Miller J B, Teal S B, Stockdale F E
Department of Medicine, Stanford University School of Medicine, California 94305-5306.
J Biol Chem. 1989 Aug 5;264(22):13122-30.
A cDNA expression strategy was used to localize amino acid sequences which were specific for fast, as opposed to slow, isoforms of the chicken skeletal muscle myosin heavy chain (MHC) and which were conserved in vertebrate evolution. Five monoclonal antibodies (mAbs), termed F18, F27, F30, F47, and F59, were prepared that reacted with all of the known chicken fast MHC isoforms but did not react with any of the known chicken slow nor with smooth muscle MHC isoforms. The epitopes recognized by mAbs F18, F30, F47, and F59 were on the globular head fragment of the MHC, whereas the epitope recognized by mAb F27 was on the helical tail or rod fragment. Reactivity of all five mAbs also was confined to fast MHCs in the rat, with the exception of mAb F59, which also reacted with the beta-cardiac MHC, the single slow MHC isoform common to both the rat heart and skeletal muscle. None of the five epitopes was expressed on amphioxus, nematode, or Dictyostelium MHC. The F27 and F59 epitopes were found on shark, electric ray, goldfish, newt, frog, turtle, chicken, quail, rabbit, and rat MHCs. The epitopes recognized by these mAbs were conserved, therefore, to varying degrees through vertebrate evolution and differed in sequence from homologous regions of a number of invertebrate MHCs and myosin-like proteins. The sequence of those epitopes on the head were mapped using a two-part cDNA expression strategy. First, Bal31 exonuclease digestion was used to rapidly generate fragments of a chicken embryonic fast MHC cDNA that were progressively deleted from the 3' end. These cDNA fragments were expressed as beta-galactosidase/MHC fusion proteins using the pUR290 vector; the fusion proteins were tested by immunoblotting for reactivity with the mAbs; and the approximate locations of the epitopes were determined from the sizes of the cDNA fragments that encoded a particular epitope. The epitopes were then precisely mapped by expression of overlapping cDNA fragments of known sequence that covered the approximate location of the epitopes. With this method, the epitope recognized by mAb F59 was mapped to amino acids 211-231 of the chicken embryonic fast MHC and the three distinct epitopes recognized by mAbs F18, F30, and F47 were mapped to amino acids approximately 65-92. Each of these epitope sequences is at or near the ATPase active site.
采用cDNA表达策略定位鸡骨骼肌肌球蛋白重链（MHC）快速亚型（与慢速亚型相对）所特有的、且在脊椎动物进化过程中保守的氨基酸序列。制备了5种单克隆抗体（mAb），分别命名为F18、F27、F30、F47和F59，它们与所有已知的鸡快速MHC亚型发生反应，但不与任何已知的鸡慢速MHC亚型或平滑肌MHC亚型发生反应。mAb F18、F30、F47和F59识别的表位位于MHC的球状头部片段，而mAb F27识别的表位位于螺旋尾部或杆状片段。除mAb F59外，所有5种mAb的反应性也仅限于大鼠的快速MHC，mAb F59还与β-心脏MHC发生反应，β-心脏MHC是大鼠心脏和骨骼肌共有的单一慢速MHC亚型。5个表位在文昌鱼、线虫或盘基网柄菌的MHC上均未表达。在鲨鱼、电鳐、金鱼、蝾螈、青蛙、乌龟、鸡、鹌鹑、兔子和大鼠的MHC上发现了F27和F59表位。因此，这些mAb识别的表位在脊椎动物进化过程中不同程度地保守，并且与许多无脊椎动物MHC和肌球蛋白样蛋白的同源区域序列不同。使用两部分cDNA表达策略绘制头部这些表位的序列图谱。首先，使用Bal31核酸外切酶消化快速生成鸡胚胎快速MHC cDNA的片段，这些片段从3'端逐渐缺失。使用pUR290载体将这些cDNA片段表达为β-半乳糖苷酶/MHC融合蛋白；通过免疫印迹检测融合蛋白与mAb的反应性；并根据编码特定表位的cDNA片段大小确定表位的大致位置。然后通过表达覆盖表位大致位置的已知序列的重叠cDNA片段精确绘制表位图谱。用这种方法，mAb F59识别的表位被定位到鸡胚胎快速MHC的第211 - 231位氨基酸，mAb F18、F30和F47识别的3个不同表位被定位到大约第65 - 92位氨基酸。这些表位序列中的每一个都位于ATP酶活性位点或其附近。
