NGAL的结构与功能研究进展

NGAL（neutrophil gelatinase-associated lipocalin)是一种新的lipocalin,其结构与功能的研究进展为人瞩目,目前认为NGAL具有运输疏水性分子,调节明胶酶B（gelatinaseB或matrix metalloproteinase-9,MMP-9)活性等功能,并可能参与免疫炎症反应以及肿瘤的发生发展,特别是肿瘤的浸润转移等过程,最近,NGAL蛋白的空间结构已被研究清楚,这对于促进NGAL生物学作用或功能的研究。特别是对其新的生物学作用或功能的探索,比如与肿瘤的关系（NGAL可能是一种新的癌基因或促癌基因）将具有十分重要的意义。对此作者进行了综合评述。     

Interactions between neutrophil gelatinase-associated lipocalin and natural lipophilic ligands

Neutrophils are activated by both paracrine molecules, e.g. platelet activating factor (PAF) and leukotriene B4 (LTB4), and the bacterial hydrophobic peptide N-formyl-Met-Leu-Phe (fMLP). Several mechanisms are involved in regulation of the activation, including receptor endocytosis and ligand breakdown. The interactions between the specific granule protein neutrophil gelatinase-associated lipocalin (NGAL), expressed in human neutrophils, and fMLP, PAF and LTB4, were investigated by weak affinity chromatography. NGAL was immobilised to a silica matrix and packed in a micro-column and the retention times of retarded ligands were measured and used to calculate the strength of the interactions. The association constants for fMLP were K(ass) = 0.85 x 10(3) M(-1) at 20 degrees C and 0.77 x 10(3) M(-1) at 37 degrees C, for LTB4 were K(ass) = 4.37 x 10(3) M(-1) at 20 degrees C and 3.27 x 10(3) M(-1) at 37 degrees C and for PAF were K(ass) = 25.4 x 10(3) M(-1) at 20 degrees C and 10.5 x 10(3) M(-1) at 37 degrees C. Other methods of detecting the interactions such as gel filtration, immunoprecipitation, photoactivated ligands and fluorescence quenching proved to be insufficient. The results demonstrate the superiority of weak affinity chromatography as a method of studying the interactions of the specific granule protein NGAL.     

Expression of neutrophil gelatinase-associated lipocalin regulates epithelial morphogenesis in vitro

Growth factors such as hepatocyte growth factor (HGF) are highly up-regulated during development and following renal injury and are known to induce marked morphogenic actions in cultured tubular epithelial cells, including scattering, migration, single cell branching morphogenesis, and multicellular branching tubulogenesis. In the present study, we demonstrate that HGF stimulates epithelial cells to express neutrophil gelatinase-associated lipocalin (Ngal), a member of the lipocalin family of secreted proteins that has recently been shown to participate in mesenchymal-epithelial transformation via its ability to augment cellular iron uptake. At concentrations below those found to mediate iron transport, purified Ngal can induce a promigratory and probranching effect that is dependent on ERK activation. The suppression of Ngal expression using short hairpin RNA results in increased cyst formation by tubular cells. However, the simultaneous addition of Ngal and HGF leads to direct association of the two proteins, and results in a partial inhibition of HGF-mediated activation of c-Met and the downstream MAPK and phosphatidylinositol 3-kinase signaling pathways. This inhibitory effect down-regulates HGF-stimulated single cell migration, and limits branching morphogenesis at both the single cell and multicellular level. These experiments demonstrate that the local expression of Ngal can play a regulatory role in epithelial morphogenesis by promoting the organization of cells into tubular structures while simultaneously negatively modulating the branching effects of HGF.     

The solution structure and dynamics of human neutrophil gelatinase-associated lipocalin.

