Alan J. Stewart

Structural and immunologic characterization of bovine, horse, and rabbit serum albumins.

Serum albumin (SA) is the most abundant plasma protein in mammals. SA is a multifunctional protein with extraordinary ligand binding capacity, making it a transporter molecule for a diverse range of metabolites, drugs, nutrients, metals and other molecules. Due to its ligand binding properties, albumins have wide clinical, pharmaceutical, and biochemical applications. Albumins are also allergenic, and exhibit a high degree of cross-reactivity due to significant sequence and structure similarity of SAs from different organisms. Here we present crystal structures of albumins from cattle (BSA), horse (ESA) and rabbit (RSA) sera. The structural data are correlated with the results of immunological studies of SAs. We also analyze the conservation or divergence of structures and sequences of SAs in the context of their potential allergenicity and cross-reactivity. In addition, we identified a previously uncharacterized ligand binding site in the structure of RSA, and calcium binding sites in the structure of BSA, which is the first serum albumin structure to contain metal ions.

Interdomain zinc site on human albumin

Albumin is the major transport protein in blood for Zn2+, a metal ion required for physiological processes and recruited by various drugs and toxins. However, the Zn2+-binding site(s) on albumin is ill-defined. We have analyzed the 18 x-ray crystal structures of human albumin in the PDB and identified a potential five-coordinate Zn site at the interface of domains I and II consisting of N ligands from His-67 and His-247 and O ligands from Asn-99, Asp-249, and H2O, which are the same amino acid ligands as those in the zinc enzymes calcineurin, endonucleotidase, and purple acid phosphatase. The site is preformed in unliganded apo-albumin and highly conserved in mammalian albumins. We have used 111Cd NMR as a probe for Zn2+ binding to recombinant human albumin. We show that His-67 → Ala (His67Ala) mutation strongly perturbs Cd2+ binding, whereas the mutations Cys34Ala, or His39Leu and Tyr84Phe (residues which may H-bond to Cys-34) have no effect. Weak Cl− binding to the fifth coordination site of Cd2+ was demonstrated. Cd2+ binding was dramatically affected by high fatty acid loading of albumin. Analysis of the x-ray structures suggests that fatty acid binding to site 2 triggers a spring-lock mechanism, which disengages the upper (His-67/Asn-99) and lower (His-247/Asp-249) halves of the metal site. These findings provide a possible mechanism whereby fatty acids (and perhaps other small molecules) could influence the transport and delivery of zinc in blood.

Albumin as a zinc carrier: properties of its high-affinity zinc-binding site

Although details of the molecular mechanisms for the uptake of the essential nutrient zinc into the bloodstream and its subsequent delivery to zinc-requiring organs and cells are poorly understood, it is clear that in vertebrates the majority of plasma zinc (9-14 microM; approx. 75-85%) is bound to serum albumin, constituting part of the so-called exchangeable pool. The binding of metal ions to serum albumins has been the subject of decades of studies, employing a multitude of techniques, but only recently has the identity and putative structure of the major zinc site on albumin been reported. Intriguingly, this site is located at the interface between two domains, and involves two residues from each of domains I and II. Comparisons of X-ray crystal structures of free and fatty-acid bound human serum albumin suggest that zinc binding to this site and fatty acid binding to one of the five major sites may be interdependent. Interactive binding of zinc and long-chain fatty acids to albumin may therefore have physiological implications.

