Laboratory of Chemistry of Noninfectious Immunity
Head of the Lab
Oleg V. CHERNIKOV , Ph.D.
Tel. +7 (423) 231-07-19, chernikov@piboc.dvo.ru
Irina V. Chikalovets (Senior Researcher, Ph.D.), Tatyana O. Mizgina (Junior Researcher, Ph.D.), Aleksandra S. Kuzmich (Researcher), Lidia R. Zakharkina (Laboratory Assistant).
Marine polysaccharide recently attracted attention because they are not toxic and reveal interesting biological properties (immunomodulatory, anti-inflammatory, radio-protective, antiviral, antimicrobial, anti-tumor, wound-healing and etc.). They can find application in medical practice, food and cosmetic industries.
The main goals:
The main scientific results are related to studies on the isolation, establishment of structure, study of physico-chemical properties and biological activity of lectins of marine aquatic organisms. A Gal/GalNAc-specific lectins were isolated from the mussels Crenomytilus grayanus (CGL) and Mytilus trossulus (MTL), and its physicochemical properties and biological activity were studied.
CGL and MTL have been shown to have antibacterial and fungicidal activities. Changes in lectin levels in response to bacterial infection suggest their participation in the innate immunity of shellfish. The domain organization of CGL was studied and it was shown that CGL oligomerization is essential for the manifestation of biological properties. Establishment of the amino acid sequence showed that CGL and MTL have a high degree of homology and are representatives of a new class of lectins.
The spatial organization of the MTL was studied using computer modeling. MTL dimerization has been shown to result in a total of six ligand binding sites, which may be important for the expression of its biological properties. The ability of MTL to form dimeric and oligomeric structures was confirmed by dynamic light scattering and SDS-PAGE.
The mucin-specific lectin GYL, which is a homodimeric β/α protein with a molecular weight of 36 kDa, was isolated from the bivalve Glycymeris yessoensis. The activity of GYL depends on the presence of calcium ions and is stable at pH above 8 and temperatures up to 20°C for 30 minutes. GYL is a pattern recognition receptor because it binds molecular structures associated with common pathogens, such as peptidoglycan, LPS, β-1,3-glucan, and mannan. GYL has a wide spectrum of action on microorganisms. GYL is a novel member of the C-type lectin family and may be involved in the immune response of G. yessoensis in response to bacterial challenge.
The GYL cDNA, which encodes 161 amino acids and a C-type carbohydrate recognition domain (CRD), was cloned. The resulting amino acid sequence showed similarity to other C-type lectins. GYL is a glycoprotein containing two N-glycosylation sites per subunit. N-glycans consist of xylose, mannose, β-D-glucosamine, 3-O-methylated galactose, D-quinovose, 3-O-methylated 6-deoxy-D-glucose. The potential tertiary structure of CRD GYL has the elongated two-loop structure typical of C-type lectins and includes three disulfide bridges at the base of the loops. In addition, through sequence confirmation of GYL, 8 isoforms of this lectin were identified. This fact indicates the presence of a multigene family of GYL-like C-type lectins in the bivalve G. yessoensis. Using glycoarray, natural carbohydrate ligands were identified and the glycotope for GYL was reconstructed as “Galβ1–4GlcNAcβ, necessarily containing an additional fragment”, such as a sulfate group or a methyl group of fucose or N-acetylgalactosamine residues.
The immunomodulatory properties of a polysaccharide isolated from the coral Pseudopterogorgia americana (PPA) were studied. Almost PPA has been shown to increase the expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and cyclooxygenase-2 (COX-2) in macrophages. Notably, lipopolysaccharide (LPS), but not PPA, increased the expression levels of inducible NO synthase and nitric oxide, indicating the absence of LPS contamination in the PPA sample. PPA induces the formation of reactive oxygen species (ROS), increases phosphorylation levels of protein kinase C (PKC)-α, PKC-δ and mitogen-activated protein kinases (MAPK), and activates NF-κB. ROS inhibition and PKC-α knockdown reduced PPA-mediated TNF-α and IL-6 expression, but PKC-δ knockdown significantly increased TNF-α expression. In addition, inhibition of c-Jun N-terminal kinase-1/2 and NF-κB reduces the expression of TNF-α and IL-6. In addition, inhibition of ROS, MAPK, and PKC-α/δ reduced NF-κB activation, indicating that ROS, MAPK, and PKC-α/δ function as upstream signals to NF-κB. Finally, PPA treatment reduces macrophage phagocytosis activity and cytokine expression in bacteria-infected macrophages. The findings indicate the potential for PPA to be used as an immunomodulatory drug in the future.
