Characterization of p40/GPR69A as a Peripheral Membrane Protein Related to the Lantibiotic Synthetase Component C

doi:10.1006/bbrc.2000.3260

Biochemical and Biophysical Research Communications

Volume 275, Issue 1, 18 August 2000, Pages 69-74

https://doi.org/10.1006/bbrc.2000.3260 Get rights and content

Abstract

The 40 kDa erythrocyte membrane protein p40/GPR69A, previously assigned to the G-protein-coupled receptor superfamily, was now identified by peptide-antibodies and characterized as a loosely associated peripheral membrane protein. This result is in striking contrast to the proposed seven-transmembrane protein structure and function and therefore we wish to correct our previous proposal. p40 is located at the cytoplasmic side of the membrane and is neither associated with the cytoskeleton nor lipid rafts. Refined sequence analysis revealed that p40 is related to the LanC family of bacterial membrane-associated proteins which are involved in the biosynthesis of antimicrobial peptides. Therefore, we rename p40 to LanC-like protein 1 (LANCL1) and suggest that it may play a similar role as a peptide-modifying enzyme component in eukaryotic cells.

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Cited by (48)

LanCLs add glutathione to dehydroamino acids generated at phosphorylated sites in the proteome
2021, Cell
Citation Excerpt :
The structure of human LanCL1 resembles NisC (a bacterial LanC); both structures contain two-layered α helix barrels that bind Zn2+ (Figure 1A) (Li et al., 2006; Zhang et al., 2009). LanCL1 and 2 are ubiquitously expressed in various tissues (Bauer et al., 2000; Eley et al., 2002; Mayer et al., 1998, 2001b), with the highest expression levels in brain, heart, and testes, whereas the expression pattern of LanCL3 remains to be determined due to the lack of a specific antibody. Human LanCL1 and LanCL2 bind to GSH and LanCL1 possesses weak glutathione S-transferase activity in assays with a xenobiotic substrate (Chung et al., 2007; Huang et al., 2014; Mladkova et al., 2010; Zhang et al., 2009).
Enzyme-mediated damage repair or mitigation, while common for nucleic acids, is rare for proteins. Examples of protein damage are elimination of phosphorylated Ser/Thr to dehydroalanine/dehydrobutyrine (Dha/Dhb) in pathogenesis and aging. Bacterial LanC enzymes use Dha/Dhb to form carbon-sulfur linkages in antimicrobial peptides, but the functions of eukaryotic LanC-like (LanCL) counterparts are unknown. We show that LanCLs catalyze the addition of glutathione to Dha/Dhb in proteins, driving irreversible C-glutathionylation. Chemo-enzymatic methods were developed to site-selectively incorporate Dha/Dhb at phospho-regulated sites in kinases. In human MAPK-MEK1, such “elimination damage” generated aberrantly activated kinases, which were deactivated by LanCL-mediated C-glutathionylation. Surveys of endogenous proteins bearing damage from elimination (the eliminylome) also suggest it is a source of electrophilic reactivity. LanCLs thus remove these reactive electrophiles and their potentially dysregulatory effects from the proteome. As knockout of LanCL in mice can result in premature death, repair of this kind of protein damage appears important physiologically.
LanCL1 attenuates ischemia-induced oxidative stress by Sirt3-mediated preservation of mitochondrial function
2018, Brain Research Bulletin
Citation Excerpt :
Tissue plasminogen activator is the only drug approved by the FDA for acute ischemic stroke treatment, but only 4–5% of all stroke patients can benefit from it (Docagne et al., 2015). The lanthionine synthetase C-like protein 1 (LanCL1), originally known as p40/GPR69 A, is one member of the LanC-like protein family, which are homologous to prokaryotic lanthionine synthetase component C (lanthionine cyclases) (Bauer et al., 2000). Prokaryotic lanthionine cyclases act in concert with dehydratases to facilitate intramolecular conjugation of cysteine to serine or threonine residues, yielding potent “lantibiotics”, which are not present in mammals (Bierbaum and Sahl, 2009; Chatterjee et al., 2005).
