Significance of prohormone convertase 2, PC2, mediated initial cleavage at the proglucagon interdomain site, Lys70-Arg71, to generate glucagon
Significance of prohormone convertase 2, PC2, mediated initial cleavage at the proglucagon interdomain site, Lys70-Arg71, to generate glucagon
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DOI：
10.1210/en.2004-1118
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发表时间：
2005-02-01
期刊：
影响因子：
4.8
通讯作者：
Steiner, DF
中科院分区：
文献类型：
--
作者：
Dey, A;Lipkind, GM;Steiner, DF
为了确定胰高血糖素原（PG）结构域间位点（K70-R71向下箭头）处的初始切割的生物学意义，我们创建了两个结构域间突变体，K70 Q-R71 Q和R71 A。在GH4 C1细胞中的共转染研究显示，PC 2沿着有大量的胰高血糖素产生，同时有来自野生型PG的一些胰高血糖素、胰高血糖素相关多肽-胰高血糖素（GRPP-胰高血糖素）和胃泌酸调节素。相反，较大的肽PG 33-158和少量的GRPP-胰高血糖素主要由结构域间突变体产生。HPLC分析显示，相对于结构域间突变体，PC 2从野生型PG产生的胰高血糖素增加5倍，未加工前体的积累和分泌相应降低4倍。PC 2从胰高血糖素（PG 1-69）表达质粒产生显著水平的胰高血糖素，而PC 1/3仅产生适度量的胃泌酸调节素。采用主要的PG片段（PG 72-158）表达质粒，我们表明，PC 1/3主要产生胰高血糖素样肽（GLP）-1，而PC 2只产生N-末端延伸的GLP-1。令人惊讶的是，与野生型PG相比，来自结构域间突变体的PC 1/3的GLP-1和GLP-2的产生没有显著受损。除了PC 2和PC 1/3，PC 5/6A和弗林蛋白酶也能够切割PG中的位点，K70-R71下箭头和R107-X-R-R110下箭头。我们显示弗林蛋白酶切割单碱基位点R77下箭头的能力比切割双碱基位点R124-R125下箭头的能力大得多，双碱基位点R124-R125下箭头也被PC 5/6A弱加工，表明这两种转化酶主要与PC 1/3的特异性重叠。在这里，我们提出了一个三聚体样模型的PG分子内的激素序列的空间组织中，大多数切割位点的激素原转化酶作用的可访问性是有限的，除了域间的网站，K70-R71，这是最大限度地访问。
To define the biological significance of the initial cleavage at the proglucagon (PG) interdomain site, K70-R71down arrow, we created two interdomain mutants, K70Q-R71Q and R71A. Cotransfection studies in GH4C1 cells show significant amounts of glucagon production by PC2 along with some glicentin, glicentin-related polypeptide-glucagon (GRPP-glucagon) and oxyntomodulin from wild-type PG. In contrast, a larger peptide, PG 33-158, and low amounts of GRPP-glucagon are predominantly generated from interdomain mutants. HPLC analysis shows a 5-fold increase in glucagon production by PC2 from wild-type PG and a corresponding 4-fold lower accumulation and secretion of unprocessed precursor relative to interdomain mutants. PC2 generates significant levels of glucagon from a glicentin (PG 1-69) expression plasmid, whereas PC1/3 produces only modest amounts of oxyntomodulin. Employing a major PG fragment (PG 72-158) expression plasmid, we show that PC1/3 predominantly generates glucagon-like peptide (GLP)-1, whereas PC2 produces only N-terminally extended GLP-1. Surprisingly, production of GLP-1 and GLP-2 by PC1/3 from interdomain mutants, compared with wild-type PG, is not significantly impaired. In addition to PC2 and PC1/3, PC5/6A and furin are also able to cleave the sites, K70-R71down arrow and R107-X-R-R110down arrow in PG. We show a much greater ability of furin to cleave the monobasic site, R77down arrow, than at the dibasic site, R124-R125down arrow, which is also weakly processed by PC5/6A, indicating overlapping specificities of these two convertases mainly with PC1/3. We propose here a trimer-like model of the spatial organization of the hormonal sequences within the PG molecule in which the accessibility to prohormone convertase action of most cleavage sites is restricted with the exception of the interdomain site, K70-R71, which is maximally accessible.