Iron uptake and transfer from ceruloplasmin to transferrin

Background

Dietary and recycled iron are in the Fe^2 + oxidation state. However, the metal is transported in serum by transferrin as Fe^3 +. The multi-copper ferroxidase ceruloplasmin is suspected to be the missing link between acquired Fe^2 + and transported Fe^3 +.

Methods

This study uses the techniques of chemical relaxation and spectrophotometric detection.

Results

Under anaerobic conditions, ceruloplasmin captures and oxidizes two Fe^2 +. The first uptake occurs in domain 6 (< 1 ms) at the divalent iron-binding site. It is accompanied by Fe^2 + oxidation by Cu^2 +_D6. Fe^3 + is then transferred from the binding site to the holding site. Cu⁺_D6 is then re-oxidized by a Cu^2 + of the trinuclear cluster in about 200 ms. The second Fe^2 + uptake and oxidation involve domain 4 and are under the kinetic control of a 200 s change in the protein conformation. With transferrin and in the formed ceruloplasmin–transferrin adduct, two Fe^3 + are transferred from their holding sites to two C-lobes of two transferrins. The first transfer (~ 100 s) is followed by conformation changes (500 s) leading to the release of monoferric transferrin. The second transfer occurs in two steps in the 1000–10,000 second range.

Conclusion

Fe^3 + is transferred after Fe^2 + uptake and oxidation by ceruloplasmin to the C-lobe of transferrin in a protein–protein adduct. This adduct is in a permanent state of equilibrium with all the metal-free or bounded ceruloplasmin and transferrin species present in the medium.

General significance

Ceruloplasmin is a go-between dietary or recycled Fe^2 + and transferrin transported Fe^3 +.     

Whole blood,flow-chamber studies in real-time indicate a biphasic role for thymosin β-4 in platelet adhesion

Background

Thymosin beta 4 (Tβ₄) is a major actin sequestering peptide present in most mammalian cells. It also acts as an anti-inflammatory agent and promotes corneal wound healing.

Methods

In the present study, we constructed a four channel cylindrical flow chambers out of polydimethylsiloxane (PDMS) on microscope coverslips. The platelet-binding proteins–fibrinogen and collagen–were immobilized onto the middle ~ 25% of the inner cylindrical surface. The flow method introduced here was employed to determine the effect of Tβ₄, on the deposition of ADP-activated platelets onto fibrinogen cross-linked flow chambers.

Results

The binding data from the flow chambers indicated that the both the rate constant of platelet deposition (average: 0.026 ± 0.0015 s^− 1, corresponding to a half-life of 26.7 s) and the total number of deposited platelets were independent of the platelet binding protein and the activating agent. Our results show that low concentrations of Tβ₄ (0.2 μM to 0.5 μM) increased both the rate constant of platelet deposition by ~ 1.5-fold (i.e. half-life decreased from 26.7 s to 17.6 s) and the total number of deposited platelets by ~ 3-fold. However at higher concentrations (> 1 μM) the Tβ₄-potentiating effect was diminished to near control levels. Tβ₄ did interact with fibrinogen with an estimated K_D of ~ 126 ± 18 nM or 66 ± 20 nM under equilibrium or flow, respectively.

Conclusion

These results suggest that Tβ₄ could potentially increase the affinity of platelet receptors for their ligands thus promoting platelet deposition. Tβ₄ could also bind to fibrinogen and as its concentration increased would prevent platelet–fibrinogen interactions resulting in the attenuation of platelet deposition.

General significance

This work suggests that Tβ₄ might have a dual role in platelet function.     

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion

Background

Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated proline–glycine–proline (N-ac-PGP). In the current study, we investigate whether N-ac-PGP influences β₂-integrin activation and function in neutrophilic firm adhesion to endothelium.

