Theiss P, Karpas A, Wise K S
Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia 65212, USA.
Infect Immun. 1996 May;64(5):1800-9. doi: 10.1128/iai.64.5.1800-1809.1996.
Antibodies to P29, a major lipid-modified surface protein of Mycoplasma fermentans, reveal phase variation of surface epitopes occurring with high frequency in clonal lineages of the organism. This occurs despite continuous expression of the entire epitope-bearing P29 product (detected by Western immunoblotting) and contrasts with phase variation of other surface antigens mediated by differential expression of proteins. To understand the structure and antigenic topology of P29, the single-copy p29 gene from strain PG18 was cloned and sequenced. The gene encodes a prolipoprotein containing a signal sequence predicted to be modified with lipid and cleaved at the N-terminal Cys-1 residue of the mature P29 lipoprotein. The remaining 218-residue hydrophilic sequence of P29 is predicted to be located external to the single plasma membrane. Additional Cys residues at positions 91 and 128 in the mature protein were shown to form a 36-residue disulfide loop by selectively labeling sulfhydryl groups that were liberated only after chemical reduction of monomeric P29. Two nearly identical charged amino acid sequences occurred in P29, within the disulfide loop and upstream of this structure. Two distinct epitopes binding different monoclonal antibodies were associated with opposite ends of the P29 protein, by mapping products expressed in Escherichia coli from PCR-generated 3' deletion mutations of the p29 gene. Each monoclonal antibody detected high-frequency and noncoordinate changes in accessibility of the corresponding epitopes in colony immunoblots of clonal variants, yet sequencing of the p29 gene from these variants and analysis of disulfide bonds revealed no associated changes in the primary sequence or disulfide loop structure of P29. These results suggest that P29 surface epitope variation may involve masking of selected regions of P29, possibly by other surface components undergoing phase variation by differential expression. Differential masking may be an important mechanism for altering the antigenic or functional surface topology of this mycoplasma and other wall-less mycoplasmas.
抗P29抗体可识别发酵支原体的一种主要脂质修饰表面蛋白，该抗体揭示了在该生物体的克隆谱系中高频出现的表面表位的相变。尽管整个携带表位的P29产物持续表达（通过蛋白质免疫印迹法检测），但仍会出现这种情况，这与由蛋白质差异表达介导的其他表面抗原的相变形成对比。为了解P29的结构和抗原拓扑结构，对PG18菌株的单拷贝p29基因进行了克隆和测序。该基因编码一种前脂蛋白，其包含一个预测会被脂质修饰并在成熟P29脂蛋白的N端Cys-1残基处切割的信号序列。P29剩余的218个残基的亲水性序列预计位于单个质膜外部。通过选择性标记仅在单体P29化学还原后释放的巯基，显示成熟蛋白中第91和128位的额外Cys残基形成了一个36个残基的二硫键环。在P29中，二硫键环内及其上游出现了两个几乎相同的带电荷氨基酸序列。通过对从p29基因的PCR产生的3'缺失突变体在大肠杆菌中表达的产物进行定位，发现结合不同单克隆抗体的两个不同表位与P29蛋白的相对末端相关。每种单克隆抗体在克隆变体的菌落免疫印迹中检测到相应表位可及性的高频和非协同变化，但对这些变体的p29基因进行测序并分析二硫键后发现，P29的一级序列或二硫键环结构没有相关变化。这些结果表明，P29表面表位变异可能涉及P29选定区域的掩盖，可能是由通过差异表达进行相变的其他表面成分造成的。差异掩盖可能是改变这种支原体和其他无壁支原体的抗原或功能表面拓扑结构的重要机制。
