Polysialic acid, an abundant cell surface component of the developing nervous system, which declines rapidly postnatally to virtual absence in the majority of adult tissues, is highly expressed in some malignant tumors including neuroblastoma. We found that the binding of a noncatalytic endosialidase to polysialic acid causes internalization of the complex from the surface of neuroblastoma kSK-N-SH cells, a subline of SK-N-SH, and leads to a complete relocalization of polysialic acid to the intracellular compartment. The binding and uptake of the endosialidase is polysialic acid–dependent as it is inhibited by free excess ligand or removal of polysialic acid by active endosialidase, and does not happen if catalytic endosialidase is used in place of inactive endosialidase. A fusion protein composed of the noncatalytic endosialidase and the cytotoxic portion of diphtheria toxin was prepared to investigate whether the cellular uptake observed could be used for the specific elimination of polysialic acid–containing cells. The conjugate toxin was found to be toxic to polysialic acid–positive kSK-N-SH with an IC50 of 1.0 nmol/L. Replacing the noncatalytic endosialidase with active endosialidase decreased the activity to the level of nonconjugated toxin. Normal nonmalignant cells were selectively resistant to the toxin conjugate. The results demonstrate that noncatalytic endosialidase induces a quantitative removal and cellular uptake of polysialic acid from the cell surface which, by conjugation with diphtheria toxin fragment, can be exploited for the selective elimination of polysialic acid–containing tumor cells.

中文翻译：

---

使用作用于与毒素融合的聚唾液酸的酶设计细胞毒性神经母细胞瘤靶向剂

聚唾液酸是发育中的神经系统中丰富的细胞表面成分，在大多数成人组织中会在出生后迅速下降至几乎不存在，在包括神经母细胞瘤在内的一些恶性肿瘤中高度表达。我们发现非催化性内切唾液酸酶与聚唾液酸的结合导致复合物从神经母细胞瘤 kSK-N-SH 细胞（SK-N-SH 的一个亚系）表面内化，并导致聚唾液酸完全重新定位到细胞内。隔间。内切唾液酸酶的结合和摄取是聚唾液酸依赖性的，因为它被游离过量配体抑制或被活性内切唾液酸酶去除聚唾液酸，如果使用催化内切唾液酸酶代替无活性的内切唾液酸酶，则不会发生这种情况。制备了一种由非催化性内切唾液酸酶和白喉毒素的细胞毒性部分组成的融合蛋白，以研究观察到的细胞摄取是否可用于特异性消除含聚唾液酸的细胞。发现共轭毒素对聚唾液酸阳性 kSK-N-SH 有毒，IC50 为 1.0 nmol/L。用活性内切唾液酸酶代替非催化内切唾液酸酶将活性降低到非结合毒素的水平。正常的非恶性细胞对毒素偶联物具有选择性抗性。结果表明，非催化性内切唾液酸酶诱导细胞表面定量去除和吸收聚唾液酸，通过与白喉毒素片段结合，可用于选择性消除含聚唾液酸的肿瘤细胞。