Honoring Prof. Dr. Valentin A. Stonik for His Outstanding Contribution to Marine Natural Product Chemistry on the Occasion of His 80th Birthday.

Marine natural products are a very structurally diverse group of preferably low-weight organic molecules [...].

Chilensosides E, F, and G-New Tetrasulfated Triterpene Glycosides from the Sea Cucumber Paracaudina chilensis (Caudinidae, Molpadida): Structures, Activity, and Biogenesis.

Three new tetrasulfated triterpene glycosides, chilensosides E (1), F (2), and G (3), have been isolated from the Far-Eastern sea cucumber Paracaudina chilensis (Caudinidae, Molpadida). The structures were established based on extensive analysis of 1D and 2D NMR spectra and confirmed by HR-ESI-MS data. The compounds differ in their carbohydrate chains, namely in the number of monosaccharide residues (five or six) and in the positions of sulfate groups. Chilensosides E (1) and F (2) are tetrasulfated pentaosides with the position of one of the sulfate groups at C-3 Glc3, and chilensoside G (3) is a tetrasulfated hexaoside. The biogenetic analysis of the glycosides of P. chilensis has revealed that the structures form a network due to the attachment of sulfate groups to almost all possible positions. The upper semi-chain is sulfated earlier in the biosynthetic process than the lower one. Noticeably, the presence of a sulfate group at C-3 Glc3-a terminal monosaccharide residue in the bottom semi-chain of compounds 1 and 2-excludes the possibility of this sugar chain's further elongation. Presumably, the processes of glycosylation and sulfation are concurrent biosynthetic stages. They can be shifted in time in relation to each other, which is a characteristic feature of the mosaic type of biosynthesis. The hemolytic action of compounds 1-3 against human erythrocytes and cytotoxic activities against five human cancer cell lines were tested. The compounds showed moderate hemolytic activity but were inactive against cancer cells, probably because of their structural peculiarities, such as the combination of positions of four sulfate groups.

Rhabdastrellosides A and B: Two New Isomalabaricane Glycosides from the Marine Sponge Rhabdastrella globostellata, and Their Cytotoxic and Cytoprotective Effects.

Investigation of the Vietnamese marine sponge Rhabdastrella globostellata led to the isolation of two new polar isomalabaricanes: rhabdastrellosides A (1) and B (2). Their structures and stereochemistry were elucidated with the application of 1D and 2D NMR, HRESIMS, and HRESIMS/MS methods, as well as chemical modifications and GC-MS analysis. Metabolites 1 and 2 are the first isomalabaricanes with non-oxidized cyclopentane ring in the tricyclic core system. Moreover, having a 3-O-disaccharide moiety in their structures, they increase a very rare group of isomalabaricane glycosides. We report here a weak cytotoxicity of 1 and 2 toward human neuroblastoma SH-SY5Y cells and normal rat H9c2 cardiomyocytes, as well as the cytoprotective activity of rhabdastrelloside B (2) at 1 µM evaluated using CoCl2-treated SH-SY5Y and H9c2 cells.

Sulfated Triterpene Glycosides from the Far Eastern Sea Cucumber Cucumaria djakonovi: Djakonoviosides C1, D1, E1, and F1; Cytotoxicity against Human Breast Cancer Cell Lines; Quantitative Structure-Activity Relationships.

