Carboxypeptidase O (CPO) is a member of the M14 family of metallocarboxypeptidases with a preference for the cleavage of C-terminal acidic amino acids. CPO is largely expressed in the small intestine, although it has been detected in other tissues such as the brain and ovaries. CPO does not contain a prodomain, nor is it strongly regulated by pH, and hence appears to exist as a constitutively active enzyme. The goal of this study was to investigate the intracellular distribution and activity of CPO in order to predict physiological substrates and function. The distribution of CPO, when expressed in MDCK cells, was analyzed by immunofluorescence microscopy. Soon after addition of nutrient-rich media, CPO was found to associate with lipid droplets, causing an increase in lipid droplet quantity. As media became depleted, CPO moved to a broader ER distribution, no longer impacting lipid droplet numbers. Membrane cholesterol levels played a role in the distribution and in vitro enzymatic activity of CPO, with cholesterol enrichment leading to decreased lipid droplet association and enzymatic activity. The ability of CPO to cleave C-terminal amino acids within the early secretory pathway (in vivo) was examined using Gaussia luciferase as a substrate, C-terminally tagged with variants of an ER retention signal. While no effect of cholesterol was observed, these data show that CPO does function as an active enzyme within the ER where it removes C-terminal glutamates and aspartates, as well as a number of polar amino acids.

羧肽酶O（CPO）是金属羧肽酶M14家族的成员，偏爱C末端酸性氨基酸的切割。尽管已在其他组织（例如大脑和卵巢）中检测到了CPO，但CPO主要在小肠中表达。CPO不包含前结构域，也不受pH强烈调节，因此似乎以组成型活性酶的形式存在。这项研究的目的是调查CPO的细胞内分布和活性，以预测生理底物和功能。当在MDCK细胞中表达时，通过免疫荧光显微镜分析CPO的分布。添加营养丰富的培养基后不久，发现CPO与脂滴相关联，导致脂滴量增加。随着媒体的枯竭，CPO转移到更广泛的ER分布，不再影响脂质滴数。膜胆固醇水平在分布和CPO的体外酶促活性，胆固醇富集导致脂质滴缔合和酶促活性降低。使用高斯荧光素酶作为底物检查CPO在早期分泌途径（体内）中裂解C末端氨基酸的能力，并用ER保留信号的变体在C末端标记。虽然未观察到胆固醇的作用，但这些数据表明CPO确实在ER内起活性酶的作用，从中除去C末端的谷氨酸和天冬氨酸以及许多极性氨基酸。