Han Jianting, Song Meinai, Cheng Yijia, Gong Wei, Zhang Fei, Cao Qin
Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China.
Department of General Surgery, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai Research Center of Biliary Tract Disease, Shanghai, China.
PLoS Biol. 2025 Jun 23;23(6):e3003238. doi: 10.1371/journal.pbio.3003238. eCollection 2025 Jun.
Glycoprotein 2 (GP2) and Uromodulin (UMOD) are considered as paralogs that share high sequence similarity and have similar antibacterial functions. UMOD are abundant as filaments in the urinary tract, and a high-mannose N-glycosylation site located on the N-terminal region protruding from UMOD filament core (referred to as branch) acts as an adhesion antagonist against pathogenic bacterial infections. The antibacterial function of UMOD can be eliminated by proteases, as the UMOD branch is susceptible to proteolytic activity. GP2 is expressed in the pancreas and secreted into the digestive tract. Whether GP2 executes its function in filament form and how it remains functional in the protease-enriched digestive tract is unclear. In this study, we extract GP2 filaments from surgically excised human pancreas and determined their cryo-EM structure. Our structure analysis unveiled that GP2 forms filaments with its ZP modules, composing the ZPN and ZPC domains along with a linker that connects these two domains. The N-terminal region (branch) of GP2 does not constitute the filament core and appears flexible in the cryo-EM structure. Our biochemical experiments suggested that although the GP2 branch is also protease-susceptible, additional high-mannose N-glycans were identified on the protease-resistant GP2 filament core. Consequently, the branch-free GP2 filaments retain their binding ability to the bacterial adhesin FimH, ensuring GP2's antibacterial function unaffected in the proteolytic environment. Our study provides the first experimental evidence of GP2 filament formation and reveals the molecular mechanisms underlying GP2's adaptation to a different environment compared to UMOD.
糖蛋白2（GP2）和尿调节蛋白（UMOD）被认为是旁系同源物，它们具有高度的序列相似性和相似的抗菌功能。UMOD在尿路中以细丝形式大量存在，位于从UMOD细丝核心突出的N端区域（称为分支）上的一个高甘露糖N-糖基化位点作为对抗病原菌感染的粘附拮抗剂。UMOD的抗菌功能可被蛋白酶消除，因为UMOD分支易受蛋白水解活性的影响。GP2在胰腺中表达并分泌到消化道。GP2是否以细丝形式发挥其功能以及它如何在富含蛋白酶的消化道中保持功能尚不清楚。在本研究中，我们从手术切除的人胰腺中提取了GP2细丝，并确定了它们的冷冻电镜结构。我们的结构分析表明，GP2通过其ZP模块形成细丝，与连接这两个结构域的接头一起构成ZPN和ZPC结构域。GP2的N端区域（分支）不构成细丝核心，在冷冻电镜结构中显得灵活。我们的生化实验表明，尽管GP2分支也对蛋白酶敏感，但在抗蛋白酶的GP2细丝核心上鉴定出了额外的高甘露糖N-聚糖。因此，无分支的GP2细丝保留了它们与细菌粘附素FimH的结合能力，确保GP2在蛋白水解环境中的抗菌功能不受影响。我们的研究提供了GP2细丝形成的首个实验证据，并揭示了与UMOD相比，GP2适应不同环境的分子机制。
