Proteomic identification of binding partners for the brain metabolite lanthionine ketimine (LK) and documentation of LK effects on microglia and motoneuron cell cultures

Abstract

Lanthionine ketimine (LK) represents a poorly understood class of thioethers present in mammalian CNS. Previous work has indicated high-affinity interaction of LK with synaptosomal membrane protein(s), but neither LK binding partners nor specific bioactivities have been reported. In this study, LK was chemically synthesized and used as an affinity agent to capture binding partners from mammalian brain lysate. Liquid chromatography with electrospray ionization-mass spectrometry of electrophoretically separated, LK-bound proteins identified polypeptides implicated in axon remodeling or vesicle trafficking and diseases including Alzheimer's disease and schizophrenia: collapsin response mediator protein-2/dihydropyrimidinase-like protein-2 (CRMP2/DRP2/DPYSL2), myelin basic protein, and syntaxin-binding protein-1 (STXBP1/Munc-18). Also identified was the recently discovered glutathione-binding protein lanthionine synthetase-like protein-1. Functional consequences of LK:CRMP2 interactions were probed through immunoprecipitation studies using brain lysate wherein LK was found to increase CRMP2 coprecipitation with its partner neurofibromin-1 but decreased CRMP2 coprecipitation with beta-tubulin. Functional studies of NSC-34 motor neuron-like cells indicated that a cell-permeable LK-ester, LKE, was nontoxic and protective against oxidative challenge with H(2)O(2). LKE-treated NSC-34 cells significantly increased neurite number and length in a serum concentration-dependent manner, consistent with a CRMP2 interaction. Finally, LKE antagonized the activation of EOC-20 microglia by inflammogens. The results are discussed with reference to possible biochemical origins, paracrine functions, neurological significance, and pharmacological potential of lanthionyl compounds.

Figure 1.

Structures and reactions discussed in text. A, Structures of lanthionine and lanthionine ketimine and likely biological source of lanthionine ketimine in the brain. B, Chemical synthesis of lanthionine ketimine from l-cysteine and 3-bromopyruvate. C, Conjugation of lanthionine ketimine to DADPA-agarose column by means of the Mannich reaction. The reaction is expected to proceed through the enamine tautomer as depicted.

Figure 2.

A, Coomassie Blue-stained SDS-PAGE profile of bovine brain proteins captured and released from an LK-derivatized (baited) DADPA column or an unbaited, mock-derivatized column. M, Molecular weight markers; F₀, 20 μg of bovine brain lysate before affinity chromatography; Blk, last column volume of wash (running) buffer before LK elution; − and +, protein fractions eluted with 100 mm LK from the unbaited and baited columns, respectively. Numbers adjacent to bands indicate proteins that were subjected to tryptic digestion, extraction, and mass spectrometry. Protein identities of these bands are indicated in Table 1. B, C, The + and − protein fractions were immunoblotted using antibodies against LanCL1 or DRP2/CRMP2 as an orthogonal method of MS corroboration. WB, Western blot.

Figure 3.

A, A molecular weight search probability-based protein identification output; B, a representative electrospray ionization-MS/MS of a tryptic peptide derived from in-gel digestion of the Coomassie Blue-stained band identified as LanCL1 in Figure 2.

Figure 4.

LK affects CRMP2 interaction with known protein binding partners. Whole-brain mouse protein extract was treated with or without 5 mm LK and immunoprecipitated with anti-CRMP2. A, Immunoprecipitated samples were electrophoresed under nonreducing conditions and blotted with antibodies against NF1 (∼300 kDa), β-tubulin (50 kDa), or CRMP2 (60 kDa) as indicated. B, Bands from the blots in A were quantified by densitometry, and the data were graphed as the value of each LK-treated sample, as a percentage of the mean of the respective samples that were not treated with LK. Error bars represent mean ± SD; *p < 0.05 by pairwise t test. WB, Western blot.

Figure 5.

Coomassie blue-stained one-dimensional gel showing tubulin and microfilament proteins that were coimmunoprecipitated with CRMP2 from mouse brain in the absence (−) and presence (+) of LK. Band number 5 revealed sequences corresponding to α-internexin primarily but also relatively minor but significant sequence components matched to CRMP2, which is expected to comigrate at this molecular weight. The band number 14 likely represents CRMP2 oligomers or CRMP2:anti-CRMP2 conjugates that were not separated under these electrophoretic conditions. Note the relative increase of neurofilament medium chain (NFL-M, band 15) in the LK (+) samples.

Figure 6.

Synthetic LKE is readily hydrolyzed to LK by general esterase activity. Synthetic LKE was treated with porcine esterase as described in Results and analyzed with HPLC with ultraviolet-visible spectrophotometry (UV/Vis) detection to determine whether the enzyme could hydrolyze the compound to yield LK. A peak corresponding to the retention time and UV/Vis characteristics of authentic LK was observed in esterase-treated LKE solutions. au, Arbitrary units.

Figure 7.

LK and LKE inhibit TNFα-stimulated ·NO production in EOC-20 microglia. A, Comparison of LK and LKE effects at 100 μm of each of the test compounds. B, Dose dependency of the LKE suppression of nitrite accumulation in EOC-24 cells challenged with TNFα. *p < 0.05 relative to positive control (no LK or LKE, cytokine only); **p < 0.01 relative to positive control; ^†p < 0.05 relative to LK-treated cytokine-stimulated cells. Each error bar represents mean ± SEM (n = 6); statistical significance determined by unpaired two-tailed t tests.

Figure 8.

LKE dose dependently protects NSC-34 motor neuron-like cells from toxicity caused by hydrogen peroxide. Cells were treated with the indicated concentrations of H₂O₂ in the presence or absence of various doses of LKE, and cell viability was determined by tetrazolium reduction 24 h later. Data represent mean ± SEM (n = 6). Both 100 and 500 μm LKE produced statistically significant protection as assessed by repeated-measures ANOVA.

Figure 9.

Photomicrographs of NSC-34 cells cultured under different ambient concentrations of FCS and LKE as indicated in each panel label. Images of fluorescently labeled cells were processed with computer-assisted algorithms and quantified for morphometry as described in Results.

Figure 10.

Quantitation of serum concentration and LKE-dependent effects on NSC-34 cell viability and morphological parameters. A, Effect of increasing serum on the fraction of NSC-34 cells developing neurites. B, Relative viability of NSC-34 cells as indexed by the tetrazolium reduction assay. C, Effect of FCS concentration and LKE exposure on the measured average number of neurites per cell. D, Effect of FCS concentration and LKE exposure on the average neurite length per cell. Error bars indicate mean ± SEM of six fields containing 40–100 cells per field in a typical experiment. *p < 0.05 relative to serum-free condition; ^†p < 0.05 for LKE effect, by two-tailed t test.