Cholangiocytes express an isoform of soluble adenylyl cyclase that is N-linked glycosylated and secreted in extracellular vesicles.

IF 3.6 3区 生物学 Q3 CELL BIOLOGY Traffic Pub Date : 2023-09-01 DOI:10.1111/tra.12904
Simei Go, Hang Lam Li, Jung-Chin Chang, Arthur J Verhoeven, Ronald P J Oude Elferink
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引用次数: 2

Abstract

Soluble adenylyl cyclase (sAC)-derived cAMP regulates various cellular processes; however, the regulatory landscape mediating sAC protein levels remains underexplored. We consistently observed a 85 kD (sAC85 ) or 75 kD (sAC75 ) sAC protein band under glucose-sufficient or glucose-deprived states, respectively, in H69 cholangiocytes by immunoblotting. Deglycosylation by PNGase-F demonstrated that both sAC75 and sAC85 are N-linked glycosylated proteins with the same polypeptide backbone. Deglycosylation with Endo-H further revealed that sAC75 and sAC85 carry distinct sugar chains. We observed release of N-linked glycosylated sAC (sACEV ) in extracellular vesicles under conditions that support intracellular sAC85 (glucose-sufficient) as opposed to sAC75 (glucose-deprived) conditions. Consistently, disrupting the vesicular machinery affects the maturation of intracellular sAC and inhibits the release of sACEV into extracellular vesicles. The intracellular turnover of sAC85 is extremely short (t1/2 ~30 min) and release of sACEV in the medium was detected within 3 h. Our observations support the maturation and trafficking in cholangiocytes of an N-linked glycosylated sAC isoform that is rapidly released into extracellular vesicles.

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胆管细胞表达一种可溶性腺苷酸环化酶的异构体,该酶被n链糖基化并在细胞外囊泡中分泌。
可溶性腺苷酸环化酶(sAC)衍生的cAMP调节多种细胞过程;然而,调节sAC蛋白水平的调控景观仍未得到充分探索。通过免疫印迹,我们在H69胆管细胞中分别观察到一个85 kD (sAC85)或75 kD (sAC75)的sAC蛋白带处于葡萄糖充足或葡萄糖缺乏状态。pnase - f去糖基化表明sAC75和sAC85都是具有相同多肽主链的n -链糖基化蛋白。用Endo-H去糖基化进一步揭示sAC75和sAC85携带不同的糖链。我们观察到在支持细胞内sAC85(葡萄糖充足)而不是sAC75(葡萄糖缺乏)条件下,细胞外囊泡中n -连接糖基化sAC (sACEV)的释放。一致地,破坏囊泡机制会影响细胞内sAC的成熟并抑制sACEV向细胞外囊泡的释放。sAC85的胞内转换极短(t1/2 ~ 30min),在3 h内检测到sACEV在培养基中的释放。我们的观察结果支持了一种n链糖基化sAC异构体在胆管细胞中的成熟和运输,这种异构体被迅速释放到细胞外囊泡中。
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来源期刊
Traffic
Traffic 生物-细胞生物学
CiteScore
8.10
自引率
2.20%
发文量
50
审稿时长
2 months
期刊介绍: Traffic encourages and facilitates the publication of papers in any field relating to intracellular transport in health and disease. Traffic papers span disciplines such as developmental biology, neuroscience, innate and adaptive immunity, epithelial cell biology, intracellular pathogens and host-pathogen interactions, among others using any eukaryotic model system. Areas of particular interest include protein, nucleic acid and lipid traffic, molecular motors, intracellular pathogens, intracellular proteolysis, nuclear import and export, cytokinesis and the cell cycle, the interface between signaling and trafficking or localization, protein translocation, the cell biology of adaptive an innate immunity, organelle biogenesis, metabolism, cell polarity and organization, and organelle movement. All aspects of the structural, molecular biology, biochemistry, genetics, morphology, intracellular signaling and relationship to hereditary or infectious diseases will be covered. Manuscripts must provide a clear conceptual or mechanistic advance. The editors will reject papers that require major changes, including addition of significant experimental data or other significant revision. Traffic will consider manuscripts of any length, but encourages authors to limit their papers to 16 typeset pages or less.
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