Human neutrophil gelatinase-associated lipocalin (HNGAL) is a member of the lipocalin family of extracellular proteins that function as transporters of small, hydrophobic molecules. HNGAL, a component of human blood granulocytes, binds bacterially derived formyl peptides that act as chemotactic agents and induce leukocyte granule discharge. HNGAL also forms a complex with the proenzyme form of matrix metalloproteinase-9 (pro-MMP-9, or progelatinase B) via an intermolecular disulphide bridge. This association allows the subsequent formation of ternary and quaternary metalloproteinase/inhibitor complexes that vary greatly in their metalloproteinase activities. The structure and dynamics of apo-HNGAL have been determined by NMR spectroscopy. Simulated annealing calculations yielded a set of 20 convergent structures with an average backbone RMSD from mean coordinate positions of 0. 79(+/-0.13) A over secondary structure elements. The overall rotational correlation time (13.3 ns) derived from15N relaxation data is consistent with a monomeric protein of the size of HNGAL (179 residues) under the experimental conditions (1.4 mM protein, pH 6.0, 24.5 degrees C). The structure features an eight-stranded antiparallel beta-barrel, typical of the lipocalin family. One end of the barrel is open, providing access to the binding site within the barrel cavity, while the other is closed by a short 310-helix. The free cysteine residue required for association with pro-MMP-9 lies in an inter-strand loop at the closed end of the barrel. The structure provides a detailed model of the ligand-binding site and has led to the proposal of a site for pro-MMP-9 association. Dynamic data correlate well with structural features, which has allowed us to investigate a mechanism by which a cell-surface receptor might distinguish between apo and holo-HNGAL through conformational changes at the open end of the barrel.     

Correlation between the concentrations of lactoferrin and neutrophil gelatinase-associated lipocalin in meconium

Neutrophil gelatinase-associated lipocalin (NGAL) and lactoferrin (Lf) are among the key components of the innate immune system due to their ability to bind iron with high affinity and thus control inflammation. The aim of this study was to test the use of NGAL and LF measurements in meconium for the assessment of the intrauterine homeostasis. NGAL and Lf concentrations were measured using ELISA kits in all serial meconium portions (n = 81) collected from 20 healthy neonates. Mean ± SD meconium concentration of Lf was 45.07 ± 78.53 µg/g and more than 1000-fold higher compared with that of NGAL at 1.93 ± 2.46 ng/g. The correlation between the two proteins (r = 0.83, p < 0.0001) was found only for portions with Lf concentrations > 25 μg/g. High variability of NGAL and Lf concentrations in meconium and their correlations prove their key role as biomarkers of the fetal condition in utero. NGAL and Lf measured in meconium are candidate biomarkers for fetal iron status.     

Expression of neutrophil gelatinase-associated lipocalin (NGAL) in peripheral nerve repair

Introduction: Trauma is one of the causes of peripheral nerve injuries. Free radicals increase after tissue damage. Free radicals are usually scavenged and detoxi?ed by antioxidants. In this study, we assessed the antioxidative role of the NGAL molecule in sciatic nerve repair in rats. Materials and methods: The sciatic nerves of 40 rats were crushed and the total mRNA of samples from day 1 and 3 and week 1, 3, 5 post injury was extracted. The expression of the NGAL gene was confirmed by RT-PCR. For immunohistochemistry analysis, the samples were ?xed in paraformaldehyde and cut in 20 micrometer slices by cryostat. Results: The expression of NGAL signi?cantly upregulated in day 1, 3 and week 1 following the crushing of sciatic nerves in comparison with the intact nerves. Immunohistochemistry results also confirmed the protein expression of this gene. Discussion: The NGAL molecule showed upregulation in the degeneration process after nerve injury, so it may play an important role in nerve repair.     

Extracorporeal circulation causes release of neutrophil gelatinase-associated lipocalin (NGAL)

Extracorporeal circulation (ECC) used during cardiac surgery causes activation of several inflammatory systems. These events are not fully understood but are responsible for complications during the immediate postoperative period. Neutrophil gelatinase-associated lipocalin (NGAL), a member of the expanding lipocalin family, has recently been described as an inflammatory protein. In this study, the release of NGAL into the circulation in 41 patients undergoing heart surgery with ECC was evaluated. A 4- to 5-fold elevation of the concentration of NGAL in plasma was observed during the immediate postoperative course with a rapid elimination during the first postoperative day. Four patients undergoing lung surgery (without ECC) were also studied. The plasma concentration of NGAL only increased with a factor of 1.1-2.2 over the operation. We conclude that NGAL is released into the circulation during heart surgery, probably as a result of the inflammatory activation of leukocytes initiated by the extracorporeal circulation.     

