Background
The natural course of Helicobacter pylori infection, as well as the success of antibiotic eradication is determined by the immune response to bacteria. The aim of the study is to ...investigate how different Helicobacter pylori isolates influence the dendritic cells maturation and antigen‐presenting function in order to elucidate the differences between Helicobacter pylori strains, isolated from the patients with successful antibiotic eradication therapy or repeated eradication failure.
Materials and Methods
Dendritic cells maturation and antigen presentation were monitored by flow cytometry analysis of the major histocompatibility complex class II (MHC‐II), Toll‐like receptor (TLR) and costimulatory molecules expression, and by determining cytokine secretion.
Results
Dendritic cells stimulated with Helicobacter pylori isolated from patients with repeated antibiotic eradication failure expressed less human leukocyte antigen (HLA‐DR), CD86, TLR‐2, and interleukin‐8 (IL‐8) compared to Helicobacter pylori strains susceptible to antibiotic therapy; the latter expressed lower production of IL‐10. Polymyxin B inhibition of lipopolysaccharide reduces IL‐8 secretion in the group of Helicobacter pylori strains susceptible to antibiotic therapy. The differences in IL‐8 secretion between both groups are lipopolysaccharide dependent, while the differences in secretion of IL‐10 remain unchanged after lipopolysaccharide inhibition. Inhibitor of cathepsin X Mab 2F12 reduced the secretion of IL‐6, and the secretion was significantly lower in the group of Helicobacter pylori strains isolated from patients with repeated antibiotic eradication failure.
Conclusion
Helicobacter pylori strains, susceptible/resistant to antibiotic eradication therapy, differ in their capability to induce DCs maturation and antigen‐presenting function.
Full text
Available for:
BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Syntrophins are scaffold proteins that can bind several signaling molecules and localize them to the plasma membrane. We demonstrate here that in neuroblastoma SH-SY5Y cells, brain-specific ...γ1-syntrophin binds the neurotrophic factor γ-enolase through its PDZ domain, and translocates it to the plasma membrane, as shown by immunoprecipitation, surface plasmon resonance, fluorescence colocalization and flow cytometry. Extensive colocalization of γ1-syntrophin and γ-enolase was observed in neurite growth cones in differentiated SH-SY5Y cells. Silencing of the γ1-syntrophin gene by RNA interference significantly reduced the re-distribution of γ-enolase to the plasma membrane and impaired its neurotrophic effects. We demonstrated that an intact C-terminal end of γ-enolase is essential for its γ1-syntrophin-assisted trafficking. The cleavage of two amino acids at the C-terminal end of γ-enolase by the carboxypeptidase cathepsin X prevents binding with the γ1-syntrophin PDZ domain. Collectively, these data demonstrate that γ1-syntrophin participates in γ-enolase translocation towards the plasma membrane, a pre-requisite for its neurotrophic activity. By disrupting this γ1-syntrophin-guided subcellular distribution, cathepsin X reduces γ-enolase-induced neurotrophic signaling.
Glycosaminoglycans have been shown to be important regulators of activity of several papain-like cathepsins. Binding of glycosaminoglycans to cathepsins thus directly affects catalytic activity, ...stability or the rate of autocatalytic activation of cathepsins. The interaction between cathepsin X and heparin has been revealed by affinity chromatography using heparin–Sepharose. Conformational changes were observed to accompany heparin–cathepsin X interaction by far UV-circular dichroism at both acidic (4.5) and neutral (7.4) pH. These conformational changes promoted a 4-fold increase in the dissociation constant of the enzyme-substrate interaction and increased 2.6-fold the
k
cat value also. The interaction between cathepsin X and heparin or heparan sulfate is specific since dermatan sulfate, chondroitin sulfate, and hyaluronic acid had no effect on the cathepsin X activity. Using flow cytometry cathepsin X was shown to bind cell surface heparan sulfate proteoglycans in wild-type CHO cells but not in CHO-745 cells, which are deficient in glycosaminoglycan synthesis. Moreover, fluorescently labeled cathepsin X was shown by confocal microscopy to be endocytosed by wild-type CHO cells, but not by CHO-745 cells. These results demonstrate the existence of an endocytosis mechanism of cathepsin X by the CHO cells dependent on heparan sulfate proteoglycans present at the cell surface, thus strongly suggesting that heparan sulfate proteoglycans can regulate the cellular trafficking and the enzymatic activity of cathepsin X.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Cathepsin X, a cysteine protease, has been shown to regulate an immune response by activating β-2 integrin receptors. In this study we demonstrate its role in regulating the immune response to ...infection with
H. pylori. The level of cathepsin X was determined in THP-1 monocyte cells primed with
H. pylori antigens isolated from subjects suffering from gastritis, who had either eradicated or not the disease after the antibiotic therapy. We show that the specific clinical outcome of
H. pylori eradication therapy correlates strongly with the membrane expression of cathepsin X in stimulated THP-1 cells, being significantly higher after stimulation with
H. pylori strains from those subjects who did not respond to antibiotic therapy. The same antigens elicit a more vigorous immune response, increased expression of MHC II, however trigger inadequate cytokine profile (IFN-γ and IL-4) to eradicate the pathogen. We propose that cathepsin X mediated activation of β-2 integrin receptor Mac-1 suppresses the stimulatory signal in the form of cytokines. Cathepsin X co-localizes on the membrane of THP-1 cells with Mac-1 integrin receptor and its inhibition increases homotypic aggregation and mononuclear cell proliferation, events that are associated with low Mac-1 activity. Our study highlights the diversity of the innate immune response to
H. pylori antigens leading to either successful eradication of the infection or maintenance of chronic inflammation, revealing cathepsin X location and activity as a regulator of the effectiveness of
H. pylori eradication.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Cathepsin X (Cat X) has been identified as a member of cathepsin family. Studies have shown that Cat X is involved in tumorigenesis and tumor development of various cancers. The aim of this study is ...to investigate the relationship between the clinicopathological prognosis and the levels of Cat X and cystatin C in the serum of patients with lung cancer.
