Amino Acid Sequence Determination of Human S100A12 (P6, Calgranulin C, CGRP, CAAF1) by Tandem Mass Spectrometry

doi:10.1006/bbrc.1996.1144

Biochemical and Biophysical Research Communications

Volume 225, Issue 1, 5 August 1996, Pages 146-150

https://doi.org/10.1006/bbrc.1996.1144 Get rights and content

Abstract

S100A12 has been isolated from human neutrophils. The molecular weight and the amino acid sequence of S100A12 was determined by electrospray-mass spectrometry, tandem mass spectrometry and Edman microsequence analysis. Interestingly, a sequence comparison of S100A12 with all known human S100 proteins revealed that S100A12 is the most divergent of the S100 proteins.

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Cited by (44)

S100A12 promotes Mn(II) binding to pneumococcal PsaA and staphylococcal MntC by Zn(II) sequestration
2022, Journal of Inorganic Biochemistry
Citation Excerpt :
We evaluate the ability of human S100A12 to sequester Zn(II) from pneumococcal PsaA and staphylococcal MntC, and consequently facilitate Mn(II) binding to these SBPs. Human S100A12 (calgranulin C, MRP6, EN-RAGE) is a small (10.6 kDa) α-helical Ca(II)-binding protein that is produced mostly by white blood cells [13,14]. The apo protein typically exists as a homodimer with each subunit forming a stable four-helix fold with a hydrophobic core (Fig. 1) [15].
Human S100A12 (calgranulin C, EN-RAGE) is a Zn(II)-sequestering host-defense protein that contributes to the metal-withholding innate immune response against microbial pathogens. S100A12 coordinates Zn(II) ions at two His₃Asp sites with high affinity. A similar His₃Asp site found in calprotectin (S100A8/S100A9, calgranulin A/B), a closely related human S100 protein, can sequester divalent metal ions from the solute-binding proteins (SBPs) pneumococcal PsaA (pneumococcal surface protein A) and staphylococcal MntC (manganese transport protein C). Both SBPs are components of Mn(II) transporters and capture extracellular Mn(II) ions for subsequent delivery into the bacterial cytosol. Nevertheless, PsaA and MntC exhibit a thermodynamic preference for Zn(II) over Mn(II), and Zn(II) binding can interfere with Mn(II) acquisition. In this work, we have used a biotinylated variant of S100A12 to show that S100A12 can sequester Zn(II) ions from PsaA and MntC. Moreover, electron paramagnetic resonance (EPR) spectroscopy indicates that by sequestering Zn(II) from Zn(II)-bound PsaA and MntC, S100A12 promotes Mn(II) binding to the SBPs. These results inform the function of S100A12 in Zn(II) sequestration, and further suggest that Zn(II)-sequestering S100 proteins may inadvertently protect bacterial pathogens during infection.
Myeloperoxidase-dependent lipid peroxidation promotes the oxidative modification of cytosolic proteins in phagocytic neutrophils
2015, Journal of Biological Chemistry
Background: Neutrophils generate oxidants to kill microbes ingested into phagosomes, but extraphagosomal protein oxidation may also occur.
Results: Specific neutrophil cytosolic proteins were carbonylated following phagocytosis; carbonylation was impaired by myeloperoxidase inhibitors or radical scavengers that inhibit lipid peroxidation.
Conclusion: Lipid peroxidation in the phagosomal membrane leads to oxidation of cytosolic proteins.
Significance: Protein oxidation may influence the function and fate of post-phagocytic neutrophils.
Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occurs within an intracellular phagosome, but diffusible species can react with the neutrophil and surrounding tissue. To investigate the extent of oxidative modification, we assessed the carbonylation of cytosolic proteins in phagocytic neutrophils. A 4-fold increase in protein carbonylation was measured within 15 min of initiating phagocytosis. Carbonylation was dependent on NADPH oxidase and myeloperoxidase activity and was inhibited by butylated hydroxytoluene and Trolox, indicating a role for myeloperoxidase-dependent lipid peroxidation. Proteomic analysis of target proteins revealed significant carbonylation of the S100A9 subunit of calprotectin, a truncated form of Hsp70, actin, and hemoglobin from contaminating erythrocytes. The addition of the reactive aldehyde 4-hydroxynonenal (HNE) caused carbonylation, and HNE-glutathione adducts were detected in the cytosol of phagocytic neutrophils. The post-translational modification of neutrophil proteins will influence the functioning and fate of these immune cells in the period following phagocytic activation, and provides a marker of neutrophil activation during infection and inflammation.
