Number Of Glutamate Residues Research Articles

Influence of composition of cysteine-containing peptide-based chelators on biodistribution of 99mTc-labeled anti-EGFR affibody molecules

Epidermal growth factor receptor (EGFR) is overexpressed in a number of cancers and is the molecular target for several anti-cancer therapeutics. Radionuclide molecular imaging of EGFR expression should enable personalization of anti-cancer treatment. Affibody molecule is a promising type of high-affinity imaging probes based on a non-immunoglobulin scaffold. A series of derivatives of the anti-EGFR affibody molecule ZEGFR:2377, having peptide-based cysteine-containing chelators for conjugation of 99mTc, was designed and evaluated. It was found that glutamate-containing chelators Gly-Gly-Glu-Cys (GGEC), Gly-Glu-Glu-Cys (GEEC) and Glu-Glu-Glu-Cys (EEEC) provide the best labeling stability. The glutamate containing conjugates bound to EGFR-expressing cells specifically and with high affinity. Specific targeting of EGFR-expressing xenografts in mice was demonstrated. The number of glutamate residues in the chelator had strong influence on biodistribution of radiolabeled affibody molecules. Increase of glutamate content was associated with lower uptake in normal tissues. The 99mTc-labeled variant containing the EEEC chelator provided the highest tumor-to-organ ratios. In conclusion, optimizing the composition of peptide-based chelators enhances contrast of imaging of EGFR-expression using affibody molecules.

Tunable delivery of bioactive peptides from hydroxyapatite biomaterials and allograft bone using variable‐length polyglutamate domains

Hydroxyapatite (HA) biomaterials and allograft bone are common alternatives to autogenous grafts; however, these materials lack the strong osteoinductive potential of autologous bone. Previous studies have established that polyglutamate domains, which bind selectively to HA, can be engineered onto bioactive peptides as a mechanism for coupling osteoinductive signals onto HA and allograft. In the current investigation, we adapted the polyglutamate approach to tailor delivery of a model collagen-derived peptide, Asp-Gly-Glu-Ala (DGEA), by manipulating the number of glutamates in the HA binding domain. Specifically, DGEA was modified with diglutamate (E2-DGEA), tetraglutamate (E4-DGEA), or heptaglutamate (E7-DGEA), and it was found that initial peptide binding to HA and allograft was significantly enhanced as the number of glutamates increased. We also determined that the rate of release of polyglutamate-DGEA from substrates over a 5-day interval increased proportionally as the number of glutamate residues was decreased. Additionally, we tuned the peptide release rate by creating mixtures of E2-DGEA, E4-DGEA, and E7-DGEA, and observed that release kinetics of the mixtures were distinct from pure solutions of each respective peptide. These collective results suggest that variable-length polyglutamate domains provide an effective mechanism for controlled delivery of osteoregenerative peptides on HA-containing bone graft materials.

Differential distribution of glutamylated tubulin isoforms along the sea urchin sperm axoneme.

Tubulin belongs to a highly conserved multigenic family, in which several gene products usually coexist in the same tissue or the same cell. Moreover, seven classes of post-translational modifications of these gene products lead to an amazing diversity of tubulin polypeptide chains, within the same cell type, whose physiological function remains elusive. Such diversity has been found in a very stable microtubular organelle, the sea urchin sperm flagellum, where some tubulin isoforms have been directly implicated in motility, whereas others may play a more structural role. In particular, polyglutamylated tubulin has been shown to be crucial for motility (Gagnon et al., 1996: J Cell Sci 109:1545 p). Here, we show with the GT335 antibody that polyglutamylated tubulin is distributed according to a decreasing gradient along the sea urchin sperm axoneme, since a semi-quantitative measurement of immunofluorescence intensity reveals that in its proximal half, the axoneme is sixfold more labeled than in its distal half. This gradient along the length of the axoneme is confirmed by immunogold labeling procedures which, in addition, demonstrate a uniform distribution of polyglutamylated tubulin among peripheral doublets and a lesser content in the central pair within a same section. Moreover, our data obtained with B3, an antibody that recognizes both mono- and poly-glutamylated tubulin, suggest that the number of glutamate residues in the lateral poly-glutamyl chain of tubulin varies along the whole length of the axoneme. These novel results coupled with those published earlier may be important to understand the role of polyglutamylation in flagellar motility.

