Structural and Functional Modifications of Corneal Crystallin ALDH3A1 by UVB Light

As one of the most abundantly expressed proteins in the mammalian corneal epithelium, aldehyde dehydrogenase 3A1 (ALDH3A1) plays critical and multifaceted roles in protecting the cornea from oxidative stress. Recent studies have demonstrated that one protective mechanism of ALDH3A1 is the direct absorption of UV-energy, which reduces damage to other corneal proteins such as glucose-6-phosphate dehydrogenase through a competition mechanism. UV-exposure, however, leads to the inactivation of ALDH3A1 in such cases. In the current study, we demonstrate that UV-light caused soluble, non-native aggregation of ALDH3A1 due to both covalent and non-covalent interactions, and that the formation of the aggregates was responsible for the loss of ALDH3A1 enzymatic activity. Spectroscopic studies revealed that as a result of aggregation, the secondary and tertiary structure of ALDH3A1 were perturbed. LysC peptide mapping using MALDI-TOF mass spectrometry shows that UV-induced damage to ALDH3A1 also includes chemical modifications to Trp, Met, and Cys residues. Surprisingly, the conserved active site Cys of ALDH3A1 does not appear to be affected by UV-exposure; this residue remained intact after exposure to UV-light that rendered the enzyme completely inactive. Collectively, our data suggest that the UV-induced inactivation of ALDH3A1 is a result of non-native aggregation and associated structural changes rather than specific damage to the active site Cys

BSA degradation under acidic conditions: A model for protein instability during release from PLGA delivery systems

Acidification of the internal poly(lactide- co -glycolide) (PLGA) microenvironment is considered one of the major protein stresses during controlled release from such delivery systems. A model protein, bovine serum albumin (BSA), was incubated at 37°C for 28 days to simulate the environment within the aqueous pores of PLGA during the release phase and to determine how acidic microclimate conditions affect BSA stability. Size-exclusion high performance liquid chromatography (SE-HPLC), SDS–PAGE, and infrared spectroscopy were used to monitor BSA degradation. BSA was most stable at pH 7, but rapidly degraded via aggregation and hydrolysis at pH 2. These simulated degradation products were nearly identical to that of unreleased BSA found entrapped within PLGA 50/50 millicylinders. At pH 2, changes in BSA conformation detected by various spectroscopic techniques were consistent with acid denaturation of the protein. By contrast, at pH 5 and above, damage to BSA was insufficient to explain the instability of the protein in the polymer. Thus, these data confirm the hypothesis that acid-induced unfolding is the basis of BSA aggregation in PLGA and the acidic microclimate within PLGA is indeed a dominant stress for encapsulated BSA. To increase the stability of proteins within PLGA systems, formulations must protect against potentially extreme acidification such that native structure is maintained. © 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95: 1626–1639, 2006Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55225/1/20625_ftp.pd

Human aldehyde dehydrogenase 3A1 (ALDH3A1): biochemical characterization and immunohistochemical localization in the cornea.

ALDH3A1 (aldehyde dehydrogenase 3A1) is expressed at high concentrations in the mammalian cornea and it is believed that it protects this vital tissue and the rest of the eye against UV-light-induced damage. The precise biological function(s) and cellular distribution of ALDH3A1 in the corneal tissue remain to be elucidated. Among the hypotheses proposed for ALDH3A1 function in cornea is detoxification of aldehydes formed during UV-induced lipid peroxidation. To investigate in detail the biochemical properties and distribution of this protein in the human cornea, we expressed human ALDH3A1 in Sf9 insect cells using a baculovirus vector and raised monoclonal antibodies against ALDH3A1. Recombinant ALDH3A1 protein was purified to homogeneity with a single-step affinity chromatography method using 5'-AMP-Sepharose 4B. Human ALDH3A1 demonstrated high substrate specificity for medium-chain (6 carbons and more) saturated and unsaturated aldehydes, including 4-hydroxy-2-nonenal, which are generated by the peroxidation of cellular lipids. Short-chain aliphatic aldehydes, such as acetaldehyde, propionaldehyde and malondialdehyde, were found to be very poor substrates for human ALDH3A1. In addition, ALDH3A1 metabolized glyceraldehyde poorly and did not metabolize glucose 6-phosphate, 6-phosphoglucono-delta-lactone and 6-phosphogluconate at all, suggesting that this enzyme is not involved in either glycolysis or the pentose phosphate pathway. Immunohistochemistry in human corneas, using the monoclonal antibodies described herein, revealed ALDH3A1 expression in epithelial cells and stromal keratocytes, but not in endothelial cells. Overall, these cumulative findings support the metabolic function of ALDH3A1 as a part of a corneal cellular defence mechanism against oxidative damage caused by aldehydic products of lipid peroxidation. Both recombinant human ALDH3A1 and the highly specific monoclonal antibodies described in the present paper may prove to be useful in probing biological functions of this protein in ocular tissue

Campus Reflections

Campus Reflections on convicted civility featuring: Trayvon Estey, Lauren Bournique, Sharee Nurse, Tia Etter, and Chester Chan

Fluoresence studies of UV-irradiated ALDH3A1.

<p>(<b><i>A</i></b>) Intrinsic ALDH3A1 Trp fluorescence (λ_ex = 295 nm). (<b><i>B</i></b>) Instrinsic ALDH3A1 Tyr fluorescence (λ_ex = 278 nm). (<b><i>C</i></b>) Fluorescence of the Trp degradation product, NFK (λ_ex = 315 nm). ALDH3A1 was irradiated and NFK fluorescence spectra collected at 1 mg/ml. Samples were excited at 315 nm and emission scans collected from 330 to 600 nm. The arrow indicates the trend in the change in fluorescent properties as a function of the exposure time in each case.</p

SDS-PAGE analysis of UVB-exposed ALDH3A1.

<p>ALDH3A1 was subjected to SDS-PAGE with Coomassie blue staining under non-reducing (<b><i>A</i></b>) and reducing (<b><i>B</i></b>) conditions. Each well represents 10 µg protein. Exposure times are given on the top and molecular weight markers on the left. The ALDH3A1 monomer is also indicated.</p

Sequence Coverage of ALDH3A1 By Fractionation Elution^a.

a<p>LysC digestion peptides were eluted from the C₁₈ ZipTip by acetonitrile (ACN) fractionation as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015218#s4" target="_blank">methods</a> section.</p>b<p>Expressed as monoisotopic singly charged species, [M+H]⁺.</p>c<p>Peptide masses that were detectable only using the linear mode of the indicated fraction are shown in italics.</p

Far UV CD of ALDH3A1.

<p>ALDH3A1 (0.1 mg/ml) scans were collected from 190 to 260 nm at 0.5 nm intervals. Data transformed to mean residue ellipticity. Each spectrum is an average of three independent samples.</p