trans-membrane region. Homological modeling of representative S-layer proteins from each cluster revealed a few unique structural features. All representative S-layer proteins have a plenty of β-meander motif that exclusively composed by β-barrel structural architectures linked together by hairpin loops.展开更多
The role of S-layer proteins(SLP),which form the outermost layer of cell walls in lactic acid bacteria(LAB),plays a crucial role in regulating immune-stimulating activity,thereby closely influencing LAB's ability ...The role of S-layer proteins(SLP),which form the outermost layer of cell walls in lactic acid bacteria(LAB),plays a crucial role in regulating immune-stimulating activity,thereby closely influencing LAB's ability to boost host immunity.In this study,the heterologous expression,purification,and characterization of Lactobacillus acidophilus CICC6074-derived slpX protein and the molecular mechanism of its anti-inflammatory effect on LPS-induced RAW264.7 cells were investigated.Initially,the PCR results were shown to successfully clone the slpX DNA sequence by homologous recombination to obtain the pet-32a-slpX recombinant plasmid.SDS-PAGE results revealed that slpX protein with a molecular weight of 54 kDa was successfully obtained under 0.7 mmol/L IPTG-induced conditions,which was further demonstrated by Western blot(WB)and LC-MS/MS.ELISA,WB and molecular docking results indicated that slpX protein can inhibit LPS-induced cellular inflammatory responses by linking to TLR4 and MD2 via hydrogen bonding and increasing the levels of anti-inflammatory factor IL-10 and decreasing the levels of inflammatory factors(TNF-α,IL-6,NO)and ROS via MAPK and NF-κB signaling pathways.The study is essential for the preparation of pure slpX protein and revealing its anti-inflammatory molecular mechanism.展开更多
文摘Surface (S)-layer proteins are model systems for studying protein glycosylation in bacteria and simultaneously hold promises for the design of novel, glyco-functionalized modules for nanobiotechnology due to their 2D self-assembly capability. Understanding the mechanism governing S-layer glycan biosynthesis in the Gram-positive bacterium Paenibacillus alvei CCM 2051T is necessary for the tailored glyco-functionalization of its S-layer. Here, the putative oligosaccharyl:S-layer protein transferase WsfB from the P. alvei S-layer glycosylation gene locus is characterized. The enzyme is proposed to catalyze the final step of the glycosylation pathway, transferring the elongated S-layer glycan onto distinct tyrosine O-glycosylation sites. Genetic knock-out of WsfB is shown to abolish glycosylation of the S-layer protein SpaA but not that of other glycoproteins present in P. alvei CCM 2051T, confining its role to the S-layer glycosylation pathway. A transmembrane topology model of the 781-amino acid WsfB protein is inferred from activity measurements of green fluorescent protein and phosphatase A fused to defined truncations of WsfB. This model shows an overall number of 13 membrane spanning helices with the Wzy_C domain characteristic of O-oligosaccharyl:protein transferases (O-OTases) located in a central extra-cytoplasmic loop, which both compares well to the topology of OTases from Gram-negative bacteria. Mutations in the Wzy C motif resulted in loss of WsfB function evidenced in reconstitution experiments in P. alvei ΔWsfB cells. Attempts to use WsfB for transferring heterologous oligosaccharides to its native S-layer target protein in Escherichia coli CWG702 and Salmonella enterica SL3749, which should provide lipid-linked oligosaccharide substrates mimicking to some extent those of the natural host, were not successful, possibly due to the stringent function of WsfB. Concluding, WsfB has all features of a bacterial O-OTase, making it the most probable candidate for the oligosaccharyl:S-layer protein transferase of P. alvei, and a promising candidate for the first O-OTase reported in Gram-positives.
文摘Surface layer (S-layer) proteins are one of the most commonly observed cell envelope components in both Archaea and Bacteria. It has versatile functions and holds considerable application potential in biotechnology. Bifidobacteria are representative probiotics conferring health promoting properties. However, there is little study of S-layer in bifidobacteria yet. The distribution and characteristics of S-layer in bifidobacteria are unknown. In this study, search for S-layer protein in the identical protein groups in NCBI yielded 49 hits belonging to bifidobacteria. These proteins were annotated as either “S-layer (domain) protein” or “putative S-layer (y) domain protein” that distributed among 26 species of Bifidobacterium genus. Multiple alignments suggest S-layer proteins are relatively conservative. Phylogenetic analysis of 24 S-layer (domain) protein sequences groups them into three distinct clusters, with the majority species in Cluster-2. S-layer (domain) protein has a universe motif DUF4381, though its function is unknown. Meanwhile, two other motifs CARDB and EphA2_TM involved in cell adhesion and cell signaling respectively, presented in most S-layer (domain) protein in bifidobacteria. All S-layer proteins have a typical N-terminal Sec-dependent signal peptide and a C-terminal trans-membrane region. Homological modeling of representative S-layer proteins from each cluster revealed a few unique structural features. All representative S-layer proteins have a plenty of β-meander motif that exclusively composed by β-barrel structural architectures linked together by hairpin loops.
基金supported by the National Natural Science Foundation of China(32272339)National Key R&D Program of China(2021YFD2100104)。
文摘The role of S-layer proteins(SLP),which form the outermost layer of cell walls in lactic acid bacteria(LAB),plays a crucial role in regulating immune-stimulating activity,thereby closely influencing LAB's ability to boost host immunity.In this study,the heterologous expression,purification,and characterization of Lactobacillus acidophilus CICC6074-derived slpX protein and the molecular mechanism of its anti-inflammatory effect on LPS-induced RAW264.7 cells were investigated.Initially,the PCR results were shown to successfully clone the slpX DNA sequence by homologous recombination to obtain the pet-32a-slpX recombinant plasmid.SDS-PAGE results revealed that slpX protein with a molecular weight of 54 kDa was successfully obtained under 0.7 mmol/L IPTG-induced conditions,which was further demonstrated by Western blot(WB)and LC-MS/MS.ELISA,WB and molecular docking results indicated that slpX protein can inhibit LPS-induced cellular inflammatory responses by linking to TLR4 and MD2 via hydrogen bonding and increasing the levels of anti-inflammatory factor IL-10 and decreasing the levels of inflammatory factors(TNF-α,IL-6,NO)and ROS via MAPK and NF-κB signaling pathways.The study is essential for the preparation of pure slpX protein and revealing its anti-inflammatory molecular mechanism.
文摘边界层吸入(BLI,boundary layer ingestion)式风扇进口存在复杂的畸变流场,现有研究大多局限于传统的三维模拟方式,计算耗时长。本文以DGEN380风扇转子为研究对象,设计了一款与之匹配的S型进气道以吸入来流边界层,并采用基于体积力模型(BFM,body force modelling)的计算方法,计算结果的参数变化趋势与非定常计算结果吻合良好,风扇入口的旋流角及无量纲总压值的周向均值与非定常结果偏差在6%以内,计算时间为非定常方法的1/50。基于此模型探究了15%、30%、60%进口高度下3种不同厚度BLI对于风扇转子进出口流场的影响。结果表明:针对风扇转子入口,在不同叶高处总压畸变及旋流畸变程度随着BLI厚度的增加均有所增加,且旋流分布呈现对涡形式,在经过风扇转子通道叶片作用之后,对涡逐渐转化为单向涡,风扇转子出口气流落后角减小,BLI区风扇功系数值较均匀流工况有所提升。
