Lysine Dipeptide Research Articles

Lysine Dipeptide Enhances Gut Structure and Whole-Body Protein Synthesis in Neonatal Piglets with Intestinal Atrophy

BackgroundParenteral nutrition (PN) is often a necessity for preterm infants; however, prolonged PN leads to gut atrophy, weakened gut barrier function, and a higher risk of intestinal infections. Peptide transporter-1 (PepT1) is a di- or tripeptide transporter in the gut and, unlike other nutrient transporters, its activity is preserved with the onset of intestinal atrophy from PN. As such, enteral amino acids in the form of dipeptides may be more bioavailable than free amino acids when atrophy is present. ObjectivesIn Yucatan miniature piglets with PN-induced intestinal atrophy, we sought to determine the structural and functional effects of enteral refeeding with lysine as a dipeptide, compared to free L-lysine. MethodsPiglets aged 7-8 days were PN-fed for 4 days to induce intestinal atrophy, then were refed with enteral diets with equimolar lysine supplied as lysyl-lysine (Lys-Lys; n = 7), free lysine (n = 7), or Lys-Lys with glycyl-sarcosine (n = 6; to determine whether competitive inhibition of Lys-Lys uptake would abolish PepT1-mediated effects). The diets provided lysine at 75% of the requirement and were gastrically delivered for a total of 18 hours. Whole-body and tissue-specific protein synthesis, as well as indices for gut structure and barrier function, were measured. ResultsThe villus height, mucosal weight, and free lysine concentration were higher in the Lys-Lys group compared to the other 2 groups (P < 0.05). Lysyl-lysine led to greater whole-body protein synthesis compared to free lysine (P < 0.05). Mucosal myeloperoxidase activity was lower in the Lys-Lys group (P < 0.05), suggesting less inflammation. The inclusion of glycyl-sarcosine with Lys-Lys abolished the dipeptide effects on whole-body and tissue-specific protein synthesis (P < 0.05), suggesting that improved lysine availability was mediated by PepT1. ConclusionsImproved intestinal structure and whole-body protein synthesis suggests that feeding strategies designed to exploit PepT1 may help to avoid adverse effects when enteral nutrition is reintroduced into the compromised guts of neonatal piglets.

Deciphering the structural preference encoded in amide-I vibrations of lysine dipeptide in gas phase and in aqueous solution.

Protein's biological function is critically associated with its structural feature, which is encoded in its amino acid sequence. For evaluation of conformational fluctuation and folding mechanism, DFT calculations were performed on the model compound - lysine dipeptide (LYSD) in gas phase to demonstrate the correlation between amide-I vibrations and secondary structure. Molecular dynamics simulations were carried out for the structural dynamics of LYSD in aqueous solution. The results show that LYSD tends form C7eq, C5, β, PPII and α conformations in the gas phase and primarily presented PPII and α conformations in aqueous solution. The obtained amide-I vibrational frequencies of LYSD conformers were assigned, thus build the correlations between amide-I probes and secondary structure of LYSD. These results provide theoretical insights into the structural feature of LYSD through amide-I vibrations, and would shed light on site specific structural prediction of polypeptides.

Probabilistic model to treat flexibility in molecular contacts

ABSTRACTEvaluating accessible conformational space is computationally expensive and thermal motions are partly neglected in computer models of molecular interactions. This produces error into the estimates of binding strength. We introduce a method for modelling interactions so that structural flexibility is inherently taken into account. It has a statistical model for three-dimensional (3D) properties of non-local contacts and a physics-based description of local interactions, based on mechanical torque. The form of the torque barrier is derived using a representation of the local electronic structure, which is presumed to improve transferability, compared to traditional force fields. The non-local contacts are more distant than 1–4 interactions and target atoms are represented by 3D probability densities. Probability mass quantifies the strength of contact and is calculated as an overlap integral. Repulsion is described by negative probability density, allowing probability mass to be used as the descriptor of contact preference. As a result, we are able to transform the high-dimensional problem into a simpler evaluation of 3D integrals. We outline how this scoring function gives a tool to study the enthalpy–entropy compensation and demonstrate the feasibility of our approach by evaluating numerical probability masses for chosen side chain to main chain contacts in a lysine dipeptide structure.

