Human Milk Lactoferrin and Antibodies: Catalytic Activities, Complexes, and Other Features

Human milk is a source of biologically active proteins, including lactoferrin (LF) and antibodies (Abs). These proteins are considered as the most polyfunctional proteins of human milk. Apparently, human milk is not a simple mixture of proteins and peptides: recently it was shown that human milk contains stable supramolecular protein complex, composed of LF, α‐lactalbumin, milk albumin, β‐casein, IgG, and sIgA molecules. We believe that the whole set of different biological functions of the individual milk proteins is significantly supplemented by features of their complexes

Milk Exosomes: Isolation, Biochemistry, Morphology, and Perspectives of Use

Cells of the multicellular organisms communicate with each other in many different ways, among which extracellular vesicles play a unique role. Almost all cell types secrete vesicles into the extracellular space and deliver their contents to recipient cells. Today, one of the groups of extracellular vesicles that is of particular interest for studying is exosomes—membrane vesicles with a diameter of 40–100 nm. Exosomes are secreted by cells and found in various biological fluids—blood, tears, saliva, urine, cerebrospinal fluid, and milk. Exosomes provide not only targeted delivery of molecular signals to recipient cells but also carry unique markers, which makes them a promising substrate in diagnostic studies, primarily due to their small RNA and protein contents. The milk of cows, horses, humans, and other mammals is a unique source of exosomes since these organisms can produce liters of milk per day, which is much higher than the volume of exosomes produced in cell culture fluid or blood plasma. Unfortunately, milk exosomes are currently much less studied than exosomes of blood or culture fluid. This review examines the methods of the isolation, biochemical analysis (composition of proteins, lipids, and nucleic acids), morphology, and prospects for the use of milk exosomes

Natural Antibodies Produced in Vaccinated Patients and COVID-19 Convalescents Recognize and Hydrolyze Oligopeptides Corresponding to the S-Protein of SARS-CoV-2

The S-protein is the major antigen of the SARS-CoV-2 virus, against which protective antibodies are generated. The S-protein gene was used in adenoviral vectors and mRNA vaccines against COVID-19. While the primary function of antibodies is to bind to antigens, catalytic antibodies can hydrolyze various substrates, including nucleic acids, proteins, oligopeptides, polysaccharides, and some other molecules. In this study, antibody fractions with affinity for RBD and S-protein (RBD-IgG and S-IgG) were isolated from the blood of COVID-19 patients vaccinated with Sputnik V. The fractions were analyzed for their potential to hydrolyze 18-mer oligopeptides corresponding to linear fragments of the SARS-CoV-2 S-protein. Here, we show that the IgG antibodies hydrolyze six out of nine oligopeptides efficiently, with the antibodies of COVID-19-exposed donors demonstrating the most significant activity. The IgGs of control donors not exposed to SARS-CoV-2 were found to be inactive in oligopeptide hydrolysis. The antibodies of convalescents and vaccinated patients were found to hydrolyze oligopeptides in a wide pH range, with the optimal pH range between 6.5 and 7.5. The hydrolysis of most oligopeptides by RBD-IgG antibodies is inhibited by thiol protease inhibitors, whereas S-IgG active centers generally combine several types of proteolytic activities. Ca2+ ions increase the catalytic activity of IgG preparations containing metalloprotease-like active centers. Thus, the proteolytic activity of natural antibodies against the SARS-CoV-2 protein is believed to be due to the similarity of catalytic antibodies’ active centers to canonical proteases. This work raises the question of the possible physiological role of proteolytic natural RBD-IgG and S-IgG resulting from vaccination and exposure to COVID-19

Human Placenta Exosomes: Biogenesis, Isolation, Composition, and Prospects for Use in Diagnostics

Exosomes are 40–100 nm nanovesicles participating in intercellular communication and transferring various bioactive proteins, mRNAs, miRNAs, and lipids. During pregnancy, the placenta releases exosomes into the maternal circulation. Placental exosomes are detected in the maternal blood even in the first trimester of pregnancy and their numbers increase significantly by the end of pregnancy. Exosomes are necessary for the normal functioning of the placenta and fetal development. Effects of exosomes on target cells depend not only on their concentration but also on their intrinsic components. The biochemical composition of the placental exosomes may cause various complications of pregnancy. Some studies relate the changes in the composition of nanovesicles to placental dysfunction. Isolation of placental exosomes from the blood of pregnant women and the study of protein, lipid, and nucleic composition can lead to the development of methods for early diagnosis of pregnancy pathologies. This review describes the biogenesis of exosomes, methods of their isolation, analyzes their biochemical composition, and considers the prospects for using exosomes to diagnose pregnancy pathologies

Human milk sIgA molecules contain various combinations of different antigen-binding sites resulting in a multiple binding specificity of antibodies and enzymatic activities of abzymes.