Usefulness of S100A12 as a prognostic biomarker for adverse events in patients with heart failure
2015, Clinical Biochemistry
Citation Excerpt :
S100A12, a member of S100 calcium-binding protein family, is mainly secreted from these cells [4] and is recognized as potentially playing a key role in inflammation [5]. It is a close homologue to two other S100 proteins, S100A8 and S100A9 [6], collectively termed calgranulins or myeloid-related proteins. The interaction between S100A12 and the receptor for advanced glycation end products (RAGE) mediates the proinflammatory properties of this protein.
S100A12 has been proposed as a novel pivotal factor in inflammation produced by granulocytes. The purpose of this study was to investigate the relationship between S100A12 and chronic heart failure (CHF).
One hundred and seventy-seven patients with CHF and 66 subjects without CHF were included in this study. Plasma levels of S100A12 and high-sensitivity C-reactive protein (hs-CRP) were measured in all participants. After a follow-up period of 18 months for CHF patients, major cardiovascular events (MCE), including cardiac death and rehospitalization for heart failure, were recorded.
Plasma levels of S100A12 were significantly higher in CHF patients than in control subjects (P < 0.001) and positively correlated with hs-CRP (r = 0.316, P < 0.001). S100A12 levels were also higher in MCE patients than in MCE-free patients. The occurrence of MCE increased with advancing plasma S100A12 levels by stratification according to quartiles (Q4 vs Q1, P = 0.015). Cox proportional hazards regression analysis revealed that S100A12 was an independent risk factor for MCE in CHF patients (P = 0.009).
S100A12 is a potential biomarker of CHF that may provide important information regarding the prediction of MCE in patients with CHF.
Development and analytic validation of an immunoassay for the quantification of canine S100A12 in serum and fecal samples and its biological variability in serum from healthy dogs
2011, Veterinary Immunology and Immunopathology
S100A12 (calgranulin C) is a Ca²⁺-binding protein that has been proposed to play a central role in both innate and acquired immune responses. In humans, S100A12 has been reported to be increased in serum and/or plasma in patients with various inflammatory disorders, and this protein has been suggested to be a sensitive and specific marker for inflammatory bowel disease (IBD). An immunoassay for S100A12 is currently available for use in humans, but antibodies against the human protein do not cross-react with canine S100A12 (cS100A12). Both sensitive and specific markers for canine patients with systemic or localized inflammatory diseases are currently lacking, thus the aim of this study was to develop and analytically validate a radioimmunoassay (RIA) for the quantification of cS100A12 in serum and fecal specimens and to determine the biological variation of cS100A12 in serum from healthy dogs.
A competitive liquid-phase RIA was developed and analytically validated by determining assay working range, dilutional parallelism, spiking recovery, and intra- and inter-assay variability. Reference intervals for serum and fecal concentrations of cS100A12 were established from 124 and 65 healthy dogs, respectively, and components of variation for serum cS100A12 were determined from 11 dogs over 2.6 months.
The working range of the assay was 0.6–432.7 μg/L. No cross-reactivity was observed with the cS100A8/A9 protein complex, the closest structural analogues available. Observed-to-expected ratios (O/E) for the serial dilution of serum and fecal extracts ranged from 97.2 to 146.8% and from 75.3 to 129.8%, respectively. O/E for spiking recovery for serum and fecal extracts ranged from 87.8 to 130.4% and from 84.8 to 143.8%, respectively. Coefficients of variation (CV) for intra- and inter-assay variability for sera were ≤8.1% and ≤7.8%, respectively, and were ≤7.8% and ≤8.7%, respectively, for fecal extracts. Reference intervals for serum and fecal cS100A12 were 33.2–225.1 μg/L and <24−745 ng/g, respectively. For biological variability testing, analytical, intra-individual, inter-individual, and total CV were 5.7, 29.2, 31.2, and 66.0%, respectively, yielding an index of individuality of 0.95 and a minimum critical difference (p < 0.05) for sequential values of 84.9%.
