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Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/?-albumin.

ABSTRACT: Wnt plays important role during development and in various diseases. Because Wnts are lipidated and highly hydrophobic, they can only be purified in the presence of detergents, limiting their use in various in vitro and in vivo assays. We purified N-terminally tagged recombinant Wnt3a secreted from cells and accidentally discovered that Wnt3a co-purified with a glycoprotein afamin derived from the bovine serum included in the media. Wnt3a forms a 1:1 complex with afamin, which remains soluble in aqueous buffer after isolation, and can induce signaling in various cellular systems including the intestical stem cell growth assay. By co-expressing with afamin, biologically active afamin-Wnt complex can be easily obtained in large quantity. As afamin can also solubilize Wnt5a, Wnt3, and many more Wnt subtypes, afamin complexation will open a way to put various Wnt ligands and their signaling mechanisms under a thorough biochemical scrutiny that had been difficult for years.

SUBMITTER: Mihara E

PROVIDER: S-EPMC4775226 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin.

Mihara Emiko E Hirai Hidenori H Yamamoto Hideki H Tamura-Kawakami Keiko K Matano Mami M Kikuchi Akira A Sato Toshiro T Takagi Junichi J

eLife 20160223

Wnt plays important role during development and in various diseases. Because Wnts are lipidated and highly hydrophobic, they can only be purified in the presence of detergents, limiting their use in various in vitro and in vivo assays. We purified N-terminally tagged recombinant Wnt3a secreted from cells and accidentally discovered that Wnt3a co-purified with a glycoprotein afamin derived from the bovine serum included in the media. Wnt3a forms a 1:1 complex with afamin, which remains soluble in ...[more]

PMID: 26902720

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Project description:Afamin, which is a human blood plasma glycoprotein, a putative multifunctional transporter of hydrophobic molecules and a marker for metabolic syndrome, poses multiple challenges for crystallographic structure determination, both practically and in analysis of the models. Several hundred crystals were analysed, and an unusual variability in cell volume and difficulty in solving the structure despite an ∼34% sequence identity with nonglycosylated human serum albumin indicated that the molecule exhibits variable and context-sensitive packing, despite the simplified glycosylation in insect cell-expressed recombinant afamin. Controlled dehydration of the crystals was able to stabilize the orthorhombic crystal form, reducing the number of molecules in the asymmetric unit from the monoclinic form and changing the conformational state of the protein. An iterative strategy using fully automatic experiments available on MASSIF-1 was used to quickly determine the optimal protocol to achieve the phase transition, which should be readily applicable to many types of sample. The study also highlights the drawback of using a single crystallographic structure model for computational modelling purposes given that the conformational state of the binding sites and the electron density in the binding site, which is likely to result from PEGs, greatly varies between models. This also holds for the analysis of nonspecific low-affinity ligands, where often a variety of fragments with similar uncertainty can be modelled, inviting interpretative bias. As a promiscuous transporter, afamin also seems to bind gadoteridol, a magnetic resonance imaging contrast compound, in at least two sites. One pair of gadoteridol molecules is located near the human albumin Sudlow site, and a second gadoteridol molecule is located at an intermolecular site in proximity to domain IA. The data from the co-crystals support modern metrics of data quality in the context of the information that can be gleaned from data sets that would be abandoned on classical measures.

Project description:ObjectivesThe association between biomarkers and the risk of gestational diabetes mellitus (GDM) or preeclampsia (PE) has been extensively studied. However, there is still a lack of convenient, specific, and sensitive indicators for early identification of GMD and PE. Therefore, we conducted a meta-analysis of published articles to investigate the value of afamin circulating levels in the early diagnosis of GDM and PE.MethodsWe searched the PubMed, Embase, Cochrane Library, and Web of Science databases for English studies published before November 16, 2022, that examined the association between afamin and GDM or PE. In addition, we searched Clinicaltrials.gov for the relevant completed and ongoing clinical trials. Pooled standard mean differences (SMDs) and weighted mean differences (WMDs) with 95% confidence intervals (CIs) were used to compare the levels of afamin in different groups.ResultsEleven studies were included in our analysis (N = 3047 participants: 1195 GDM, 1407 non-GDM, 195 PE, and 250 non-PE). Subgroup analysis based on different blood collection periods found that the plasma afamin levels in pregnant women with GDM in the first trimester were significantly higher than those in healthy pregnant women (SMD = 0.481, 95% CI: 0.280-0.682), but the analysis showed the opposite results in the second and late stages (SMD = 0.292, 95% CI: -0.092-0.676). The plasma afamin levels of pregnant women with PE in the first trimester (SMD = 0.808, 95% CI: 0.558-1.059) and second/third trimesters (SMD = 0.904, 95% CI: 0.570-1.239) were significantly higher than those in healthy pregnant women.ConclusionThe plasma afamin levels in pregnant women with GDM in the first trimester were significantly higher than those in healthy pregnant women, but the analysis showed the opposite results in the second and third trimesters. The plasma afamin levels in pregnant women with PE in the first, second, and third trimesters were significantly higher than those in healthy pregnant women. Additional large-scale prospective studies are desired to verify these findings, and it is recommended that afamin should be included as a routine diagnostic test for women with GDM and PE.Systematic review registrationhttps://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=339171, identifier CRD42022339171.

Dataset Information

Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/?-albumin.

Publications

Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin.

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