Four new mono- and trisulfated triterpene penta- and tetraosides, djakonoviosides C1 (1), D1 (2), E1 (3), and F1 (4) were isolated from the Far Eastern sea cucumber Cucumaria djakonovi (Cucumariidae, Dendrochirotida), along with six known glycosides found earlier in other Cucumaria species. The structures of unreported compounds were established on the basis of extensive analysis of 1D and 2D NMR spectra as well as by HR-ESI-MS data. The set of compounds contains six different types of carbohydrate chains including two new ones. Thus, djakonovioside C1 (1) is characterized by xylose as the second residue, that was a branchpoint in the pentasaccharide chain. Meanwhile, only quinovose and rarely glucose have been found earlier in pentasaccharide chains branched at C-2 of the second sugar unit. Djakonovioside E1 (3) is characterized by a tetrasaccharide trisulfated chain, with glucose as the second residue. So, in the series of isolated glycosides, three types of sugars in the second position were presented: the most common, quinovose-in six compounds; glucose-in three substances; and the rare xylose-in one glycoside. The set of aglycones was composed of holostane- and non-holostane-type polycyclic systems; the latter comprised normal and reduced side chains. Noticeably, isokoreoside A (9), isolated from C. djakonovi, was a single glycoside having a 9(11)-double bond, indicating two oxidosqualenecyclases are operating in the process of the biosynthesis of aglycones. Some of the glycosides from C. djakonovi, which were characterized by pentasaccharide branched chains containing one to three sulfate groups, are chemotaxonomic features of the representatives of the genus Cucumaria. The assortment of sugar parts of Cucumaria's glycosides was broadened with previously undescribed penta- and tetrasaccharide moieties. The metabolic network of sugar parts and aglycones is constructed based on biogenetic relationships. The cytotoxic action of compounds 1-10, isolated from C. djakonovi, against human breast cancer cell lines was investigated along with the hemolytic activity. Erythrocytes were, as usual, more sensitive to the membranolytic action of the glycosides than cancer cells. The triple-negative breast cancer MDA-MB-231 cell line was more vulnerable to the action of glycosides in comparison with the other tested cancer cells, while the MCF-7 cell line was less susceptible to cytotoxic action. Djakonovioside E1 (3) demonstrated selective action against ER-positive MCF-7 and triple-negative MDA-MB-231 cell lines, while the toxic effect in relation to normal mammary epithelial cells (MCF-10A) was absent. Cucumarioside A2-5 (6) inhibited the formation and growth of colonies of cancer cells to 44% and tumor cell migration to 85% of the control. Quantitative structure-activity relationships (QSAR) were calculated on the basis of the correlational analysis of the physicochemical properties and structural features of the glycosidic molecules and their membranolytic activity. QSAR revealed the extremely complex nature of such relationships, but these calculations correlated well with the observed SAR.

New Rare Triterpene Glycosides from Pacific Sun Star, Solaster pacificus, and Their Anticancer Activity.

Six previously unknown triterpene glycosides, pacificusosides L-Q (1-6), and two previously known triterpene glycosides, cucumariosides B1 (7) and A5 (8), were isolated from an alcoholic extract of Pacific sun star, Solaster pacificus. The structures of 1-6 were determined using 1D and 2D NMR, ESIMS, and chemical modifications. Compound 1 is a rare type of triterpene glycoside with non-holostane aglycon, having a linear trisaccharide carbohydrate chain. Pacificusosides M-P (2-5) have new structures containing a Δ8(9)-3,16,18-trihydroxy tetracyclic triterpene moiety. This tetracyclic fragment in sea star or sea cucumber triterpene glycosides was described for the first time. All the compounds under study exhibit low or moderate cytotoxic activity against colorectal carcinoma HCT 116 cells, and breast cancer MDA-MB-231 cells were assessed by MTS assay. Compound 2 effectively suppresses the colony formation of cancer cells at a non-toxic concentration, using the soft-agar assay. A scratch assay has shown a significant anti-invasive potential of compound 2 against HCT 116 cells, but not against MDA-MB-231 cells.

Recombinant Analogs of Sea Anemone Kunitz-Type Peptides Influence P2X7 Receptor Activity in Neuro-2a Cells.

