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ZAG is a multifunctional glycoprotein with a class I MHC-like protein fold and an α1-α2 lipid-binding groove. The intrinsic ZAG ligand is unknown. Our previous studies showed that ZAG binds the dansylated C11 fatty acid, DAUDA, differently to the boron dipyrromethane C16 fatty acid, C16 -BODIPY. Here, the molecular basis for this difference was elucidated. Multi-wavelength analytical ultracentrifugation confirmed that DAUDA and C16 -BODIPY individually bind to ZAG and compete for the same binding site. Molecular docking of lipid-binding in the structurally related CD1-proteins predicted nine conserved ligand contact residues in ZAG. Twelve mutants were accordingly created by alanine scanning site directed mutagenesis for characterisation. Mutation of Y12 caused ZAG to misfold. Mutation of K147, R157 and A158 abrogated C16 -BODIPY but not DAUDA binding. L69 and T169 increased the fluorescence emission intensity of C16 -BODIPY but not of DAUDA compared to wild-type ZAG and showed that C16 -BODIPY binds close to T169 and L69. Distance measurements of the crystal structure revealed K147 forms a salt bridge with D83. A range of bioactive bulky lipids including phospholipids and sphingolipids displaced DAUDA from the ZAG binding site but unexpectedly did not displace C16 -BODIPY. We conclude that the ZAG α1-α2 groove contains separate but overlapping sites for DAUDA and C16 -BODIPY and is involved in binding to a bulkier and wider repertoire of lipids than previously reported. This work suggested that the in vivo activity of ZAG may be dictated by its lipid ligand. (262 words).

The human complement Factor H–related 5 protein (FHR5) antagonizes the main circulating complement regulator Factor H, resulting in the deregulation of complement activation. FHR5 normally contains nine short complement regulator (SCR) domains, but a FHR5 mutant has been identified with a duplicated N-terminal SCR-1/2 domain pair that causes CFHR5 nephropathy. To understand how this duplication causes disease, we characterized the solution structure of native FHR5 by analytical ultracentrifugation and small-angle X-ray scattering. Sedimentation velocity and X-ray scattering indicated that FHR5 was dimeric, with a radius of gyration (R(g)) of 5.5 ± 0.2 nm and a maximum protein length of 20 nm for its 18 domains. This result indicated that FHR5 was even more compact than the main regulator Factor H, which showed an overall length of 26–29 nm for its 20 SCR domains. Atomistic modeling for FHR5 generated a library of 250,000 physically realistic trial arrangements of SCR domains for scattering curve fits. Only compact domain structures in this library fit well to the scattering data, and these structures readily accommodated the extra SCR-1/2 domain pair present in CFHR5 nephropathy. This model indicated that mutant FHR5 can form oligomers that possess additional binding sites for C3b in FHR5. We conclude that the deregulation of complement regulation by the FHR5 mutant can be rationalized by the enhanced binding of FHR5 oligomers to C3b deposited on host cell surfaces. Our FHR5 structures thus explained key features of the mechanism and pathology of CFHR5 nephropathy.

Human zinc-α2-glycoprotein (ZAG) is a 42 kDa adipokine which regulates body fat mass and is associated with cachexia and obesity. ZAG belongs to the major histocompatibility complex class I protein family and binds long-chain polyunsaturated fatty acids in its groove formed from the α1 and α2 domains. To identify the molecular basis of its lipid-binding function, we determined the first crystal structure at 2.49 Å resolution for fatty acid-bound ZAG, where the ligand was the fluorescent 11-(dansylamino)undecanoic acid (DAUDA). The 192 kDa crystallographic asymmetric unit contained six ZAG and eight fatty acid molecules in unique conformations. Six fatty acid molecules were localised to the ZAG grooves, where their tails were bound in two distinct conformations. The carboxylate groups of three fatty acids projected out of the groove, while the fourth was hydrogen bonded with R73 inside the groove. Other ligand-residue contacts were primarily hydrophobic. A new fatty acid site was revealed for two further DAUDA molecules at the ZAG α3 domains. Following conformational changes from unbound ZAG, the α3 domains formed tetrameric β-barrel structures lined by fatty acid molecules that doubled the binding capacity of ZAG. Analytical ultracentrifugation revealed that ZAG in solution was a monomer in the absence of DAUDA, but formed small amounts of tetramers with DAUDA. By showing that ZAG binds fatty acids in different locations, we demonstrate an augmented mechanism for fatty acid binding in ZAG that is distinct from other known fatty acid binding proteins, and may be relevant to cachexia.

Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigated. Analytical ultracentrifugation (AUC) and CD showed that zinc, but not other divalent metals, causes ZAG to oligomerize in solution. Thus ZAG dimers and trimers were observed in the presence of 1 and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting that the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG–zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid). Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid-binding groove in ZAG contains at least two distinct fatty acid-binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally related immune protein CD1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid-binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homoeostasis occurs.

