Andrew Plumridge

Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor

Albumin is the most abundant protein in blood where it has a pivotal role as a transporter of fatty acids and drugs. Like IgG, albumin has long serum half-life, protected from degradation by pH-dependent recycling mediated by interaction with the neonatal Fc receptor, FcRn. Although the FcRn interaction with IgG is well characterized at the atomic level, its interaction with albumin is not. Here we present structure-based modelling of the FcRn–albumin complex, supported by binding analysis of site-specific mutants, providing mechanistic evidence for the presence of pH-sensitive ionic networks at the interaction interface. These networks involve conserved histidines in both FcRn and albumin domain III. Histidines also contribute to intramolecular interactions that stabilize the otherwise flexible loops at both the interacting surfaces. Molecular details of the FcRn–albumin complex may guide the development of novel albumin variants with altered serum half-life as carriers of drugs.

Impact of the unfolded protein response upon genome-wide expression patterns, and the role of Hac1 in the polarized growth, of Candida albicans.

The unfolded protein response (UPR) regulates the expression of genes involved in the protein secretory pathway and in endoplasmic reticulum (ER) stress in yeasts and filamentous fungi. We have characterized the global transcriptional response of Candida albicans to ER stresses (dithiothreitol and tunicamycin) and established the impact of the transcription factor Hac1 upon this response. Expression of C. albicans Hac1, which is the functional homologue of Saccharomyces cerevisiae Hac1p, is predicted to be translationally regulated via an atypical mRNA splicing event during ER stress. C. albicans genes involved in secretion, vesicle trafficking, stress responses and cell wall biogenesis are up-regulated in response to ER stress, and translation and ribosome biogenesis genes are down-regulated. Hac1 is not essential for C. albicans viability, but plays a major role in this stress-related transcriptional response and is required for resistance to ER stress. In addition, we show that Hac1 plays an important role in regulating the morphology of C. albicans and in the expression of genes encoding cell surface proteins during ER stress, factors that are important in virulence of this fungal pathogen.

Extending Serum Half-life of Albumin by Engineering Neonatal Fc Receptor (FcRn) Binding

Background: FcRn controls the long serum half-life of albumin. Results: A single amino acid substitution of albumin considerably improved binding to FcRn and extended serum half-life in mice and rhesus monkeys. Conclusion: Serum half-life of albumin may be tailored by engineering the FcRn-albumin interaction. Significance: This study reports on engineered albumin that may be attractive for improving the serum half-life of biopharmaceuticals. A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals.

The Weak Acid Preservative Sorbic Acid Inhibits Conidial Germination and Mycelial Growth of Aspergillus niger through Intracellular Acidification

ABSTRACT The growth of the filamentous fungus Aspergillus niger, a common food spoilage organism, is inhibited by the weak acid preservative sorbic acid (trans-trans-2,4-hexadienoic acid). Conidia inoculated at 105/ml of medium showed a sorbic acid MIC of 4.5 mM at pH 4.0, whereas the MIC for the amount of mycelia at 24 h developed from the same spore inoculum was threefold lower. The MIC for conidia and, to a lesser extent, mycelia was shown to be dependent on the inoculum size. A. niger is capable of degrading sorbic acid, and this ability has consequences for food preservation strategies. The mechanism of action of sorbic acid was investigated using 31P nuclear magnetic resonance (NMR) spectroscopy. We show that a rapid decline in cytosolic pH (pHcyt) by more than 1 pH unit and a depression of vacuolar pH (pHvac) in A. niger occurs in the presence of sorbic acid. The pH gradient over the vacuole completely collapsed as a result of the decline in pHcyt. NMR spectra also revealed that sorbic acid (3.0 mM at pH 4.0) caused intracellular ATP pools and levels of sugar-phosphomonoesters and -phosphodiesters of A. niger mycelia to decrease dramatically, and they did not recover. The disruption of pH homeostasis by sorbic acid at concentrations below the MIC could account for the delay in spore germination and retardation of the onset of subsequent mycelial growth.