Methods

Human polymorphonuclear leukocytes (PMNs) were isolated from fresh human blood. Subsequently, a transmigration assay was performed to evaluate the active migration of PMNs towards N-ac-PGP. Furthermore, the effect of the tripeptide on β₂-integrin activation was assessed by performing the adhesion assay using fibrinogen as a ligand. To determine whether this effect was due to conformational change of β₂-integrins, antibodies against CD11b and CD18 were used in the adhesion assay and the expression pattern of CD11b was determined.

Results

Human neutrophils transmigrated through an endothelial cell layer in response to basolateral N-ac-PGP. N-ac-PGP induced also a neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that CD11b/CD18 (Mac-1) was responsible for the N-ac-PGP-induced firm adhesion of neutrophils to fibrinogen. Pertussis toxin decreased the Mac-1 activation indicating the involvement of G-proteins. N-ac-PGP most likely activated Mac-1 by initiating a conformational change, since the expression pattern of Mac-1 on the cell surface did not change significantly.

Conclusions

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion.

General significance

This is the first study to describe that the chemo-attractant N-ac-PGP also activates Mac-1 on the surface of neutrophils, which can additionally contribute to neutrophilic transmigration into the lung tissue during lung inflammation.     

Methylenetetrahydrofolate reductase C677T variant in Moroccan patients with inflammatory bowel disease

Background

The association of genetic polymorphisms related to metabolism of homocysteine and folate with inflammatory bowel disease has been evidenced. Several studies have identified genetic variants of MTHFR as significant susceptibility loci for Crohn's disease (CD) and ulcerative colitis (UC). The C677T genetic polymorphism in the MTHFR gene is found to be associated with a thermolabile variant enzyme that shows a reduced activity. Therefore, we investigated whether the C677T variant confers genetic susceptibility to CD or UC and evaluated the genotype–phenotype associations in the Moroccan population.

Methods

The present study included 96 inflammatory bowel disease patients (68 patients with CD and 28 with UC) and 182 healthy controls. DNA samples were genotyped for the MTHFR (C677T) mutation by the PCR-RFLP method. Statistical analyzes were performed using MedCalc software, Chi square test and Fisher test.

Results

The respective odds ratio for CD, UC and control group were, 1.55 (CI 95%: 0.53–4.53, P = 0.52); 0.50 (CI 95%: 0.06–4.15, P = 0.52) and 0.50 (CI 95%: 0.06–4.15, P = 0.52). Thus, no statistically significant association with the disease was observed in frequency of the TT variant in comparison to healthy controls. Stratification of IBD patients on the basis of CD or UC showed that individuals carrying at least one T allele are not protected against Crohn's disease. Furthermore, clinical features of the disease did not show any significant association.

Conclusion

In conclusion, the present study indicates that the genetic risk for IBD is not modulated by MTHFR C677T polymorphism in Moroccan population.     

A rubber transfer gasket to improve the throughput of liquid–liquid extraction in 96-well plates: Application to vitamin D testing

Background

The unmitigated rise in demand for the assessment of vitamin D status has taxed the ability of clinical mass spectrometry laboratories to preserve turn-around times. We aimed to improve the throughput of liquid–liquid extraction of plasma/serum for the assay of 25-hydroxy vitamin D.

Methods

We designed and fabricated a flexible rubber gasket that seals two 96-well plates together to quantitatively transfer the contents of one plate to another. Using the transfer gasket and a dry-ice acetone bath to freeze the aqueous infranatant, we developed a novel liquid–liquid extraction workflow in a 96-well plate format. We applied the technology to the mass spectrometric quantification of 25-hydroxy vitamin D.

Results

Cross-contamination between wells was ≤0.13%. The interassay imprecision over 132 days of clinical implementation was less than 10%. The method compared favorably to a standard liquid–liquid extraction in glass tubes (Deming slope = 1.018, S_x|y = 0.022). The accuracy of the assay was 102–105% as assessed with the recently released control materials from NIST.

Conclusions

The development of a plate-sealing gasket permits the liquid–liquid extraction of clinical specimens in a moderate-throughput workflow and the reliable assay of vitamin D status. In the future, the gasket may also prove useful in other sample preparation techniques for HPLC or mass spectrometry.     

