Comer Jason E, Marshall Mark A, Blanch Vincent J, Deal Carolyn D, Castric Peter
Department of Biological Sciences, Duquesne University, Pittsburgh, Pennsylvania 15282, USA.
Infect Immun. 2002 Jun;70(6):2837-45. doi: 10.1128/IAI.70.6.2837-2845.2002.
Previous work (P. Castric, F. J. Cassels, and R. W. Carlson, J. Biol. Chem. 276:26479-26485, 2001) has shown the Pseudomonas aeruginosa 1244 pilin glycan to be covalently bound to a serine residue. N-terminal sequencing of pilin fragments produced from endopeptidase treatment and identified by reaction with a glycan-specific monoclonal antibody indicated that the glycan was present between residue 75 and the pilin carboxy terminus. Further sequencing of these peptides revealed that serine residues 75, 81, 84, 105, 106, and 108 were not modified. Conversion of serine 148, but not serine 118, to alanine by site-directed mutagenesis, resulted in loss of the ability to carry out pilin glycosylation when tested in an in vivo system. These results showed the pilin glycan to be attached to residue 148, the carboxy-terminal amino acid. The carboxy-proximal portion of the pilin disulfide loop, which is adjacent to the pilin glycan, was found to be a major linear B-cell epitope, as determined by peptide epitope mapping analysis. Immunization of mice with pure pili produced antibodies that recognized the pilin glycan. These sera also reacted with P. aeruginosa 1244 lipopolysaccharide as measured by Western blotting and enzyme-linked immunosorbent assay.
先前的研究工作（P. 卡斯特里克、F. J. 卡塞尔和R. W. 卡尔森，《生物化学杂志》276:26479 - 26485，2001年）表明，铜绿假单胞菌1244菌毛聚糖与一个丝氨酸残基共价结合。对内肽酶处理产生的菌毛片段进行N端测序，并通过与聚糖特异性单克隆抗体反应进行鉴定，结果表明聚糖存在于第75位残基和菌毛羧基末端之间。对这些肽段的进一步测序显示，丝氨酸残基75、81、84、105、106和108未被修饰。通过定点诱变将丝氨酸148而非丝氨酸118转化为丙氨酸，在体内系统中进行测试时，导致了进行菌毛糖基化的能力丧失。这些结果表明菌毛聚糖附着于第148位残基，即羧基末端氨基酸。通过肽表位图谱分析确定，与菌毛聚糖相邻的菌毛二硫键环的羧基近端部分是一个主要的线性B细胞表位。用纯菌毛免疫小鼠产生了识别菌毛聚糖的抗体。通过蛋白质印迹法和酶联免疫吸附测定法检测，这些血清也与铜绿假单胞菌1244脂多糖发生反应。
