Chronic Disease, Ottawa Hospital Research Institute, University of Ottawa, Ottawa, Ontario, Canada.
J Cell Physiol. 2011 Nov;226(11):2817-26. doi: 10.1002/jcp.22626.
Proprotein convertase subtilisin/kexin 4 (PCSK4) is implicated for sperm fertilizing ability, based on studies using Pcsk4-null mice. Herein we demonstrated proprotein convertase (PC) activity in intact sperm and acrosomal vesicles. To determine whether this activity was important for sperm fertilizing ability, a peptide inhibitor was designed based on PCSK4 prodomain sequence (proPC4(75-90)), which contains its primary autocatalytic cleavage site. ProPC4(75-90) inhibited recombinant PCSK4's activity with a K(i) value of 5.4 µM, and at 500 µM, it inhibited sperm PC activity almost completely. Treatment of sperm with proPC4(75-90) inhibited their egg fertilizing ability in a dose dependent manner. Correlation between sperm PC activity and fertilizing ability showed a high co-efficient value (>0.9), indicating the importance of sperm PC activity in fertilization. In particular, sperm PC activity was important for capacitation and zona pellucida (ZP)-induced acrosome reaction, since proPC4(75-90) -treated sperm showed markedly decreased rates in these two events. These results were opposite to those observed in Pcsk4-null sperm, which contained higher PC activity than wild type sperm, possibly due to overcompensation by PCSK7, the other PCSK enzyme found in sperm. ADAM2 (45 kDa), a sperm plasma membrane protein, involved in sperm-egg plasma membrane interaction, was also processed into a smaller form (27 kDa) during capacitation at a much reduced level in proPC4(75-90) -treated sperm. This result suggested that ADAM2 may be a natural substrate of sperm PCSK4 and its cleavage by the enzyme during acrosome reaction may be relevant to the fertilization process.
脯氨酸内切酶枯草溶菌素/克胰蛋白酶 4（PCSK4）被认为与精子的受精能力有关，这是基于使用 Pcsk4 基因敲除小鼠的研究。在此，我们证明了完整精子和顶体小泡中存在脯氨酸内切酶（PC）活性。为了确定这种活性对精子受精能力是否重要，我们根据 PCSK4 前导肽序列（proPC4（75-90））设计了一种肽抑制剂，该抑制剂包含其主要的自身切割位点。ProPC4（75-90）对重组 PCSK4 的抑制作用具有 5.4 μM 的 K(i)值，并且在 500 μM 时，它几乎完全抑制了精子 PC 活性。proPC4（75-90）处理精子会导致其卵子受精能力呈剂量依赖性下降。精子 PC 活性与受精能力之间的相关性显示出高相关系数值（>0.9），表明精子 PC 活性在受精过程中非常重要。特别是，精子 PC 活性对获能和透明带（ZP）诱导的顶体反应非常重要，因为 proPC4（75-90）处理的精子在这两个事件中的发生率明显下降。这些结果与 Pcsk4 基因敲除精子相反，后者的 PC 活性高于野生型精子，这可能是由于另一种存在于精子中的 PCSK 酶 PCSK7 的过度补偿所致。ADAM2（45 kDa）是一种参与精子-卵子质膜相互作用的精子质膜蛋白，在获能过程中也被加工成较小的形式（27 kDa），在 proPC4（75-90）处理的精子中，其水平显著降低。这一结果表明，ADAM2 可能是精子 PCSK4 的天然底物，其在顶体反应过程中的裂解可能与受精过程有关。
