Conformational studies of a monoclonal antibody, IgG1, by chemical oxidation: structural analysis by ultrahigh-pressure LC-electrospray ionization time-of-flight MS and multivariate data analysis

We describe the development of a method in which protein oxidation by H₂O₂ followed by ultrahigh-pressure liquid chromatography (UHPLC) coupled with electrospray ionization time-of-flight mass spectrometry (ESI-ToFMS) and multivariate analysis are used to detect alterations in conformational states of proteins. In the study reported here, an IgG1 monoclonal antibody in native and denatured conformational states was oxidized by treatment with hydrogen peroxide. Peptide fragments generated by tryptic digestion were then analyzed by UHPLC-ESI-ToFMS. After reducing noise and extracting peaks from the LC–MS data using MzExplorer, software developed in-house and based on Matlab, we were able to distinguish peptides arising from the native and denatured states of the oxidized protein by principal component analysis. Peptides containing residues, which are inclined to undergo oxidation, such as methionine, are founded to be particularly important in this approach. We believe that the methodology could facilitate attempts to characterize the conformational states of recombinant monoclonal antibodies and other proteins.     

An anaerobic bacterial MsrB model reveals catalytic mechanisms, advantages, and disadvantages provided by selenocysteine and cysteine in reduction of methionine-R-sulfoxide

We verified and generalized the catalytic features that selenocysteine (Sec) and cysteine (Cys) contribute to the reduction of methionine-R-sulfoxide using an anaerobic bacterial MsrB from Clostridium sp. OhILA as a model protein. The Sec-containing Clostridium MsrB form exhibited 100-fold higher activity than its Cys-containing form, revealing that Sec provided the catalytic advantage of higher activity. However, a resolving Cys was required for the thioredoxin (Trx)-dependent recycling process of the Sec-containing form. Thus, Trx could reduce the selenenylsulfide bond, but its Trx-dependent recycling process was much less efficient compared to that for the disulfide bond in the Cys-containing form, demonstrating an obvious catalytic disadvantage. These data agreed well with our previous data on mammalian MsrBs, and therefore suggested that the catalytic mechanisms, as well as the catalytic advantages and disadvantages provided by the Sec and Cys residues, are most likely conserved from anaerobic bacteria to mammals. Taken together, we propose that the use of Sec in MsrB may depend on a balance between the catalytic advantage of higher activity and the disadvantage of a less efficient regeneration process provided by this residue.     

Thioredoxin as a reducing agent for mammalian methionine sulfoxide reductases B lacking resolving cysteine

Previous reports described thioredoxin (Trx) as a very poor reductant for mammalian MsrB2 and MsrB3, which lack a resolving Cys residue. In contrast, we here report that Trx could reduce both MsrB2 and MsrB3 enzymes, similarly to the reduction of mammalian MsrA. We demonstrated that functional Trx is required for the reduction of these enzymes. We further identified MsrB2- or MsrB3-Trx complexes formed through intermolecular disulfide bonds involving catalytic residue of Trx. The present study provides evidence that the sulfenic acid intermediate of oxidized MsrBs lacking resolving Cys could interact with Trx and be directly reduced by this protein.     

Constitutive behavior of methionyl-tRNA synthetase compared to repressible behavior of methionine adenosyltransferase in mammalian cells

Methionine adenosyltransferase, one of the two major enzymes utilizing methionine, is regulated by the levels of methionine in the growth medium (Jacobsen, S.J., Hoffman, R.M. and Erbe, R.W. (1980) J. Natl. Cancer Inst. 65, 1237–1244, and Caboche, M. and Mulsant, P. (1978) Somatic Cell Genet. 4, 407–421). We report here that methionyl-tRNA synthetase, unlike methionine adenosyltransferase, behaves in a constitutive manner with respect to the concentration of methionine in the culture medium. This behavior is seen in Chinese hamster ovary cells and in normal diploid and SV 40-transformed human fibroblasts. Although the kinetics of regulation of methionine adenosyltransferase and methionyl-tRNA synthetase by exogenous methionine are clearly different, the levels of the two enzymes in the human cell lines are similar.     