A new β-chain haemoglobin variant with increased oxygen affinity: Hb Roma [β115(g17)Ala → Val]

Background

Haemoglobin Roma [β115(G17)Ala → Val] is a new adult haemoglobin variant found in a patient presenting a mild hypochromia and microcytosis. We studied this previously uncharacterised variant in order to evaluate the effect on the structural and funcional properties of the Ala → Val substitution at the α1β1 interface.

Methods and results

The variant chain was identified by direct DNA sequencing of the β-globin gene, which revealed a GCC → GTC mutation in codon 115. This mutation was confirmed by mass spectrometric analysis of the tetramers and peptides. The oxygen-binding properties of the haemoglobin A/haemoglobin Roma mixture, in which the variant makes up 25% of the haemoglobins, showed a significant increase in oxygen affinity with respect to normal haemoglobin A, both in the absence and presence of 2,3-bisphosphoglycerate. The role of the βG17 position, situated at the α₁β₁ interface, has been examined using computational models of haemoglobin Roma and other known βG17 variants, in comparison with normal haemoglobin A.

Conclusions

This study suggests that the β115(G17)Ala → Val substitution at the α1β1 interface is responsible for increased oxygen affinity and mild destabilisation of the haemoglobin Roma.

General significance

An amino acid substitution at the G17 position of the α1β1 interface may result in stabilisation of the high affinity R-state of the haemoglobin molecule.     

Single nucleotide polymorphism of CD40 in the 5′-untranslated region is associated with ischemic stroke

Objectives

Ischemic stroke is influenced by both environmental and genetic factors. The CD40/CD40L system is related to proinflammatory and prothrombogenic responses, which are involved in the pathophysiology of ischemic stroke. The aim of this study was to evaluate association between the CD40 -1C/T single nucleotide polymorphism (SNP) and ischemic stroke in a Chinese population.

Methods

We conducted a case–control study including 286 ischemic stroke patients and 336 controls. CD40 -1C/T SNP was genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing methods, and evaluated its relevance to ischemic stroke susceptibility.

Results

Significantly increased ischemic stroke risk was found to be associated with the T allele of CD40 -1C/T (OR = 1.273, 95% CI = 1.016–1.594). The frequencies of CT and TT/CT genotypes of CD40 -1C/T in ischemic stroke patients were significantly higher than those of controls, respectively (for CT: OR = 2.350, 95% CI = 1.601–3.449; for TT/CT: OR = 2.148, 95% CI = 1.479–3.119). And, similar results were obtained after adjusting non-matched variables. We found that the frequency of carried T genotypes (TT and TT/CT) was significantly increased in patients with history of stroke compared with patients without (for TT: OR = 6.538, 95%CI = 1.655–25.833; for TT/CT: OR = 3.469, 95%CI = 1.031–11.670), respectively.

Conclusions

The findings suggested that the CD40 -1C/T polymorphism might contribute to the susceptibility to ischemic stroke in the Chinese population, and might be associated with history of previous stroke.     

Three single nucleotide variants of the HDAC gene are associated with type 2 diabetes mellitus in a Chinese population: A community-based case–control study

Objectives

There are no data regarding the possible role of the single nucleotide polymorphism (SNP) of class I histone deacetylases (HDACs) in type 2 diabetes mellitus (DM). We designed this study to examine whether polymorphisms of HDACs can be implicated in that disease.

Methods

A community-based, case–control study was conducted, with a total of 568 subjects (284 patients and 284 controls) enrolled. Four polymorphisms of HDAC1 (rs1741981) and HDAC3 (rs11741808, rs2547547, rs2530223) were examined by the use of TaqMan technology.