Lanthionine synthetase C-like protein 1 (LanCL1) is homologous to prokaryotic lanthionine cyclases, and has been shown to have novel functions in neuronal redox homeostasis. A recent study showed that LanCL1 expression was developmental and activity-dependent regulated, and LanCL1 transgene protected neurons against oxidative stress. In the present study, the potential protective effects of LanCL1 against ischemia was investigated in an in vitro model mimicked by oxygen and glucose deprivation (OGD) in neuronal HT22 cells. We found that OGD exposure induced a temporal increase and persistent decreases in the expression of LanCL1 at both mRNA and protein levels. Overexpression of LanCL1 by lentivirus (LV-LanCL1) transfection preserved cell viability, reduced lactate dehydrogenase (LDH) release and attenuated apoptosis after OGD. These protective effects were accompanied by decreased protein radical formation, lipid peroxidation and mitochondrial dysfunction. In addition, LanCL1 significantly stimulated mitochondrial enzyme activities and SOD2 deacetylation in a Sirt3-dependent manner. The results of western blot analysis showed that LanCL1-induced activation of Sirt3 was dependent on Akt-PGC-1α pathway. Knockdown of PGC-1α expression using small interfering RNA (siRNA) or blocking Akt activation using specific antagonist partially prevented the protective effects of LanCL1 in HT22 cells. Taken together, our results show that LanCL1 protects against OGD through activating the Akt-PGC-1α-Sirt3 pathway, and may have potential therapeutic value for ischemic stroke.
Identification of a high affinity binding site for abscisic acid on human lanthionine synthetase component C-like protein 2
2018, International Journal of Biochemistry and Cell Biology
Citation Excerpt :
The lanthionine synthetase C-like (LANCL) enzyme family comprises the eukaryotic homologues of the prokaryotic lanthionine synthetase component C (LanC) protein, a zinc-containing enzyme involved in the modification of peptides and lanthionines (Bauer et al., 2000).
Lanthionine synthetase component C-like protein 2 (LANCL2) has been identified as the mammalian receptor mediating the functional effects of the universal stress hormone abscisic acid (ABA) in mammals. ABA stimulates insulin independent glucose uptake in myocytes and adipocytes via LANCL2 binding in vitro, improves glucose tolerance in vivo and induces brown fat activity in vitro and in vivo. The emerging role of the ABA/LANCL2 system in glucose and lipid metabolism makes it an attractive target for pharmacological interventions in diabetes mellitus and the metabolic syndrome. The aim of this study was to investigate the presence of ABA binding site(s) on LANCL2 and identify the amino acid residues involved in ABA binding. Equilibrium binding assays ([³H]-ABA saturation binding and surface plasmon resonance analysis) suggested multiple ABA-binding sites, prompting us to perform a computational study that indicated one putative high-affinity and two low-affinity binding sites. Site-directed mutagenesis (single mutant R118I, triple mutants R118I/R22I/K362I and R118I/S41A/E46I) and equilibrium binding experiments on the mutated LANCL2 proteins identified a high-affinity ABA-binding site involving R118, with a K_D of 2.6 nM ± 1.2 nM, as determined by surface plasmon resonance. Scatchard plot analysis of binding curves from both types of equilibrium binding assays revealed a Hill coefficient >1, suggesting cooperativity of ABA binding to LANCL2. Identification of the high-affinity ABA-binding site is expected to allow the design of ABA agonists/antagonists, which will help to understand the role of the ABA/LANCL2 system in human physiology and disease.
Developmental and Activity-Dependent Expression of LanCL1 Confers Antioxidant Activity Required for Neuronal Survival
2014, Developmental Cell
