Wong W Y, Irvin R T, Paranchych W, Hodges R S
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Protein Sci. 1992 Oct;1(10):1308-18. doi: 10.1002/pro.5560011010.
The C-terminal region of Pseudomonas aeruginosa strain K (PAK) pilin comprises both an epitope for the strain-specific monoclonal antibody PK99H, which blocks pilus-mediated adherence, and the adherence binding domain for buccal and tracheal epithelial cells. The PK99H epitope was located in sequence 134-140 (Asp-Glu-Gln-Phe-Ile-Pro-Lys) by using a single alanine replacement analysis on the 17-residue synthetic peptide corresponding to the PAK C-terminal sequence 128-144. Indeed, a 7-residue peptide corresponding to this sequence was shown to have a similar binding affinity to that of the native conformationally constrained (disulfide bridged) 17-residue peptide. This epitope was found to contain two critical residues (Phe137 and Lys140) and one nonessential residue (Gln136). Interestingly, the peptide, Phe-Ile-Pro-Lys, which constitutes the four most important side chains for antibody binding did not bind to PK99H. It was of interest to investigate the structural basis of the strain-specificity of PK99H utilizing naturally occurring pilin sequences. Therefore, all different residues found in the sequence corresponding to the PK99H epitope of the four other strains (PAO, CD4, K122-4, and KB7) were substituted one at a time in the PAK sequence and the changes in binding affinity of these analogs to the antibody PK99H were determined by competitive ELISA. The strain-specificity of PK99H for strains PAO, K122-4, and KB7 can be explained by the accumulated sequence changes in these strains, and at least two amino acid changes were required to explain the strain-specificity of PK99H. Similarly, cross-reactivity of PK99H with CD4 can be explained by the fact that there was only one side chain responsible for decreasing binding affinity compared to the PAK sequence.
铜绿假单胞菌K株（PAK）菌毛蛋白的C末端区域既包含菌株特异性单克隆抗体PK99H的表位，该表位可阻断菌毛介导的黏附，也包含与颊和气管上皮细胞的黏附结合结构域。通过对与PAK C末端序列128 - 144相对应的17个残基合成肽进行单个丙氨酸替代分析，将PK99H表位定位在序列134 - 140（天冬氨酸-谷氨酸-谷氨酰胺-苯丙氨酸-异亮氨酸-脯氨酸-赖氨酸）。实际上，与该序列相对应的一个7个残基的肽显示出与天然构象受限（二硫键桥连）的17个残基肽具有相似的结合亲和力。发现该表位包含两个关键残基（苯丙氨酸137和赖氨酸140）和一个非必需残基（谷氨酰胺136）。有趣的是，构成抗体结合的四个最重要侧链的肽苯丙氨酸-异亮氨酸-脯氨酸-赖氨酸并不与PK99H结合。利用天然存在的菌毛蛋白序列研究PK99H菌株特异性的结构基础很有意义。因此，在PAK序列中一次一个地替换在其他四个菌株（PAO、CD4、K122 - 4和KB7）与PK99H表位相对应的序列中发现的所有不同残基，并通过竞争性酶联免疫吸附测定法确定这些类似物与抗体PK99H结合亲和力的变化。PK99H对PAO、K122 - 4和KB7菌株的菌株特异性可以通过这些菌株中积累的序列变化来解释，并且至少需要两个氨基酸变化来解释PK99H的菌株特异性。同样，PK99H与CD4的交叉反应性可以通过以下事实来解释：与PAK序列相比，只有一个侧链负责降低结合亲和力。
