Laursen Lisbeth S, Overgaard Michael T, Weyer Kathrin, Boldt Henning B, Ebbesen Peter, Christiansen Michael, Sottrup-Jensen Lars, Giudice Linda C, Oxvig Claus
Department of Molecular Biology, Science Park, University of Aarhus, Aarhus C, Denmark.
J Biol Chem. 2002 Dec 6;277(49):47225-34. doi: 10.1074/jbc.M209155200. Epub 2002 Oct 4.
The activities of insulin-like growth factor (IGF)-I and -II are regulated by IGF-binding proteins (IGFBPs). Cleavage of IGFBP-4 by the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) causes release of bound IGF and has been established in several biological systems including the human reproductive system. Using flow cytometry, we first demonstrate that PAPP-A reversibly binds to the cell surface of several cell types analyzed. Heparin and heparan sulfate, but not dermatan or chondroitin sulfate, effectively compete for PAPP-A surface binding, and because incubation of cells with heparinase abrogated PAPP-A adhesion, binding is probably mediated by a cell surface heparan sulfate proteoglycan. Furthermore, the proteolytic activity of PAPP-A is preserved while bound to cells, suggesting that adhesion functions to target its activity to the vicinity of the IGF receptor, decreasing the probability that released IGF is captured by another IGFBP molecule before receptor binding. This mechanism potentially functions in both autocrine and paracrine regulation, as PAPP-A need not be synthesized in a cell to which it adheres. A truncated PAPP-A variant without the five short consensus repeats in the C-terminal third of the 1547-residue PAPP-A subunit, lacked surface binding. We also show that PAPP-A2, a recently discovered IGFBP-5 proteinase with homology to PAPP-A, does not bind cells. This finding allowed further mapping of the PAPP-A adhesion site to short consensus repeat modules 3 and 4 by the expression and analysis of nine PAPP-A/PAPP-A2 chimeras. Interestingly, the proteolytically inactive, disulfide-bound complex of PAPP-A and the proform of eosinophil major basic protein (proMBP), PAPP-A.proMBP, shows only weak surface binding, probably because the adhesion site of PAPP-A is occupied by heparan sulfate, known to be covalently bound to proMBP. This hypothesis was further substantiated by demonstrating that heparinase treatment of PAPP-A.proMBP restores surface binding. We finally propose a model in which IGF bioactivity is regulated by reversible cell surface binding of PAPP-A, which in turn is regulated by proMBP.
胰岛素样生长因子（IGF）-I和-II的活性受IGF结合蛋白（IGFBP）调节。金属蛋白酶妊娠相关血浆蛋白-A（PAPP-A）对IGFBP-4的切割会导致结合的IGF释放，这已在包括人类生殖系统在内的多个生物系统中得到证实。我们首先通过流式细胞术证明，PAPP-A能可逆地结合到所分析的几种细胞类型的细胞表面。肝素和硫酸乙酰肝素能有效竞争PAPP-A的表面结合，而硫酸皮肤素或硫酸软骨素则不能，并且由于用肝素酶处理细胞可消除PAPP-A的黏附，所以这种结合可能是由细胞表面的硫酸乙酰肝素蛋白聚糖介导的。此外，PAPP-A与细胞结合时其蛋白水解活性得以保留，这表明黏附作用可将其活性靶向至IGF受体附近，从而降低释放的IGF在受体结合前被另一个IGFBP分子捕获的可能性。由于PAPP-A无需在所黏附的细胞中合成，所以这种机制可能在自分泌和旁分泌调节中均发挥作用。在由1547个残基组成的PAPP-A亚基的C末端三分之一区域中没有五个短共有重复序列的截短型PAPP-A变体缺乏表面结合能力。我们还表明，PAPP-A2是一种最近发现的与PAPP-A具有同源性的IGFBP-5蛋白酶，它不结合细胞。通过九个PAPP-A/PAPP-A2嵌合体的表达和分析，这一发现进一步将PAPP-A的黏附位点定位到短共有重复模块3和4。有趣的是，PAPP-A与嗜酸性粒细胞主要碱性蛋白原（proMBP）的蛋白水解无活性、二硫键结合的复合物PAPP-A.proMBP仅表现出较弱的表面结合，这可能是因为PAPP-A的黏附位点被已知与proMBP共价结合的硫酸乙酰肝素占据。通过证明用肝素酶处理PAPP-A.proMBP可恢复表面结合，这一假设得到了进一步证实。我们最终提出了一个模型，其中IGF的生物活性受PAPP-A可逆性细胞表面结合的调节，而PAPP-A又受proMBP的调节。
