LOCALIZATION OF BINDING-SITES FOR CARBOXYL TERMINAL SPECIFIC ANTI-RHODOPSIN MONOCLONAL-ANTIBODIES USING SYNTHETIC PEPTIDES
LOCALIZATION OF BINDING-SITES FOR CARBOXYL TERMINAL SPECIFIC ANTI-RHODOPSIN MONOCLONAL-ANTIBODIES USING SYNTHETIC PEPTIDES
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DOI：
10.1021/bi00321a041
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发表时间：
1984-01-01
期刊：
影响因子：
2.9
通讯作者：
MOLDAY, RS
中科院分区：
文献类型：
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作者：
MACKENZIE, D;ARENDT, A;MOLDAY, RS
4个单克隆抗体rho 1D4、rho 3C2、rho 3A6和rho 1C5的结合位点定位于牛视紫红质c端：Asp18′-Glu-Ala16′-Ser-Thr-Val12′-Ser-Lys-Thr-Glu8′-Thr-Ser-Gln-Val4′-Ala-Pro-Ala1′。抗体结合位点通过合成c端肽结合固相竞争抑制测定和有限蛋白水解酶切紫红质结合电泳免疫印迹技术进行定位。rho 1D4和rho 3C2抗体与固定视紫红质的结合被长度为1''-8‘’或更长的肽段所抑制。抗体rho 1D4的结合不受2′-13′和3′-18′肽段的抑制，表明需要紫红质c端丙氨酸残基。类似的竞争性抑制研究表明，抗体rho 3A6需要长度为1“-12”或更长的肽，而rho 1C5需要长度为1“-18”的肽。肽3''-18‘’与1''-18‘’同样有效地抑制rho 3A6与视紫红质的结合，但用谷氨酰胺取代8‘’位置的谷氨酸会消除竞争。这种取代对抗体rho 1C5的结合影响不大。因此，Glu8’对于rho 3A6的结合是必需的，而对于rho 1C5抗体的结合则不是必需的。用金黄色葡萄球菌V8蛋白酶切割红紫质的7个氨基酸c端，并进一步切割F1 （MW 25,000）和F2 （MW 12,000）片段，可以消除rho 1D4抗体与膜结合的红紫质片段的结合。然而，rho 3A6和rho 1C5抗体被发现结合了缺乏7个氨基酸c端和相应的F2片段的视紫红质。这些结果表明，rho 1D4和rho 3C2抗体结合需要一个长度大于1“-6”的肽，rho 3A6抗体结合需要8“-12”的肽，rho 1C5抗体需要9“-17”或9“-18”的肽。由于较长的肽和视紫红质是更有效的竞争者，其他因素如构象也会影响结合反应性。这些研究以及这些抗体与膜结合和洗涤剂溶解的漂白和未漂白的视紫红质的结合的相关研究表明，这些免疫探针可以高度接近视紫红质的羧基末端1“-18”段，并且对视紫红质的漂白和增溶状态相对不敏感。研究了这些单克隆抗体与牛视紫红质其他区域的交叉反应性。在猪、狗、猫、大鼠、兔、蛙的视紫红质中，视紫红质的c端片段（由rho 1D4和rho 3C2抗体指定）、F1-F2连接区和n端区域高度保守。由rho 3A6和rho 1C5抗体指定的距离c端更远的片段更可变。
The binding sites for 4 monoclonal antibodies, rho 1D4, rho 3C2, rho 3A6 and rho 1C5, were localized within the C-terminal region of bovine rhodopsin: Asp18''-Glu-Ala16''-Ser-Thr-Val12''-Ser-Lys-Thr-Glu8''-Thr-Ser-Gln-Val4''-Ala-Pro-Ala1''. Antibody binding sites were localized by using synthetic C-terminal peptides in conjunction with solid-phase competitive inhibition assays and limited proteolytic digestion of rhodopsin in conjunction with electrophoretic immunoblotting techniques. Binding of the rho 1D4 and rho 3C2 antibodies to immobilized rhodopsin was inhibited with peptides of length 1''-8'' and longer. Antibody rho 1D4 binding was not inhibited by peptides 2''-13'' or 3''-18'', indicating that the C-terminal alanine residue of rhodopsin was required. Similar competitive inhibition studies indicated that the antibody rho 3A6 required peptides of length 1''-12'' and longer whereas rho 1C5 required peptide 1''-18''. Peptide 3''-18'' was as effective as 1''-18'' in inhibiting rho 3A6 binding to rhodopsin, but replacement of glutamic acid in position 8'' with glutamine abolished competition. This substitution had little effect on the binding of antibody rho 1C5. Thus, Glu8'' was essential for rho 3A6 binding but not for the binding of the rho 1C5 antibody. Cleavage of the 7 amino acid C-terminus from rhodopsin and further cleavage to F1 (MW 25,000) and F2 (MW 12,000) fragments with Staphylococcus aureus V8 protease abolished binding of rho 1D4 antibody to the membrane-bound rhodopsin fragments. Antibodies rho 3A6 and rho 1C5, however, were found to bind both rhodopsin lacking the 7 amino acid C-terminus and the corresponding F2 fragment. These results indicate that a peptide longer than 1''-6'' is required for antibodies rho 1D4 and rho 3C2 binding, 8''-12'' for antibody rho 3A6 binding, and 9''-17'' or 9''-18'' for antibody rho 1C5. Since longer peptides and rhodopsin are more effective competitors, other factors such as conformation also affect binding reactivity. These studies in conjunction with related studies on the binding of these antibodies to membrane-bound and detergent solubilized bleached and unbleached rhodopsin indicate that the carboxyl-terminal 1''-18'' segment of rhodopsin is highly accessible to these immunological probes and relatively insensitive to the state of bleaching and solubilization of rhodopsin. The cross-reactivity of these monoclonal antibodies as well as monoclonal antibodies against other regions of bovine rhodopsin was also studied. The C-terminal segment (as specified by the rho 1D4 and rho 3C2 antibodies), the F1-F2 linking region, and the N-terminal region of rhodopsin are highly conserved in pig, dog, cat, rat, rabbit and frog rhodopsins. Segments farther in from the C-terminus as specified by the rho 3A6 and rho 1C5 antibodies are more variable.