The New Zealand Institute for Plant and Food Research Limited, Private Bag 92169, Auckland 1142, New Zealand; Polymer Biointerface Centre, School of Chemical Sciences, The University of Auckland, Auckland 1023, New Zealand; MacDiarmid Institute for Advanced Materials and Nanotechnology, Wellington 6140, New Zealand.
The New Zealand Institute for Plant and Food Research Limited, Private Bag 92169, Auckland 1142, New Zealand.
Biochim Biophys Acta Gen Subj. 2022 Nov;1866(11):130205. doi: 10.1016/j.bbagen.2022.130205. Epub 2022 Jul 28.
Lipocalins are a large family of proteins, which possess a highly conserved eight-stranded antiparallel beta-barrel structure as distinctive trait. This family includes Major Urinary Proteins (MUPs) from rats and mouse, studied for their role in urinary protein-mediated chemosignalling. Vulpeculin has been identified as the most abundant protein in the urine of the common brushtail possum, Trichosurus vulpecula. On the basis of high similarity with other MUPS, we hypothesised that vulpeculin might have a role in possum chemosignalling and investigated its stability and binding ability.
We expressed and purified vulpeculin using an E.coli-based system and confirmed correct folding by circular dichroism (CD) spectroscopy. Thermal stability was studied by CD and binding properties were investigated using two optical probes N-phenyl-naphthylamine (NPN) and 8-anilino-1-naphthalene sulphonic acid (ANS).
CD revealed a secondary structure typical of a predominantly β-sheet protein, consistent with the beta barrel structure of the lipocalin family. Vulpeculin showed a high level of thermostability, as assessed by CD, exhibiting a small shift in the secondary structure even at 95 °C. Binding assays indicated that vulpeculin cannot accommodate the NPN ligand but can bind ANS.
The urinary secretion, high degree of sequence similarity with other lipocalins, its beta sheet structure assessed by CD and potential to bind hydrophobic ligands in the hydrophobic cavity or an external hydrophobic pocket, suggest vulpeculin may be involved in possum chemosignalling.
This work represents a first step towards the further investigation of the newly discovered lipocalin and its role in possum chemosignalling.
脂联素是一个庞大的蛋白质家族，具有高度保守的八链反平行β-桶结构，这是其独特的特征。这个家族包括大鼠和小鼠的主要尿蛋白（MUPs），它们因其在尿蛋白介导的化学信号中的作用而被研究。Vulpeculin 已被确定为普通帚尾袋貂（Trichosurus vulpecula）尿液中最丰富的蛋白质。基于与其他 MUPS 的高度相似性，我们假设 vulpeculin 可能在袋貂的化学信号中发挥作用，并研究了其稳定性和结合能力。
我们使用基于大肠杆菌的系统表达和纯化 vulpeculin，并通过圆二色性（CD）光谱证实正确折叠。通过 CD 研究热稳定性，并使用两种光学探针 N-苯基-萘胺（NPN）和 8-苯胺-1-萘磺酸（ANS）研究结合特性。
CD 显示出二级结构典型的主要β-折叠蛋白，与脂联素家族的β-桶结构一致。Vulpeculin 表现出高水平的热稳定性，如 CD 评估所示，即使在 95°C 时，二级结构也只有很小的移动。结合实验表明，vulpeculin 不能容纳 NPN 配体，但可以结合 ANS。
尿液分泌、与其他脂联素高度相似的序列、CD 评估的β-折叠结构以及在疏水性腔或外部疏水性口袋中结合疏水性配体的潜力，表明 vulpeculin 可能参与袋貂的化学信号传递。
这项工作代表了对新发现的脂联素及其在袋貂化学信号传递中的作用的进一步研究的第一步。