Interleukin-10-induced neutrophil gelatinase-associated lipocalin production in macrophages with consequences for tumor growth

Tumor cell-derived factors, such as interleukin 10 (IL-10), polarize macrophages toward a regulatory M2 phenotype, characterized by the expression of anti-inflammatory cytokines and protumorigenic mediators. Here we explored molecular mechanisms allowing IL-10 to upregulate the protumorigenic protein NGAL in primary human macrophages. Reporter assays of full-length or deletion constructs of the NGAL promoter provided evidence that NGAL production is STAT3 dependent, activated downstream of the IL-10-Janus kinase (Jak) axis, as well as being C/EBPβ dependent. The involvement of STAT3 and C/EBPβ was shown by chromatin immunoprecipitation (ChIP) and ChIP-Western analysis, as well as decoy oligonucleotides scavenging both STAT3 and C/EBPβ in human macrophages. Furthermore, the production of NGAL in macrophages in response to IL-10 induces cellular growth and proliferation of MCF-7 breast cancer cells. We conclude that both STAT3 and C/EBPβ are needed to elicit IL-10-mediated NGAL expression in primary human macrophages. Macrophage-secreted NGAL shapes the protumorigenic macrophage phenotype to promote growth of MCF-7 breast cancer cells. Our data point to a macrophage-dependent IL-10-STAT3-NGAL axis that might contribute to tumor progression.     

A novel alternative spliced variant of neutrophil gelatinase-associated lipocalin receptor in oesophageal carcinoma cells

Recent studies suggest that NGAL (neutrophil gelatinase-associated lipocalin) is a novel iron transporter with functions distinct from that of transferrin and mediates a new iron-delivery pathway. To get a better understanding of NGAL function in oesophageal carcinoma, we analysed the expression of NGAL receptors in oesophageal carcinoma cells and identified a novel spliced variant designated NgalR-3. When expressed in a heterologous system, the protein produced from this novel spliced variant exhibits the biochemical characteristics of interaction and co-localization with NGAL protein in vivo. This new finding suggests that NgalR-3 may act as a potential NGAL receptor and play a role in NGAL-mediated iron transport in oesophageal carcinoma.     

Delayed graft function and chronic allograft nephropathy: diagnostic and prognostic role of neutrophil gelatinase-associated lipocalin

Context: Available markers are not reliable parameters to early detect kidney injury in transplanted patients.

Objective: Examine neutrophil gelatinase associated lipocalin (NGAL) in early detection of delayed graft function (DGF) and as a long-term predictor of graft outcome.

Patients and methods: NGAL was evaluated in 124 transplanted patients.

Results: Urinary NGAL levels were associated to a 10% (HR: 1.10; 95% CI: 1.04–1.25; p?<?0.001) and 15% (HR: 1.15; 95% CI: 1.09–1.26; p?<?0.001) increased risk of DGF and allograft nephropathy progression, respectively.

Conclusion: NGAL reflects the entity of renal impairment in transplanted patients, representing a biomarker and an independent risk factor for DGF and chronic allograft nephropathy progression.     

N-acetylcysteine decreases urinary level of neutrophil gelatinase-associated lipocalin in deceased-donor renal transplant recipients: a randomized clinical trial

Context: Acute kidney injury (AKI) is a common complication after kidney transplantation (KT), especially in recipients from deceased donors. Urinary neutrophil gelatinase-associated lipocalin (u-NGAL) is an early and sensitive marker of AKI after transplantation.

Objectives: We assessed the renoprotective effect of N-acetylcysteine (NAC) on u-NGAL levels as an early prognostic marker of graft function immediately after transplantation.

Materials and methods: A double-blind, randomized, placebo-controlled trial was conducted on 70 deceased-donor KT recipients (www.irct.ir, trial registration number: IRCT2014090214693N4). Patients received 600?mg oral NAC or placebo twice daily from day 0 to 5 and urine samples were taken before, and on the first and fifth days after transplantation. U-NGAL and early graft function were compared between the two groups.

Results: NAC significantly reduced u-NGAL levels compared to placebo (p value?=?0.02), while improvement in early graft function with NAC did not reach statistical significance.

Conclusions: This study showed that NAC administration in deceased-donor KT recipients can reduce tubular kidney injury, evidenced by u-NGAL measurements. Improvement in early graft function needs a larger sample size to reach a statistical conclusion.     

Iron depletion strategy for targeted cancer therapy: utilizing the dual roles of neutrophil gelatinase-associated lipocalin protein

Decreasing iron uptake and increasing iron efflux may result in cell death by oxidative inactivation of vital enzymes. Applying the dual function of neutrophil gelatinase-associated lipocalin (NGAL) could achieve the goal of iron depletion in the cancer cells. Tyr106, Lys125 or Lys134 was the key binding site for NGAL protein to sequester iron-chelating siderophores. In this study, we employed all bioactive peptides in peptide databank to dock with the siderophore-binding sites of NGAL protein by virtual screening. In addition, we performed molecular dynamics (MD) simulation to observe the molecular character and structural variation of ligand-protein interaction. Glu-Glu-Lys-Glu (EEKE), Glu-Glu-Asp-Cys-Lys (EEDCK), and Gly-Glu-Glu-Cys-Asp (GEECD) were selected preliminarily by rigorous scoring functions for further investigation. GEECD was excluded due to higher binding total energy than the others. Moreover, we also excluded EEKE due to larger influence to the stability of binding residues by the information of root mean square fluctuation (RMSF) and principal component analysis (PCA). Thus, we suggested that EEDCK was the potential bioactive peptide which had been proved to inhibit malignant cells for targeted cancer therapy.