Results

We found a significant association with risk of type 2 DM for three SNPs of HDAC3, including rs11741808 [odds ratio (OR) = 0.53, 95% confidence interval (CI): 0.35–0.81], rs2547547 [OR = 1.72, 95% CI: 1.13–2.64], and rs2530223 [OR = 1.39; 95% CI: 1.01–1.91]. Subgroup analysis showed that BMI ≥ 23 kg/m², high triglyceride and high blood pressure, together with the rs11741808AG genotype, were associated with a significantly decreased risk for type 2 DM, with ORs of 0.50 (95% CI: 0.27–0.91), 0.38 (95% CI: 0.20–0.71) and 0.43 (95% CI: 0.24–0.76) compared with the AA genotype, respectively. In a population with normal total cholesterol, the AG genotype yielded a significantly decreased risk of type 2 DM risk, with an OR of 0.42 (95% CI: 0.25–0.70) when compared with the persons of the AA genotype. For rs2547547, in a population with normal total cholesterol and triglyceride, the AG genotype was associated with a significantly increased risk of type 2 DM, with ORs of 1.92 (95% CI: 1.17–3.15) and 2.24 (95% CI: 1.28–3.94) when compared with the population carrying the AA genotype.

Conclusions

The results suggest that variants of HDAC3 contribute to an increased prevalence of type 2 DM in the Chinese Han population.     

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7

Background

Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (C_P) it lacks the resolving Cys (C_R), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.

Methods

Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.

Results

Oxidation of the C_P is fast (k_+ 1 > 10³ M^− 1 s^− 1), however the rate of reduction by GSH is slow (k′_+ 2 = 12.6 M^− 1 s^− 1) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized C_P can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k_+ 1 > 10³ M^− 1 s^− 1), but not by Trx. By surface plasmon resonance analysis, a K_D = 5.2 μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical C_R in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.

Conclusions

GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.

General significance

In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.     

Calcium binding studies of peptides of human phospholipid scramblases 1 to 4 suggest that scramblases are new class of calcium binding proteins in the cell

Background

Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca²⁺ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca²⁺/Mg²⁺ binding to human scramblases and conformational changes taking place in them remains unknown.

Methods

In the present study, we analyzed the Ca²⁺ and Mg²⁺ binding to the calcium binding motifs of hPLSCR1–4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry.

Results

The results in this study show that (i) affinities of the peptides are in the order hPLSCR1  > hPLSCR3 > hPLSCR2 > hPLSCR4 for Ca²⁺ and in the order hPLSCR1 > hPLSCR2 > hPLSCR3 > hPLSCR4 for Mg²⁺, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca²⁺ and Mg²⁺ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families.

Conclusions

Based on the above results, we hypothesize that the Ca²⁺ binding motif of hPLSCR1 is a novel type of Ca²⁺ binding motif.

General significance

Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function.     

Polymorphisms of the TLR4 gene and risk of gastric cancer

Objective

Toll-like receptor 4 (TLR4) is an important lipo-polysaccharide (LPS) receptor in gastric epithelial cell signaling transduction and plays critical roles in the development and progression of gastric cancer (GC). We investigated the effects of TLR4 gene polymorphisms and gene–environmental interactions on the risk of GC in Northeastern China.

Methods

We genotyped two single-nucleotide polymorphisms (SNPs) in TLR4 (rs10116253 and rs1927911) in 217 GC patients and 294 cancer-free controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Odds ratio (OR) and 95% confidence intervals (CIs) were estimated by unconditional logistic-regression models.

Results

Individuals carrying CC genotype of rs10116253 and TT genotype of rs1927911 had a significantly decreased risk of GC (adjusted OR = 0.33, 95% CI 0.18–0.60, P < 0.001 and adjusted OR = 0.37, 95% CI 0.21–0.67, P = 0.001 respectively), compared with TT genotype of rs10116253 and CC genotype of rs1927911. In addition, the SNP effects were additive to the effects of some known environmental factors without any interaction between them in the susceptibility to GC.

Conclusion

Our data suggested that TLR4 gene polymorphisms may be associated with a decreased risk of GC in Chinese population. And these SNPs and their combined effects with environmental factors may be associated with the risk of GC.     

