A New Practice to Monitor the Fabrication Process of Fab-Targeting Ligands from Bevacizumab by LC-MS: Preparation and Analytical Characterization

The objective of this study was to qualitatively evaluate a Fab-targeting ligand preparation containing free thiol groups in the hinge region by using bevacizumab as a model. The evaluation focused on the purification of fragments through a nonaffinity-based process using a centrifugal ultrafiltration technique and mild reduction conditions for the intact production of F(ab’) fragments with specific inter-heavy-chain disulfide bonds cleavage. Under these conditions, F(ab’) fragments with a defined chemical composition were successfully obtained via proteolytic digestion followed by a controlled reduction reaction process maintaining the integrity of the binding sites. The ultrafiltration purification technique appears to be suitable for the removal of the digestive enzyme but inefficient for the removal of Fc fragments, thus requiring additional processing. A suitable analytical strategy was developed, allowing us to demonstrate the reformation of disulfide bridges between the two reduced cysteines within F(ab’) fragments.

Lasso Proteins—Unifying Cysteine Knots and Miniproteins

Complex lasso proteins are a recently identified class of biological compounds that are present in considerable fraction of proteins with disulfide bridges. In this work, we look at complex lasso proteins as a generalization of well-known cysteine knots and miniproteins (lasso peptides). In particular, we show that complex lasso proteins with the same crucial topological features—cysteine knots and lasso peptides—are antimicrobial proteins, which suggests that they act as a molecular plug. Based on an analysis of the stability of the lasso piercing residue, we also introduce a method to determine which lasso motif is potentially functional. Using this method, we show that the lasso motif in antimicrobial proteins, as well in that in cytokines, is functionally relevant. We also study the evolution of lasso motifs, their conservation, and the usefulness of the lasso fingerprint, which extracts all topologically non-triviality concerning covalent loops. The work is completed by the presentation of extensive statistics on complex lasso proteins to analyze, in particular, the strange propensity for “negative” piercings. We also identify 21 previously unknown complex lasso proteins with an ester and a thioester bridge.

The Major Peanut Allergen Ara h 2 Produced in Nicotiana benthamiana Contains Hydroxyprolines and Is a Viable Alternative to the E. Coli Product in Allergy Diagnosis

Peanut allergy is a potentially life-threatening disease that is mediated by allergen-specific immunoglobulin E (IgE) antibodies. The major peanut allergen Ara h 2, a 2S albumin seed storage protein, is one of the most dangerous and potent plant allergens. Ara h 2 is posttranslationally modified to harbor four disulfide bridges and three hydroxyprolines. These hydroxyproline residues are required for optimal IgE-binding to the DPYSPOHS motifs representing an immunodominant IgE epitope. So far, recombinant Ara h 2 has been produced in Escherichia coli, Lactococcus lactis, Trichoplusia ni insect cell, and Chlamydomonas reinhardtii chloroplast expression systems, which were all incapable of proline hydroxylation. However, molecular diagnosis of peanut allergy is performed using either natural or E. coli-produced major peanut allergens. As IgE from the majority of patients is directed to Ara h 2, it is of great importance that the recombinant Ara h 2 harbors all of its eukaryotic posttranslational modifications. We produced hydroxyproline-containing and correctly folded Ara h 2 in the endoplasmic reticulum of leaf cells of Nicotiana benthamiana plants, using the plant virus-based magnICON® transient expression system with a yield of 200 mg/kg fresh biomass. To compare prokaryotic with eukaryotic expression methods, Ara h 2 was expressed in E. coli together with the disulfide-bond isomerase DsbC and thus harbored disulfide bridges but no hydroxyprolines. The recombinant allergens from N. benthamiana and E. coli were characterized and compared to the natural Ara h 2 isolated from roasted peanuts. Natural Ara h 2 outperformed both recombinant proteins in IgE-binding and activation of basophils via IgE cross-linking, the latter indicating the potency of the allergen. Interestingly, significantly more efficient IgE cross-linking by the N. benthamiana-produced allergen was observed in comparison to the one induced by the E. coli product. Ara h 2 from N. benthamiana plants displayed a higher similarity to the natural allergen in terms of basophil activation due to the presence of hydroxyproline residues, supporting so far published data on their contribution to the immunodominant IgE epitope. Our study advocates the use of N. benthamiana plants instead of prokaryotic expression hosts for the production of the major peanut allergen Ara h 2.

