Plasma levels of alpha1-antichymotrypsin and secretory leukocyte proteinase inhibitor in healthy and chronic obstructive pulmonary disease (COPD) subjects with and without severe α1-antitrypsin deficiency

Hollander, Camilla; Westin, Ulla; Wallmark, Anders; Piitulainen, Eeva; Sveger, Tomas; Janciauskiene, Sabina

doi:10.1186/1471-2466-7-1

Cited by 19 publications

(15 citation statements)

References 36 publications

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“…A total of 57 MRMs were monitored by the 4000 QTRAP in triple quadrupole mode, including the AAC-1 peptide and a series of peptides representing other high abundance plasma proteins such as albumin. As an unfractionated control, a sample containing the unfractionated digest of 14 nl of human plasma diluted to 10 l in 70% acetonitrile, 0.1% formic acid (ϳ1 g of total peptide; no antibodies or beads) was injected through the same plumbing system and diluted prior to capture on the C 18 PepMap trap to mimic the procedure used for the SISCAPA run. Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The mass spectrometer was an Applied Biosystems/Sciex 4000 QTRAP controlled by Analyst software v1.4. The bead trap SISCAPA method consisted of four phases: 1) assembly of the SISCAPA binding reaction mixture and incubation, 2) loading and washing beads in the bead trap, 3) elution of peptides from the beads and transfer to the PepMap C 18 Prior to use, the Dynal beads were washed three times by dilution into 5 ml of 0.1 M ammonium acetate, 0.5 M NaCl, 0.1% CHAPS, pH 7.5, and vortexing for 30 s followed by magnetic recovery after which the final bead mass was resuspended in a volume of PBS equal to the volume taken from the original product vial. Plasma digest, antibody, and detergent were typically mixed and incubated for 1-8 h followed by addition of beads and further incubation for 1 h on the microplate shaker.…”

Section: Lc-ms/ms System and Protocols-mentioning

confidence: 99%

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SISCAPA Peptide Enrichment on Magnetic Beads Using an In-line Bead Trap Device

Anderson¹,

Jackson

Smith

et al. 2009

Molecular & Cellular Proteomics

137

111

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A SISCAPA (stable isotope standards and capture by antipeptide antibodies) method for specific antibody-based capture of individual tryptic peptides from a digest of whole human plasma was developed using a simplified magnetic bead protocol and a novel rotary magnetic bead trap device. MS is the method of choice for identification of peptides in digests of biological samples based on the power of MS to detect the chemically well defined masses of both peptides and their fragments produced by processes such as CID. This high level of structural specificity is also critical in improving peptide (and protein) quantitation because it overcomes the well known problems inherent in classical immunoassays related to limited antibody specificity, dynamic range, and multiplexability. In principle, a quantitative peptide assay using MRM 1 detection in a triple quadrupole mass spectrometer should have nearly absolute structural specificity, a dynamic range of ϳ1eϩ4, and the ability to multiplex measurements of hundreds of peptides per sample (1). These properties suggest that MS-based methods could ultimately replace classical immunoassay technologies in many research and clinical applications.An important limitation of present peptide MRM measurements is sensitivity. The most sensitive widely used quantitative MS platforms use nanoflow chromatography and ESI to deliver trace amounts of peptides to the mass spectrometer. However, these processes are limited in the total amount of peptide that can be applied while retaining maximum sensitivity (typically limited to ϳ1 g of total peptide sample, i.e. the product obtained from digesting ϳ14 nl of plasma). The lower cutoff for detecting proteins in a digest of unfractionated plasma by this approach appears to be in the neighborhood of 1-20 g/ml plasma concentration, which would restrict analysis to the top 100 or so proteins in plasma (1).The sensitivity of MS assays can be substantially increased by fractionating the sample at the level of intact proteins, the tryptic peptides derived from them, or both. For example, immunodepletion of the six most abundant plasma proteins, removes ϳ85% of the protein mass (2) and results in an increase of ϳ7-fold in the signal-to-noise of MRM measurements of peptides from the remaining proteins after digestion (1). Similarly chromatographic fractionation by strong cation exchange provides another major improvement in sensitivity (3). However, increased sample fractionation brings with it the disadvantages of increased cost and time, the risk of losing specific components, and the continued requirement for very high resolution (lengthy, low throughput) reversed phase nanoflow chromatography en route to the ESI source.An alternative fractionation approach, used in the SISCAPA method, enriches specific target peptides through capture by anti-peptide antibodies, thus circumventing these disadvantages for preselected targets (4). In its initial implementation, SISCAPA used very small (ϳ10-nl) columns of POROS chro-