mTORC1 stimulates cell growth through SAM synthesis and m6A mRNA-dependent control of protein synthesis

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Binding of [H][D-Ala, MePhe, Gly-ol] Enkephalin, [H][D-Pen, D-Pen]Enkephalin, and [H]U-69,593 to Airway and Pulmonary Tissues of Normal and Sensitized Rats

Bhargava, H. N., V. M. Villar, J. Cortijo and E. J. Morcillo. Binding of [³H][D-Ala², MePhe⁴, Gly-ol⁵]enkephalin, [³H][D-Pen², D-Pen⁵]enkephalin, and [³H]U-69,593 to airway and pulmonary tissues of normal and sensitized rats. Peptides 18(10) 1603–1608, 1997.—The role of endogenous opioid peptides in the regulation of bronchomotor tone, as well as in the pathophysiology of asthma is uncertain. We have studied the binding of highly selective [³H]labeled ligands of μ-([D-Ala², MePhe⁴, Gly-ol⁵]enkephalin; DAMGO), δ ([D-Pen², D-Pen⁵]enkephalin; DPDPE), and κ-(U-69,593) opioid receptors to membranes of trachea, main bronchus, lung parenchyma and pulmonary artery obtained from normal (unsensitized) and actively IgE-sensitized rats acutely challenged with the specific antigen. [³H]DAMGO, [³H]DPDPE and [³H]U-69,593 bound to membranes of normal and sensitized tissues at a saturable, single high-affinity site. The rank order of receptor densities in normal tissues was δ- ≥ κ- ≥ μ-, with lung parenchyma exhibiting the greatest binding capacity for δ- and μ- receptors compared to the other regions examined. The K_d values showed small differences between ligands and regions tested. The μ- and δ-opioid receptor densities were decreased in sensitized main bronchus and lung parenchyma, respectively, compared to normal tissues. By contrast, κ-opioid receptor density was augmented in sensitized lung parenchyma but an increase in K_d values was also observed. These differential changes in the density and affinity of opioid receptor types may be related to alterations in endogenous opioid peptides during the process of sensitization.     

Heme crevice disorder after sixth ligand displacement in the cytochrome c-551 family

1H NMR and visible absorption spectroscopy were used to monitor sixth ligand methionine displacement reactions in four members of the ferricytochrome c-551 family from Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas stutzeri substrain ZoBell, and Nitrosomonas europae. Potassium cyanide displaces the methionine ligand with very modest changes in the visible spectra, but profound changes in the NMR spectra. The initial product formed kinetically, designated complex I, changes with time and/or heating to a more thermodynamically favored product termed complex II. Spectra indicate that both I and II are actually a family of closely related conformational isomers. Low temperature NMR spectra of complex II indicate that some of the isomers are in chemical exchange on the NMR time scale. High pH also displaces the methionine ligand in a manner similar to the well-known alkaline transition of mitochondrial cytochrome c. However, the reaction occurs at higher pH values and over a narrower pH range for the c-551 family, and the transition pH range is different for the different proteins studied. The final alkaline forms also show peak widths and a number of peaks indicative of multiple conformational isomers.     

Formation of methionine sulfoxide during glycoxidation and lipoxidation of ribonuclease A