Analysis of Folate Form Distribution by Affinity Followed by Reversed-Phase Chromatography with Electrochemical Detection

Naturally occurring folates exist in multiple forms, differing in pteridine ring structure and number of glutamate residues. The ability to measure these folate coenzymes in tissues and cells gives important information about in vivo folate metabolism. Folates were heat-extracted from biological samples. A two-column HPLC system with four-channel coulometric electrochemical detection was used for analysis. An affinity column was used first to purify folates from the extract. Purified folates were eluted from the affinity column onto a phenyl analytical column, utilizing a switching valve, and folate forms were separated using an acetonitrile gradient. Folate forms differing in pteridine ring structure and number of glutamate chain residues were identified by retention time and characteristic response across the channels of the detector. Folates were quantified by comparison to an external calibration mixture. Limits of detection for pentaglutamyl folates ranged from 0.21 pmol for tetrahydrofolate to 0.41 pmol for 5-methyltetrahydrofolate. CVs (n = 5) for peaks containing 9-67 pmol of folate were 0.6-6.4% (within day) and 5.2-8. 4% (between days). CVs (n = 5) for peaks containing 0.9-3.5 pmol folate were 5.7-16% (within day) and 8.4-13% (between days). This automated HPLC system allows the simultaneous determination of polyglutamyl forms of folates from biological samples, including tetrahydrofolate, 5-methyltetrahydrofolate, formylated folates, and pteroylglutamate. The low detection limits allow analysis of folate form distribution in human samples such as erythrocytes and lymphocytes.

In vitro and in vivo evaluation of progesterone implants based on new biodegradable poly(glutamic and glutamate esters) copolymers.

New biodegradable polypeptidic copolymers of glutamic acid and tert-butyloxycarbonylmethyl glutamate were evaluated as implantable drug delivery systems. Three copolymers varying in degrees of esterification, which is inversely proportional to the number of glutamic residues, were selected for their range of hydrophilicity and biodegradation rates. Progesterone-containing implants were then prepared by means of an extrusion process and both in vitro and in vivo evaluations were performed. The effects of drug loading, nature, and pH of release medium were investigated. In vitro/in vivo correlations were established for some types of implants. Finally, mathematical fitting of the data, using published models, helped to better understand the mechanisms governing release.

Combined affinity and ion pair column chromatographies for the analysis of food folate

Folate in nature exists in a variety of forms that differ by the state of oxidation, one carbon substitution of the pteridine ring, and by the number of glutamate residues. A method was developed in our laboratory (Selhub, 1989) that uses affinity chromatography followed by ion-pair high performance liquid chromatography (HPLC) for the analysis of folate distribution in tissues with simultaneous information on the structural nature at both ends of the folate molecule. The purpose of this study was to determine if this (affinity/HPLC) method is also suitable for the analysis of food folates. A total of 10 food products were analyzed. The food items were suspended in 10 volumes of 2% ascorbate-10 mmol/L 2-mercaptoethanol at pH 7.8 and heat extracted in an autoclave for 30 min. After centrifugation, folate in the supernatant fraction was purified by affinity chromatography and analyzed by ion-pair reverse HPLC using a diode array UV detector. Results showed variability of folate distribution in the various products ranging from a single derivative of 5-methylH 4PteGlu found in egg yolk to more complex mixtures of pentaglutamyl folates in lima beans, a series of methylated tetrahydrofolates in banana, and a multiplicity of forms in yeast extracts. The method appears to be reliable, as the measured variability amounted to an average of 10%, while total folates obtained by integrating the concentration of individual folates were comparable to total folates estimated using the more traditional microbial assay method.