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Section: Resultsmentioning

confidence: 99%

Section: Lc-ms/ms System and Protocols-mentioning

confidence: 99%

SISCAPA Peptide Enrichment on Magnetic Beads Using an In-line Bead Trap Device

Anderson¹,

Jackson

Smith

et al. 2009

Molecular & Cellular Proteomics

137

111

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“…This pathway of CatG in contributing to plasma LDL level changes may not operate in the presence of plasma protease inhibitors — such as α1-antichymotrypsin, an endogenous inhibitor of CatG (and of neutrophil elastase and mast cell chymase) derived from mast cells, neutrophils [47], hepatocytes, bronchial epithelial cells, and neuronal cells [48, 49]. But several lines of evidence support the hypothesis that plasma α1-anti-chymotrypsin [50, 51] has limited inhibitory activity on CatG-mediated LDL catabolism in vivo. First, a fraction of CatG from human polymorphonuclear neutrophils [52], and probably other inflammatory cells and vascular cells [53], may remain bound to cell-surface negatively charged heparin proteoglycans.…”

Section: Discussionmentioning

confidence: 99%

Cathepsin G activity lowers plasma LDL and reduces atherosclerosis

Wang

Sjöberg

Tang

et al. 2014

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

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Cathepsin G (CatG), a serine protease present in mast cells and neutrophils, can produce angiotensin-II (Ang-II) and degrade elastin. Here we demonstrate increased CatG expression in smooth muscle cells (SMCs), endothelial cells (ECs), macrophages, and T cells from human atherosclerotic lesions. In low-density lipoprotein (LDL) receptor-deficient (Ldlr−/−) mice, the absence of CatG reduces arterial wall elastin degradation and attenuates early atherosclerosis when mice consume a Western diet for 3 months. When mice consume this diet for 6 months, however, CatG deficiency exacerbates atherosclerosis in aortic arch without affecting lesion inflammatory cell content or extracellular matrix accumulation, but raises plasma total cholesterol and LDL levels without affecting high-density lipoprotein (HDL) or triglyceride levels. Patients with atherosclerosis also have significantly reduced plasma CatG levels that correlate inversely with total cholesterol (r= −0.535, P<0.0001) and LDL cholesterol (r= −0.559, P<0.0001), but not with HDL cholesterol (P=0.901) or triglycerides (P=0.186). Such inverse correlations with total cholesterol (r= −0.504, P<0.0001) and LDL cholesterol (r= −0.502, P<0.0001) remain significant after adjusting for lipid lowering treatments among this patient population. Human CatG degrades purified human LDL, but not HDL. This study suggests that CatG promotes early atherogenesis through its elastinolytic activity, but suppresses late progression of atherosclerosis by degrading LDL without affecting HDL or triglycerides.

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“…In our experiments, A1AT decreased AFC to 12%, which is similar to the baseline value of Planes and colleagues (1). The structural properties that allow A1AT to inhibit proteases are susceptible to post-translational modifications by oxidation, nitration, and polymerization (49,50). Neutrophils and macrophages, which are capable of secreting high levels of oxidants at sites of inflammation, have been shown to oxidize two of the nine methionines in A1AT (358 and 351), leading to loss of antielastase activity (51).…”