Purinergic P2X7 receptors (P2X7) have now been proven to play an important role and represent an important therapeutic target in many pathological conditions including neurodegeneration. Here, we investigated the impact of peptides on purinergic signaling in Neuro-2a cells through the P2X7 subtype in in vitro models. We have found that a number of recombinant peptides, analogs of sea anemone Kunitz-type peptides, are able to influence the action of high concentrations of ATP and thereby reduce the toxic effects of ATP. The influx of calcium, as well as the fluorescent dye YO-PRO-1, was significantly suppressed by the studied peptides. Immunofluorescence experiments confirmed that the peptides reduce the P2X7 expression level in neuronal Neuro-2a cells. Two selected active peptides, HCRG1 and HCGS1.10, were found to specifically interact with the extracellular domain of P2X7 and formed stable complexes with the receptor in surface plasmon resonance experiments. The molecular docking approach allowed us to establish the putative binding sites of the most active HCRG1 peptide on the extracellular domain of the P2X7 homotrimer and propose a mechanism for regulating its function. Thus, our work demonstrates the ability of the Kunitz-type peptides to prevent neuronal death by affecting signaling through the P2X7 receptor.

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.

EAE of Mice: Enzymatic Cross Site-Specific Hydrolysis of H2A Histone by IgGs against H2A, H1, H2B, H3, and H4 Histones and Myelin Basic Protein.

Histones play vital roles in chromatin function and gene transcription; however, they are very harmful in the intercellular space because they stimulate systemic inflammatory and toxic responses. Myelin basic protein (MBP) is the major protein of the axon myelin-proteolipid sheath. Antibodies-abzymes with various catalytic activities are specific features of some autoimmune diseases. IgGs against individual histones (H2A, H1, H2B, H3, and H4) and MBP were isolated from the blood of experimental-autoimmune-encephalomyelitis-prone C57BL/6 mice by several affinity chromatographies. These Abs-abzymes corresponded to various stages of EAE development: spontaneous EAE, MOG, and DNA-histones accelerated the onset, acute, and remission stages. IgGs-abzymes against MBP and five individual histones showed unusual polyreactivity in the complex formation and enzymatic cross-reactivity in the specific hydrolysis of the H2A histone. All the IgGs of 3-month-old mice (zero time) against MBP and individual histones demonstrated from 4 to 35 different H2A hydrolysis sites. The spontaneous development of EAE over 60 days led to a significant change in the type and number of H2A histone hydrolysis sites by IgGs against five histones and MBP. Mice treatment with MOG and the DNA-histone complex changed the type and number of H2A hydrolysis sites compared to zero time. The minimum number (4) of different H2A hydrolysis sites was found for IgGs against H2A (zero time), while the maximum (35) for anti-H2B IgGs (60 days after mice treatment with DNA-histone complex). Overall, it was first demonstrated that at different stages of EAE evolution, IgGs-abzymes against individual histones and MBP could significantly differ in the number and type of specific sites of H2A hydrolysis. The possible reasons for the catalytic cross-reactivity and great differences in the number and type of histone H2A cleavage sites were analyzed.

Isolation of Extracellular Vesicles of Holothuria (Sea Cucumber Eupentacta fraudatrix).

Extracellular vesicles (EVs), carriers of molecular signals, are considered a critical link in maintaining homeostasis in mammals. Currently, there is growing interest in studying the role of EVs, including exosomes (subpopulation of EVs), in animals of other evolutionary levels, including marine invertebrates. We have studied the possibility of obtaining appropriate preparations of EVs from whole-body extract of holothuria Eupentacta fraudatrix using a standard combination of centrifugation and ultracentrifugation. However, the preparations were heavily polluted, which did not allow us to conclude that they contained vesicles. Subsequent purification by FLX gel filtration significantly reduced the pollution but did not increase vesicle concentration to a necessary level. To detect EVs presence in the body of holothurians, we used transmission electron microscopy of ultrathin sections. Late endosomes, producing the exosomes, were found in the cells of the coelom epithelium covering the gonad, digestive tube and respiratory tree, as well as in the parenchyma cells of these organs. The study of purified homogenates of these organs revealed vesicles (30-100 nm) morphologically corresponding to exosomes. Thus, we can say for sure that holothurian cells produce EVs including exosomes, which can be isolated from homogenates of visceral organs.