Pre-eclampsia is characterized by placental hypoxia and dyslipidemia. Arachidonic and docosahexanoic acids are essential maternal nutrients for fetal development. They are transported via placental trophoblast cells by membrane and cytosolic fatty acid binding proteins. Others report the expressions of these proteins which are increased in hypoxic trophoblasts. Using bioinformatics, BeWo cells, reporter assays, quantitative real-time PCR and immunoblotting we tested the hypothesis that hypoxia inducible factors 1α (HIF-1α) and/or 2α (HIF-2α) regulate the expressions of FABP1, FABP3, FABP4 and FATP2 proteins. Three hypoxia responsive elements (HRE) were identified in FABP1 which cumulatively responded strongly to HIF-1α and weakly to HIF-2α. FABP3 expression partially responded to HIF-1α. Two putative HRE were validated in FABP4 both of which responded weakly to HIF-1α and HIF-2α. FATP2 protein expression reacted positively to hypoxia. Thus, fetal essential fatty acid supply via the placenta is protected under hypoxia. It will be interesting to determine if our findings are replicated in human pre-eclamptic placenta.

Polyproteins comprise long polypeptides that are post-translationally cleaved into proteins of different function, or tandemly repetitive polypeptides which are processed into multiple versions of proteins which are presumed to have the same function. In the latter case the individual units of the polyprotein can differ substantially in sequence. Identity of function between the different units therefore cannot be assumed. Here we have examined the ABA-1 polyprotein allergen of the parasitic nematode Ascarissuum and found it to contain units which show a 50% difference in amino acid sequence. The parasite therefore produces at least two radically different forms of the allergen encoded within the polyprotein array. In fluorescence-based ligand-binding assays, recombinant polypeptides representing the two forms (designated ABA-1A1 and ABA-1B1) showed similar binding affinities for a range of fluorescent active-site probes [retinol, dansylundecanoic acid, dansyl-DL-α-amino-octanoic acid, cis-parinaric acid (cPnA)] and for the non-specific hydrophobic surface probe 8-anilinonaphthalene-1-sulphonic acid. However, the molecular environments in the active sites are markedly different, as indicated by disparate fluorescence emission peaks and intensities of bound probes. CD showed that the proteins have similar secondary structures but differ in susceptibility to chemical denaturation/unfolding by guanidinium chloride. Both retain a single conserved tryptophan residue in a characteristic non-polar environment, as revealed by extreme fluorescence blue shift. Thus the gross differences in sequence of the two proteins are not reflected in their ligand-binding specificities but in their binding-site environments.

Parasitic nematodes have recently been found to produce proteins which represent two new classes of fatty acid and retinoid binding protein. The first is the nematode polyprotein allergens/antigens (NPAs) which, as their name suggests, are synthesised as large polyproteins which are subsequently cleaved at regularly spaced sites to form multiple copies of a fatty acid binding protein of approximately 14.5 kDa. Binding studies using molecular environment-sensitive fluorescent ligands have shown that the binding site is highly unusual, producing blue-shifting in fluorescence to an unprecedented degree, suggesting a remarkably non-polar environment and isolation from solvent water. Computer-based structural predictions and biophysical observations have identified the NPAs as highly helical proteins which might form a four helix bundle, so constitute a new class of lipid binding protein from animals. The second class, like the NPAs, binds both fatty acids and retinol, but with a higher affinity for the latter. These are also highly helical but are structurally distinct from the NPAs. The biological function of these new classes of protein are discussed in the context of both the metabolic requirements of the parasites and the possible role of the proteins in control of the immune and inflammatory environment of the tissue sites parasitised.

Ov20 is a major antigen of the parasitic nematodeOnchocerca volvulus, the causative agent of river blindness in humans, and the protein is secreted into the tissue occupied by the parasite. DNA encoding Ov20 was isolated, and the protein was expressed in Escherichia coli. Fluorescence-based ligand binding assays show that the protein contains a high affinity binding site for retinol, fluorescent fatty acids (11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid, dansyl-dl-α-aminocaprylic acid, and parinaric acid) and, by competition, oleic and arachidonic acids, but not cholesterol. The fluorescence emission of dansylated fatty acids is significantly blue-shifted upon binding in comparison to similarly sized β-sheet-rich mammalian retinol- and fatty acid-binding proteins. Secondary structure prediction algorithms indicate that a α-helix predominates in Ov20, possibly in a coiled coil motif, with no evidence of β structures, and this was confirmed by circular dichroism. The protein is highly stable in solution, requiring temperatures in excess of 90 °C or high denaturant concentrations for unfolding. Ov20 therefore represents a novel class of small retinol-binding protein, which appears to be confined to nematodes. The retinol binding activity of Ov20 could possibly contribute to the eye defects associated with onchocerciasis and, because there is no counterpart in mammals, represents a strategic target for chemotherapy.

Zinc-α2-glycoprotein (ZAG) is an adipokine with an MHC class I-like protein fold. Even though zinc causes ZAG to precipitate from plasma during protein purification, no zinc binding has been identified to date. Using mass spectrometry, we demonstrated that ZAG contains one strongly bound zinc ion, predicted to lie close to the α1 and α2 helical groove. UV, CD and fluorescence spectroscopies detected weak zinc binding to holo-ZAG, which can bind up to 15 zinc ions. Zinc binding to 11-(dansylamino) undecanoic acid was enhanced by holo-ZAG. Zinc binding may be important for ZAG binding to fatty acids and the β-adrenergic receptor. © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.