Masure S, Paemen L, Van Aelst I, Fiten P, Proost P, Billiau A, Van Damme J, Opdenakker G
Rega Institute for Medical Research, Laboratory of Molecular Immunology, University of Leuven, Belgium.
Eur J Biochem. 1997 Feb 15;244(1):21-30. doi: 10.1111/j.1432-1033.1997.00021.x.
Gelatinase B is a matrix metalloproteinase involved in tissue remodelling. When mouse cells are triggered in vitro with interleukin-1, bacterial endotoxin, virus-mimicking double-stranded RNA or cytokine inducers, they produce gelatinase B. To test the effects of gelatinase B in vivo, the enzyme was expressed in Chinese hamster ovary (CHO) cells. Hybrid genomic DNA-cDNA constructs under the control of two constitutive viral promoters were generated by PCR-mediated exon amplification. In vitro transcription and translation of the mRNA in reticulocyte lysate yielded the correct 79-kDa protein, and expression in CHO cells resulted in an intact glycosylated 110-kDa gelatinase B which was enzymically active. However, the production yields of recombinant enzyme from 50 tested clones were low and cell-culture supernatants contained significant amounts of copurifiable endogenous CHO gelatinase B. Therefore, the enzyme was expressed in the yeast Pichia pastoris. Recombinant proenzyme was secreted and recovered from the yeast culture medium at 10 mg/l. Amino-terminal sequence analysis indicated that affinity purification of the recombinant protein on gelatin-Sepharose yielded the expected N-glycosylated proenzyme form (110 kDa) in addition to an amino-terminally truncated unglycosylated variant (69 kDa). Both forms had gelatinolytic activity on zymography. The recombinant mouse gelatinase B was used to determine its pharmacokinetics and its haematological effects in vivo. After intravenous injection in rabbits, gelatinase B disappeared from the circulation within 6 h. In addition to a transient leukopenia, we observed a rapid increase in leukocytosis, which indicates that gelatinase B might be a factor involved in the desorption of adherent leukocytes from the vascular bed and in the release of leukocytes from the bone marrow. Gelatinase B secretion and activation might well be one of the crucial molecular mechanisms explaining leukocytosis which is associated with infections and almost all types of inflammation.
明胶酶B是一种参与组织重塑的基质金属蛋白酶。当小鼠细胞在体外被白细胞介素-1、细菌内毒素、模拟病毒的双链RNA或细胞因子诱导剂触发时，它们会产生明胶酶B。为了测试明胶酶B在体内的作用，该酶在中国仓鼠卵巢（CHO）细胞中表达。通过PCR介导的外显子扩增，在两个组成型病毒启动子的控制下生成了杂交基因组DNA-cDNA构建体。网织红细胞裂解物中mRNA的体外转录和翻译产生了正确的79 kDa蛋白，在CHO细胞中的表达产生了完整的糖基化110 kDa明胶酶B，其具有酶活性。然而，50个测试克隆中重组酶的产量较低，细胞培养上清液中含有大量可共纯化的内源性CHO明胶酶B。因此，该酶在酵母毕赤酵母中表达。重组酶原被分泌并从酵母培养基中以10 mg/l的浓度回收。氨基末端序列分析表明，在明胶-琼脂糖上对重组蛋白进行亲和纯化，除了产生预期的N-糖基化酶原形式（110 kDa）外，还产生了氨基末端截短的非糖基化变体（69 kDa）。两种形式在酶谱分析中均具有明胶水解活性。重组小鼠明胶酶B被用于确定其体内药代动力学和血液学效应。在兔子静脉注射后，明胶酶B在6小时内从循环中消失。除了短暂的白细胞减少外，我们还观察到白细胞增多迅速增加，这表明明胶酶B可能是参与粘附白细胞从血管床解吸附以及白细胞从骨髓释放的一个因素。明胶酶B的分泌和激活很可能是解释与感染和几乎所有类型炎症相关的白细胞增多的关键分子机制之一。