Research fields
Marine biological resources are rich of new physiologically active substances including polysaccharides of various chemical structure and lectins. Lectins are carbohydrate-binding proteins specifically recognizing and reversibly binding to certain carbohydrate glycoconjugate structures without change in covalent structure of the recognized glycosyl ligands. This distinguishes lectins from other carbohydrate binding proteins and enzymes, and makes them invaluable tools in biomedical and glycoconjugate research. Lectins are involved in various biological processes, such as fertilization, cell adhesion, cell signaling, cell growth and differentiation. It is usually assumed that the lectins perform a protective function in the system of innate immunity. Marine invertebrates lectins exhibit various biological activities such as antifungal, mytogenic, antiproliferative and antiviral. They can be used as diagnostic tools for the investigation of the early cell-membrane alterations and carbohydrate changes that accompany tumor and immune processes.
Marine polysaccharide recently attracted attention because they are not toxic and reveal interesting biological properties (immunomodulatory, anti-inflammatory, radio-protective, antiviral, antimicrobial, anti-tumor, wound-healing and etc.). They can find application in medical practice, food and cosmetic industries.
Our Lab mainly focuses on isolation of the lectins from marine invertebrates. We investigate the chemical and biological properties. Study the role in marine organisms.
The main goals:
The main scientific results are related to studies on the isolation, establishment of structure, study of physico-chemical properties and biological activity of lectins of marine aquatic organisms. A Gal/GalNAc-specific lectins were isolated from the mussels Crenomytilus grayanus (CGL) and Mytilus trossulus (MTL), and its physicochemical properties and biological activity were studied.
CGL and MTL have been shown to have antibacterial and fungicidal activities. Changes in lectin levels in response to bacterial infection suggest their participation in the innate immunity of shellfish. The domain organization of CGL was studied and it was shown that CGL oligomerization is essential for the manifestation of biological properties. Establishment of the amino acid sequence showed that CGL and MTL have a high degree of homology and are representatives of a new class of lectins.
The spatial organization of the MTL was studied using computer modeling. MTL dimerization has been shown to result in a total of six ligand binding sites, which may be important for the expression of its biological properties. The ability of MTL to form dimeric and oligomeric structures was confirmed by dynamic light scattering and SDS-PAGE.
The mucin-specific lectin GYL, which is a homodimeric β/α protein with a molecular weight of 36 kDa, was isolated from the bivalve Glycymeris yessoensis. The activity of GYL depends on the presence of calcium ions and is stable at pH above 8 and temperatures up to 20°C for 30 minutes. GYL is a pattern recognition receptor because it binds molecular structures associated with common pathogens, such as peptidoglycan, LPS, β-1,3-glucan, and mannan. GYL has a wide spectrum of action on microorganisms. GYL is a novel member of the C-type lectin family and may be involved in the immune response of G. yessoensis in response to bacterial challenge.
The GYL cDNA, which encodes 161 amino acids and a C-type carbohydrate recognition domain (CRD), was cloned. The resulting amino acid sequence showed similarity to other C-type lectins. GYL is a glycoprotein containing two N-glycosylation sites per subunit. N-glycans consist of xylose, mannose, β-D-glucosamine, 3-O-methylated galactose, D-quinovose, 3-O-methylated 6-deoxy-D-glucose. The potential tertiary structure of CRD GYL has the elongated two-loop structure typical of C-type lectins and includes three disulfide bridges at the base of the loops. In addition, through sequence confirmation of GYL, 8 isoforms of this lectin were identified. This fact indicates the presence of a multigene family of GYL-like C-type lectins in the bivalve G. yessoensis. Using glycoarray, natural carbohydrate ligands were identified and the glycotope for GYL was reconstructed as “Galβ1–4GlcNAcβ, necessarily containing an additional fragment”, such as a sulfate group or a methyl group of fucose or N-acetylgalactosamine residues.
The immunomodulatory properties of a polysaccharide isolated from the coral Pseudopterogorgia americana (PPA) were studied. Almost PPA has been shown to increase the expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and cyclooxygenase-2 (COX-2) in macrophages. Notably, lipopolysaccharide (LPS), but not PPA, increased the expression levels of inducible NO synthase and nitric oxide, indicating the absence of LPS contamination in the PPA sample. PPA induces the formation of reactive oxygen species (ROS), increases phosphorylation levels of protein kinase C (PKC)-α, PKC-δ and mitogen-activated protein kinases (MAPK), and activates NF-κB. ROS inhibition and PKC-α knockdown reduced PPA-mediated TNF-α and IL-6 expression, but PKC-δ knockdown significantly increased TNF-α expression. In addition, inhibition of c-Jun N-terminal kinase-1/2 and NF-κB reduces the expression of TNF-α and IL-6. In addition, inhibition of ROS, MAPK, and PKC-α/δ reduced NF-κB activation, indicating that ROS, MAPK, and PKC-α/δ function as upstream signals to NF-κB. Finally, PPA treatment reduces macrophage phagocytosis activity and cytokine expression in bacteria-infected macrophages. The findings indicate the potential for PPA to be used as an immunomodulatory drug in the future.