Munehiro Nakata

A Novel Human Serum Lectin with Collagen- and Fibrinogen-like Domains That Functions as an Opsonin

Collectins are C-type animal lectins with both collagenous and carbohydrate recognition domains and are involved in the first line host defense against pathogens. We report here a novel Ca2+-dependent and GlcNAc-binding lectin consisting of subunits of 35 kDa (P35) with a collagen-like sequence. When P35 is isolated from human serum, it forms a homopolymer by means of intermolecular disulfide bonding, as is the case with collectins. P35 cDNA was cloned from a human liver cDNA library, and the deduced amino acid sequence of 313 residues revealed that the mature form of P35 consists mainly of collagen- and fibrinogen-like domains. The latter contained two potential Ca2+-binding sites that may be involved in carbohydrate binding. The overall sequence of P35 was highly homologous to porcine ficolins α and β. Northern blots of various human tissues showed that the major product of the 1.3-kilobase-long P35 transcript is expressed in liver. P35 enhanced phagocytosis of Salmonella typhimurium by neutrophils, suggesting an opsonic effect via the collagen region. P35 was found to bind to GlcNAc-conjugated bovine serum albumin, a neoglycoprotein, as well as to neoglycolipids containing complex-type oligosaccharides derived from glycoproteins, suggesting that P35 recognizes GlcNAc residues such as those found in microbial glycoconjugates and complex-type oligosaccharides. Therefore, P35 represents a new type of GlcNAc-binding lectin with structural and functional similarities to collectins involved in innate immunity.

Human M-Ficolin Is a Secretory Protein That Activates the Lectin Complement Pathway

Three types of ficolins have been identified in humans: L-ficolin, M-ficolin, and H-ficolin. Similar to mannose-binding lectin, L-ficolin and H-ficolin are the recognition molecules in the lectin complement pathway. Another human ficolin, M-ficolin, is a nonserum ficolin that is expressed in leukocytes and lung; however, little is known about its physiologic roles. In this study, we report the characterization of M-ficolin in terms of its protein localization and lectin activity. M-ficolin was localized in secretory granules in the cytoplasm of neutrophils, monocytes, and type II alveolar epithelial cells in lung. M-ficolin precipitated with mannose-binding lectin-associated serine proteases (MASP)-1 and MASP-2 in a coimmunoprecipitation assay, indicating that M-ficolin forms complexes with MASP-1 and MASP-2. M-ficolin-MASP complexes activated complement on N-acetylglucosamine (GlcNAc)-coated microplates in a C4 deposition assay. M-ficolin bound to several neoglycoproteins bearing GlcNAc, N-acetylgalactosamine, and sialyl-N-acetyllactosamine, suggesting that M-ficolin can recognize the common carbohydrate residues found in microbes. Indeed, M-ficolin bound to Staphylococcus aureus through GlcNAc. These results indicate that M-ficolin, like its family members, functions as a recognition molecule of the lectin complement pathway and plays an important role in innate immunity.

Antitumor activity of extracts and compounds from the skin of the toad Bufo bufo gargarizans Cantor.

The skin of the toad Bufo bufo gargarizans Cantor is known to be rich in bufadienolides, peptides and alkaloids. It has been found to be a source of some extracts and biologically active compounds with antitumor activity. Cinobufacini (Huachansu), a Chinese medicine prepared from the dried toad skin, has been widely used in clinical therapy for various cancers in China. Bufadienolides, such as bufalin, cinobufagin, resibufogenin, and telocinobufagin, are the major active compounds derived from the toad skin. They are the maker biologically active compounds of cinobufagin while the antitumor activity of cinobufagin may be due to this kind of components. Experimental research has suggested that cinobufacini and its active compounds (e.g. bufalin and cinobufagin) exhibit significant antitumor activity, including inhibition of cell proliferation, induction of cell differentiation, induction of apoptosis, disruption of the cell cycle, inhibition of cancer angiogenesis, reversal of multi-drug resistance, and regulation of the immune response. Clinical data have indicated that cinobufacini may have effective anticancer activity with low toxicity and few side effects. Data to date suggest it may also enhance quality of life for patients with cancer. Thus, this review briefly summarizes recent studies on the anticancer activity of cinobufacini and some of its active compounds from the skin of the toad Bufo bufo gargarizans Cantor. This might provide additional evidence for further study of the extracts and active compounds from the toad skin in cancer treatment.