Production of reactive oxygen species (ROS) increases with neuronal activity that accompanies synaptic development and function. Transcription-related factors and metabolic enzymes that are expressed in all tissues have been described to counteract neuronal ROS to prevent oxidative damage. Here, we describe the antioxidant gene LanCL1 that is prominently enriched in brain neurons. Its expression is developmentally regulated and induced by neuronal activity, neurotrophic factors implicated in neuronal plasticity and survival, and oxidative stress. Genetic deletion of LanCL1 causes enhanced accumulation of ROS in brain, as well as development-related lipid, protein, and DNA damage; mitochondrial dysfunction; and apoptotic neurodegeneration. LanCL1 transgene protects neurons from ROS. LanCL1 protein purified from eukaryotic cells catalyzes the formation of thioether products similar to glutathione S-transferase. These studies reveal a neuron-specific glutathione defense mechanism that is essential for neuronal function and survival.
LANCL2 is necessary for abscisic acid binding and signaling in human granulocytes and in rat insulinoma cells
2009, Journal of Biological Chemistry
Abscisic acid (ABA) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. Recently, ABA was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells, and by human and murine pancreatic β cells. ABA autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin-sensitive receptor/G-protein complex, cAMP, CD38-produced cADP-ribose and intracellular calcium. Here we show that the lanthionine synthetase C-like protein LANCL2 is required for ABA binding on the membrane of human granulocytes and that LANCL2 is necessary for transduction of the ABA signal into the cell-specific functional responses in granulocytes and in rat insulinoma cells. Co-expression of LANCL2 and CD38 in the human HeLa cell line reproduces the ABA-signaling pathway. Results obtained with granulocytes and CD38⁺/LANCL2⁺ HeLa transfected with a chimeric G-protein (Gα_q/i) suggest that the pertussis toxin-sensitive G-protein coupled to LANCL2 is a G_i. Identification of LANCL2 as a critical component of the ABA-sensing protein complex will enable the screening of synthetic ABA antagonists as prospective new anti-inflammatory and anti-diabetic agents.
Criteria for confirming sequence periodicity identified by Fourier transform analysis: Application to GCR2, a candidate plant GPCR?
2008, Biophysical Chemistry
Methods to determine peridiodicity in protein sequences are useful for inferring function. Fourier transformation is one approach but care is required to ensure the periodicity is genuine. Here we have shown that empirically-derived statistical tables can be used as a measure of significance. Genuine protein sequences data rather than randomly generated sequences were used as the statistical backdrop. The method has been applied to G-protein coupled receptor (GPCR) sequences, by Fourier transformation of hydrophobicity values, codon frequencies and the extent of over-representation of codon pairs; the latter being related to translational step times. Genuine periodicity was observed in the hydrophobicity whereas the apparent periodicity (as inferred from previously reported measures) in the translation step times was not validated statistically. GCR2 has recently been proposed as the plant GPCR receptor for the hormone abscisic acid. It has homology to the Lanthionine synthetase C-like family of proteins, an observation confirmed by fold recognition. Application of the Fourier transform algorithm to the GCR2 family revealed strongly predicted seven fold periodicity in hydrophobicity, suggesting why GCR2 has been reported to be a GPCR, despite negative indications in most transmembrane prediction algorithms. The underlying multiple sequence alignment, also required for the Fourier transform analysis of periodicity, indicated that the hydrophobic regions around the 7 GXXG motifs commence near the C-terminal end of each of the 7 inner helices of the α-toroid and continue to the N-terminal region of the helix. The results clearly explain why GCR2 has been understandably but erroneously predicted to be a GPCR.

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Regular ArticleCharacterization of p40/GPR69A as a Peripheral Membrane Protein Related to the Lantibiotic Synthetase Component C

Abstract

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Gene

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Curr. Opin. Immunol.

Immunol. Today

The G-Protein Linked Receptor Facts Book

Regular Article
Characterization of p40/GPR69A as a Peripheral Membrane Protein Related to the Lantibiotic Synthetase Component C