Decarboxylation of Sorbic Acid by Spoilage Yeasts Is Associated with the PAD1 Gene

ABSTRACT The spoilage yeast Saccharomyces cerevisiae degraded the food preservative sorbic acid (2,4-hexadienoic acid) to a volatile hydrocarbon, identified by gas chromatography mass spectrometry as 1,3-pentadiene. The gene responsible was identified as PAD1, previously associated with the decarboxylation of the aromatic carboxylic acids cinnamic acid, ferulic acid, and coumaric acid to styrene, 4-vinylguaiacol, and 4-vinylphenol, respectively. The loss of PAD1 resulted in the simultaneous loss of decarboxylation activity against both sorbic and cinnamic acids. Pad1p is therefore an unusual decarboxylase capable of accepting both aromatic and aliphatic carboxylic acids as substrates. All members of the Saccharomyces genus (sensu stricto) were found to decarboxylate both sorbic and cinnamic acids. PAD1 homologues and decarboxylation activity were found also in Candida albicans, Candida dubliniensis, Debaryomyces hansenii, and Pichia anomala. The decarboxylation of sorbic acid was assessed as a possible mechanism of resistance in spoilage yeasts. The decarboxylation of either sorbic or cinnamic acid was not detected for Zygosaccharomyces, Kazachstania (Saccharomyces sensu lato), Zygotorulaspora, or Torulaspora, the genera containing the most notorious spoilage yeasts. Scatter plots showed no correlation between the extent of sorbic acid decarboxylation and resistance to sorbic acid in spoilage yeasts. Inhibitory concentrations of sorbic acid were almost identical for S. cerevisiae wild-type and Δpad1 strains. We concluded that Pad1p-mediated sorbic acid decarboxylation did not constitute a significant mechanism of resistance to weak-acid preservatives by spoilage yeasts, even if the decarboxylation contributed to spoilage through the generation of unpleasant odors.

The Weak-Acid Preservative Sorbic Acid Is Decarboxylated and Detoxified by a Phenylacrylic Acid Decarboxylase, PadA1, in the Spoilage Mold Aspergillus niger

ABSTRACT Resistance to sorbic and cinnamic acids is mediated by a phenylacrylic acid decarboxylase (PadA1) in Aspergillus niger. A. niger ΔpadA1 mutants are unable to decarboxylate sorbic and cinnamic acids, and the MIC of sorbic acid required to inhibit spore germination was reduced by ∼50% in ΔpadA1 mutants.

Single-chain Variable Fragment Albumin Fusions Bind the Neonatal Fc Receptor (FcRn) in a Species-dependent Manner IMPLICATIONS FOR IN VIVO HALF-LIFE EVALUATION OF ALBUMIN FUSION THERAPEUTICS

Background: Albumin is utilized as carrier of biopharmaceuticals. FcRn binding regulates its long half-life. Results: ScFv fusion to HSA only slightly reduces human FcRn binding, whereas HSA and scFv-HSA fusions have very weak binding to rodent FcRn. Conclusion: Rodents have limitations for preclinical evaluation of HSA fusions. Significance: We illuminate design of HSA fusions and highlight cross-species differences to consider prior to preclinical evaluation. Albumin has a serum half-life of 3 weeks in humans. This has been utilized to extend the serum persistence of biopharmaceuticals that are fused to albumin. In light of the fact that the neonatal Fc receptor (FcRn) is a key regulator of albumin homeostasis, it is crucial to address how fusion of therapeutics to albumin impacts binding to FcRn. Here, we report on a detailed molecular investigation on how genetic fusion of a short peptide or an single-chain variable fragment (scFv) fragment to human serum albumin (HSA) influences pH-dependent binding to FcRn from mouse, rat, monkey, and human. We have found that fusion to the N- or C-terminal end of HSA only slightly reduces receptor binding, where the most noticeable effect is seen after fusion to the C-terminal end. Furthermore, in contrast to the observed strong binding to human and monkey FcRn, HSA and all HSA fusions bound very poorly to mouse and rat versions of the receptor. Thus, we demonstrate that conventional rodents are limited as preclinical models for analysis of serum half-life of HSA-based biopharmaceuticals. This finding is explained by cross-species differences mainly found within domain III (DIII) of albumin. Our data demonstrate that although fusion, particularly to the C-terminal end, may slightly reduce the affinity for FcRn, HSA is versatile as a carrier of biopharmaceuticals.

The decarboxylation of the weak-acid preservative, sorbic acid, is encoded by linked genes in Aspergillus spp.