Analysis of a gene family for PDF-like peptides from Arabidopsis

Plant defensins are small, basic peptides that have a characteristic three-dimensional folding pattern which is stabilized by four disulfide bridges. We show here that Arabidopsis contains in addition to the proper plant defensins a group of 9 plant defensin-like (PdfL) genes. They are all expressed at low levels while GUS fusions of the promoters showed expression in most tissues with only minor differences. We produced two of the encoded peptides in E. coli and tested the antimicrobial activity in vitro. Both were highly active against fungi but had lower activity against bacteria. At higher concentrations hyperbranching and swollen tips, which are indicative of antimicrobial activity, were induced in Fusarium graminearum by both peptides. Overexpression lines for most PdfL genes were produced using the 35S CaMV promoter to study their possible in planta function. With the exception of PdfL4.1 these lines had enhanced resistance against F. oxysporum. All PDFL peptides were also transiently expressed in Nicotiana benthamiana leaves with agroinfiltration using the pPZP3425 vector. In case of PDFL1.4 this resulted in complete death of the infiltrated tissues after 7 days. All other PDFLs resulted only in various degrees of small necrotic lesions. In conclusion, our results show that at least some of the PdfL genes could function in plant resistance.

Enhanced Thermostability of D-Psicose 3-Epimerase from Clostridium bolteae through Rational Design and Engineering of New Disulfide Bridges

D-psicose 3-epimerase (DPEase) catalyzes the isomerization of D-fructose to D-psicose (aka D-allulose, a low-calorie sweetener), but its industrial application has been restricted by the poor thermostability of the naturally available enzymes. Computational rational design of disulfide bridges was used to select potential sites in the protein structure of DPEase from Clostridium bolteae to engineer new disulfide bridges. Three mutants were engineered successfully with new disulfide bridges in different locations, increasing their optimum catalytic temperature from 55 to 65 °C, greatly improving their thermal stability and extending their half-lives (t1/2) at 55 °C from 0.37 h to 4–4.5 h, thereby greatly enhancing their potential for industrial application. Molecular dynamics simulation and spatial configuration analysis revealed that introduction of a disulfide bridge modified the protein hydrogen–bond network, rigidified both the local and overall structures of the mutants and decreased the entropy of unfolded protein, thereby enhancing the thermostability of DPEase.

FT-Raman Spectroscopy as a Tool to Study the Secondary Structures of Wheat Gliadin Proteins

Raman spectroscopy is a useful method in biological, biomedical, food, and agricultural studies, allowing the simultaneous examination of various chemical compounds and evaluation of molecular changes occurring in tested objects. The purpose of our research was to explain how the elimination of ω-fractions from the wheat gliadin complex influences the secondary structures of the remaining αβγ-gliadins. To this aim, we analyzed the endosperm of wheat kernels as well as gliadin proteins extracted from two winter wheat genotypes: wasko.gl+ (control genotype containing the full set of gliadins) and wasko.gl− (modified genotype lacking all ω-gliadins). Based on the decomposition of the amide I band, we observed a moderate increase in β-forms (sheets and turns) at the expense of α-helical and random coil structures for gliadins isolated from the flour of the wasko.gl− line. Since ω-gliadins contain no cysteine residues, they do not participate in the formation of the disulfide bridges that stabilize the protein structure. However, they can interact with other proteins via weak, low-energetic hydrogen bonds. We conclude that the elimination of ω-fractions from the gliadin complex causes minor modifications in secondary structures of the remaining gliadin proteins. In our opinion, these small, structural changes of proteins may lead to alterations in gliadin allergenicity.