SAR Development of Lysine-Based Irreversible Inhibitors of Transglutaminase 2 for Huntington's Disease.

We report a series of irreversible transglutaminase 2 inhibitors starting from a known lysine dipeptide bearing an acrylamide warhead. We established new SARs resulting in compounds demonstrating improved potency and better physical and calculated properties. Transglutaminase selectivity profiling and in vitro ADME properties of selected compounds are also reported.

Characterization by mass spectrometry and IRMPD spectroscopy of the sulfoxide group in oxidized methionine and related compounds

Methionine protein residues are prone to oxidation. To unravel the controversy about the mechanism of its one-electron oxidation, we characterised the main biological product, methionine sulfoxide, using mass spectrometry and IR multiple photon dissociation spectroscopy. Gas phase IR spectra in the 800–2000 cm −1 range of protonated methionine and its sulfoxide were recorded and compared to those computed for the lowest energy structures. The signature of the S O bond was clearly identified at around 1000 cm −1. Oxidation of methionine–lysine dipeptide by OH radicals in the presence of catalase revealed the formation of methionine sulfoxide upon one-electron oxidation.

Flow-injection amperometry at microfabricated silicon-based μ-liquid–liquid interface arrays

Geometrically regular silicon membrane-based micropore arrays were employed for defined arrays of micrometer-sized interfaces between two immiscible electrolyte solutions (μITIES). These were incorporated into a poly(tetrafluoroethylene) (PTFE) hydrodynamic cell. Electrochemistry at the μITIES array was undertaken following gellification of the organic phase using polyvinyl chloride (PVC) and flowing an aqueous phase over the array surface. Cyclic voltammetric characterization of asymmetric diffusion profiles on either side of the μITIES was accomplished under flowing conditions using positively and negatively charged (TEA + and 4-OBSA −, respectively) model analyte species. Incorporation of an ionophore (dibenzo-18-crown-6 ether) into the organogel allowed the ion-transfer detection of two oligopeptides (phenylalanine dipeptide and lysine dipeptide) within the available potential window under stationary and flowing conditions. Flow rate studies with TEA + indicated that the amperometric peak currents do not obey the Levich equation, due to diffusion dominating the mass transport, as opposed to convection. The influence of the applied potential ( Δ O W ϕ ) on the amperometric response of the oligopeptides was studied and hydrodynamic voltammograms (HDVs) for the individual oligopeptides were subsequently constructed. The data presented provide a basis for the use of silicon membrane-based μITIES arrays in flow analytical methods.

Tissue Transglutaminase and the Progression of Human Renal Scarring

Experimental renal scarring indicates that tissue transglutaminase (tTg) may be associated with the accumulation of extracellular matrix (ECM), both indirectly via TGF-beta1 activation and directly by the formation of epsilon(gamma-glutamyl) lysine dipeptide bonds within the ECM. The latter potentially accelerates deposition and confers the ECM with resistance to proteolytic digestion. Studied were 136 human renal biopsy samples from a range of chronic renal diseases (CRD) to determine changes in tTg and epsilon(gamma-glutamyl) lysine crosslinking. Immunofluorescence for insoluble tTg showed a 14-fold increase in the kidneys of CRD patients (5.3 +/- 0.5 versus 76 +/- 54 mV/cm(2)), which was shown to be active by a similar 11-fold increase in the epsilon(gamma-glutamyl) lysine crosslink (1.8 +/- 0.2 versus 19.3 +/- 14.2 mV/cm(2)). Correlations were obtained with renal function for tTg and crosslink. In situ hybridization for tTg mRNA showed that tubular epithelial cells were the major source of tTg; however, both mesangial and interstitial cells also contributed to elevated levels in CRD. This mRNA pattern was consistent with immunohistochemistry for soluble tTg. Changes in renal tTg and its product, the epsilon(gamma-glutamyl) lysine crosslink, occur in progressive renal scarring in humans independently of the original etiology and in a similar manner to experimental models. tTg may therefore play a role in the pathogenesis of renal scarring and fibrosis in patients with CRD and can therefore be considered a potential therapeutic target.

