The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG.
The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG.
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DOI：
10.1186/1475-2859-11-15
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发表时间：
2012-02-01
影响因子：
6.4
通讯作者：
Vanderleyden J
中科院分区：
文献类型：
--
作者：
Lebeer S;Claes IJ;Balog CI;Schoofs G;Verhoeven TL;Nys K;von Ossowski I;de Vos WM;Tytgat HL;Agostinis P;Palva A;Van Damme EJ;Deelder AM;De Keersmaecker SC;Wuhrer M;Vanderleyden J
虽然糖蛋白的发生、生物合成和可能的功能越来越多地被记录在病原体中，但糖蛋白在益生菌中还没有被广泛描述。然而，蛋白质糖基化的知识具有重要的潜力，更好地了解特定的聚糖介导的益生菌的相互作用和食品级微生物中的糖工程。在这里，我们提供的证据表明，主要分泌蛋白Msp 1/p75的益生菌鼠李糖乳杆菌GG糖基化。Msp 1被证明是染色阳性与酸性希夫染色，是容易发生化学去糖基化，并结合甘露糖特异性伴刀豆球蛋白A（刀豆球蛋白A）凝集素。在大肠杆菌中的重组表达导致分子量的显着降低，ConA反应性的损失和对链霉蛋白酶E和蛋白酶K的敏感性增加。质谱分析表明Msp 1是O-糖基化的，并鉴定了一个糖肽TVETPSSA（氨基酸101-108），该糖肽含有可能与丝氨酸残基连接的己糖。有趣的是，这些丝氨酸残基不存在于测试的几种干酪乳杆菌菌株的同源蛋白中，其也不与ConA结合。还研究了聚糖取代在Msp 1已知功能中的作用。糖基化似乎对Msp 1的肽聚糖水解酶活性没有显著影响。此外，聚糖链似乎不是Msp 1激活肠上皮细胞中Akt信号传导所必需的。另一方面，对不同细胞提取物的检查表明，Msp 1在上清液中是糖基化蛋白，但在细胞壁和胞质溶胶部分中不是糖基化蛋白，这表明糖基化和这种蛋白质的分泌之间的联系。在这项研究中，我们提供了第一个证据的蛋白质O-糖基化的益生菌鼠李糖乳杆菌GG。主要分泌蛋白Msp 1在丝氨酸残基106和107处被ConA反应性糖糖基化。糖基化不是Msp 1的肽聚糖水解酶活性所必需的，也不是上皮细胞中Akt活化能力所必需的，但似乎对其稳定性和对蛋白酶的保护是重要的。
Although the occurrence, biosynthesis and possible functions of glycoproteins are increasingly documented for pathogens, glycoproteins are not yet widely described in probiotic bacteria. Nevertheless, knowledge of protein glycosylation holds important potential for better understanding specific glycan-mediated interactions of probiotics and for glycoengineering in food-grade microbes. Here, we provide evidence that the major secreted protein Msp1/p75 of the probiotic Lactobacillus rhamnosus GG is glycosylated. Msp1 was shown to stain positive with periodic-acid Schiff staining, to be susceptible to chemical deglycosylation, and to bind with the mannose-specific Concanavalin A (ConA) lectin. Recombinant expression in Escherichia coli resulted in a significant reduction in molecular mass, loss of ConA reactivity and increased sensitivity towards pronase E and proteinase K. Mass spectrometry showed that Msp1 is O-glycosylated and identified a glycopeptide TVETPSSA (amino acids 101-108) bearing hexoses presumably linked to the serine residues. Interestingly, these serine residues are not present in the homologous protein of several Lactobacillus casei strains tested, which also did not bind to ConA. The role of the glycan substitutions in known functions of Msp1 was also investigated. Glycosylation did not seem to impact significantly on the peptidoglycan hydrolase activity of Msp1. In addition, the glycan chain appeared not to be required for the activation of Akt signaling in intestinal epithelial cells by Msp1. On the other hand, examination of different cell extracts showed that Msp1 is a glycosylated protein in the supernatant, but not in the cell wall and cytosol fraction, suggesting a link between glycosylation and secretion of this protein. In this study we have provided the first evidence of protein O-glycosylation in the probiotic L rhamnosus GG. The major secreted protein Msp1 is glycosylated with ConA reactive sugars at the serine residues at 106 and 107. Glycosylation is not required for the peptidoglycan hydrolase activity of Msp1 nor for Akt activation capacity in epithelial cells, but appears to be important for its stability and protection against proteases.
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影响因子：
3.7
作者：
Claes IJ;Schoofs G;Regulski K;Courtin P;Chapot-Chartier MP;Rolain T;Hols P;von Ossowski I;Reunanen J;de Vos WM;Palva A;Vanderleyden J;De Keersmaecker SC;Lebeer S
通讯作者：
Lebeer S
影响因子：
3.6
作者：
Grass, S;Buscher, AZ;St Geme, JW
通讯作者：
St Geme, JW
影响因子：
4.4
作者：
Lebeer, Sarah;Verhoeven, Tine L. A.;De Keersmaecker, Sigrid C. J.
通讯作者：
De Keersmaecker, Sigrid C. J.
影响因子：
1.2
作者：
Baeuerl, Christine;Perez-Martinez, Gaspar;Monedero, Vicente
通讯作者：
Monedero, Vicente
影响因子：
3.2
作者：
Lebeer, Sarah;De Keersmaecker, Sigrid C. J.;Vanderleyden, Jos
通讯作者：
Vanderleyden, Jos