The RIA for cS100A12 measurement described here is analytically sensitive and specific, linear, accurate, precise, and reproducible, and will facilitate further research into the clinical utility of quantifying serum and fecal cS100A12 in canine patients with inflammatory diseases. Moderate changes in serum cS100A12 concentrations may be clinically relevant; however, the use of a population-based reference interval may require caution.
Purification and partial characterization of canine S100A12
2010, Enfermedades Infecciosas y Microbiologia Clinica
Citation Excerpt :
In human medicine, S100A12 has been reported to be a very sensitive and specific marker of localized inflammatory processes, such as gastrointestinal inflammation [19,20], and to be increased in plasma/serum in patients with various inflammatory disorders [18,21,22]. To the authors’ knowledge, only human, porcine, bovine, and rabbit S100A12 have been purified to date [1,3,23–26], and an immunoassay for the quantification of S100A12 is available only for use in human patients. A high sequence divergence has been reported for S100A12 proteins from different species, thus further substantiating the necessity of using species-specific immunologic methods for the detection of S100A12 [7].
Canine S100A12 (cS100A12) is a calcium-binding protein of the S100 superfamily of EF-hand proteins, and its expression is restricted to neutrophils and monocytes. Interaction of S100A12 with the receptor for advanced glycation end products (RAGE) has been suggested to play a central role in inflammation. Moreover, S100A12 has been shown to represent a sensitive and specific marker for gastrointestinal inflammation in humans. Only human, porcine, bovine, and rabbit S100A12 have been purified to date, and an immunoassay for the quantification of S100A12 is available only for humans. Therefore, the aim of this study was to develop a protocol for the purification of S100A12 and to partially characterize this protein in the dog (Canis lupus familiaris) as a prelude to the development of an immunologic method for its detection and quantification in canine serum and fecal specimens. Leukocytes were isolated from canine whole blood by dextran sedimentation, and canine S100A12 was extracted from the cytosol fraction of these cells. Further purification of cS100A12 comprised of ammonium sulfate precipitation, hydrophobic interaction chromatography, and strong cation- and anion-exchange column chromatography. Canine S100A12 was successfully purified from canine whole blood. The relative molecular mass of the protein was estimated at 10,379.5 and isoelectric focusing revealed an isoelectric point of 6.0. The approximate specific absorbance of cS100A12 at 280 nm was determined to be 1.78 for a 1 mg/ml solution. The N-terminal AA sequence of the first 15 residues of cS100A12 was Thr-Lys-Leu-Glu-Asp-His-X-Glu-Gly-Ile-Val-Asp-Val-Phe-His, and revealed 100% identity with the predicted protein sequence available through the canine genome project. Sequence homology for the 14 N-terminal residues identified for cS100A12 with those of feline, bovine, porcine, and human S100A12 was 78.6%. We conclude that canine S100A12 can be successfully purified from canine whole blood using the described methods.
Binding of S100 proteins to RAGE: An update
2009, Biochimica et Biophysica Acta - Molecular Cell Research
The Receptor for Advanced Glycation Endproducts (RAGE) is a multi-ligand receptor of the immunoglobulin family. RAGE interacts with structurally different ligands probably through the oligomerization of the receptor on the cell surface. However, the exact mechanism is unknown. Among RAGE ligands are members of the S100 protein family. S100 proteins are small calcium binding proteins with high structural homology. Several members of the family have been shown to interact with RAGE in vitro or in cell-based assays. Interestingly, many RAGE ligands appear to interact with distinct domains of the extracellular portion of RAGE and to trigger various cellular effects. In this review, we summarize the modes of S100 protein–RAGE interaction with regard to their cellular functions.

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^☆: The sequence of human S100A12 was submitted to the EMBL data library and is available under the accession number P80511.

²: The first two authors contributed equally to this work.

³: To whom correspondence should be addressed. Fax: +411 266 71 69.

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Regular ArticleAmino Acid Sequence Determination of Human S100A12 (P6, Calgranulin C, CGRP, CAAF1) by Tandem Mass Spectrometry☆

Abstract

Regular Article
Amino Acid Sequence Determination of Human S100A12 (P6, Calgranulin C, CGRP, CAAF1) by Tandem Mass Spectrometry☆