Section: Discussionmentioning

confidence: 99%

α₁-Antitrypsin Inhibits Epithelial Na⁺ Transport In Vitro and In Vivo

Lazrak¹,

Nita²,

Subramaniyam³

et al. 2009

Am J Respir Cell Mol Biol

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A variety of studies have shown that Na 1 reabsorption across epithelial cells depends on the protease-antiprotease balance. Herein, we investigate the mechanisms by which a 1 -antitrypsin (A1AT), a major anti-serine protease in human plasma and lung epithelial fluid and lacking a Kunitz domain, regulates amiloridesensitive epithelial Na 1 channel (ENaC) function in vitro and in vivo. A1AT (0.05 mg/ml 5 1 mM) decreased ENaC currents across Xenopus laevis oocytes injected with human a,b,g-ENaC (hENaC) cRNAs, and human lung Clara-like (H441) cells expressing native ENaC, in a partially irreversible fashion. A1AT also decreased ENaC singlechannel activity when added in the pipette but not in the bath solutions of ENaC-expressing oocytes patched in the cell-attached mode. Incubation of A1AT with peroxynitrite (ONOO 2 ), an oxidizing and nitrating agent, abolished its antiprotease activity and significantly decreased its ability to inhibit ENaC. Intratracheal instillation of normal but not ONOO 2 -treated A1AT (1 mM) in C57BL/6 mice also decreased Na 1 -dependent alveolar fluid clearance to the same level as amiloride. Incubation of either H441 cells or ENaC-expressing oocytes with normal but not ONOO 2 -treated A1AT decreased their ability to cleave a substrate of serine proteases. A1AT had no effect on amiloride-sensitive currents of oocytes injected with hENaC bearing Liddle mutations, presumably because these channels remain at the surface longer than the wild-type channels. These data indicate that A1AT may be an important modulator of ENaC activity and of Na 1 -dependent fluid clearance across the distal lung epithelium in vivo by decreasing endogenous protease activity needed to activate silent ENaC.Keywords: alveolar fluid clearance; serine proteases; H441 cells; Xenopus oocytes; ENaCThe amiloride-sensitive epithelial Na 1 channels (ENaCs) play an important role in Na 1 and fluid homeostasis across a number of epithelial cells, including those in airway and alveolar spaces (1, 2). ENaC mRNA, protein levels, and ENaC activity are regulated by a variety of hormones (such as aldosterone and dexamethasone), second messengers cAMP/protein kinase A, and reactive intermediates (2-4). In addition, endogenous channelactivating proteases (CAPs), as well as proteases released by inflammatory cells (trypsin, elastase), activate ENaC either by cleaving critical amino acids in a-and g-ENaC subunits, or by activating signaling pathways (1, 2, 5-7).There has been considerable interest in identifying the mechanisms by which endogenous and exogenous proteases activate ENaC. Aprotinin, a potent and reversible Kunitz-type inhibitor of several serine proteases, including trypsin, plasmin, and kallikreins (8), has been reported to inhibit sodium transport among a variety of epithelial cells, including A6 (9), human bronchial epithelial (HBE) cells (10, 11), and rat and mouse lung alveolar epithelial cells (1). Other Kunitz-type serine protease inhibitors, such as hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2 (plac...

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Plasma levels of alpha1-antichymotrypsin and secretory leukocyte proteinase inhibitor in healthy and chronic obstructive pulmonary disease (COPD) subjects with and without severe α1-antitrypsin deficiency

Cited by 19 publications

References 36 publications

SISCAPA Peptide Enrichment on Magnetic Beads Using an In-line Bead Trap Device

SISCAPA Peptide Enrichment on Magnetic Beads Using an In-line Bead Trap Device

Cathepsin G activity lowers plasma LDL and reduces atherosclerosis

α₁-Antitrypsin Inhibits Epithelial Na⁺ Transport In Vitro and In Vivo

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Plasma levels of alpha1-antichymotrypsin and secretory leukocyte proteinase inhibitor in healthy and chronic obstructive pulmonary disease (COPD) subjects with and without severe α1-antitrypsin deficiency

Cited by 19 publications

References 36 publications

SISCAPA Peptide Enrichment on Magnetic Beads Using an In-line Bead Trap Device

SISCAPA Peptide Enrichment on Magnetic Beads Using an In-line Bead Trap Device

Cathepsin G activity lowers plasma LDL and reduces atherosclerosis

α1-Antitrypsin Inhibits Epithelial Na+ Transport In Vitro and In Vivo

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α₁-Antitrypsin Inhibits Epithelial Na⁺ Transport In Vitro and In Vivo