In the classic paradigm, immunoglobulins are monospecific molecules that have stable structures and two or more identical antigen-binding sites. However, we show here for the first time that the sIgA pool of human milk contains, depending on the donor, only 35±5% λ-sIgAs, 48±7% κ-sIgAs, and 17±4% of chimeric λ-κ-sIgAs. sIgA preparations contained no traces of canonical enzymes. However, all sIgA fractions eluted from several specific affinity sorbents under the conditions destroying even strong immune complexes demonstrated high catalytic activities in hydrolysis of ATP, DNA, and oligosaccharides, and phosphorylation of proteins, lipids, and oligosaccharides. Sequential re-chromatographies of the sIgA fractions with high affinity to one affinity sorbents on the second, third and then fourth affinity sorbents bearing other immobilized antigens led to the distribution of Abs and all catalytic activities all over the profiles of these chromatographies; in all cases some fractions eluted from affinity sorbents only under the conditions destroying strong immune complexes. In vitro, only an addition of reduced glutathione and milk plasma containing no Abs to two sIgA fractions with different affinity for DNA-cellulose led to a transition of up to 11-20% of Ab from one fraction to the other. Our data are indicative of the possibility of half-molecule exchange between different IgA and sIgA molecules. In addition, it cannot be excluded that during the penetration of IgAs through the specific milk barrier, the secretory component (S) and the join chain (J) can combine molecules of dimeric H(2)L(2) λ-IgAs and κ-IgAs against different antigens forming many different variants of H(4)L(4)SJ sIgA molecules. Therefore, some chimeric molecules of sIgA can contain from two to four HL-fragments to various antigens interacting with high affinity with different sorbents and catalyzing various chemical reactions. Our data essentially expand the ideas concerning explanation of the phenomenon of polyspecificity and cross-reactivity of Abs

Plant Growth-Promoting Soil Bacteria: Nitrogen Fixation, Phosphate Solubilization, Siderophore Production, and Other Biological Activities

This review covers the literature data on plant growth-promoting bacteria in soil, which can fix atmospheric nitrogen, solubilize phosphates, produce and secrete siderophores, and may exhibit several different behaviors simultaneously. We discuss perspectives for creating bacterial consortia and introducing them into the soil to increase crop productivity in agrosystems. The application of rhizosphere bacteria—which are capable of fixing nitrogen, solubilizing organic and inorganic phosphates, and secreting siderophores, as well as their consortia—has been demonstrated to meet the objectives of sustainable agriculture, such as increasing soil fertility and crop yields. The combining of plant growth-promoting bacteria with mineral fertilizers is a crucial trend that allows for a reduction in fertilizer use and is beneficial for crop production

Bacterial Siderophores: Classification, Biosynthesis, Perspectives of Use in Agriculture

Siderophores are synthesized and secreted by many bacteria, yeasts, fungi, and plants for Fe (III) chelation. A variety of plant-growth-promoting bacteria (PGPB) colonize the rhizosphere and contribute to iron assimilation by plants. These microorganisms possess mechanisms to produce Fe ions under iron-deficient conditions. Under appropriate conditions, they synthesize and release siderophores, thereby increasing and regulating iron bioavailability. This review focuses on various bacterial strains that positively affect plant growth and development through synthesizing siderophores. Here we discuss the diverse chemical nature of siderophores produced by plant root bacteria; the life cycle of siderophores, from their biosynthesis to the Fe–siderophore complex degradation; three mechanisms of siderophore biosynthesis in bacteria; the methods for analyzing siderophores and the siderophore-producing activity of bacteria and the methods for screening the siderophore-producing activity of bacterial colonies. Further analysis of biochemical, molecular–biological, and physiological features of siderophore synthesis by bacteria and their use by plants will allow one to create effective microbiological preparations for improving soil fertility and increasing plant biomass, which is highly relevant for sustainable agriculture

Identification of Antibody-Mediated Hydrolysis Sites of Oligopeptides Corresponding to the SARS-CoV-2 S-Protein by MALDI-TOF Mass Spectrometry

Antibodies recognizing RBD and the S-protein have been previously demonstrated to be formed in humans after SARS-CoV-2 infection and vaccination with the Sputnik V adenovirus vaccine. These antibodies were found to be active when hydrolyzing FITC-labeled oligopeptides corresponding to linear epitopes of the S-protein. The thin-layer chromatography method allows the relative accumulation of the reaction product to be estimated but cannot identify hydrolysis sites. This study used the MALDI-TOF MS method to establish oligopeptide hydrolysis sites. Using the MALDI-TOF MS method in combination with the analysis of known hydrolysis sites characteristic of canonical proteases allowed us to establish the unique hydrolysis sites inherent only to catalytically active antibodies. We have discovered two 12-mer oligopeptides to have six hydrolysis sites equally distributed throughout the oligopeptide. The other three oligopeptides were found to have two to three closely spaced hydrolysis sites. In contrast to trypsin and chymotrypsin proteases, the catalytically active antibodies of COVID-19 patients have their peptide bond hydrolyzed mainly after proline, threonine, glycine, or serine residues. Here, we propose a new high-throughput experimental method for analyzing the proteolytic activity of natural antibodies produced in viral pathology