Identification of Cross-linked 46 KDa Protein in Experimentally Induced Silicotic Nodule in Rat Lung

OBJECTIVES: This study was conducted in order to understand the cellular events associated with silica-induced pathogenesis of the rat lung.METHODS: Silicosis was induced by an intratracheal instillation of 50 mg of silica (SiO2, 0.15 - 10 micrometer) suspended in 500 microliter of a sterile saline solution in Sprague-Dawley rats weighing 200g. Silicotic nodules were excised from the rat lungs 4 weeks after silica instillation, then boiled for 4 days at 110 degrees in solution containing 2% SDS, 10 M urea and 40 mM DTT. The insoluble cellular encapsulates were electrophoresed on 4-12 % gradient SDS-PAGE, and the amino acid composition was analyzed. Affinity chromatographies of the homogenate supernatants of the control lung, silicotic nodule, and normal rat plasma were performed using rabbit IgG, anti-rat, cross-linked protein from the silicotic nodule. The amounts of N epsilon-(gamma-glutamyl) lysine cross-linked in the control lungs and silicotic nodules were determined using HPLC analysis.RESULTS: The remaining cross-linked protein was insoluble in the 10 M urea and 40 mM sulfhydryl reagents even under prolonged boiling conditions. The encapsulate revealed the retention of silica particles within the protein whose amino acid composition showed a high percentage of alanine, leucine and glycine. A 46 KDa protein was identified as a cross-linked protein in the silicotic nodule by affinity chromatography. The level of N epsilon-(gamma-glutamyl) lysine dipeptide in the nodule digest was prominently increased compared with that in the control lung.CONCLUSIONS: Transglutaminase (TGase)-catalyzed cross-linking appears to be involved in the silicotic nodule formation, and the 46 KDa protein may be cross-linked to itself and other extracellular matrix proteins during fibrosis and the formation of eventually insoluble nodule.

Pellicle precursor protein crosslinking characterization of an adduct between acidic proline-rich protein (PRP-1) and statherin generated by transglutaminase.

Recent work with oral transglutaminase indicated that this enzyme, derived from oral epithelial cells, crosslinked pellicle precursor proteins which may be important in the formation of the acquired enamel pellicle. The purpose of this study was to investigate whether purified acidic PRP-1 can form crosslinks with statherin, and whether such a crosslink is derived from a transglutaminase-catalyzed reaction between glutaminyl and lysyl side-chains, leading to a covalent bond formation. Enzymatic reaction products were analyzed by SDS-PAGE and reverse-phase HPLC. The SDS electrophoretogram revealed a protein band with an apparent molecular weight of 32 kDa, which is consistent with the combined apparent molecular weight of acidic PRP-1 (24 kDa) and statherin (8 kDa). A reaction product isolated by HPLC was characterized by amino acid analysis, which showed a stoichiometry consistent with being an adduct composed of one molecule of acidic PRP-1 and one molecule of statherin. In negative control experiments, it could be shown that this adduct was not detected when the lysines of both substrates were modified by reductive methylation prior to the enzymatic reaction. In addition, amino acid analysis and mass spectrometry confirmed the presence of a gamma-glutamyl-epsilon-lysine dipeptide after enzymatic hydrolysis and the absence of this dipeptide after acid hydrolysis. Analysis of the data obtained indicates that oral transglutaminase is capable of crosslinking acidic PRP-1 and statherin in vitro. In addition, this finding exemplifies the potential of post-secretory processing of salivary proteins, which may represent an additional mechanism to generate new protein species.

Synthesis and folding of native collagen III model peptides.