Structure, Properties, and Engineering of the Major Zinc Binding Site on Human Albumin

Most blood plasma zinc is bound to albumin, but the structure of the binding site has not been determined. Zn K-edge extended x-ray absorption fine structure spectroscopy and modeling studies show that the major Zn2+ site on albumin is a 5-coordinate site with average Zn-O/N distances of 1.98 Å and a weak sixth O/N bond of 2.48 Å, consistent with coordination to His67 and Asn99 from domain I, His247 and Asp249 from domain II (residues conserved in all sequenced mammalian albumins), plus a water ligand. The dynamics of the domain I/II interface, thought to be important to biological function, are affected by Zn2+ binding, which induces cooperative allosteric effects related to those of the pH-dependent neutral-to-base transition. N99D and N99H mutations enhance Zn2+ binding but alter protein stability, whereas mutation of His67 to alanine removes an interdomain H-bond and weakens Zn2+ binding. Both wild-type and mutant albumins promote the safe management of high micromolar zinc concentrations for cells in cultures.

GPR39: a Zn2+-activated G protein-coupled receptor that regulates pancreatic, gastrointestinal and neuronal functions

GPR39 is a vertebrate G protein-coupled receptor related to the ghrelin/neurotensin receptor subfamily. The receptor is expressed in a range of tissues including the pancreas, gut/gastrointestinal tract, liver, kidney and in some regions of the brain. GPR39 was initially thought to be the cognitive receptor for the peptide hormone, obestatin. However, subsequent in vitro studies have failed to demonstrate binding of this peptide to the receptor. Zn2+ has been shown to be a potent stimulator of GPR39 activity via the Gαq, Gα12/13 and Gαs pathways. The potency and specificity of Zn2+ in activating GPR39 suggest it to be a physiologically important agonist. GPR39 is now emerging as an important transducer of autocrine and paracrine Zn2+ signals, impacting upon cellular processes such as insulin secretion, gastric emptying, neurotransmission and epithelial repair. This review focuses on the molecular, structural and biological properties of GPR39 and its various physiological functions.

Human PHOSPHO1 exhibits high specific phosphoethanolamine and phosphocholine phosphatase activities

Human PHOSPHO1 is a phosphatase enzyme for which expression is upregulated in mineralizing cells. This enzyme has been implicated in the generation of P(i) for matrix mineralization, a process central to skeletal development. PHOSPHO1 is a member of the haloacid dehalogenase (HAD) superfamily of Mg2+-dependent hydrolases. However, substrates for PHOSPHO1 are, as yet, unidentified and little is known about its activity. We show here that PHOSPHO1 exhibits high specific activities toward phosphoethanolamine (PEA) and phosphocholine (PCho). Optimal enzymic activity was observed at approx. pH 6.7. The enzyme shows a high specific Mg2+-dependence, with apparent K(m) values of 3.0 microM for PEA and 11.4 microM for PCho. These results provide a novel mechanism for the generation of P(i) in mineralizing cells from PEA and PCho.

Role of Tyr84 in controlling the reactivity of Cys34 of human albumin.

Cys34 in domain I of the three‐domain serum protein albumin is the binding site for a wide variety of biologically and clinically important small molecules, provides antioxidant activity, and constitutes the largest portion of free thiol in blood. Analysis of X‐ray structures of albumin reveals that the loop containing Tyr84 occurs in multiple conformations. In structures where the loop is well defined, there appears to be an H‐bond between the OH of Tyr84 and the sulfur of Cys34. We show that the reaction of 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB) with Tyr84Phe mutant albumin is approximately four times faster than with the wild‐type protein between pH 6 and pH 8. In contrast, the His39Leu mutant reacts with DTNB more slowly than the wild‐type protein at pH < 8, but at a similar rate at pH 8. Above pH 8 there is a dramatic increase in reactivity for the Tyr84Phe mutant. We also report 1H NMR studies of disulfide interchange reactions with cysteine. The tethering of the two loops containing Tyr84 and Cys34 not only appears to control the redox potential and accessibility of Cys34, but also triggers the transmission of information about the state of Cys34 throughout domain I, and to the domainI/II interface.

