DiStefano P S, Chelsea D M, Schick C M, McKelvy J F
Neuroscience Research Division, Abbott Laboratories, Abbott Park, Illinois 60064.
J Neurosci. 1993 Jun;13(6):2405-14. doi: 10.1523/JNEUROSCI.13-06-02405.1993.
The mechanism of low-affinity NGF receptor (LNGFR) truncation was investigated in cultured Schwann cells. Affinity labeling of Schwann cells with 125I-NGF or metabolic labeling with 35S-cysteine showed that truncated NGF receptor (NGF-Rt) was derived from the cell surface form of the receptor. Addition of full-length, exogenous NGF receptor (M(r) = 80 kDa) to Schwann cell membranes resulted in cleavage of the exogenous substrate to NGF-Rt. Investigations into the mechanism of truncation revealed that metalloprotease inhibitors such as phenanthroline, bathophenanthroline, and 8-hydroxyquinoline (8-OHQ) blocked LNGFR truncation in a concentration-dependent fashion. Inhibitors of other protease classes had no effect on truncation. In addition, truncation did not occur at 4 degrees C. It was found that truncation could also occur in Schwann cell membrane preparations, indicating that the putative protease was membrane bound and closely associated with the LNGFR. Metal reconstitution experiments revealed a strong preference toward zinc for the truncating activity, with iron and manganese having slight reconstitution activity in phenanthroline-quenched membranes. To determine if apparent truncation could be inhibited in vivo, the metalloprotease inhibitor 8-OHQ was administered to neonatal rats. 8-OHQ resulted in decreased urine and blood NGF-Rt levels and increased the sciatic nerve LNGFR content; this effect was dose dependent. In adult rats with sciatic nerve crush lesions, 8-OHQ (30-300 mg/kg, t.i.d.) significantly enhanced the rate of sensory neuron regeneration as assessed by the nerve pinch assay. This was accompanied by increased levels of LNGFR in distal nerve segments. These results suggest that Schwann cells possess a metalloprotease-like activity that serves to cleave LNGFR from the surface of these cells. We propose that the putative metalloprotease represents a novel mechanism by which the Schwann cell regulates this particular cell surface protein. Furthermore, increasing the amount of Schwann cell surface LNGFR appears to be of functional significance in that sensory nerve regeneration can be enhanced by inhibition of truncation.
在培养的雪旺细胞中研究了低亲和力神经生长因子受体（LNGFR）截短的机制。用125I-NGF对雪旺细胞进行亲和标记或用35S-半胱氨酸进行代谢标记表明，截短的神经生长因子受体（NGF-Rt）源自受体的细胞表面形式。向雪旺细胞膜中添加全长的外源性神经生长因子受体（分子量=80 kDa）导致外源性底物裂解为NGF-Rt。对截短机制的研究表明，金属蛋白酶抑制剂如菲咯啉、bathophenanthroline和8-羟基喹啉（8-OHQ）以浓度依赖性方式阻断LNGFR截短。其他蛋白酶类别的抑制剂对截短没有影响。此外，在4℃时不发生截短。发现截短也可发生在雪旺细胞膜制剂中，表明假定的蛋白酶是膜结合的且与LNGFR紧密相关。金属重构实验表明，截短活性对锌有强烈偏好，铁和锰在菲咯啉淬灭的膜中具有轻微的重构活性。为了确定在体内是否可以抑制明显的截短，将金属蛋白酶抑制剂8-OHQ给予新生大鼠。8-OHQ导致尿液和血液中NGF-Rt水平降低，并增加坐骨神经LNGFR含量；这种作用是剂量依赖性的。在患有坐骨神经挤压损伤的成年大鼠中，通过神经捏压试验评估，8-OHQ（30-300 mg/kg，每日三次）显著提高了感觉神经元再生的速率。这伴随着远端神经节段中LNGFR水平的增加。这些结果表明，雪旺细胞具有一种金属蛋白酶样活性，可从这些细胞表面裂解LNGFR。我们提出，假定的金属蛋白酶代表了雪旺细胞调节这种特定细胞表面蛋白的一种新机制。此外，增加雪旺细胞表面LNGFR的量似乎具有功能意义，因为通过抑制截短可以增强感觉神经再生。