Solid-phase synthesis of triple-helical peptides, including native collagen III sequences, was started with a trimeric branch, based upon the lysine dipeptide [Fields, C. G., Mickelson, D. J., Drake, S. L., McCarthy, J. B., and Fields, G. B. (1993) J. Biol. Chem. 268, 14153-14160]. Branch synthesis was modified, using TentaGel R as resin, p-hydroxybenzyl alcohol (HMP) as linker, Dde as N(epsilon)-protective group, and HATU/HOAT as coupling reagent. Three homotrimeric sequences, each containing the Gly 606-Gly 618 portion of human type III collagen, were added to the amino functions of the branch. The final incorporation of GlyProHyp triplets, stabilizing the collagen III triple helix, was performed by using protected GlyProHyp tripeptides, each containing tert-butylated hydroxyproline [P(tBu)] instead of hydroxyproline (P). Among the protected tripeptides FmocP(tBu)PG, FmocPP(tBu)G, and FmocGPP(tBu), prepared manually on a chlorotrityl resin, incorporation of FmocPP(tBu)Gly was best suited for synthesis of large and stable peptides, such as PPG(8), containing 8 (PPG)(3) trimers (115 residues, 10 610 Da). The structures of five peptides, differing from each other by the type and number of the triplets incorporated, were verified by MALDI-TOF-MS. Their conformations and thermodynamic data were studied by circular dichroism and differential scanning calorimetry. Except for PPG(8), containing 8 (PPG)(3) trimers with hydroxyproline in the X position and adopting a polyproline II structure, all peptides were triple-helical in 0.1 M acetic acid and their thermal stabilities ranged from t(1/2) = 39. 4 to t(1/2) = 62.5 degrees C, depending on the identity and number of the triplets used. Similar values of the van't Hoff enthalpy, DeltaH(vH), derived from melting curves, and the calorimetric enthalpy, DeltaH(cal), obtained from heat capacity curves, indicate a cooperative ratio of CR = DeltaH(vH)/DeltaH(cal) = 1, establishing a two-state process for unfolding of THP(III) peptides. The independence of the transition temperatures t(1/2) on peptide concentration as well as equilibrium centrifugation data indicate monomolecular dimer(f) to dimer(u) (F(2) <--> U(2)) transitions and, in addition, bimolecular dimer(f) to monomer(u) transitions (F(2) <--> 2U). The dominance of the concentration-independent monomolecular reaction over the concentration-dependent bimolecular reaction makes thermal unfolding of THP(III) peptides appear to be monomolecular. If one designates the molecularity described by the term pseudomonomolecular, unfolding of the dimeric peptides PPG(6-8) follows a two-state, pseudomonomolecular reaction.

Transglutaminase Effects on Low Temperature Gelation of Fish Protein Sols

ABSTRACTMyosin polymerization and formation of ɛ‐(γ‐glutamyl)lysine linkages were quantified in Alaska pollock surimi gels which contained no additive (control), or a commercial microbial transglutaminase (MTGase). As preincubation (“setting”) time at 25°C was increased, the gel strength of control and 0.2% MTGase‐added samples increased, with greater increases at higher MTGase levels. SDS‐PAGE and HPLC analyses showed increasing nondisulfide polymerization and ɛ‐(γ‐glutamyl)lysine dipeptide content, with increasing setting time and/or added MTGase. Content of ɛ‐(γ‐glutamyl)lysine dipeptide correlated with gel strength (shear stress) and shear modulus at failure (Gf) for these gels. Higher stresses were measured in samples containing 0.2% MTGase than in controls at corresponding levels of ɛ‐(γ‐glutamyl)lysine dipeptide, indicating that rate of myosin polymerization may affect ultimate gel strength.

