Daubenspeck James M, Zeng Huadong, Chen Ping, Dong Shengli, Steichen Christopher T, Krishna N Rama, Pritchard David G, Turnbough Charles L
Department of Microbiology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
J Biol Chem. 2004 Jul 23;279(30):30945-53. doi: 10.1074/jbc.M401613200. Epub 2004 May 19.
Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a prominent loose fitting layer called the exosporium. The exosporium consists of a basal layer and an external hairlike nap. The filaments of the nap are composed of a highly immunogenic glycoprotein called BclA, which has a long, central collagen-like region with multiple XXG repeats. Most of the triplet repeats are PTG, and nearly all of the triplet repeats contain a threonine residue, providing multiple potential sites for O-glycosylation. In this study, we demonstrated that two O-linked oligosaccharides, a 715-Da tetrasaccharide and a 324-Da disaccharide, are released from spore- and exosporium-associated BclA by hydrazinolysis. Each oligosaccharide is probably attached to BclA through a GalNAc linker, which was lost during oligosaccharide release. We found that multiple copies of the tetrasaccharide are linked to the collagen-like region of BclA, whereas the disaccharide may be attached outside of this region. Using NMR, mass spectrometry, and other analytical techniques, we determined that the structure of the tetrasaccharide is 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-beta-d-glucopyranosyl-(1-->3)-alpha-l-rhamnopyranosyl-(1-->3)-alpha-l-rhamnopyranosyl-(1-->2)-l-rhamnopyranose. The previously undescribed nonreducing terminal sugar (i.e. 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-d-glucose) was given the trivial name anthrose. Anthrose was not found in spores of either Bacillus cereus or Bacillus thuringiensis, two species that are the most phylogenetically similar to B. anthracis. Thus, anthrose may be useful for species-specific detection of B. anthracis spores or as a new target for therapeutic intervention.
炭疽病的病原体炭疽芽孢杆菌的孢子被一层称为芽孢外膜的显著宽松外层所包裹。芽孢外膜由一个基底层和一个外部毛发状绒毛层组成。绒毛的细丝由一种高度免疫原性的糖蛋白BclA组成，它有一个长的、中央的胶原样区域，带有多个XXG重复序列。大多数三联体重复序列是PTG，几乎所有的三联体重复序列都含有一个苏氨酸残基，为O-糖基化提供了多个潜在位点。在本研究中，我们证明了两种O-连接寡糖，一种715Da的四糖和一种324Da的二糖，通过肼解从与孢子和芽孢外膜相关的BclA中释放出来。每种寡糖可能通过一个N-乙酰半乳糖胺连接子连接到BclA上，该连接子在寡糖释放过程中丢失。我们发现四糖的多个拷贝与BclA的胶原样区域相连，而二糖可能连接在该区域之外。使用核磁共振、质谱和其他分析技术，我们确定了四糖的结构为2-O-甲基-4-(3-羟基-3-甲基丁酰胺基)-4,6-二脱氧-β-D-吡喃葡萄糖基-(1→3)-α-L-鼠李吡喃糖基-(1→3)-α-L-鼠李吡喃糖基-(1→2)-L-鼠李吡喃糖。这个以前未描述的非还原末端糖（即2-O-甲基-4-(3-羟基-3-甲基丁酰胺基)-4,6-二脱氧-D-葡萄糖）被赋予了俗名炭疽糖。在蜡状芽孢杆菌或苏云金芽孢杆菌的孢子中未发现炭疽糖，这两个物种在系统发育上与炭疽芽孢杆菌最相似。因此，炭疽糖可能有助于炭疽芽孢杆菌孢子的物种特异性检测，或作为治疗干预的新靶点。
