S-layer, Surface-Accessible, and Concanavalin A Binding Proteins of <i>Methanosarcina acetivorans</i> and <i>Methanosarcina mazei</i>

The outermost cell envelope structure of many archaea and bacteria contains a proteinaceous lattice termed the surface layer or S-layer. It is typically composed of only one or two abundant, often post-translationally modified proteins that self-assemble to form the highly organized arrays. Surprisingly, over 100 proteins were annotated to be S-layer components in the archaeal species Methanosarcina acetivorans C2A and Methanosarcina mazei Gö1, reflecting limitations of current predictions. An in vivo biotinylation methodology was devised to affinity tag surface-exposed proteins while overcoming unique challenges in working with these fragile organisms. Cells were adapted to growth under N2 fixing conditions, thus, minimizing free amines reactive to the NHS-label, and high pH media compatible with the acylation chemistry was used. A 3-phase separation procedure was employed to isolate intact, labeled cells from lysed-cell derived proteins. Streptavidin affinity enrichment followed by stringent wash conditions removed nonspecifically bound proteins. This methodology revealed S-layer proteins in M. acetivorans C2A and M. mazei Gö1 to be MA0829 and MM1976, respectively. Each was demonstrated to exist as multiple glycosylated forms using SDS-PAGE coupled with glycoprotein-specific staining, and by interaction with the lectin, Concanavalin A. A number of additional surface-exposed proteins and glycoproteins were identified and included all three subunits of the thermosome: the latter suggests that the chaperonin complex is both surface- and cytoplasmically localized. This approach provides an alternative strategy to study surface proteins in the archaea.许多古菌与细菌的最外层细胞被膜结构含有一种被称为表面层（S-layer，surface layer）的蛋白质晶格。该结构通常仅由1~2种丰度较高的蛋白质构成，这类蛋白质往往经过翻译后修饰，并可自组装形成高度有序的阵列。令人意外的是，在古菌物种产甲烷八叠球菌C2A（Methanosarcina acetivorans C2A）与迷宫产甲烷八叠球菌Gö1（Methanosarcina mazei Gö1）中，已有超过100种蛋白质被注释为S层组分，这反映出当前预测方法存在局限性。 研究人员开发了一种体内生物素标记法（in vivo biotinylation），用于对表面暴露蛋白进行亲和标签标记，同时攻克了针对这类脆弱微生物开展实验的独特难题。实验中，将细胞置于固氮条件下培养，以最大限度减少可与NHS标记物（NHS-label）反应的游离胺，并使用与酰化反应兼容的高pH培养基。研究采用三相分离流程，从裂解细胞来源的蛋白质中分离得到完整的标记细胞；随后通过链霉亲和素亲和富集，配合严格的洗涤条件去除非特异性结合的蛋白质。 该方法揭示，产甲烷八叠球菌C2A与迷宫产甲烷八叠球菌Gö1的S层蛋白分别为MA0829与MM1976。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）结合糖蛋白特异性染色（glycoprotein-specific staining），以及与凝集素（lectin）刀豆球蛋白A（Concanavalin A）的相互作用实验，证实这两种蛋白均存在多种糖基化形式。研究还鉴定出多种其他表面暴露蛋白与糖蛋白，其中包括热激体（thermosome）的全部三个亚基；该结果表明，这类伴侣蛋白复合体（chaperonin complex）同时定位于细胞表面与细胞质中。 本研究开发的方法为古菌表面蛋白的研究提供了一种替代性策略。