High-performance liquid chromatographicdetermination of n- ε-(2-propenal)lysine in biological samples after derivatization with diethylethoxymethylenemalonate

A recently reported methodology for amino acid analysis by HPLC has been adapted for quantification of N- ε-(2-propenal)lysine (a modified lysine by reaction with malondialdehyde that has been found in enzymatic digests of foods and in urine) in biological samples. We describe its use for investigating the in vitro degradation of N- ε-(2-propenal)lysine using rat tissue homogenates. Lysine dipeptide, used as a control in the incubation mixtures, and the lysine released by the hydrolytic action of the homogenates in the in vitro incubations are quantified in the same way. The samples are subjected to a cleanup prederivatization step using PD-10 disposable columns (Pharmacia). This allows precolumn derivatization with diethylethoxymethylenemalonate (50 min, 50°C) and resolution of the derivatives of the compounds of interest by reversed-phase HPLC (binary gradient, 45 min) with quantification based on the uv absorption of the derivatives at 280 nm (detection limits below 1 pmol). The entire analysis takes 110 min. This method can be of general use for the determination of N- ε-(2-propenal)lysine in the context of research dealing with protein deterioration by reaction with malondialdehyde in biological systems and in foods. A method for the synthesis of N- ε-(2-propenal)lysine, used as external standard for the HPLC analysis, is described.

Fluorescent color factor calculation using dBASE-II.

A software system utilizing dBASE-II operating on a dual-drive Apple II+ computer is described. Color factors and retention times for 15 amino acids and epsilon-(gamma-glutamyl)lysine dipeptide are calculated following high performance liquid chromatography. The software package produces a listing of acceptable limits for these parameters calculated as plus and minus 2 standard deviations of the mean. The code is distributed in source form.

High-pressure liquid chromatographic procedure for the determination of ϵ-(γ-glutamyl)lysine in proteins

A high-pressure liquid chromatographic method for measuring the ϵ-(γ-glutamyl)lysine dipeptide which is formed postranslationally in many biological systems as a result of protein crosslinking by transglutaminases was developed. Precolumn derivatization with o-phthalaldehyde was carried out for 2 min in 0.4 m potassium borate, pH 10.4, which was diluted with methanol (1:1) and to which 2-mercaptoethanol was added to a concentration of 2% (v/v). Separation of the peptide product was achieved on a Zorbax C 8 reverse-phase column by a linear gradient of 20 to 95% (v/v) methanol in 0.02 m potassium acetate (pH 5.5), with 1% (v/v) tetrahydrofuran, at a flow rate of 2 ml/min. The peptide derivative was eluted at 11.2 min, between tyrosine (9.7 min) and methionine (12.5 min), and a linear response of fluorescence was obtained up to at least 1.6 nmol of the peptide. The general utility, rapidity, and sensitivity of the new chromatographic technique was tested by measuring ϵ-(γ-glutamyl)lysine crosslinks in the clotted seminal vesicle secretion protein of the guinea pig. Comparison was made with an ion-exchange chromatography method using an amino acid analyzer, which also provided an excellent, albeit much slower, separation of ϵ-(γ-glutamyl)lysine. The availability of two independent analytical procedures based on different chromatographic principles should greatly aid in studying the formation of ϵ-(γ-glutamyl)lysine crosslinks in biological systems.

Tests for carbohydrates and isopeptide bonds in larval salivary proteins of Chironomus tentans

This study evaluated several aspects of the composition of the salivary polypeptides of larval Chironomus tentans. Proteins migrating with the apparent molecular weights of approximately 400,000 and 160,000 were prominent components detected in the secretion using gradient gel electrophoresis in the presence of sodium dodecyl sulphate. Several smaller components were also found. The large salivary gland proteins were found to by glycosylated. This glycosylation could influence the molecular weight estimates for these proteins. Tunicamycin suppressed attachment of fucose and glucosamine to the proteins, indicating that at least some of these carbohydrates were N-linked. To test the hypothesis that the salivary proteins are made up of small polypeptides crosslinked through lysine residues, efforts were made to detect ϵ(γ-glutamyl)lysine dipeptide in the secretion. We found no evidence for this dipeptide. Isolation of salivary gland proteins in the presence of inhibitors of formation of this bond (iodoacetamide, glycine ethyl ester) did not result in smaller proteins. No other atypical amino acid crosslinks were detected either, although lysine-threonine dipeptides were particularly resistant to proteolysis.