Blood samples were collected from 84 patients with lung cancer and 36 healthy control subjects. Cat X and cystatin C were determined by quantitative ELISA.
Cat X and cystatin C levels were significantly higher in the patients with lung cancer than that in the healthy control subjects (P<0.01). Cat X level was correlated with the pathological types of lung cancer (P=0.076). Cystatin C was positively correlated with TNM stage (P=0.01). Furthermore, cystatin C/Cat X was correlated with lymph node metastasis (P=0.058). The patients with high Cat X levels experienced significantly shorter overall survival rates compared with those with low Cat X. Univariate analysis
Full text
Available for:
FFLJ, NUK, ODKLJ, UL, UM, UPUK, VSZLJ
Cathepsin X is a widespread, abundantly expressed papain-like mammalian lysosomal cysteine protease. It exhibits carboxy-monopeptidase as well as carboxy-dipeptidase activity and shares a similar ...activity profile with cathepsin B. The latter has been implicated in normal physiological events as well as in various pathological states such as rheumatoid arthritis, Alzheimer's disease and cancer progression. Thus the question is raised as to which of the two enzyme activities has actually been monitored.
The crystal structure of human cathepsin X has been determined at 2.67 A resolution. The structure shares the common features of a papain-like enzyme fold, but with a unique active site. The most pronounced feature of the cathepsin X structure is the mini-loop that includes a short three-residue insertion protruding into the active site of the protease. The residue Tyr27 on one side of the loop forms the surface of the S1 substrate-binding site, and His23 on the other side modulates both carboxy-monopeptidase as well as carboxy-dipeptidase activity of the enzyme by binding the C-terminal carboxyl group of a substrate in two different sidechain conformations.
The structure of cathepsin X exhibits a binding surface that will assist in the design of specific inhibitors of cathepsin X as well as of cathepsin B and thereby help to clarify the physiological roles of both proteases.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In this study, we have cloned a cDNA encoding for cathepsin X (
PoCtX) from the olive flounder,
Paralichthys olivaceus. The presence of an HIP motif, which is conserved in the unique cathepsin X ...family,
PoCtX, clearly shows its relation to the cathepsin X group, apart from the cathepsin L or B subfamily. The results of RT-PCR and real-time PCR analyses revealed ubiquitous
PoCtX expression in normal and LPS-stimulated tissues. The cDNA encoding for the proenzyme of
PoCtX (proPoCtX) was expressed in
Escherichia coli as a 57 kDa fusion protein with glutathione
S-transferase. Its activity was quantified via the cleavage of the synthetic fluorogenic peptide substrate Z-Phe-Arg-AMC, and the optimal pH for the protease activity was 5. The recombinant proPoCtX was inhibited by antipain and leupeptin. The PoCtX protein from
P. olivaceus muscle extracts was purified 9.48-fold via a one-step purification process using a DEAE-Sephagel high performance liquid chromatography (HPLC) column. Western blotting and ELISA were conducted in order to evaluate the reaction ability and detection-specificity of the anti-proPoCtX polyclonal antibody to native
PoCtX and recombinant proPoCtX proteins. Our findings indicate that the
P. olivaceus cathepsin X is highly conserved within the cathepsin X subfamily in terms of its amino acid sequence, tissue expression, and biochemical activity.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The human lysosomal cysteine-type carboxypeptidase cathepsin X is mainly present in monocytes and macrophages and may be released into the circulation due to constitutive and/or regulated secretion ...by (activated) immune cells. To define its potential diagnostic value as an inflammatory marker, we have developed a highly sensitive and specific sandwich-type immunoassay (ELISA) for cathepsin X permitting both intra- and extracellular detection and quantification.
The dynamic range of the cathepsin X ELISA was determined to be 100 (detection limit) to 8000 pg/ml. Reproducibility of both within and between runs yielded coefficients of variation (CVs) of 2.7–3.5% and 6.3–7.3%, respectively. Cross-reactivity with other members (cathepsin B, L) of the thiol-dependent cathepsin family was not observed. The ELISA was used to quantify cathepsin X in leukocytes as well as in plasma of healthy volunteers and patients with multiple trauma. During the first 72 h after trauma, plasma levels of cathepsin X increased significantly, particularly in patients who died during the posttraumatic period. In comparison to the well-known inflammation marker neutrophil elastase, cathepsin X levels predicted survival with a higher significance in the later posttraumatic phase.
In conclusion, this report provides the first evidence of cathepsin X immunoreactivity not only in cell lysates but also in plasma samples. We suggest that the newly developed highly reproducible ELISA will be of great value for further evaluation of this protease as a diagnostic and/or prognostic marker in inflammatory diseases.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
PDF