Chemical modification of proteins by reactive oxygen species affects protein structure, function and turnover during aging and chronic disease. Some of this damage is direct, for example by oxidation of amino acids in protein by peroxide or other reactive oxygen species, but autoxidation of ambient carbohydrates and lipids amplifies both the oxidative and chemical damage to protein and leads to formation of advanced glycoxidation and lipoxidation end-products (AGE/ALEs). In previous work, we have observed the oxidation of methionine during glycoxidation and lipoxidation reactions, and in the present work we set out to determine if methionine sulfoxide (MetSO) in protein was a more sensitive indicator of glycoxidative and lipoxidative damage than AGE/ALEs. We also investigated the sites of methionine oxidation in a model protein, ribonuclease A (RNase), in order to determine whether analysis of the site specificity of methionine oxidation in proteins could be used to indicate the source of the oxidative damage, i.e. carbohydrate or lipid. We describe here the development of an LC/MS/MS for quantification of methionine oxidation at specific sites in RNase during glycoxidation or lipoxidation by glucose or arachidonate, respectively. Glycoxidized and lipoxidized RNase were analyzed by tryptic digestion, followed by reversed phase HPLC and mass spectrometric analysis to quantify methionine and methionine sulfoxide containing peptides. We observed that: (1) compared to AGE/ALEs, methionine sulfoxide was a more sensitive biomarker of glycoxidative or lipoxidative damage to proteins; (2) regardless of oxidizable substrate, the relative rate of oxidation of methionine residues in RNase was Met29>Met30>Met13, with Met79 being resistant to oxidation; and (3) arachidonate produced a significantly greater yield of MetSO, compared to glucose. The methods developed here should be useful for assessing a protein's overall exposure to oxidative stress from a variety of sources in vivo.     

Immunfluorescence study of neuropeptides in identified neurons of the rat auditory superior olivary complex

 The present study was conducted to investigate the distribution and immunohistochemical characteristics of ascending and descending projection neurons of the rat superior olivary complex (SOC), a group of interrelated brainstem nuclei. Ascending neurons were identified by injection of cholera toxin B subunit (CTB) into the central nucleus of the inferior colliculus (IC), descending neurons were labeled by application of Fluoro-Gold (FG) into the scala tympani of the cochlea, ipsilaterally to the IC injection. In accordance with the literature, we observed neurons innervating the IC located in the lateral superior olivary nucleus (LSO) and dorsal periolivary groups (DPO) on both sides, in the superior paraolivary nucleus (SPO) predominantly ipsilateral, as well as in the ipsilateral medial superior olivary nucleus (MSO) and the medial nucleus of the trapezoid body (MNTB). Cochlear projection neurons were found predominantly in the ipsilateral LSO as well as in the bilateral SPO, DPO, MSO and MNTB. In addition, a considerable population of neurons in the ipsilateral LSO and SPO were identified as being both ascending and descending. To further characterize these double-projecting neurons, brainstem sections were incubated in antisera directed against different neuroactive substances. The majority of ascending/descending cells in the LSO contained calcitonin gene-related peptide, but not substance P (SP), met-enkephalin (ENK) or tyrosine hydroxylase (TH). Some of these neurons apparently were contacted by ENK- or SP-immunoreactive fibers and terminals. In addition, we found TH-immunoreactive neurons in the lateral MNTB region. These neurons, which were labeled upon tracer injection into the cochlea (but not upon IC injection), probably belong to the C1 catecholaminergic cell group and may represent a division of the uncrossed olivocochlear bundle. The present results reveal the existence of a previously unknown subpopulation of SOC neurons that project to both the cochlea and the inferior colliculus. Their CGRP immunoreactivity and their uncrossed projection pattern provide evidence that they may belong to the cholinergic, putatively excitatory cell group. Received: 4 January 1999 / Accepted: 17 February 1999     

Novel reversible methionine aminopeptidase-2 (MetAP-2) inhibitors based on purine and related bicyclic templates

The natural product fumagillin 1 and derivatives like TNP-470 2 or beloranib 3 bind to methionine aminopeptidase 2 (MetAP-2) irreversibly. This enzyme is critical for protein maturation and plays a key role in angiogenesis. In this paper we describe the synthesis, MetAP-2 binding affinity and structural analysis of reversible MetAP-2 inhibitors. Optimization of enzymatic activity of screening hit 10 (IC₅₀: 1 μM) led to the most potent compound 27 (IC₅₀: 0.038 μM), with a concomitant improvement in LLE from 2.1 to 4.2. Structural analysis of these MetAP-2 inhibitors revealed an unprecedented conformation of the His339 side-chain imidazole ring being co-planar sandwiched between the imidazole of His331 and the aryl-ether moiety, which is bound to the purine scaffold. Systematic alteration and reduction of H-bonding capability of this metal binding moiety induced an unexpected 180° flip for the triazolo[1,5-a]pyrimdine bicyclic template.