Djakonoviosides A, A1, A2, B1-B4 - Triterpene Monosulfated Tetra- and Pentaosides from the Sea Cucumber Cucumaria djakonovi: The First Finding of a Hemiketal Fragment in the Aglycones; Activity against Human Breast Cancer Cell Lines.

Seven new monosulfated triterpene glycosides, djakonoviosides A (1), A1 (2), A2 (3), and B1-B4 (4-7), along with three known glycosides found earlier in the other Cucumaria species, namely okhotoside A1-1, cucumarioside A0-1, and frondoside D, have been isolated from the far eastern sea cucumber Cucumaria djakonovi (Cucumariidae, Dendrochirotida). The structures were established on the basis of extensive analysis of 1D and 2D NMR spectra and confirmed by HR-ESI-MS data. The compounds of groups A and B differ from each other in their carbohydrate chains, namely monosulfated tetrasaccharide chains are inherent to group A and pentasaccharide chains with one sulfate group, branched by C-2 Qui2, are characteristic of group B. The aglycones of djakonoviosides A2 (3), B2 (5), and B4 (7) are characterized by a unique structural feature, a 23,16-hemiketal fragment found first in the sea cucumbers' glycosides. The biosynthetic pathway of its formation is discussed. The set of aglycones of C. djakonovi glycosides was species specific because of the presence of new aglycones. At the same time, the finding in C. djakonovi of the known glycosides isolated earlier from the other species of Cucumaria, as well as the set of carbohydrate chains characteristic of the glycosides of all investigated representatives of the genus Cucumaria, demonstrated the significance of these glycosides as chemotaxonomic markers. The membranolytic actions of compounds 1-7 and known glycosides okhotoside A1-1, cucumarioside A0-1, and frondoside D, isolated from C. djakonovi against human cell lines, including erythrocytes and breast cancer cells (MCF-7, T-47D, and triple negative MDA-MB-231), as well as leukemia HL-60 and the embryonic kidney HEK-293 cell line, have been studied. Okhotoside A1-1 was the most active compound from the series because of the presence of a tetrasaccharide linear chain and holostane aglycone with a 7(8)-double bond and 16ß-O-acetoxy group, cucumarioside A0-1, having the same aglycone, was slightly less active because of the presence of branching xylose residue at C-2 Qui2. Generally, the activity of the djakonoviosides of group A was higher than that of the djakonoviosides of group B containing the same aglycones, indicating the significance of a linear chain containing four monosaccharide residues for the demonstration of membranolytic action by the glycosides. All the compounds containing hemiketal fragments, djakonovioside A2 (3), B2 (5), and B4 (7), were almost inactive. The most aggressive triple-negative MDA-MB-231 breast cancer cell line was the most sensitive to the glycosides action when compared with the other cancer cells. Okhotoside A1-1 and cucumarioside A0-1 demonstrated promising effects against MDA-MB-231 cells, significantly inhibiting the migration, as well as the formation and growth, of colonies.

EAE of Mice: Enzymatic Cross Site-Specific Hydrolysis of H2B Histone by IgGs against H1, H2A, H2B, H3, and H4 Histones and Myelin Basic Protein.