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Graphical Abstract Perspective drug design of occupying the siderophore-binding sites of NGAL outside the cell temporarily by a potential short peptide until NGAL enters into the cell, and releasing the siderophore-binding sites inside the cell.

     

NGAL对直接PCI术后对比剂肾病的早期诊断价值

目的:评价NGAL在急性心肌梗死患者直接PCI术后预测CIN的诊断作用.方法:选择2009.01～2009.12我科收治的65例行直接PCI的急性心肌梗死患者,测定介入治疗前和术后8～12h、48h的血清肌酐变化情况,确定发生对比剂肾痛的患者.分别收集术前、术后8～12h血清标本置于-80℃冰箱,采用ELISA法测定各组血清中NGAL表达水平,观察术后NGAL浓度对CIN的诊断价值.结果:①145例术前肌酐正常的PCI患者,术后共有14例患者被诊断为CIN,其中研究组有8例(12.3%)、对照组有6例(7.5%)诊断为发生了CIN.②研究组中被诊断为CIN的患者术后8～12h血清NGAL的浓度与术前比较[(9.21±0.18 vs 4.05±1.30)ng/ml,p＜0.01]有显著差异,而术后8～12h Scr、BUN、GFR等指标与术前比较差异无统计学意义,血清NGAL较Scr提前至少24h诊断CIN.ROC曲线下的面积为0.851±0.052,AUC 95%的可信区间为(0.748,0.953).根据ROC曲线结果分析,NGAL预测CIN的临界点为9.04ng/ml,对应的灵敏度为78.6%,特异度为85.2%,③单变量分析示术前NGAL水平与GFR呈负相关,与Scr、BUN、舒张压(DBP)呈正相关;术后NGAL水平与GFR呈负相关,与Scr、对比剂用量呈正相关;多元Logistic回归分析显示Scr、GFR、对比剂用量是NGAL水平的独立影响因子.结论:NGAL对于AMI患者直接PCI术后发生的CIN具有良好的预测诊断作用,为早期诊断CIN提供了新思路.     

A miniaturized immunoassay platform to measure neutrophil gelatinase-associated lipocalin (NGAL) for diagnosis of acute kidney injury

Abstract

In this study, we describe the development and evaluation of a slide-based immunoassay platform for the detection of neutrophil gelatinase associated-lipocalin (NGAL) in plasma and urine samples. The capture NGAL antibody was immobilized onto a microscope slide before an analysis of NGAL based on a sandwich immunoassay was further carried out. This assay system exhibited linearity between 50 to 1000?ng/ml of NGAL. The coefficients of variability (CVs) indicated good reproducibility and repeatability of the system. The levels of plasma NGAL measured by the slide-based system were highly correlated with those of ELISA, while this system over-predicted urine NGAL.     

Neutrophil gelatinase-associated lipocalin in dehydrated patients: a preliminary report

Background

Acute kidney injury has been recognized as a major contributor to end stage renal disease. Although neutrophil gelatinase-associated lipocalin (Ngal) has been reported as a promising biomarker for early detection of acute kidney injury, no study has yet examined its potential clinical impact in patients with normal renal function. The purpose of current study is to investigate possible difference in serum Ngal levels between dehydrated and control patients.

Findings

A total of twelve patients presented with symptoms of mild dehydration defined by history of diarrheas or vomiting and orthostatic (postural) hypotension and an age and sex matched group of twelve control patients were included. The two groups of patients did not seem to differ in basic clinical and laboratory parameters. Serum Ngal was higher in dehydrated patients when compared to control group (Ngal = 129.4 ± 25.7 ng/mL vs 60.6 ± 0.4 ng/mL, p = 0.02). Ngal was not correlated with age, hemoglobin, white blood cell count, red blood cell count, urea or creatinine.

Conclusions

The presence of elevated Ngal levels in dehydrated patients may suggest its role as a very sensitive biomarker in even minimal and "silent" prerenal kidney dysfunction