Department of Cellular and Physiological SciencesThe University of British Columbia, Vancouver, British Columbia, Canada.
Department of Cellular and Physiological SciencesThe University of British Columbia, Vancouver, British Columbia, Canada
J Mol Endocrinol. 2018 Feb;60(2):71-84. doi: 10.1530/JME-17-0234. Epub 2017 Dec 22.
Corticosteroid-binding globulin (CBG) is a plasma carrier of glucocorticoids. Human and rat CBGs have six -glycosylation sites. Glycosylation of human CBG influences its steroid-binding activity, and there are -glycosylation sites in the reactive center loops (RCLs) of human and rat CBGs. Proteolysis of the RCL of human CBG causes a structural change that disrupts steroid binding. We now show that mutations of conserved -glycosylation sites at N238 in human CBG and N230 in rat CBG disrupt steroid binding. Inhibiting glycosylation by tunicamycin also markedly reduced human and rat CBG steroid-binding activities. Deglycosylation of fully glycosylated human CBG or human CBG with only one -glycan at N238 with Endo H-reduced steroid-binding affinity, while PNGase F-mediated deglycosylation does not, indicating that steroid binding is preserved by deamidation of N238 when its -glycan is removed. When expressed in -acetylglucosaminyltransferase-I-deficient Lec1 cells, human and rat CBGs, and a human CBG mutant with only one glycosylation site at N238, have higher (2-4 fold) steroid-binding affinities than when produced by sialylation-deficient Lec2 cells or glycosylation-competent CHO-S cells. Thus, the presence and composition of an -glycan in this conserved position both appear to influence the steroid binding of CBG. We also demonstrate that neutrophil elastase cleaves the RCL of human CBG and reduces its steroid-binding capacity more efficiently than does chymotrypsin or the protease LasB. Moreover, while glycosylation of N347 in the RCL limits these activities, -glycans at other sites also appear to protect CBG from neutrophil elastase or chymotrypsin.
皮质类固醇结合球蛋白（CBG）是糖皮质激素的血浆载体。人和大鼠 CBG 有 6 个糖基化位点。人 CBG 的糖基化影响其类固醇结合活性，并且人 CBG 和大鼠 CBG 的反应中心环（RCL）中有 4 个糖基化位点。人 CBG 的 RCL 蛋白水解会导致结构改变，从而破坏类固醇结合。我们现在表明，人 CBG 的 N238 和大鼠 CBG 的 N230 处保守的 N-糖基化位点的突变会破坏类固醇结合。用衣霉素抑制糖基化也显著降低了人 CBG 和大鼠 CBG 的类固醇结合活性。对完全糖基化的人 CBG 或只有一个 N238 处 -糖基的人 CBG 进行内肽酶 H 去糖基化会降低类固醇结合亲和力，而 PNGase F 介导的去糖基化则不会，这表明当 N238 的 -糖基被去除时，去酰胺化会保留类固醇结合。当在缺乏 -乙酰氨基葡萄糖转移酶-I 的 Lec1 细胞中表达时，人 CBG、大鼠 CBG 以及只有一个 N238 糖基化位点的人 CBG 突变体的类固醇结合亲和力比在唾液酸化缺陷的 Lec2 细胞或糖基化能力强的 CHO-S 细胞中表达时高（2-4 倍）。因此，该保守位置处 -聚糖的存在和组成似乎都影响 CBG 的类固醇结合。我们还证明，中性粒细胞弹性蛋白酶裂解人 CBG 的 RCL，并比胰凝乳蛋白酶或蛋白酶 LasB 更有效地降低其类固醇结合能力。此外，虽然 RCL 中 N347 的糖基化限制了这些活性，但其他部位的 -聚糖似乎也能保护 CBG 免受中性粒细胞弹性蛋白酶或胰凝乳蛋白酶的侵害。