The ability to resist anti-microbial compounds is of key evolutionary benefit to microorganisms. Aspergillus niger has previously been shown to require the activity of a phenylacrylic acid decarboxylase (encoded by padA1) for the decarboxylation of the weak-acid preservative sorbic acid (2,4-hexadienoic acid) to 1,3-pentadiene. It is now shown that this decarboxylation process also requires the activity of a putative 4-hydroxybenzoic acid (3-octaprenyl-4-hydroxybenzoic acid) decarboxylase, encoded by a gene termed ohbA1, and a putative transcription factor, sorbic acid decarboxylase regulator, encoded by sdrA. The padA1,ohbA1 and sdrA genes are in close proximity to each other on chromosome 6 in the A. niger genome and further bioinformatic analysis revealed conserved synteny at this locus in several Aspergillus species and other ascomycete fungi indicating clustering of metabolic function. This cluster is absent from the genomes of A. fumigatus and A. clavatus and, as a consequence, neither species is capable of decarboxylating sorbic acid.

Auxotrophy for uridine increases the sensitivity of Aspergillus niger to weak-acid preservatives.

Weak-acid preservatives such as sorbic acid are added to foods to prevent fungal spoilage. The modes of action of weak-acid preservatives are only partially understood and, in this paper, further insight is presented into the mechanisms by which weak acids inhibit the growth of fungi. Uridine-requiring strains of Aspergillus niger were shown to be more sensitive to weak acids (including sorbic, acetic and benzoic acids) than wild-type (WT) strains. In contrast, sensitivity to other, non-acidic, antifungal substances was similar in mutant and WT strains. By complementing a pyrG(-) strain of A. niger with an intact pyrG gene, WT-like resistance to weak-acid preservatives was restored. Using (14)C-labelled uridine, sorbic acid was shown to completely inhibit uridine uptake in germinating conidia in a non-competitive manner. It is therefore proposed that the additional weak-acid sensitivity of the pyrG(-) strains was caused by weak-acid inhibition of uridine uptake. Several other auxotrophic strains of A. niger were screened for sensitivity to acetic, sorbic and decanoic acids. Strains auxotrophic for either adenine or uridine were found to have enhanced sensitivity but, in contrast, amino acid auxotrophs showed resistance comparable to that of the WT. Uridine auxotrophs of Saccharomyces cerevisiae were not more sensitive to weak acids compared to WT strains. In conclusion, this study describes a previously unknown mechanism of action of weak acids against the filamentous fungus A. niger, which may fundamentally affect our understanding of the preservation of food against spoilage fungi.

Mapping the structural requirements of inducers and substrates for decarboxylation of weak acid preservatives by the food spoilage mould Aspergillus niger.

Moulds are able to cause spoilage in preserved foods through degradation of the preservatives using the Pad-decarboxylation system. This causes, for example, decarboxylation of the preservative sorbic acid to 1,3-pentadiene, a volatile compound with a kerosene-like odour. Neither the natural role of this system nor the range of potential substrates has yet been reported. The Pad-decarboxylation system, encoded by a gene cluster in germinating spores of the mould Aspergillus niger, involves activity by two decarboxylases, PadA1 and OhbA1, and a regulator, SdrA, acting pleiotropically on sorbic acid and cinnamic acid. The structural features of compounds important for the induction of Pad-decarboxylation at both transcriptional and functionality levels were investigated by rtPCR and GCMS. Sorbic and cinnamic acids served as transcriptional inducers but ferulic, coumaric and hexanoic acids did not. 2,3,4,5,6-Pentafluorocinnamic acid was a substrate for the enzyme but had no inducer function; it was used to distinguish induction and competence for decarboxylation in combination with the analogue chemicals. The structural requirements for the substrates of the Pad-decarboxylation system were probed using a variety of sorbic and cinnamic acid analogues. High decarboxylation activity, ~100% conversion of 1mM substrates, required a mono-carboxylic acid with an alkenyl double bond in the trans (E)-configuration at position C2, further unsaturation at C4, and an overall molecular length between 6.5Å and 9Å. Polar groups on the phenyl ring of cinnamic acid abolished activity (no conversion). Furthermore, several compounds were shown to block Pad-decarboxylation. These compounds, primarily aldehyde analogues of active substrates, may serve to reduce food spoilage by moulds such as A. niger. The possible ecological role of Pad-decarboxylation of spore self-inhibitors is unlikely and the most probable role for Pad-decarboxylation is to remove cinnamic acid-type inhibitors from plant material and allow uninhibited germination and growth of mould spores.