Chondroadherin Fragmentation Mediated by the Protease HTRA1 Distinguishes Human Intervertebral Disc Degeneration from Normal Aging
Chondroadherin Fragmentation Mediated by the Protease HTRA1 Distinguishes Human Intervertebral Disc Degeneration from Normal Aging
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DOI：
10.1074/jbc.m112.443010
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发表时间：
2013-06-28
影响因子：
4.8
通讯作者：
Haglund, Lisbet
中科院分区：
文献类型：
--
作者：
Akhatib, Bashar;Onnerfjord, Patrik;Haglund, Lisbet
软骨粘附蛋白是富含亮氨酸的重复蛋白家族的一员，在一些青少年特发性脊柱侧凸中已被证明是碎片化的。这一观察结果使我们对成人椎间盘退变进行了研究。免疫印迹分析显示，来自三个不同年龄组的非退变椎间盘未显示软骨粘附蛋白断裂。此外，通过免疫印迹分析，比较了成人退变椎间盘和青少年脊柱侧凸椎间盘的软骨粘附蛋白片段，发现它们具有相似的大小。然后我们研究了软骨粘附素碎片是否随着椎间盘退变的严重程度而增加。从同一椎间盘中选取不同严重程度的三种不同样本，发现随着退变严重程度的增加，软骨粘附蛋白碎裂更丰富。这一观察结果使我们对观察到的切割位点产生了新表位抗体。然后我们观察到成人退变椎间盘和青少年脊柱侧凸椎间盘的切割位点与新表位抗体证实的相同。因此，有必要研究能够在该位点切割软骨粘附蛋白的蛋白酶。体外消化椎间盘组织表明，ADAMTS-4和-5；组织蛋白酶K、B、L；MMP-3、-7、-12和-13不能在这个位点切割软骨粘附蛋白，HTRA1确实是唯一能切割软骨粘附蛋白的蛋白酶。此外，通过免疫印迹显示，在退行性椎间盘组织中，HTRA1加工形式的蛋白水平升高。结果表明，软骨粘附蛋白断裂可以作为区分椎间盘退变和正常衰老过程的生物标志物。
Chondroadherin, a member of the leucine-rich repeat family, has previously been demonstrated to be fragmented in some juveniles with idiopathic scoliosis. This observation led us to investigate adults with disc degeneration. Immunoblotting analysis demonstrated that non-degenerate discs from three different age groups show no chondroadherin fragmentation. Furthermore, the chondroadherin fragments in adult degenerate disc and the juvenile scoliotic disc were compared via immunoblot analysis and appeared to have a similar size. We then investigated whether or not chondroadherin fragmentation increases with the severity of disc degeneration. Three different samples with different severities were chosen from the same disc, and chondroadherin fragmentation was found to be more abundant with increasing severity of degeneration. This observation led us to the creation of a neoepitope antibody to the cleavage site observed. We then observed that the cleavage site in adult degenerate discs and juvenile scoliotic discs was identical as confirmed by the neoepitope antibody. Consequently, investigation of the protease capable of cleaving chondroadherin at this site was necessary. In vitro digests of disc tissue demonstrated that ADAMTS-4 and -5; cathepsins K, B, and L; and MMP-3, -7, -12, and -13 were incapable of cleavage of chondroadherin at this site and that HTRA1 was indeed the only protease capable. Furthermore, increased protein levels of the processed form of HTRA1 were demonstrated in degenerate disc tissues via immunoblotting. The results suggest that chondroadherin fragmentation can be used as a biomarker to distinguish the processes of disc degeneration from normal aging.