Søe Rikke, Overgaard Michael T, Thomsen Anni R, Laursen Lisbeth S, Olsen Inger M, Sottrup-Jensen Lars, Haaning Jesper, Giudice Linda C, Conover Cheryl A, Oxvig Claus
Department of Molecular and Structural Biology, Science Park, University of Aarhus, Denmark.
Eur J Biochem. 2002 Apr;269(8):2247-56. doi: 10.1046/j.1432-1033.2002.02883.x.
Murine pregnancy-associated plasma protein-A (PAPP-A) cDNA encoding a 1545 amino-acid protein has been cloned. We have also identified and cloned cDNA that encodes a novel variant of PAPP-A, PAPP-Ai, carrying a 29-residue highly basic insert. The point of insertion corresponds to a junction between two exons in the human PAPP-A gene. The human intron flanked by these exons does not encode a homologous corresponding insert, which is unique to the mouse. The overall sequence identity between murine and human PAPP-A is 91%, and murine PAPP-A contains sequence motifs previously described in the sequence of human PAPP-A. Through expression in mammalian cells, we show that murine PAPP-A and PAPP-Ai are active metalloproteinases, both capable of cleaving insulin-like growth factor binding protein (IGFBP)-4 and -5. Cleavage of IGFBP-4 is dramatically enhanced by the addition of IGF, whereas cleavage of IGFBP-5 is slightly inhibited by IGF, as previously established with human PAPP-A. Surprisingly, however, quantitative analyses demonstrate that the murine PAPP-Ai cleaves IGFBP-4 very slowly compared to PAPP-A, even though its ability to cleave IGFBP-5 is unaffected by the presence of the insert. By RT-PCR analysis, we find that both variants are expressed in several tissues. The level of mRNA in the murine placenta does not exceed the levels of other tissues analyzed. Furthermore, the IGFBP-4-proteolytic activity of murine pregnancy serum is not elevated. This is in striking contrast to the increase seen in human pregnancy serum, and the expression of PAPP-A in the human placenta, which exceeds other tissues at least 250-fold. Interestingly, the position of the insert of PAPP-Ai, within the proteolytic domain, lies in close proximity to the cysteine residue, which in human PAPP-A forms a disulfide bond with the proform of eosinophil major basic protein (proMBP). ProMBP functions as a proteinase inhibitor in the PAPP-A-proMBP complex, but whether any mechanistic parallel on regulation of proteolytic activity can be drawn between the insert of PAPP-Ai and the linkage to proMBP is not known. Importantly, these data support the development of the mouse as a model organism for the study of PAPP-A, which must take into account the differences between the mouse and the human.
编码一种含1545个氨基酸的蛋白质的小鼠妊娠相关血浆蛋白-A（PAPP-A）cDNA已被克隆。我们还鉴定并克隆了编码PAPP-A新变体PAPP-Ai的cDNA，该变体带有一个29个残基的高度碱性插入片段。插入点对应于人PAPP-A基因中两个外显子之间的连接处。位于这些外显子两侧的人内含子并不编码同源的相应插入片段，这是小鼠所特有的。小鼠和人PAPP-A之间的总体序列同一性为91%，并且小鼠PAPP-A含有先前在人PAPP-A序列中描述过的序列基序。通过在哺乳动物细胞中的表达，我们表明小鼠PAPP-A和PAPP-Ai都是有活性的金属蛋白酶，两者都能够切割胰岛素样生长因子结合蛋白（IGFBP）-4和-5。如先前用人PAPP-A所证实的那样，添加IGF可显著增强IGFBP-4的切割，而IGF可轻微抑制IGFBP-5的切割。然而，令人惊讶的是，定量分析表明，尽管小鼠PAPP-Ai切割IGFBP-5的能力不受插入片段存在的影响，但与PAPP-A相比，它切割IGFBP-4的速度非常慢。通过RT-PCR分析，我们发现这两种变体在几种组织中都有表达。小鼠胎盘内的mRNA水平不超过所分析的其他组织的水平。此外，小鼠妊娠血清的IGFBP-4蛋白水解活性并未升高。这与在人妊娠血清中所见的增加以及人胎盘中PAPP-A的表达形成鲜明对比，人胎盘中PAPP-A的表达比其他组织至少高250倍。有趣的是，PAPP-Ai的插入片段在蛋白水解结构域内的位置紧邻半胱氨酸残基，在人PAPP-A中，该半胱氨酸残基与嗜酸性粒细胞主要碱性蛋白（proMBP）的前体形成二硫键。ProMBP在PAPP-A-proMBP复合物中作为蛋白酶抑制剂发挥作用，但尚不清楚PAPP-Ai的插入片段与proMBP的连接之间是否能在蛋白水解活性调节方面得出任何机制上的相似之处。重要的是，这些数据支持将小鼠开发为研究PAPP-A的模式生物，这必须考虑到小鼠和人之间的差异。