An Ancient Lectin-Dependent Complement System in an Ascidian: Novel Lectin Isolated from the Plasma of the Solitary Ascidian, Halocynthia roretzi

Mannose-binding lectin (MBL) is a C-type lectin involved in the first line of host defense against pathogens and it requires MBL-associated serine protease (MASP) for activation of the complement lectin pathway. To elucidate the origin and evolution of MBL, MBL-like lectin was isolated from the plasma of a urochordate, the solitary ascidian Halocynthia roretzi, using affinity chromatography on a yeast mannan-Sepharose. SDS-PAGE of the eluted proteins revealed a major band of ∼36 kDa (p36). p36 cDNA was cloned from an ascidian hepatopancreas cDNA library. Sequence analysis revealed that the carboxy-terminal half of the ascidian lectin contains a carbohydrate recognition domain (CRD) that is homologous to C-type lectin, but it lacks a collagen-like domain that is present in mammalian MBLs. Purified p36 binds specifically to glucose but not to mannose or N-acetylglucosamine, and it was designated glucose-binding lectin (GBL). The two ascidian MASPs associated with GBL activate ascidian C3, which had been reported to act as an opsonin. The removal of GBL-MASPs complex from ascidian plasma using Ab against GBL inhibits C3-dependent phagocytosis. These observations strongly suggest that GBL acts as a recognition molecule and that the primitive complement system, consisting of the lectin-proteases complex and C3, played a major role in innate immunity before the evolution of an adaptive immune system in vertebrates.

Cinobufacini, an aqueous extract from Bufo bufo gargarizans Cantor, induces apoptosis through a mitochondria-mediated pathway in human hepatocellular carcinoma cells.

AIM OF THE STUDY Cinobufacini (Huachansu), an aqueous extract from the skin and parotid venom glands of Bufo bufo gargarizans Cantor, is a traditional Chinese medicine widely used in clinical cancer therapy in China. The present study sought to investigate the possible signaling pathway implicated in cinobufacini-induced apoptosis in the hepatocellular carcinoma cell lines HepG(2) and Bel-7402. MATERIALS AND METHODS The effects of cinobufacini on cell proliferation of HepG(2) and Bel-7402 cells were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assays. Cell apoptosis was detected by Hoechst 33258 staining and flow cytometry analysis. The mitochondrial membrane potential (Deltapsim) and caspase-9 and -3 activity were detected using MitoCapture reagent staining and colorimetric assays, respectively. The expression of apoptosis-related proteins and release of cytochrome c were assessed by Western blot analysis. RESULTS Cinobufacini significantly inhibited cell proliferation of both cell lines in a dose- and time-dependent manner. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after cinobufacini treatment. The protein expression of Bax increased whereas that of Bcl-2 decreased, leading to an increase in the Bax/Bcl-2 ratio. Subsequently, cinobufacini disrupted the mitochondrial membrane potential (Deltapsim) and resulted in the release of cytochrome c, activation of both caspase-9 and -3, and cleavage of poly (ADP-ribose) polymerase (PARP). CONCLUSION The present study indicated that cinobufacini can induce apoptosis of HepG(2) and Bel-7402 cells through a mitochondria-mediated apoptosis pathway.

Molecular and cellular basis for pathogenicity of autoantibodies: lessons from murine monoclonal autoantibodies

The pathogenesis of autoantibody-mediated cellular and tissue lesions in autoimmune diseases is most straightforwardly attributable to the combined action of self-antigen binding properties and effector functions associated with the Fc regions of the different immunoglobulin (Ig) isotypes. The analysis of two different sets of monoclonal autoantibodies derived from lupus-prone mice revealed remarkable differences in the pathogenic potentials of different IgG subclasses: (1) the IgG2a and IgG2b subclasses of anti-red blood cell (RBC) autoantibodies are the most pathogenic and efficiently activate two classes of activating IgG Fc receptors (FcγRIII and FcγRIV) and complement; (2) the IgG3 subclass is less pathogenic and activate only complement; and (3) the IgG1 subclass is the least pathogenic and interact only with FcγRIII. In addition, because of the unique property of IgG3 to form self-associating complexes and generate cryoglobulins, this subclass of rheumatoid factor and anti-DNA autoantibodies became highly pathogenic and induced lupus-like nephritis and/or vasculitis. Since the switch to IgG2a and IgG3 is promoted by Th1 cytokine interferon γ, these results strongly suggest that Th1 autoimmune responses could be critically involved in the generation of more pathogenic autoantibodies in systemic lupus erythematosus. This finding is consistent with the observation that the progression of murine lupus nephritis is correlated with the relative dominance of Th1 autoimmune responses. Finally, the analysis of IgG glycosylation pattern revealed that more sialylated IgG autoantibodies remained poorly pathogenic because of limited Fc-associated effector functions and loss of cryoglobulin activity. This suggests that the terminal sialylation of the oligosaccharide side chains of IgG could be a significant factor determining the pathogenic potential of autoantibodies. Our results thus underline the importance of subpopulations of autoantibodies, induced by the help of Th1 cells, in the pathogenesis of autoantibody-mediated cellular and tissue injuries.

