Parhami-Seren B, Keel T, Reed G L
Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
J Mol Biol. 1997 Aug 22;271(3):333-41. doi: 10.1006/jmbi.1997.1174.
Though streptokinase (SK) is widely used to treat humans with thrombotic disease, it is antigenic and anti-SK antibody causes allergic reactions and neutralizes SK's therapeutic effects. To pinpoint the fine structure of two immunodominant, continuous epitopes in SK, we used unconstrained 15 and 6-mer random peptide libraries displayed on phage (theoretical complexity of 3.2 x 10(19) and 0.64 x 10(8) unique sequences). The first epitope, recognized by both human Ab and murine monoclonal (m)Abs, was previously localized to the amino terminus of SK. Repeated panning and selection experiments against a 15-mer peptide phage library, using a representative mAb (A2.5) to this epitope, identified a dominant structural motif (GP[R/L]WL) corresponding to amino acids 3 to 7 of native SK, which was consistent with previous epitope mapping. These findings were further confirmed by: (1) the fact that a synthetic peptide spanning the epitope of A2.5 (AGPEWLL) specifically inhibited the binding of A2.5 to SK and (2) the finding that mAb 9D10, which competes with mAb A2.5 for binding to SK, independently selected, from a different random hexamer library, an epitope sequence spanning residues 4 to 9 that overlaps the A2.5 epitope. Similar studies of the second epitope in SK, which is immunodominant for murine but not human antibodies, identified a consensus sequence KS(K/L)P(F/Y) corresponding to amino acids 59 to 63 of SK; this was confirmed by epitope peptide binding experiments. This epitope is cleaved and destroyed when SK reacts with human but not murine plasminogen. Thus, pinpointing the sequences of antigenic epitopes of SK: (1) provides a potential explanation for species differences in SK's antigenicity, (2) demonstrates the overlapping fine structure of epitopes recognized by competitive mAbs, (3) confirms previous epitope mapping studies and (4) has the potential to identify antigenic sequences that lead to allergic reactions in patients treated with SK.
尽管链激酶（SK）被广泛用于治疗患有血栓性疾病的人类，但它具有抗原性，抗SK抗体可引起过敏反应并中和SK的治疗效果。为了确定SK中两个免疫显性连续表位的精细结构，我们使用了展示在噬菌体上的无约束15肽和6肽随机肽库（理论复杂度分别为3.2×10¹⁹和0.64×10⁸个独特序列）。第一个表位，可被人源抗体和鼠源单克隆抗体识别，先前已定位到SK的氨基末端。使用针对该表位的代表性单克隆抗体（A2.5），对15肽噬菌体库进行反复淘选和筛选实验，确定了一个与天然SK氨基酸3至7相对应的显性结构基序（GP[R/L]WL），这与先前的表位定位一致。这些发现通过以下方式进一步得到证实：（1）一条跨越A2.5表位的合成肽（AGPEWLL）特异性抑制A2.5与SK的结合；（2）发现与单克隆抗体A2.5竞争结合SK的单克隆抗体9D10，从不同的随机六肽库中独立选择了一个跨越残基4至9的表位序列，该序列与A2.5表位重叠。对SK中第二个表位的类似研究，该表位对鼠源抗体而非人源抗体具有免疫显性，确定了一个与SK氨基酸59至63相对应的共有序列KS(K/L)P(F/Y)；这通过表位肽结合实验得到了证实。当SK与人源而非鼠源纤溶酶原反应时，这个表位会被切割并破坏。因此，确定SK抗原表位的序列：（1）为SK抗原性的物种差异提供了潜在解释；（2）证明了竞争性单克隆抗体识别的表位具有重叠的精细结构；（3）证实了先前的表位定位研究；（4）有可能识别出在用SK治疗的患者中导致过敏反应的抗原序列。