Controlling Assembly of Mixed Thiol Monolayers on Silver Nanoparticles to Tune Their Surface Properties

Modifying the surfaces of metal nanoparticles with self-assembled monolayers of functionalized thiols provides a simple and direct method to alter their surface properties. Mixed self-assembled monolayers can extend this approach since, in principle, the surfaces can be tuned by altering the proportion of each modifier that is adsorbed. However, this works best if the composition and microstructure of the monolayers can be controlled. Here, we have modified preprepared silver colloids with binary mixtures of thiols at varying concentrations and modifier ratios. Surface-enhanced Raman spectroscopy was then used to determine the effect of altering these parameters on the composition of the resulting mixed monolayers. The data could be explained using a new model based on a modified competitive Langmuir approach. It was found that the composition of the mixed monolayer only reflected the ratio of modifiers in the feedstock when the total amount of modifier was sufficient for approximately one monolayer coverage. At higher modifier concentrations the thermodynamically favored modifier dominated, but working at near monolayer concentrations allowed the surface composition to be controlled by changing the ratios of modifiers. Finally, a positively charged porphyrin probe molecule was used to investigate the microstructure of the mixed monolayers, i.e., homogeneous versus domains. In this case the modifier domains were found to be <2 nm.

The presence of PHOSPHO1 in matrix vesicles and its developmental expression prior to skeletal mineralization

PHOSPHO1 is a phosphoethanolamine/phosphocholine phosphatase that has previously been implicated in generating inorganic phosphate (P(i)) for matrix mineralization. In this study, we have investigated PHOSPHO1 mRNA expression during embryonic development in the chick. Whole-mount in situ hybridization indicated that PHOSPHO1 expression occurred prior to E6.5 and was initially restricted to the bone collar within the mid-shaft of the diaphysis of long bones but by E11.5 expression was observed over the entire length of the diaphysis. Alcian blue/alizarin red staining revealed that PHOSPHO1 expression seen in the primary regions of ossification preceded the deposition of mineral, suggesting that it is involved in the initial events of mineral formation. We isolated MVs from growth plate chondrocytes and confirmed the presence of high levels of PHOSPHO1 by immunoblotting. Expression of PHOSPHO1, like TNAP activity, was found to be up-regulated in MVs isolated from chondrocytes induced to differentiate by the addition of ascorbic acid. This suggests that both enzymes may be regulated by similar mechanisms. These studies provide for the first time direct evidence that PHOSPHO1 is present in MVs, and its developmental expression pattern is consistent with a role in the early stages of matrix mineralization.

Retention and Silencing of Prepro-GnRH-II and Type II GnRH Receptor Genes in Mammals

The decapeptide hypothalamic-pituitary gonadotrophin-releasing hormone (GnRH)-I and the type I GnRH receptor drive the reproductive hormonal cascade in mammals by stimulating synthesis and secretion of luteinising hormone (LH) and follicle stimulating hormone (FSH). Mammals possess a second GnRH system composed of a related hormone, GnRH-II (differing from GnRH-I by three amino acid residues), and the type II GnRH receptor. In many mammalian species, one or both of the GnRH-II system genes are disrupted or deleted, rendering their products non-functional. This includes humans who possess a gene encoding GnRH-II but lack a functional type II GnRH receptor. Here we examined the genes encoding prepro-GnRH-II (GnRH2) and the type II GnRH receptor (GnRHR2) in more than 20 mammalian species, encompassing 10 orders, to determine whether they encode functional proteins. The structural organisation of both genes in most mammalian genome sequence assemblies was poorly annotated or incompletely described. Our findings show significant variation in the DNA sequence conservation and functional status of each gene, even between closely related species. Prepro-GnRH-II was functionally compromised in 12/22 species and the type II GnRH receptor gene was disrupted in 14/22 species. Retention of large sections of each gene in most mammalian genomes suggests that mammalian ancestors had a functional GnRH-II system. Gene disruptions were due to a spectrum of mutations which must have occurred independently after the evolutionary divergence of mammals from ancestral animals. The genetic information will be useful for understanding the physiological role of the GnRH-II system and establishing animal models for functional studies.