Interaction of heparin and heparin-derived oligosaccharides with synthetic peptide analogues of the heparin-binding domain of heparin/heparan sulfate-interacting protein

Background

Although protamine is effective as an antidote of heparin, there is a need to replace protamine due to its side effects. HIP peptide has been reported to neutralize the anticoagulant activity of heparin. The interaction of HIP analog peptides with heparin and heparin-derived oligosaccharides is investigated in this paper.

Methods

Seven analogues of the heparin-binding domain of heparin/heparan sulfate-interacting protein (HIP) were synthesized, and their interaction with heparin was characterized by heparin affinity chromatography, isothermal titration calorimetry, and NMR.

Results

NMR results indicate the imidazolium groups of the His side chains of histidine-containing Hip analog peptide interact site-specifically with heparin at pH 5.5. Heparin has identical affinities for HIP analog peptides of opposite chirality. Analysis by counterion condensation theory indicates the peptide AC-SRPKAKAKAKAKDQTK-NH₂ makes on average ∼ 3 ionic interactions with heparin that result in displacement of ∼ 2 Na⁺ ions, and ionic interactions account for ∼ 46% of the binding free energy at a Na⁺ concentration of 0.15 M.

Conclusions

The affinity of heparin for the peptides is strongly dependent on the nature of the cationic side chains and pH. The thermodynamic parameters measured for the interaction of HIP peptide analogs with heparin are strongly dependent on the peptide sequence and pH.

General significance

The information obtained in this research will be of use in the design of new agents for neutralization of the anticoagulant activity of heparin. The site-specific binding of protonated histidine side chains to heparin provides a molecular-level explanation for the pH-dependent binding of β-amyloid peptides by heparin and heparan sulfate proteoglycan and may have implications for amyloid formation.     

Inhibition by polyamines of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid

Background

Viroids are the smallest pathogens known to date. They infect plants and cause considerable economic losses. The members of the Avsunviroidae family are known for their capability to form hammerhead ribozymes (HHR) that catalyze self-cleavage during their rolling circle replication.

Methods

In vitro inhibition assays, based on the self-cleavage kinetics of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid (CChMVd-HHR) were performed in the presence of various putative inhibitors.

Results

Aminated compounds appear to be inhibitors of the self-cleavage activity of the CChMVd HHR. Surprisingly the spermine, a known activator of the autocatalytic activity of another hammerhead ribozyme in the presence or absence of divalent cations, is a potent inhibitor of the CChMVd-HHR with K_i of 17 ± 5 μM. Ruthenium hexamine and TMPyP4 are also efficient inhibitors with K_i of 32 ± 5 μM and IC₅₀ of 177 ± 5 nM, respectively.

Conclusions

This study shows that polyamines are inhibitors of the CChMVd-HHR self-cleavage activity, with an efficiency that increases with the number of their amino groups.

General significance

This fundamental investigation is of interest in understanding the catalytic activity of HHR as it is now known that HHR are present in the three domains of life including in the human genome. In addition these results emphasize again the remarkable plasticity and adaptability of ribozymes, a property which might have played a role in the early developments of life and must be also of significance nowadays for the multiple functions played by non-coding RNAs.     

Genetic polymorphisms in the DNA repair gene XRCC1 and susceptibility to glioma in a Han population in northeastern China: A case–control study

Background

Several single nucleotide polymorphisms (SNPs) in the X-ray cross-complementing group 1 (XRCC1) gene have been shown to influence DNA repair and to modify cancer susceptibility. To investigate the role of these loci further, we examined the association of three XRCC1 polymorphisms with the risk of gliomas in a Han population in northeastern China.

Methods

Using a PCR–RFLP method, XRCC1 Arg194Trp, Arg280His and Arg399Gln were genotyped in 624 glioma patients and 580 healthy controls.

