Structure of non-(1-84) PTH fragments secreted by parathyroid glands in primary and secondary hyperparathyroidism
Structure of non-(1-84) PTH fragments secreted by parathyroid glands in primary and secondary hyperparathyroidism
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DOI：
10.1111/j.1523-1755.2005.00493.x
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发表时间：
2005-09-01
影响因子：
19.6
通讯作者：
Zahradnik, RJ
中科院分区：
文献类型：
--
作者：
D'Amour, P;Brossard, JH;Zahradnik, RJ
背景非（1-84）甲状旁腺激素（PTH）片段是具有部分保留的氨基末端（N）结构的大循环羧基末端（C）片段。hPTH（7-84）是一种合成替代物，已被证明通过C-PTH受体在体内和体外发挥与hPTH（1-34）对PTH/PTHrP I型受体相反的生物学作用。我们想确定非（1-84）PTH片段的N结构。甲状旁腺细胞分离的腺体在手术中获得的原发性甲状旁腺功能亢进症和继发性甲状旁腺功能亢进症的三名患者与S-35-蛋氨酸孵育，内部标记其分泌产物。在患者离子钙浓度和各种蛋白酶抑制剂存在下孵育8小时。通过高效液相色谱法（HPLC）分级分离上清液，并用分别具有（1至4）和（12至23）个表位的PTH测定法分析级分。对每例患者的血清进行类似分析。将鉴定的免疫反应性峰进行序列分析，以恢复位置8和18处的S-35-甲硫氨酸残基。用PTH测定法鉴定了三个感兴趣的区域。它们对应于非（1-84）PTH片段（进一步分为区域3和4）、迁移到hPTH（1-84）前面的N-PTH峰（区域2）和对应于hPTH（1-84）洗脱位置的免疫反应性峰（区域1）。最后一个对应于从位置1开始的单个序列。区域2在所有情况下给出了类似的结果（在位置1处开始的主要信号），但有时也在位置4或7处开始的次要序列。区域3和4总是鉴定出从位置7开始的主要序列和从位置8、10和15开始的次要序列。令人惊讶的是，在位置1开始的主要信号也存在于区域3中。从给定患者的甲状旁腺细胞中获得的HPLC图谱与在每种情况下用他/她的血清获得的图谱在定性上相似。这些结果表明，非（1-84）PTH片段由一个片段家族组成，该家族可能是由N区的特异性或进行性切割产生的。最长的片段在位置4开始，最短的在位置15。起始于位置7的肽作为非（1-84）PTH片段的主要组分出现。产生过程与多年前描述的较小C-PTH片段的产生过程相似，表明所有C-PTH片段的产生机制和来源相似。
Background. Non-(1-84) parathyroid hormone (PTH) fragments are large circulating carboxyl-terminal (C) fragments with a partially preserved amino-terminal (N) structure. hPTH (7-84), a synthetic surrogate, has been demonstrated to exert biologic effects in vivo and in vitro which are opposite to those of hPTH (1-34) on the PTH/PTHrP type I receptor through a C-PTH receptor. We wanted to determine the N structure of non-(1-84) PTH fragments.Methods. Parathyroid cells isolated from glands obtained at surgery from three patients with primary hyperparathyroidism and three patients with secondary hyperparathyroidism were incubated with S-35-methionine to internally label their secretion products. Incubations were performed for 8 hours at the patient-ionized calcium concentration and in the presence of various protease inhibitors. The supernatant was fractionated by high-performance liquid chromatography (HPLC) and fractions were analyzed with PTH assays having (1 to 4) and (12 to 23) epitopes, respectively. The serum of each patient was similarly analyzed. Peaks of immunoreactivity identified were submitted to sequence analysis to recover the S-35-methionine residues in positions 8 and 18.Results. Three regions of interest were identified with PTH assays. They corresponded to non-(1-84) PTH fragments (further divided in regions 3 and 4), a peak of N-PTH migrating in front of hPTH (1-84) (region 2) and a peak of immunoreactivity corresponding to the elution position of hPTH (1-84) (region 1). The last corresponded to a single sequence starting at position 1. Region 2 gave similar results in all cases (a major signal starting at position 1) but also sometimes minor sequences starting at position 4 or 7. Regions 3 and 4 always identified a major sequence starting at positions 7 and minor sequences starting at positions 8, 10, and 15. Surprisingly, a major signal starting at position 1 was also present in region 3. The HPLC profile obtained from a given patient's parathyroid cells was qualitatively similar to the one obtained with his/her serum in each case.Conclusion. These results indicate that non-(1-84) PTH fragments are composed of a family of fragments which may be generated by specific or progressive cleavage at the N region. The longest fragment starts at position 4 and the shortest at position 15. A peptide starting at position 7 appears as the major component of non-(1-84) PTH fragments. The generation process is similar to the one described for smaller C-PTH fragments a number of years ago, suggesting a similar production mechanism and source for all C-PTH fragments.