Dey Arunangsu, Lipkind Gregory M, Rouillé Yves, Norrbom Christina, Stein Jeffrey, Zhang Chunling, Carroll Raymond, Steiner Donald F
Department of Biochemistry and Molecular Biology, Uni-versity of Chicago, Chicago, Illinois 60637, USA.
Endocrinology. 2005 Feb;146(2):713-27. doi: 10.1210/en.2004-1118. Epub 2004 Nov 4.
To define the biological significance of the initial cleavage at the proglucagon (PG) interdomain site, K70-R71 downward 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-R71 downward arrow and R107-X-R-R110 downward arrow in PG. We show a much greater ability of furin to cleave the monobasic site, R77 downward arrow, than at the dibasic site, R124-R125 downward 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.
为了确定胰高血糖素原（PG）结构域间位点K70-R71处初始切割的生物学意义，我们构建了两个结构域间突变体，K70Q-R71Q和R71A。在GH4C1细胞中的共转染研究表明，野生型PG的PC2能产生大量胰高血糖素，同时还产生一些甘丙素、甘丙素相关多肽-胰高血糖素（GRPP-胰高血糖素）和胃动素。相比之下，结构域间突变体主要产生一种更大的肽，PG 33-158，以及少量的GRPP-胰高血糖素。高效液相色谱分析显示，与结构域间突变体相比，野生型PG的PC2产生的胰高血糖素增加了5倍，未加工前体的积累和分泌相应减少了4倍。PC2从甘丙素（PG 1-69）表达质粒中产生大量胰高血糖素，而PC1/3仅产生少量胃动素。利用一个主要的PG片段（PG 72-158）表达质粒，我们发现PC1/3主要产生胰高血糖素样肽（GLP）-1，而PC2仅产生N端延伸的GLP-1。令人惊讶的是，与野生型PG相比，PC1/3从结构域间突变体产生GLP-1和GLP-2的能力并未受到显著损害。除了PC2和PC1/3外，PC5/6A和弗林蛋白酶也能够切割PG中的K70-R71和R107-X-R-R110位点。我们发现，弗林蛋白酶切割单碱性位点R77的能力比切割双碱性位点R124-R125的能力强得多，PC5/6A对双碱性位点的切割作用较弱，这表明这两种转化酶的特异性主要与PC1/3重叠。我们在此提出一个PG分子内激素序列空间组织的三聚体样模型，其中除了结构域间位点K70-R71可最大限度地被激素原转化酶作用外，大多数切割位点对激素原转化酶作用的可及性受到限制。
