Substrates of the \u3cem\u3eArabidopsis thaliana\u3c/em\u3e Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning

The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when PIMT protein binding was poisoned with S-adenosyl homocysteine. After four rounds, phage titer plateaued in AdoMet-containing pans, whereas titer declined in both controls. This strategy identified 17 in-frame PIMT target proteins, including a cupin-family protein similar to those identified previously using on-blot methylation. All recovered phage had at least one susceptible Asp or Asn residue. Five targets were recovered independently. Two in-frame targets were produced in Escherichia coli as recombinant proteins and shown by on-blot methylation to acquire isoAsp, becoming a PIMT target. Both gained isoAsp rapidly in solution upon thermal insult. Mutant analysis of plants deficient in any of three in-frame PIMT targets resulted in demonstrable phenotypes. An over-representation of clones encoding proteins involved in protein production suggests that the translational apparatus comprises a subgroup for which PIMT-mediated repair is vital for orthodox seed longevity. Impaired PIMT activity would hinder protein function in these targets, possibly resulting in poor seed performance

A sterospecific colorimetric assay for (s,s)-Adenosylmethionine quantification based on thiopurine methyltransferase-catalyzed thiol methylation

S-Adenosyl-L-methionine (AdoMet or SAM) that is biologically synthesized by AdoMet synthetase bears an s-configuration at the sulfur atom. The chiral sulfonium spontaneously racemizes to form a mixture of s-and R-isomers of AdoMet under physiological conditions or normal storage conditions. The chirality of AdoMet greatly affects its activity; the R-isomer is not accepted as a substrate for AdoMet-dependent methyltransferases. We report a stereospecific colorimetric assay for (s,s)-adenosylmethionine quantification based on an enzyme-coupled reaction in which (s,s)-AdoMet reacts with 2-nitro-5-thiobenzoic acid (TNB) to form AdoHcy and 2-nitro-5-methylthiobenzoic acid. The transformation is catalyzed by recombinant human thiopurine S-methyltransferase (TPMT, EC 2.1.1.67), and is associated with a large spectral change at 410 nm. Accumulation of the S-adenosylhomocysteine (AdoHcy) product, a feedback inhibitor of TPMT, slows down the assay. AdoHcy nucleosidase (EC 3.2.2.9) irreversibly cleaves AdoHcy to adenine and s-ribosylhomocysteine, significantly shortening the assay time to less than 10 min. The assay is linear from 5 to at least 60 f.lM (s,s)AdoMet

Synthesis and characterization of <i>Se</i>-adenosyl-L-selenohomocysteine selenoxide

<div><p>Selenium is an essential micronutrient in humans due to the important roles of the selenocysteine-containing selenoproteins. Organoselenium metabolites are generally found to be substrates for the biochemical pathways of their sulfur analogs, and the redox chemistry of selenomethionine and some other metabolites have been previously reported. We now report the first synthesis and characterization of <i>Se</i>-adenosylselenohomocysteine selenoxide (SeAHO) prepared via hydrogen peroxide oxidation of <i>Se</i>-adenosylselenohomocysteine. The selenoxide SeAHO, in contrast to its corresponding sulfoxide <i>S</i>-adenosylhomocysteine (SAHO), can form hydrate, has an electrostatic interaction between the α-amino acid moiety and the highly polar selenoxide functional group, and readily oxidizes glutathione (GSH) and cysteine thiols.</p></div

Comparison of Protein N-Homocysteinylation in Rat Plasma under Elevated Homocysteine Using a Specific Chemical Labeling Method

Elevated blood concentrations of homocysteine have been well established as a risk factor for cardiovascular diseases and neuropsychiatric diseases, yet the etiologic relationship of homocysteine to these disorders remains poorly understood. Protein N-homocysteinylation has been hypothesized as a contributing factor; however, it has not been examined globally owing to the lack of suitable detection methods. We recently developed a selective chemical method to label N-homocysteinylated proteins with a biotin-aldehyde tag followed by Western blotting analysis, which was further optimized in this study. We then investigated the variation of protein N-homocysteinylation in plasma from rats on a vitamin B12 deficient diet. Elevated “total homocysteine” concentrations were determined in rats with a vitamin B12 deficient diet. Correspondingly, overall levels of plasma protein N-homocysteinylation displayed an increased trend, and furthermore, more pronounced and statistically significant changes (e.g., 1.8-fold, p-value: 0.03) were observed for some individual protein bands. Our results suggest that, as expected, a general metabolic correlation exists between “total homocysteine” and N-homocysteinylation, although other factors are involved in homocysteine/homocysteine thiolactone metabolism, such as the transsulfuration of homocysteine by cystathionine β-synthase or the hydrolysis of homocysteine thiolactone by paraoxonase 1 (PON1), may play more significant or direct roles in determining the level of N-homocysteinylation