Histones have vital roles in chromatin functioning and gene transcription. At the same time, they are pernicious in intercellular space because they stimulate systemic inflammatory and toxic responses. Myelin basic protein (MBP) is the major protein of the axon myelin-proteolipid sheath. Antibody-abzymes with various catalytic activities are specific features of some autoimmune diseases. IgGs against five individual histones (H2B, H1, H2A, H3, and H4) and MBP were isolated from the blood of experimental autoimmune encephalomyelitis-prone C57BL/6 mice by affinity chromatography. Abzymes corresponding to various stages of EAE development, including spontaneous EAE, myelin oligodendrocyte glycoprotein (MOG)- and DNA-histone complex-accelerated onset, as well as acute and remission stages, were analyzed. IgG-abzymes against MBP and five individual histones showed unusual polyreactivity in complex formation and enzymatic cross-reactivity in the specific hydrolysis of H2B histone. All IgGs against MBP and individual histones in 3-month-old mice (zero time) demonstrated from 4 to 11 different H2B hydrolysis sites. Spontaneous development of EAE during 60 days led to a significant change in the type and number of H2B hydrolysis sites by IgGs against the five histones and MBP. Mouse treatment with MOG and DNA-histone complex changed the type and number of H2B hydrolysis sites compared to zero time. The minimum number (3) of different H2B hydrolysis sites was found for IgGs against H3 20 days after mouse immunization with DNA-histone complex, whereas the maximum number (33) for anti-H2B IgGs was found 60 days after mouse treatment with DNA-histone complex. Overall, this is the first study to demonstrate that at different stages of EAE evolution, IgG-abzymes against five individual histones and MBP could significantly differ in the specific sites and number of H2B hydrolysis sites. Possible reasons for the catalytic cross-reactivity and significant differences in the number and type of histone H2B cleavage sites were analyzed.

Application of MS-Based Metabolomic Approaches in Analysis of Starfish and Sea Cucumber Bioactive Compounds.

Today, marine natural products are considered one of the main sources of compounds for drug development. Starfish and sea cucumbers are potential sources of natural products of pharmaceutical interest. Among their metabolites, polar steroids, triterpene glycosides, and polar lipids have attracted a great deal of attention; however, studying these compounds by conventional methods is challenging. The application of modern MS-based approaches can help to obtain valuable information about such compounds. This review provides an up-to-date overview of MS-based applications for starfish and sea cucumber bioactive compounds analysis. While describing most characteristic features of MS-based approaches in the context of starfish and sea cucumber metabolites, including sample preparation and MS analysis steps, the present paper mainly focuses on the application of MS-based metabolic profiling of polar steroid compounds, triterpene glycosides, and lipids. The application of MS in metabolomics studies is also outlined.

Structure of the Lipooligosaccharide from the Deep-Sea Marine Bacterium Idiomarina zobellii KMM 231T, Isolated at a Depth of 4000 Meters.

The structural characterization of lipopolysaccharides has critical implications for some biomedical applications, and marine bacteria are an inimitable source of new glyco-structures potentially usable in medicinal chemistry. On the other hand, lipopolysaccharides of marine Gram-negative bacteria present certain structural features that can help the understanding of the adaptation processes. The deep-sea marine Gram-negative bacterium Idiomarina zobellii KMM 231T, isolated from a seawater sample taken at a depth of 4000 m, represents an engaging microorganism to investigate in terms of its cell wall components. Here, we report the structural study of the R-type lipopolysaccharide isolated from I. zobellii KMM 231T that was achieved through a multidisciplinary approach comprising chemical analyses, NMR spectroscopy, and MALDI mass spectrometry. The lipooligosaccharide turned out to be characterized by a novel and unique pentasaccharide skeleton containing a very short mono-phosphorylated core region and comprising terminal neuraminic acid. The lipid A was revealed to be composed of a classical disaccharide backbone decorated by two phosphate groups and acylated by i13:0(3-OH) in amide linkage, i11:0 (3-OH) as primary ester-linked fatty acids, and i11:0 as a secondary acyl chain.

In Vitro Anticancer and Cancer-Preventive Activity of New Triterpene Glycosides from the Far Eastern Starfish Solaster pacificus.