Determination of tissue folate composition by affinity chromatography followed by high-pressure ion pair liquid chromatography

A recent report from this laboratory described the use of affinity chromatography for the isolation of pure folates from tissue extracts (J. Selhub, B. Darcy-Vrillon, and D. Fell (1988) Anal. Biochem. 168, 247–251). The present study was undertaken to develop chromatographic procedures for quantitative analysis of the individual folates in the affinity-purified mixture. Methods were devised whereby mixtures containing pteroylglutamates (PteGlu 1–7) were batch reduced to the dihydro, H 2PteGlu 1–7, and tetrahydro, H 4PteGlu 1–7, forms. The 5-methylH 4PteGlu 1–7 and the 10-formylH 4PteGlu 1–7 series were prepared from H 4PteGlu 1–7. These compounds were used to calibrate a liquid chromatographic system for the resolution of folate mixtures. This system included reverse-phase ion pair chromatography and a diode array detector. A mixture containing oxidized and reduced PteGlu 1–7, a total of 35 derivatives, was separated into seven clusters arranged in an order of increasing number of glutamate residues. Each cluster was represented by two or more peaks which were due to folates that differed in the pteridine ring structure but had the same number of glutamate residues. In clusters containing mono and diglutamyl derivatives the 10-formyltetrahydro-, the tetrahydro-, and the dihydrofolate forms appeared as separate peaks while those representing folic acid and 5-methyltetrahydrofolate derivatives eluted in coinciding peaks. This hierarchy was maintained in the following clusters except for increasing tendency of the former three forms of folates to elute in the same peak. The number of glutamate residues of any eluting folate can be determined on the basis of retention time in relation to those of the clusters. The pteridine ring structure of that same folate can be determined on the basis of its elution position within that cluster and spectral characteristics determined by the diode array detection system. If that position is common for more than one derivative then identification is based on differential spectral properties. Using uv absorption signals at 280 nm to determine indiscriminate folate activity, absorption signals at 350 nm are used to identify folic acid and dihydrofolate derivatives and signals at 258 nm are used to identify 10-formyltetrahydrofolate derivatives. These principles were incorporated into mathematic expressions which were used for quantitative resolution of simulated mixtures containing oxidized and reduced PteGlu 5 and for the analysis of folate composition in rat liver, human milk, and cows milk.

The identification of the intracellular folate coenzymes of different cell types.

Publisher Summary This chapter discusses the identification of the intracellular folate coenzymes of different cell types. Folate polyglutamates occur within cells in low concentration. In two instances, it has been possible to isolate sufficient folate to carry out chemical analysis on the number of glutamate residues. Yeast had been shown to contain exclusively folate heptaglutamate and Clostridium acidi-urici only folate triglutamate. Two methods have been developed to examine folate polyglutamate distributions. One approach separates the intact folate polyglutamates by molecular size on sephadex G-25. This method has been shown to be unsatisfactory due to the adsorption to the gel. The other approach developed in the laboratory depends on cleavage of the C-9-N-10 bond and subsequent separation of the resulting p -aminobenzoylpolyglutamates, by anion-exchange chromatography, with suitable standards. The C-9-N-10 bond can be cleaved chemically by two main methods oxidatively with potassium permanganate or reductively with a zinc catalyst. In case of bacterial and yeast samples, the extraction procedures for folate monoglutamates and folate polyglutamates are the same.

Conformations of branched poly(α‐amino acids) in solution and at the air—water interface

AbstractThe validity of Silberberg's hypothesis concerning the conformation of branched polypeptides was tested by studies on solutions and on monolayers. The poly(α‐amino acids) investigated consisted of poly‐L‐lysine backbones with side chains of poly(gamma;‐benzyl L‐gutamate) and poly(β‐benzyl L‐aspartate). In solution, the conformation was studied in the mixed solvent chloroform–dichloroacetic acid. Optical rotatory dispersion measurements demonstrated the existence of a helix–coil transition which is a function of the length of the side chain. A significant “transition curve” appeared between 9 and 15 residues. From the results, it may be assumed that the poly‐L‐lysine backbone is extended in a coiled conformation and the side chains are coiled or helical, depending on the number of glutamate residues. At the air–water interface, a discontinuity in the surface‐pressure curve appears between 9 and 15 residues per side chain. The similarity with the results obtained in solution appears significant; however, this has to be confirmed with methods more specific for structural modifications.