When Good Intentions Go Awry: Modification of a Recombinant Monoclonal Antibody in Chemically Defined Cell Culture by Xylosone, an Oxidative Product of Ascorbic Acid

With the advent of new initiatives to develop chemically defined media, cell culture scientists screen many additives to improve cell growth and productivity. However, the introduction or increase of supplements, typically considered beneficial or protective on their own, to the basal media or feed stream may cause unexpected detrimental consequences to product quality. For instance, because cultured cells are constantly under oxidative stress, ascorbic acid (vitamin C, a potent natural reducing agent) is a common additive to cell culture media. However, as reported herein, a recombinant monoclonal antibody (adalimumab) in cell culture was covalently modified by xylosone (molecular weight 148), an oxidative product of ascorbate. Containing reactive carbonyl groups, xylosone modifies various amines (e.g., the N-termini of the heavy and light chains and susceptible lysines), forming either hemiaminal (+148 Da) or Schiff base (imine, +130 Da) products. Our findings show, for the first time, that ascorbate-derived xylosone can contribute to an increase in molecular heterogeneity, such as acidic species. Our work serves as a reminder that additives to cell culture and their metabolites may become reactive and negatively impact the overall product quality and should be carefully monitored with any changes in cell culture conditions

Discovery of a Chemical Modification by Citric Acid in a Recombinant Monoclonal Antibody

Recombinant therapeutic monoclonal antibodies exhibit a high degree of heterogeneity that can arise from various post-translational modifications. The formulation for a protein product is to maintain a specific pH and to minimize further modifications. Generally Recognized as Safe (GRAS), citric acid is commonly used for formulation to maintain a pH at a range between 3 and 6 and is generally considered chemically inert. However, as we reported herein, citric acid covalently modified a recombinant monoclonal antibody (IgG1) in a phosphate/citrate-buffered formulation at pH 5.2 and led to the formation of so-called “acidic species” that showed mass increases of 174 and 156 Da, respectively. Peptide mapping revealed that the modification occurred at the N-terminus of the light chain. Three additional antibodies also showed the same modification but displayed different susceptibilities of the N-termini of the light chain, heavy chain, or both. Thus, ostensibly unreactive excipients under certain conditions may increase heterogeneity and acidic species in formulated recombinant monoclonal antibodies. By analogy, other molecules (e.g., succinic acid) with two or more carboxylic acid groups and capable of forming an anhydride may exhibit similar reactivities. Altogether, our findings again reminded us that it is prudent to consider formulations as a potential source for chemical modifications and product heterogeneity

Protein Isoaspartate Methyltransferase-Mediated ¹⁸O-Labeling of Isoaspartic Acid for Mass Spectrometry Analysis

Arising from spontaneous aspartic acid (Asp) isomerization or asparagine (Asn) deamidation, isoaspartic acid (isoAsp, isoD, or beta-Asp) is a ubiquitous nonenzymatic modification of proteins and peptides. Because there is no mass difference between isoaspartyl and aspartyl species, sensitive and specific detection of isoAsp, particularly in complex samples, remains challenging. Here we report a novel assay for Asp isomerization by isotopic labeling with ¹⁸O via a two-step process: the isoAsp peptide is first specifically methylated by protein isoaspartate methyltransferase (PIMT, EC 2.1.1.77) to the corresponding methyl ester, which is subsequently hydrolyzed in ¹⁸O-water to regenerate isoAsp. The specific replacement of ¹⁶O with ¹⁸O at isoAsp leads to a mass shift of 2 Da, which can be automatically and unambiguously recognized using standard mass spectrometry, such as collision-induced dissociation (CID), and data analysis algorithms. Detection and site identification of several isoAsp peptides in a monoclonal antibody and the β-delta sleep-inducing peptide (DSIP) are demonstrated