Sea stars or starfish (class Asteroidea) and holothurians or sea cucumbers (class Holothuroidea), belonging to the phylum Echinodermata (echinoderms), are characterized by different sets of glycosidic metabolites: the steroid type in starfish and the triterpene type in holothurians. However, herein we report the isolation of eight new triterpene glycosides, pacificusosides D−K (1−3, 5−9) along with the known cucumarioside D (4), from the alcoholic extract of the Far Eastern starfish Solaster pacificus. The isolated new compounds are closely related to the metabolites of sea cucumbers, and their structures of 1−3 and 5−9 were determined by extensive NMR and ESIMS techniques. Compounds 2, 5, and 8 have a new type of tetrasaccharide chain with a terminal non-methylated monosaccharide unit. Compounds 3, 6, and 9 contain another new type of tetrasaccharide chain, having 6-O-SO3-Glc as one of the sugar units. The cytotoxic activity of 1−9 against non-cancerous mouse epidermal cells JB6 Cl41 and human melanoma cell lines SK-MEL-2, SK-MEL-28, and RPMI-7951 was determined by MTS assay. Compounds 1, 3, 4, 6, and 9 showed potent cytotoxicity against these cell lines, but the cancer selectivity (SI > 9) was observed only against the SK-MEL-2 cell line. Compounds 1, 3, 4, 6, and 9 at the non-toxic concentration of 0.1 µM significantly inhibited neoplastic cell transformation of JB6 Cl41 cells induced by chemical carcinogens (EGF, TPA) or ionizing radiation (X-rays and UVB). Moreover, compounds 1 and 4 at the non-toxic concentration of 0.1 µM possessed the highest inhibiting activity on colony formation among the investigated compounds and decreased the colonies number of SK-MEL-2 cells by 64% and 70%, respectively. Thus, triterpene glycosides 1 and 4 can be considered as prospective cancer-preventive and anticancer-compound leaders.

Structures and Biologic Activity of Chitonoidosides I, J, K, K1 and L-Triterpene Di-, Tri- and Tetrasulfated Hexaosides from the Sea Cucumber Psolus chitonoides.

Five new triterpene di-, tri- and tetrasulfated hexaosides (chitonoidosides I (1), J (2), K (3), K1 (4) and L (5)) were isolated from the Far-Eastern sea cucumber Psolus chitonoides, collected near Bering Island (Commander Islands) from a depth of 100-150 m. The structural variability of the glycosides concerned both the aglycones (with 7(8)- or 9(11)-double bonds) and carbohydrate chains differing from each other by the third sugar residue (Xyl or sulfated by C-6 Glc) and/or by the fourth-terminal in the bottom semi-chain-residue (Glc or sulfated by C-6 MeGlc) as well as by the positions of a sulfate group at C-4 or C-6 in the sixth-terminal in the upper semi-chain-residue (MeGlc). Hemolytic activities of these compounds 1-5 against human erythrocytes as well as cytotoxicity against human cancer cell lines, HeLa, DLD-1 and HL-60, were studied. The hexaosides, chitonoidosides K (3) and L (5) with four sulfate groups, were the most active against tumor cells in all the tests. Noticeably, the sulfate group at C-4 of MeGlc6 did not decrease the membranolytic effect of 5 as compared with 3, having the sulfate group at C-6 of MeGlc6. Erythrocytes were, as usual, more sensitive to the action of the studied glycosides than cancer cells, although the sensitivity of leukemia promyeloblast HL-60 cells was higher than that of other tumor cells. The glycosides 1 and 2 demonstrated some weaker action in relation to DLD-1 cells than against other tumor cell lines. Chitonoidoside K1 (4) with a hydroxyl at C 25 of the aglycone was not active in all the tests. The metabolic network formed by the carbohydrate chains of all the glycosides isolated from P. chitonoides as well as the aglycones biosynthetic transformations during their biosynthesis are discussed and illustrated with schemes.

New Guanidine Alkaloids Batzelladines O and P from the Marine Sponge Monanchora pulchra Induce Apoptosis and Autophagy in Prostate Cancer Cells.

