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
1研究背景。广东拥有超高密度的高层建筑群、现代化的综合交通运输体系、世界级港口群、空港群,高速公路、铁路、城市轨道交通总里程位居全国前列,具有人口密度高,经济体量大,社会影响广的特点。同时,广东经济最为发达的珠江三角洲地区...1研究背景。广东拥有超高密度的高层建筑群、现代化的综合交通运输体系、世界级港口群、空港群,高速公路、铁路、城市轨道交通总里程位居全国前列,具有人口密度高,经济体量大,社会影响广的特点。同时,广东经济最为发达的珠江三角洲地区位于华南沿海地震带中段的构造断陷盆地,盆地内部及边缘区NE向、NW向以及NEE向断裂互相切割且具有多期活动的特点(林纪曾等,1980;Lee and Lawver,1995;魏柏林等,2001);广东近海展布着曾发生多次强震的滨海断裂带,滨海断裂带是南海北部的重要控震构造和发震构造(刘以宣等,1981;赵明辉等,2003,2004;徐辉龙等,2006;孙金龙等,2012;曹敬贺等,2014),2015年发布的《中国地震动参数区划图》曾将珠江口列为7.5级潜在震源区。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.
文摘1研究背景。广东拥有超高密度的高层建筑群、现代化的综合交通运输体系、世界级港口群、空港群,高速公路、铁路、城市轨道交通总里程位居全国前列,具有人口密度高,经济体量大,社会影响广的特点。同时,广东经济最为发达的珠江三角洲地区位于华南沿海地震带中段的构造断陷盆地,盆地内部及边缘区NE向、NW向以及NEE向断裂互相切割且具有多期活动的特点(林纪曾等,1980;Lee and Lawver,1995;魏柏林等,2001);广东近海展布着曾发生多次强震的滨海断裂带,滨海断裂带是南海北部的重要控震构造和发震构造(刘以宣等,1981;赵明辉等,2003,2004;徐辉龙等,2006;孙金龙等,2012;曹敬贺等,2014),2015年发布的《中国地震动参数区划图》曾将珠江口列为7.5级潜在震源区。
