Processing of mutated proinsulin with tetrabasic cleavage sites to mature insulin in non-endocrine nell lines

胰岛素在胰腺β细胞中作为前体胰岛素原产生，其包含由两对碱性残基按以下顺序连接的三个肽：B链-Arg-Arg-C肽-Lys-Arg-A链。胰岛素原通过高尔基体转运至未成熟型分泌囊泡时，C肽在其邻近的二元残基处被从胰岛素原上切割下来。从胰岛素原到胰岛素的转化是内分泌细胞的独特功能。许多前肽激素cDNA，包括人胰岛素原cDNA，已被引入到内分泌和非内分泌细胞中，并且在内分泌细胞中表达的那些通常被正确加工，而在非内分泌细胞中表达的其他前肽组成性地分泌为非切割的前肽。然而，非内分泌细胞无法将胰岛素原转化为胰岛素并不意味着它们无法将其他前肽加工成更小的生物活性肽。非内分泌细胞，包括成纤维细胞 ...更多信息 s、肝细胞和淋巴细胞产生无生物活性的前肽，并通过切割独特的共有序列-Arg^<-4>-X^<-3>-Lys/Arg^<-2>-Arg^<-1>*X^&lt;+1&gt;-将其转化为生物活性肽。该序列被枯草杆菌蛋白酶样内切蛋白酶弗林蛋白酶很好地切割，这是非内分泌细胞的特征。因此，我们创建了突变胰岛素原DNA，其中肽结构由B链和A链按照以下顺序通过一对四碱基残基连接到C肽：B链-Arg-Arg-Lys-Arg-C肽-Arg-Arg-Lys-Arg-A链。当突变体胰岛素表达时，约60%的总免疫反应性胰岛素在培养基中以成熟胰岛素的形式出现。天然和突变胰岛素原均在以下四种细胞系中表达：猴肾衍生细胞系COS-7、中国仓鼠卵巢衍生细胞系CHO、人肝癌衍生细胞系HepG 2和小鼠成纤维细胞样细胞系NIH 3 T3。我们使用这些细胞系，因为它们含有不同数量的弗林蛋白酶mRNA，排名如下：当突变胰岛素在这些细胞中表达时，NIH 3 T3中胰岛素原转化为成熟胰岛素的转化率约为85%，HepG 2中约为70%，COS中约为60%，在CHO中转化率为50%。转化率与每个细胞系中弗林蛋白酶的表达密切相关，如通过其北方印迹带的密度所测量的。此外，在CHO中，具有最低弗林蛋白酶表达的细胞系，突变体胰岛素原与弗林蛋白酶的共表达导致胰岛素原完全转化为成熟胰岛素。COS细胞中产生的胰岛素通过将3-O-[^3H]甲基-D-葡萄糖掺入脂肪细胞的能力而呈现出与合成人胰岛素相同的生物活性。少

Insulin is produced in the pancreatic beta-cells as a precursor proinsulin that comprises of the three peptides linked by two pairs of basic residues in the following order : B chain-Arg-Arg-C peptide-Lys-Arg-A chain. C peptide is cleaved off from proinsulin at the dibasic residues adjacent to C peptide during its transport through the trans-Golgi networks to immature type secretory vesicles. The conversion from proinsulin to insulin is a unique function of endocrine cells. A number of propeptide hormone cDNAs, including a human proinsulin cDNA,have been introduced into both endocrine and non-endocrine cells, and those expressed in endocrine cells were generally processed correctly, while others expressed in non-endocrine cells were secreted constitutively as non-cleaved propeptides. However, inability of non-endocrine cells to convert proinsulin to insulin does not mean their inability to process other propeptides to smaller bioactive peptides. Non-endocrine cells including fibroblast … More s, hepatocytes, and lymphocytes produce biologically inactive propeptides and convert them to bioactive peptides by cleaving a unique consensus sequence -Arg^<-4>-X^<-3>-Lys/Arg^<-2>-Arg^<-1>*X^<+1>-. This sequence is well cleaved by the subtilisin-like endoprotease furin that is characteristic of non-endocrine cells. Thus, we created a mutant proinsulin DNA where peptide structure was comprized of B and A chains linked to C peptide by a pair of tetrabasic residues in the following order : B chain-Arg-Arg-Lys-Arg-C peptide-Arg-Arg-Lys-Arg-A chain. When mutant insulin was expressed, approximately 60% of the total immunoreactive insulin appeared as mature insulin in the culture medium. Both the native and mutant proinsulins were expressed in the following four cell lines : a monkey kidney-derived cell line COS-7, a Chinese hamster ovary-derived cell line CHO,a human liver cancer-derived cell line HepG2 and a mouse fibroblast-like cell line NIH3T3. We used these cell lines because they contain different quantities of furin mRNA,ranking as follows : NIH3T3>HepG2>COS>CHO.When mutant insulin was expressed in these cells, the conversion of proinsulin to mature insulin was approximately 85% in NIH3T3,70% in HepG2,60% in COS,and 50% in CHO.The conversion correlated well with the furin expression in each cell line as measured by the density of its Northern blot band. Moreover, in CHO,the cell line with the lowest furin expression, co-expression of mutant proinsulin with furin resulted in complete conversion of proinsulin to mature insulin. The insulin produced in COS cells presented an identical biological activity to a synthetic human insulin by the capability of incorporating 3-O-[^3H] methyl-D-glucose into adipocytes. Less