Two new guanidine alkaloids, batzelladines O (1) and P (2), were isolated from the deep-water marine sponge Monanchora pulchra. The structures of these metabolites were determined by NMR spectroscopy, mass spectrometry, and ECD. The isolated compounds exhibited cytotoxic activity in human prostate cancer cells PC3, PC3-DR, and 22Rv1 at low micromolar concentrations and inhibited colony formation and survival of the cancer cells. Batzelladines O (1) and P (2) induced apoptosis, which was detected by Western blotting as caspase-3 and PARP cleavage. Additionally, induction of pro-survival autophagy indicated as upregulation of LC3B-II and suppression of mTOR was observed in the treated cells. In line with this, the combination with autophagy inhibitor 3-methyladenine synergistically increased the cytotoxic activity of batzelladines O (1) and P (2). Both compounds were equally active in docetaxel-sensitive and docetaxel-resistant prostate cancer cells, despite exhibiting a slight p-glycoprotein substrate-like activity. In combination with docetaxel, an additive effect was observed. In conclusion, the isolated new guanidine alkaloids are promising drug candidates for the treatment of taxane-resistant prostate cancer.

Secretory immunoglobulin A from human milk hydrolyzes 5 histones and myelin basic protein.

Mother's milk provides newborns with various nutrients (e.g., enzymes, proteins, peptides, hormones, antibodies) that help babies grow and protect them from bacterial and viral infections. The functions of many components of breast milk can be very different from their corresponding functions in body fluids of healthy adults. Catalytic antibodies (abzymes) that hydrolyze peptides, proteins, DNA, RNA, and oligosaccharides were detected not only in human milk, but also in the blood sera of autoimmune patients. However, abzymes with unexpected synthetic activities (lipids, oligosaccharides, and protein kinase activities) were revealed in milk that were not found in the blood of autoimmune patients. The nutrition of infants with fresh milk has a very specific role; newborns are well protected by antibodies of mother's milk (passive immunity). Protease abzymes were found in the blood sera of autoimmune patients, whereas healthy humans usually do not contain such autoantibodies. Here, we present the first evidence that the milk of healthy mothers contains secretory (s)IgA that effectively hydrolyze 5 histones (e.g., H1, H2A, H2B, H3, and H4) and myelin basic protein (MBP). Several rigid criteria were applied to show that protease activity is an intrinsic property of sIgA. Milk abzymes against 5 histones cannot hydrolyze different control proteins except histones and MBP, whereas autoantibodies against MBP split this protein and 5 histones. Antibodies against histones and MBP exhibit complexation polyreactivity as well as specific and unusual catalytic cross-reactivity. With some exceptions, the specific sites of hydrolysis of H1, H2A, and H2B by sIgA against histones do not coincide with the sites of hydrolysis by abzymes against MBP. On the whole, fresh human milk is a very specific source of many of the most unusual antibodies and abzymes.

Protease and DNase Activities of a Very Stable High-Molecular-Mass Multiprotein Complex from Sea Cucumber Eupentacta fraudatrix.

Only some human organs, including the liver, are capable of very weak self-regeneration. Some marine echinoderms are very useful for studying the self-regeneration processes of organs and tissues. For example, sea cucumbers Eupentacta fraudatrix (holothurians) demonstrate complete restoration of all organs and the body within several weeks after their division into two parts. Therefore, these cucumbers are a prospective model for studying the general mechanisms of self-regeneration. However, there is no data available yet concerning biomolecules of holothurians, which can stimulate the processes of organ and whole-body regeneration. Investigation of these restoration mechanisms is very important for modern medicine and biology because it can help to understand which hormones, nucleic acids, proteins, enzymes, or complexes play an essential role in self-regeneration. It is possible that stable, polyfunctional, high-molecular-weight protein complexes play an essential role in these processes. It has recently been shown that sea cucumbers Eupentacta fraudatrix contain a very stable multiprotein complex of about 2000 kDa. The first analysis of possible enzymatic activities of a stable protein complex was carried out in this work, revealing that the complex possesses several protease and DNase activities. The complex metalloprotease is activated by several metal ions (Zn2+ > Mn2+ > Mg2+). The relative contribution of metalloproteases (~63.4%), serine-like protease (~30.5%), and thiol protease (~6.1%) to the total protease activity of the complex was estimated. Metal-independent proteases of the complex hydrolyze proteins at trypsin-specific sites (after Lys and Arg). The complex contains both metal-dependent and metal-independent DNases. Mg2+, Mn2+, and Co2+ ions were found to strongly increase the DNase activity of the complex.