Investigation of the complex biocatalytic and ultrafiltration action on the whey protein cluster in the production of biofilms

Для изготовления биоразлагаемых пленок и покрытий широко используются сырьевые материалы на основе белков растительного и животного происхождения. Белки молочной сыворотки, а также их гидролизаты могут успешно использоваться для создания пищевых пленок с функциональными свойствами. Существующие недостатки в отношении механических свойств упаковочных пленок и покрытий могут быть преодолены с помощью физических или биохимических методов воздействия на белковый кластер благодаря способности глобулярных белков разворачиваться и связываться с новыми полимерными структурами при определенных условиях. Согласно литературным источникам, это происходит благодаря способности тиоловых групп остатков цистеина образовывать как внутри, так и межмолекулярные дисульфидные мостики. В статье приведены результаты исследования зависимости прочностных свойств пленок на основе модифицированных сывороточных белков от профиля молекулярно-массового распределения пептидов. Приведены данные по способу получения гидролизата сывороточных белков с высоким содержанием низкомолекулярных пептидов размером менее 5 кДа. Получены зависимости глубины гидролиза сывороточных белков от продолжительности ферментативной конверсии и дальнейшего ультрафильтрационного концентрирования. Последовательное воздействие ферментативной обработки и ультрафильтрационного концентрирования на белковый кластер способствовало получению пермеата с содержанием белка 2 % и долей низкомолекулярных пептидов 95 %. Показано, что наличие в составе пленок низкомолекулярных пептидов способствует повышению разрушающего напряжения при разрыве и относительного удлинения на 86 и 50 % соответственно по сравнению с пленкой на основе негидролизованных сывороточных белков. For the manufacture of biodegradable films and coatings, raw materials based on plant and animal proteins are widely used. Whey proteins, as well as their hydrolysates, can be successfully used to create food films with functional properties. The existing disadvantages with respect to the mechanical properties of packaging films and coatings can be overcome using physical or biochemical methods of influencing the protein cluster due to the ability of globular proteins to unfold and bind to new polymer structures under certain conditions. According to the literature, this is due to the ability of thiol groups of cysteine residues to form both intra- and intermolecular disulfide bridges. The article presents the results of a study of the dependence of the strength properties of films based on modified whey proteins on the molecular weight distribution profile of peptides. The data on the method of obtaining a hydrolyzate of whey proteins with a high content of low molecular weight peptides less than 5 kDa in size are presented. The dependences of the depth of hydrolysis of whey proteins on the duration of enzymatic conversion and subsequent ultrafiltration concentration were obtained. The successive effect of enzymatic treatment and ultrafiltration concentration on the protein cluster resulted in the production of a permeate with a protein content of 2 % and a fraction of peptides with a low molecular weight of 95 %. It was shown that the presence of low molecular weight peptides in the films promotes an increase in the breaking stress at break and elongation by 86 and 50 %, respectively, in comparison with the film based on non-hydrolyzed whey proteins.

Autosomal dominant familial neurohypophyseal diabetes insipidus caused by a novel missense mutation in AVP gene in a large Italian kindred

Purpose Familial neurohypophysial diabetes insipidus (FNDI), commonly caused by autosomal dominant arginine vasopressin (AVP) mutations, is a rare condition in which vasopressin fails in regulating body’s level of water with final polyuria and polydipsia. Genetic testing in familial cases of FNDI should be carry out to ensure adequate treatments and avoid disease manifestations especially in infants. Methods In this study, we investigated three-generations of a large Italian family with clinical diagnosis of familial central diabetes insipidus for the presence of potential pathogenic mutations in the AVP gene. Results We identified a heterozygous missense mutation (c.154 T > A; p.C52S) in AVP gene in all affected members studied of a large Italian family. In silico tools were used to investigate the pathogenic role of the mutation and three-dimensional protein structure predicted that the p.C52S impairs disulfide bridges formation resulting in misfolding of the protein. Conclusions This is the first study that identified a novel missense p.C52S mutation as causative of central diabetes insipidus in a large Italian pedigree.