Results

Significant differences in the distribution of the Arg399Gln allele were detected between glioma patients and healthy controls by a logistic regression analysis (OR = 1.35, 95%CI 1.17–1.68, P = 0.001). Our data also revealed that the Arg399Gln variant (allele A) carriers had an increased glioma risk compared to the wild-type (allele G) homozygous carriers (OR = 1.40, 95%CI 1.12–1.76, P = 0.003).

Conclusions

These results suggest that the XRCC1 Arg399Gln might influence the risk of developing glioma in a Han population in northeastern Chinese.     

Up-regulation of adenylylcyclases I and II induced by long-term adaptation of rats to morphine fades away 20 days after morphine withdrawal

Background

Activation of adenylyl cyclase (AC) by prolonged exposure of mammalian organism to morphine was demonstrated in previous studies of mechanism of action of this drug. However, expression level of individual AC isoforms was not analyzed in crucial cell structure, plasma membrane (PM).

Methods

Rats were adapted to morphine for 10 days and sacrificed 24 h (group + M10) or 20 days (+ M10/−M20) after the last dose. Control animals were sacrificed in parallel with morphine-treated (groups − M10 and (− M10/−M20)). Percoll®-purified PM were isolated from brain cortex and analyzed by immunoblotting and specific radioligand binding.

Results

ACI (ACII) was increased 8× (2.5×) in morphine-adapted rats (+ M10) when compared with controls (− M10). Increase of ACI and II by long-term adaptation to increasing doses of morphine represented a specific effect as the amount of ACIII–ACIX, of prototypical PM marker, Na, K-ATPase and of trimeric G protein α and β subunits was unchanged. Increase of ACI and II was not detected in PM isolated from group (+ M10/−M20). Thus, the marked increase of ACI and ACII faded away 20 days since the last dose of morphine.

Conclusions

We assume that the specific increase in expression level of ACI and ACII in brain cortex of morphine-adapted rats proceeds as a compensatory, homeostatic response to prolonged exposure to inhibitory drug, morphine.

General significance

Our findings demonstrate that the dramatic and specific change of the crucial component of the opioid receptor cascade in brain cortex, manifested as an increase in PM level of ACI and II, is reversible.     

Gliadin T cell epitope selection by tissue transglutaminase in celiac disease. Role of enzyme specificity and pH influence on the transamidation versus deamidation process

Tissue transglutaminase (TG2) can modify proteins by transamidation or deamidation of specific glutamine residues. TG2 has a major role in the pathogenesis of celiac disease as it is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides that are recognized by CD4(+), DQ2-restricted T cells from the celiac lesions. Capillary electrophoresis with fluorescence-labeled gliadin peptides was used to separate and quantify deamidated and transamidated products. In a competition assay, the affinity of TG2 to a set of overlapping gamma-gliadin peptides was measured and compared with their recognition by celiac lesion T cells. Peptides differed considerably in their competition efficiency. Those peptides recognized by intestinal T cell lines showed marked competition indicating them as excellent substrates for TG2. The enzyme fine specificity of TG2 was characterized by synthetic peptide libraries and mass spectrometry. Residues in positions -1, +1, +2, and +3 relative to the targeted glutamine residue influenced the enzyme activity, and proline in position +2 had a particularly positive effect. The characterized sequence specificity of TG2 explained the variation between peptides as TG2 substrates indicating that the enzyme is involved in the selection of gluten T cell epitopes. The enzyme is mainly localized extracellularly in the small intestine where primary amines as substrates for the competing transamidation reaction are present. The deamidation could possibly take place in this compartment as an excess of primary amines did not completely inhibit deamidation of gluten peptides at pH 7.3. However, lowering of the pH decreased the reaction rate of the TG2-catalyzed transamidation, whereas the rate of the deamidation reaction was considerably increased. This suggests that the deamidation of gluten peptides by TG2 more likely takes place in slightly acidic environments.     

Single amino acid substitutions in recombinant plant-derived human α1-proteinase inhibitor confer enhanced stability and functional efficacy

Background

Human α₁-proteinase inhibitor (α₁-PI) is the most abundant serine protease inhibitor in the blood and the heterologous expression of recombinant α₁-PI has great potential for possible therapeutic applications. However, stability and functional efficacy of the recombinant protein expressed in alternate hosts are of major concern.