Carbohydrate-binding specificities of mouse ficolin A, a splicing variant of ficolin A and ficolin B and their complex formation with MASP-2 and sMAP.

Ficolins are a group of proteins mainly consisting of collagen-like and fibrinogen-like domains and are thought to play a role in innate immunity via their carbohydrate-binding activities. Two types of ficolins have been identified in mice, ficolin A, and ficolin B. However, their structure and function are not fully understood. In this study, we isolated the cDNA encoding a novel variant of ficolin A having a shorter collagen-like domain and a longer gap sequence, which was generated from the ficolin A gene by alternative splicing. We delineated the structure and function of mouse ficolins, including this splicing variant, by preparing the respective recombinants. Recombinant ficolin A, its splicing variant, and ficolin B showed multimeric structures and revealed binding to both N-acetylglucosamine and N-acetylgalactosamine. Interestingly, ficolin B specifically recognized sialic acid residues. Ficolin A and its variant, but not ficolin B, bound to mannose-binding lectin (MBL)-associated serine protease-2 (Masp-2) and small MBL-associated protein (smap), and the resulting complexes showed a potent complement activating capacity. In addition, smap competed with Masp-2 in association with ficolin A and its variant, and inhibited the complement activation by the ficolin A (or ficolin A variant)/MASP-2 complex, indicating its regulatory role in the lectin pathway. These results suggest that ficolin A and its variant function as recognition molecules of the lectin pathway, and ficolin B plays a distinct role through its unique carbohydrate-binding specificity.

Lectin Pathway of Bony Fish Complement: Identification of Two Homologs of the Mannose-Binding Lectin Associated with MASP2 in the Common Carp (Cyprinus carpio)

The lectin pathway of complement is considered to be the most ancient complement pathway as inferred from identification of ancient homologs of mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs) in some invertebrates. MBL homologs with galactose selectivity and an MASP3-like sequence also occur in bony fish, linking the evolution of the lectin complement pathway from invertebrates to higher vertebrates. However, these cannot be considered authentic complement components until confirmatory functional evidence is obtained. Here, we report the isolation and characterization of two MBL homologs from a cyprinid teleost, the common carp, Cyprinus carpio. One, designated GalBL, corresponds to the MBL-like molecule with the galactose specificity. The other is an authentic MBL with mannose specificity. Both were found to associate with a serine protease that cleaves native human C4 into C4b but not C4i with a hydrolyzed thioester. Molecular cloning and phylogenetic analysis revealed this C4-activating protease to be carp MASP2, indicating that MASP2 arose before the emergence of bony fish. Database mining of MBL-like genes reveals that MBL and GalBL genes are arranged in tandem in the zebrafish genome and that both lectins are conserved in the distantly related puffer fish. These results imply that bony fish have developed a diverged set of MBL homologs that function in the lectin complement pathway.

Oligomannose-coated liposomes as an adjuvant for the induction of cell-mediated immunity

The effect of the coating of ovalbumin‐reconstituted liposomes with various oligosaccharides on their immunogenicity was investigated in mice. The coating of liposomes with oligomannose or yeast mannan drastically enhanced their ability to induce an ovalbumin‐specific delayed‐type footpad swelling response with a peak at 24 to 48 h post‐challenge. Among various oligosaccharides tested, only those with mannose residue at the non‐reducing termini manifested the activity when applied to liposomes. Since such oligosaccharides are ubiquitously found in the body, these results suggested the usefulness of oligomannose‐coated liposomes as a safe adjuvant for the induction of cell‐mediated immunity.

Rapid and simple preparation of N-linked oligosaccharides by cellulose-column chromatography.

As a means of preparing N-linked oligosaccharides from hydrazinolysates of glycoproteins in a rapid and simple manner, a method has been developed using cellulose-column chromatography. Hydrazinolysates of human IgG, containing a series of biantennary complex type oligosaccharides, were applied to a cellulose column equilibrated with (4:1:1, v/v) 1-butanol-ethanol-water. The N-linked oligosaccharides were eluted with (1:1, v/v) ethanol-water, and analyzed by HPLC in combination with sequential glycosidase digestion. The oligosaccharides, with or without sialic acid, were quantitatively recovered in the fraction eluted with (1:1, v/v) ethanol-water without UV-detectable contamination by impurities derived from protein or the cellulose. Other types of N-linked oligosaccharides of alpha1-acid glycoprotein (tetraantennary complex-type), ovalbumin (hybrid-type), and ribonuclease B (high mannose-type) were also quantitatively prepared from the hydrazinolysates by elution of the cellulose column with (1:1, v/v) ethanol-water and these had as high a quality as those prepared by conventional paper chromatography.