Nandi A, Suguna K, Surolia A, Visweswariah S S
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore.
Protein Sci. 1998 Oct;7(10):2175-83. doi: 10.1002/pro.5560071015.
Guanylyl cyclase C (GCC) is the receptor for the gastrointestinal hormones, guanylin, and uroguanylin, in addition to the bacterial heat-stable enterotoxins, which are one of the major causes of watery diarrhea the world over. GCC is expressed in intestinal cells, colorectal tumor tissue and tumors originating from metastasis of the colorectal carcinoma. We have earlier generated a monoclonal antibody to human GCC, GCC:B10, which was useful for the immunohistochemical localization of the receptor in the rat intestine (Nandi A et al., 1997, J Cell Biochem 66:500-511), and identified its epitope to a 63-amino acid stretch in the intracellular domain of GCC. In view of the potential that this antibody has for the identification of colorectal tumors, we have characterized the epitope for GCC:B10 in this study. Overlapping peptide synthesis indicated that the epitope was contained in the sequence HIPPENIFPLE. This sequence was unique to GCC, and despite a short stretch of homology with serum amyloid protein and pertussis toxin, no cross reactivity was detected. The core epitope was delineated using a random hexameric phage display library, and two categories of sequences were identified, containing either a single, or two adjacent proline residues. No sequence identified by phage display was identical to the epitope present in GCC, indicating that phage sequences represented mimotopes of the native epitope. Alignment of these sequences with HIPPENIFPLE suggested duplication of the recognition motif, which was confirmed by peptide synthesis. These studies allowed us not only to define the requirements of epitope recognition by GCC:B10 monoclonal antibody, but also to describe a novel means of epitope recognition involving topological mimicry and probable duplication of the cognate epitope in the native guanylyl cyclase C receptor sequence.
鸟苷酸环化酶C（GCC）是胃肠激素鸟苷素和尿鸟苷素的受体，此外还是细菌热稳定肠毒素的受体，而细菌热稳定肠毒素是全球水样腹泻的主要原因之一。GCC在肠道细胞、结直肠肿瘤组织以及源自结直肠癌转移的肿瘤中表达。我们之前制备了一种针对人GCC的单克隆抗体GCC:B10，它可用于在大鼠肠道中对该受体进行免疫组织化学定位（Nandi A等人，1997年，《细胞生物化学杂志》66:500 - 511），并确定其表位位于GCC细胞内结构域的一段63个氨基酸的序列中。鉴于该抗体在识别结直肠肿瘤方面的潜力，我们在本研究中对GCC:B10的表位进行了表征。重叠肽合成表明该表位包含在序列HIPPENIFPLE中。此序列是GCC所特有的，尽管与血清淀粉样蛋白和百日咳毒素有一小段同源性，但未检测到交叉反应。使用随机六聚体噬菌体展示文库确定了核心表位，鉴定出两类序列，一类含有单个脯氨酸残基，另一类含有两个相邻的脯氨酸残基。通过噬菌体展示鉴定出的序列与GCC中存在的表位均不相同，表明噬菌体序列代表天然表位的模拟表位。这些序列与HIPPENIFPLE的比对表明识别基序存在重复，这通过肽合成得到了证实。这些研究不仅使我们能够确定GCC:B10单克隆抗体识别表位所需的条件，还描述了一种涉及拓扑模拟和天然鸟苷酸环化酶C受体序列中同源表位可能重复的新型表位识别方式。
