探討O-連接型醣基化對V-ATPase活性的影響

Abstract

原發性骨質疏鬆症是一種潛在且慢性的疾病，其主要特徵為雌激素缺乏導致的骨密度降低，進而增加骨折風險。在正常骨組織中，骨質動態由蝕骨細胞（osteoclasts）與成骨細胞（osteoblasts）協同維持，兩者在骨吸收與骨生成間保持平衡。蝕骨細胞負責骨質降解，藉由分泌酸與蛋白酶來溶解礦化基質與膠原蛋白，V-ATPase（質子幫浦）在此過程中扮演關鍵角色。V-ATPase 是一種 ATP 驅動的氫離子幫浦，主要分布於蝕骨細胞的皺摺緣（ruffled border）與溶酶體中。於皺摺緣，V-ATPase 將氫離子（H⁺）輸送至蝕骨陷窩（resorption lacunae），造成局部酸化以促進骨基質的溶解；而在細胞內，則調控溶酶體 pH，進而影響蛋白酶活性與胞器運輸，調節蝕骨細胞功能。氧型醣基化（O-glycosylation）是一種影響蛋白質摺疊、定位、穩定性與活性的醣修飾機制。Core 1 β1,3-半乳糖基轉移酶（C1GALT1）為參與氧型醣基化的重要酵素，負責催化 Core 1 結構的形成，該修飾主要發生於細胞膜蛋白上。本研究旨在探討氧型醣基化酵素 C1GALT1 對 V-ATPase 活性及其細胞內運輸的調控角色。我們在 HeLa 細胞中敲低 C1GALT1 表現，並以長柔毛野豌豆（VVA）凝集素進行 lectin pull-down 分析。結果顯示，C1GALT1 調控 ATP6AP1 的氧型醣基化，卻不影響 ATP6V1A 或 ATP6VoA1。為進一步探討 C1GALT1 在蝕骨細胞中對 V-ATPase 的影響，我們利用 C1GALT1 條件式剔除小鼠之骨髓細胞進行蝕骨細胞分化。免疫螢光染色結果顯示，C1GALT1 缺失不影響 V-ATPase 的組裝與亞基交互作用，但會干擾 ATP6V1A 與 ATP6VoA3 正確定位至早期內體與溶酶體，並降低這些亞基（ATP6V1A、ATP6VoA3、ATP6AP1）於細胞膜上的分布。此外，C1GALT1 抑制亦導致內體和溶酶體聚集，改變其細胞內分布。綜合而言，我們的研究發現 C1GALT1 透過調控 ATP6AP1 的氧型醣基化，參與 V-ATPase 的細胞內運輸與功能。儘管 C1GALT1 缺失不影響 V-ATPase 的亞基表現與組裝，卻會干擾其定位與溶酶體酸化，導致溶酶體 pH 升高，影響蛋白酶活性，進而抑制蝕骨細胞的骨吸收能力。

Primary osteoporosis, a major public health concern, is characterized by excessive osteoclast-mediated bone resorption, leading to decreased bone density and an increased risk of fracture. Osteoclasts degrade bone matrix through vacuolar-type H⁺-ATPase (V-ATPase), an ATP-dependent proton pump that is essential for acidifying the resorption lacuna. Proper V-ATPase localization and function rely on protein modifications, yet the role of O-glycosylation in this process remains poorly understood. Core 1 β1,3-galactosyltransferase (C1GALT1), a key enzyme in O-linked glycosylation, is crucial for protein stability, trafficking, and function. While glycosylation defects are implicated in lysosomal dysfunction, the specific impact of C1GALT1 mediated O-glycosylation on osteoclast activity and V-ATPase regulation is unclear. Our previous findings indicated that C1GALT1 deficiency leads to multivesicular body accumulation and increased lysosomal pH, suggesting a role in regulating lysosomal acidification. Here, we investigate whether C1GALT1 regulates osteoclast function by modulating V-ATPase trafficking. Using HeLa cells and VVA lectin pull-down assays, we found that C1GALT1 is required for ATP6AP1 O-glycosylation, whereas ATP6V1A and ATP6VoA1 do not exhibit detectable O-glycosylation. Despite this, C1GALT1 knockdown does not alter V-ATPase subunit mRNA and protein expression levels or assembly but instead disrupts proper subunit trafficking and localization. To further assess its role in osteoclastogenesis, we differentiated bone marrow-derived osteoclasts from C1GALT1 flox/ flox LysM-Cre mice. Immunofluorescence staining showed that C1GALT1 deficiency did not affect the assembly of V-ATPase or the interaction between its subunits. However, it impaired the proper localization of ATP6V1A and ATP6VoA3 to early endosomes and lysosomes, and reduced the membrane distribution of these subunits, including ATP6V1A, ATP6VoA3, and ATP6AP1. Our findings demonstrate that C1GALT1 plays a critical role in osteoclast-mediated bone resorption by regulating the intracellular trafficking of V-ATPase. Specifically, C1GALT1-mediated O-glycosylation of ATP6AP1 is essential for the proper localization of V-ATPase subunits, which in turn facilitates lysosomal acidification and function in osteoclasts. Although C1GALT1 deficiency does not alter the expression or assembly of V-ATPase subunits, it disrupts their trafficking and impairs lysosomal function, ultimately leading to reduced bone resorption. These results reveal a novel function for O-glycosylation in osteoclast activity and suggest that targeting C1GALT1-mediated O glycosylation could be a potential therapeutic strategy for osteoporosis.