THE HETEROGENEITY OF HUMAN CHORIONIC-GONADOTROPIN (HCG) .3. THE OCCURRENCE AND BIOLOGICAL AND IMMUNOLOGICAL ACTIVITIES OF NICKED HCG
THE HETEROGENEITY OF HUMAN CHORIONIC-GONADOTROPIN (HCG) .3. THE OCCURRENCE AND BIOLOGICAL AND IMMUNOLOGICAL ACTIVITIES OF NICKED HCG
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DOI：
10.1210/endo-129-3-1559
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发表时间：
1991-09-01
期刊：
影响因子：
4.8
通讯作者：
BIRKEN, S
中科院分区：
文献类型：
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作者：
COLE, LA;KARDANA, A;BIRKEN, S
在hCG β-亚基残基44和45之间以及残基47和48之间发现了缺口或缺失的肽键。 我们检测了缺口hCG的发生及其生物学和免疫学活性。序列分析显示，CR 127标准hCG约有20%是缺口的，一半在β-44-45，一半在β-47-48。 用人白细胞弹性蛋白酶处理增加了CR 127标准hCG的切口程度。 CR 127标准品与人白细胞弹性蛋白酶的孵育时间越长（0、2和21 h），缺口hCG的程度越大（20%、46%和89%）。 随着切口程度的增加，受体结合能力减弱，体外大鼠黄体细胞刺激孕酮产生的能力也减弱（分别为0.9、0.74和0.29 μ g/μ g hCG）。 在回归分析中，切口程度和受体结合值之间（97%相关性）以及切口程度和体外类固醇生成活性之间（99%相关性）存在线性关系。 从回归线的截距，据估计，缺口减少受体结合的11倍，减少类固醇生成活性的人绒毛膜促性腺激素的5倍，我们检查了8个人绒毛膜促性腺激素制剂，三个纯化从妊娠尿，三个从患者的尿与葡萄胎，和两个从女性绒毛膜癌的尿液。 按降序排列，8份单独hCG制备物为100%、100%、85%、76%、42%、41%、0%和0%完整。 尽管未观察到完整百分比与8个单独样品在结合CG/LH受体中置换50% [I-125]hCG的能力之间的相关性（r < 0.5），但观察到完整百分比与体外类固醇生成活性之间的密切相关性（98%相关性）。 这分离的切口对受体结合和类固醇生成活性的影响，并表明，虽然多种因素影响受体结合，只有切口抑制类固醇生成活性的结合hCG。我们研究了不同的hCG免疫测定的识别切口hCG分子。 Hybritech Tandem测定法测量总hCG，并且不能区分有切口的和完整的hCG分子（在回归分析中，免疫活性相对于完整的hCG百分比，r < 0.5）。 相反，使用B109 hCG二聚体特异性单克隆抗体和抗β-过氧化物酶的免疫测定法仅检测到hCG的完整组分（在回归分析中，免疫反应性与完整hCG百分比，98%相关性）。 我们一起使用这些测定来估计完整hCG的百分比并推断切口的程度。 在38个平行组的血清和尿液样本中，20个来自妊娠，18个来自滋养层疾病患者，完整hCG的百分比检测到广泛的变化（范围，6-100%）。 血清和尿液中完整hCG百分比的平均值分别为72 +/- 22%和76 +/-35%（平均值+/- SD; P > 0.5）。 妊娠样本（71 +/- 26%）和滋养层疾病样本（77 +/- 33%）中完整hCG的百分比没有显着差异（P > 0.3）。 我们的结论是，缺口hCG帐户，平均而言，约四分之一的总hCG在血清和urine.The发现的主要hCG成分，只有最小的类固醇生成活性是新的，并提出了有关的生理或调节功能的缺口和其变化的医学意义的问题。 此外，使用二聚体特异性单克隆抗体的hCG测定法仅测量完整hCG的发现对目前使用这种类型抗体的许多测定法的结果提出了质疑。
Nicks, or missing peptide linkages, have been found in hCG beta-subunit between residues 44 and 45 and between residues 47 and 48. We examined the occurrence and biological and immunological activities of nicked hCG.As shown by sequence analysis, CR127 standard hCG is approximately 20% nicked, half at beta-44-45 and half at beta-47-48. Treatment with human leukocyte elastase increased the extent of nicking of CR127 standard hCG. The longer the incubation of CR127 standard with human leukocyte elastase (0, 2, and 21 h), the greater the extent of nicked hCG (20%, 46%, and 89%). As the extent of nicking increased, the receptor-binding ability diminished, as did the ability to stimulate progesterone production by rat corpus luteal cells in vitro (0.9, 0.74, and 0.29-mu-g/mu-g hCG, respectively). In a regression analysis, a linear relationship was indicated between the extent of nicking and receptor binding values (97% correlation) and between the extent of nicking and steroidogenic activity in vitro (99% correlation). From the intercepts of the regression lines, it was estimated that nicks reduced receptor binding by 11-fold and reduced the steroidogenic activity of hCG by 5-fold.We examined eight individual hCG preparations, three purified from pregnancy urine, three from urine from patients with hydatidiform mole, and two from urine from women with choriocarcinoma. In descending order, the eight individual hCG preparations were 100%, 100%, 85%, 76%, 42%, 41%, 0%, and 0% intact. Although no correlation was observed between the percent intact and the ability of the eight individual samples to displace 50% [I-125]hCG in binding CG/LH receptor (r < 0.5), a close correlation was noted between the percent intact and the steroidogenic activity in vitro (98% correlation). This separated the effects of nicking on receptor binding and steroidogenic activities and indicated that while multiple factors influence receptor binding, only nicking suppresses the steroidogenic activity of bound hCG.We examined the recognition of nicked hCG molecules by different hCG immunoassays. The Hybritech Tandem assay measured total hCG and did not distinguish nicked and intact hCG molecules (in a regression analysis, immunoactivity vs. percent intact hCG, r < 0.5). In contrast, the immunometric assay using B109 hCG dimer-specific monoclonal antibody and anti-beta-peroxidase only detected the intact component of hCG (in a regression analysis, immunoreactivity vs. percent intact hCG, 98% correlation). We used these assays together to estimate the percentage of intact hCG and to deduce the extent of nicking. In 38 parallel sets of serum and urine samples, 20 from pregnancy and 18 from patients with trophoblast disease, wide variation was detected in the percentage of intact hCG (range, 6-100%). Mean values for the percentage of intact hCG in serum and urine were 72 +/- 22% and 76 +/- 35%, respectively (mean +/- SD; P > 0.5). The percentage of intact hCG was not significantly different in pregnancy (71 +/- 26%) and trophoblast disease (77 +/- 33%) samples (P > 0.3). We concluded that nicked hCG accounts, on the average, for approximately one quarter of the total hCG in serum and urine.The finding of a major hCG component with only minimal steroidogenic activity is novel and raises issues concerning the physiological or regulatory function of nicking and the medical significance of variations thereof. Also, the finding that a hCG assay using a dimer-specific monoclonal antibody only measures intact hCG calls into question the results from many assays in current use that employ this type of antibody.