Experimental Autoimmune Encephalomyelitis of Mice: Enzymatic Cross Site-Specific Hydrolysis of H4 Histone by IgGs against Histones and Myelin Basic Protein.

Histones play vital roles in chromatin functioning and gene transcription, but in intercellular space, they are harmful due to stimulating systemic inflammatory and toxic responses. Myelin basic protein (MBP) is the most important protein of the axon myelin-proteolipid sheath. Antibodies-abzymes with different catalytic activities are critical and specific features of some autoimmune diseases. Five IgG preparations against histones (H4, H1, H2A, H2B, and H3) and against MBP corresponding to different spontaneous, MOG (myelin oligodendrocyte glycoprotein of mice), and DNA-histones that accelerated onset, acute, and remission stages of experimental autoimmune encephalomyelitis (EAE; model of human multiple sclerosis) development were obtained from EAE-prone C57BL/6 mice by several affinity chromatographies. IgG-abzymes against five histones and MBP possess unusual polyreactivity in complexation and catalytic cross-reactivity in the hydrolysis of histone H4. IgGs against five histones and MBP corresponding to 3 month-old mice (zero time) in comparison with Abs corresponding to spontaneous development of EAE during 60 days differ in type and number of H4 sites for hydrolysis. Immunization of mice with MOG and DNA-histones complex results in an acceleration of EAE development associated with an increase in the activity of antibodies in H4 hydrolysis. Twenty days after mouse immunization with MOG or DNA-histones complex, the IgGs hydrolyze H4 at other additional sites compared to zero time. The maximum number of different sites of H4 hydrolysis was revealed for IgGs against five histones and MBP at 60 days after immunization of mice with MOG and DNA-histones. Overall, it first showed that at different stages of EAE development, abzymes could significantly differ in specific sites of H4 hydrolysis.

Analysis of Proteins and Peptides of Highly Purified CD9+ and CD63+ Horse Milk Exosomes Isolated by Affinity Chromatography.

Exosomes are nanovesicles with a 40-150 nm diameter and are essential for communication between cells. Literature data suggest that exosomes obtained from different sources (cell cultures, blood plasma, urea, saliva, tears, spinal fluid, milk) using a series of centrifugations and ultracentrifugations contain hundreds and thousands of different protein and nucleic acid molecules. However, most of these proteins are not an intrinsic part of exosomes; instead, they co-isolate with exosomes. Using consecutive ultracentrifugation, gel filtration, and affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we isolated highly purified vesicle preparations from 18 horse milk samples. Gel filtration of the initial preparations allowed us to remove co-isolating proteins and their complexes and to obtain highly purified vesicles morphologically corresponding to exosomes. Using affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we obtained extra-purified CD9+ and CD63+ exosomes, which simultaneously contain these two tetraspanins, while the CD81 tetraspanin was presented in a minor quantity. SDS-PAGE and MALDI analysis detected several major proteins with molecular masses over 10 kDa: CD9, CD63, CD81, lactadherin, actin, butyrophilin, lactoferrin, and xanthine dehydrogenase. Analysis of extracts by trifluoroacetic acid revealed dozens of peptides with molecular masses in the range of 0.8 to 8.5 kDa. Data on the uneven distribution of tetraspanins on the surface of horse milk exosomes and the presence of peptides open new questions about the biogenesis of these extracellular vesicles.