Methods

Five variants of plant-expressed recombinant α₁-PI protein were developed by incorporating single amino acid substitutions at specific sites, namely F51C, F51L, A70G, M358V and M374I. Purified recombinant α₁-PI variants were analyzed for their expression, biological activity, oxidation-resistance, conformational and thermal stability by DAC-ELISA, porcine pancreatic elastase (PPE) inhibition assays, transverse urea gradient (TUG) gel electrophoresis, fluorescence spectroscopy and far-UV CD spectroscopy.

Results

Urea-induced unfolding of recombinant α₁-PI variants revealed that the F51C mutation shifted the mid-point of transition from 1.4 M to 4.3 M, thus increasing the conformational stability close to the human plasma form, followed by F51L, A70G and M374I variants. The variants also exhibited enhanced stability for heat denaturation, and the size-reducing substitution at Phe51 slowed down the deactivation rate ~ 5-fold at 54 °C. The M358V mutation at the active site of the protein did not significantly affect the conformational or thermal stability of the recombinant α₁-PI but provided enhanced resistance to oxidative inactivation.

Conclusions

Our results suggest that single amino acid substitutions resulted in improved stability and oxidation-resistance of the plant-derived recombinant α₁-PI protein, without inflicting the inhibitory activity of the protein.

General significance

Our results demonstrate the significance of engineered modifications in plant-derived recombinant α₁-PI protein molecule for further therapeutic development.     

A novel bifunctional peptidic aspartic protease inhibitor inhibits chitinase A from Serratia marcescens: Kinetic analysis of inhibition and binding affinity

Background

Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API (Aspartic Protease Inhibitor), isolated from Bacillus licheniformis that inhibits chitinase A (ChiA) from Serratia marcescens.

Methods

The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA.

Results and conclusions

The peptide has an amino acid sequence N-Ile¹-Cys²-Glu³-Ala⁴-Glu⁵-His⁶-Lys⁷-Trp⁸-Gly⁹-Asp¹⁰-Tyr¹¹-Leu¹²-Asp¹³-C. The ChiA–API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I₅₀ = 600 nM and K_i = 510 nM in the presence of chromogenic substrate p-nitrophenyl-N,N′-diacetyl-β-chitobioside[p-NP-(GlcNAc)₂]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k₅ = 8.7 ± 1 × 10^− 3 s^− 1 and k₆ = 7.3 ± 0.6 × 10^− 5 s^− 1. CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme.

General significance

The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.     

The contribution of platelet glycoproteins (GPIa C807T and GPIba C-5T) and cyclooxygenase 2 (COX-2G-765C) polymorphisms to platelet response in patients treated with aspirin

Objective

Aspirin is an antiplatelet agent commonly used in treatment of patients with high risk to develop stroke and myocardial infarction. However, inter-individual variability regarding the inhibition of platelet function by aspirin is well documented. In this study, the correlation between platelet glycoproteins (GPIa C807T and GPIba C-5T) and cyclooxygenase 2 (COX-2G-765C) polymorphisms and antiplatelet response in patients treated with aspirin was investigated.

Methods

Jordanian adult patients (n = 584) who are taking aspirin as an antiplatelet agent participated in the study. Platelet aggregation response was measured using Multiplate Analyzer® system. Polymerase chain reaction–restriction fragment length polymorphism assay (PCR–RFLP) was used for genotyping of the examined polymorphisms.

Results

Aspirin resistance was found in 15.8% of patients. Response to aspirin was significantly associated with GPIba C-5T polymorphism (P < 0.05). However, the GPIa C807T and COX-2G-765C polymorphisms were not related to aspirin resistance (P > 0.05).

Conclusion

A considerable fraction of the Jordanian population is resistant to the antiplatelet effect of aspirin, which might be related to GPIba C-5T polymorphism.