血管新生抑制因子软骨调节素I前体蛋白的剪切加工研究

目的研究参与血管新生抑制因子软骨调节素I(ChM-I)前体加工的前蛋白转化酶(PCs)的种类及其作用,探讨ChM-I蛋白的翻译后加工成熟机制。方法构建ChM-I前体蛋白真核表达质粒并转染多种细胞,Western Blot检测细胞裂解液和培养基上清中ChM-I的表达变化;将ChM-I前体蛋白表达质粒与5种不同前蛋白转化酶表达质粒在ChM-I前体加工缺陷型细胞COS7中共表达,Western Blot检测细胞裂解液和培养上清中ChM-I蛋白的表达。结果 ChM-I前体荧光表达质粒构建成功,可在多种细胞系中表达ChM-I前体蛋白;ChM-I前体蛋白在COS7细胞中无法被有效地剪切加工成成熟ChM-I蛋白,可作为ChM-I加工缺陷型细胞使用;除PACE4以外,Furin、ΔFurin、PC5A和PC7均能显著提高培养液上清中ChM-I的含量,并缓解过量表达的ChM-I前体在细胞中的堆积。结论PCs中的Furin、PC5A和PC7能将过表达的ChM-I前体酶切加工为成熟ChM-I,提示Furin、PC5A和PC7可能在ChM-I蛋白剪切加工成熟过程中发挥重要作用。

The research on processing of anti-angiogenesis factor-Chondromoduin-I precursor

Objective To investigate which kind of proprotein convertases involved into the processing of a new anti - angiogenesis factor - Chondromoduin - I and the mechanism. Methods The ChM - I expressing plasmid was constructed,and then co - expressed with different PCs（including Furin,PACE4,PC5A and PC7）in COS7 cells which is deficient in processing of ChM - I precursor. Subsequently,the mature ChM - I in the medium was determined by Western blot. Results The ChM - I precursor expression plasmid was constructed successfully. The plasmid could express ChM - I precursor after transfected into several kind of cells. When co - expressing PCs and ChM - I precursor in the COS7 cells,all other PCs increased the concentration of mature ChM - I in the culture medium except for the PACE4,as well as decreased the accumulation of ChM - I precursor in the cell lysate. Conclusion Furin,PC5A and PC7 in the PCs family can convert ChM - I precursor into mature ChM - I in vitro,which indicated that these PCs may play an important role in the processing of ChM - I precursor in vivo.