Sarkar Anindya, Wintrode Patrick L
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106.
Int J Mass Spectrom. 2011 Apr;302(1-3):69-75. doi: 10.1016/j.ijms.2010.08.003.
Protein glycosylation commonly stabilizes proteins thereby increasing protein half-lives and protecting against denaturation or proteolytic degradation. While generally beneficial, such stabilization is potentially disadvantageous in the case of inhibitory serpins. These protease inhibitors are metastable and a conformational transition to a more stable form is key to their function. Instability is therefore essential for these inhibitory serpins and mutagenesis has demonstrated that substantial stabilization results in compromised function. We have used optical spectroscopy and hydrogen/deuterium exchange and mass spectrometry to investigate the effects of glycosylation on the human serpin alpha-1 antitrypsin (α(1)-AT). Previous studies found that unglycosylated recombinant α(1)-AT populates a molten globule at low denaturant and that the ability to populate this state is correlated with efficient protease inhibition. Further, a high degree of conformational flexibility was found in several important regions. Guanidine hydrochloride denaturation monitored by circular dichroism indicates that plasma α(1)-AT, which is glycosylated at 3 sites, is substantially stabilized relative to the unglycosylated form. However, hydrogen exchange reveals complete loss of protection in plasma α(1)-AT above 1 M GuHCl, similar to what is seen for the recombinant form. Sugars therefore appear to stabilize the compact denatured state of α(1)-AT without significant stabilization of the folded state. Native state hydrogen exchange reveals minor perturbations to native flexibility, but high flexibility in key regions such as the f helix is conserved. β-strand 1c is stabilized in plasma α(1)-AT, which may confer increased resistance to forming pathogenic polymers. Overall, our results indicate that glycosylation of inhibitory serpins does not interfere with either native state flexibility or the native instability that is required for efficient function, though it may confer resistance to degradation by proteases and thus extend the half-life of circulating serpins.
蛋白质糖基化通常会使蛋白质稳定，从而延长蛋白质半衰期，并防止其变性或被蛋白水解降解。虽然这种稳定作用总体上是有益的，但在抑制性丝氨酸蛋白酶抑制剂（serpin）的情况下，这种稳定作用可能是不利的。这些蛋白酶抑制剂是亚稳态的，向更稳定形式的构象转变是其发挥功能的关键。因此，不稳定性对于这些抑制性丝氨酸蛋白酶抑制剂至关重要，诱变实验表明，显著的稳定作用会导致功能受损。我们利用光谱学、氢/氘交换和质谱技术来研究糖基化对人丝氨酸蛋白酶抑制剂α1抗胰蛋白酶（α(1)-AT）的影响。先前的研究发现，未糖基化的重组α(1)-AT在低变性剂浓度下会形成熔球态，且形成这种状态的能力与有效的蛋白酶抑制作用相关。此外，在几个重要区域发现了高度的构象灵活性。通过圆二色性监测的盐酸胍变性实验表明，在3个位点进行糖基化的血浆α(1)-AT相对于未糖基化形式有显著的稳定作用。然而，氢交换实验表明，在1 M盐酸胍以上，血浆α(1)-AT的保护作用完全丧失，这与重组形式的情况类似。因此，糖类似乎稳定了α(1)-AT的紧密变性状态，而对折叠状态没有显著的稳定作用。天然状态下的氢交换实验表明，对天然灵活性有微小扰动，但关键区域（如f螺旋）的高灵活性得以保留。血浆α(1)-AT中的β链1c是稳定的，这可能使其对形成致病聚合物的抵抗力增强。总体而言，我们的结果表明，抑制性丝氨酸蛋白酶抑制剂的糖基化既不干扰天然状态的灵活性，也不干扰有效发挥功能所需的天然不稳定性，尽管它可能赋予对蛋白酶降解的抵抗力，从而延长循环丝氨酸蛋白酶抑制剂的半衰期。
