Fungal beta glucan protects radiation induced DNA damage in human lymphocytes.

BACKGROUND: Ganoderma lucidum (Ling Zhi), a basidiomycete white rot macrofungus has been used extensively for therapeutic use in China, Japan, Korea and other Asian countries for 2,000 years. The present study is an attempt to investigate its DNA protecting property in human lymphocytes. MATERIALS AND METHODS: Beta glucan (BG) was isolated by standard procedure and the structure and composition were studied by infrared radiation (IR) and nuclear magnetic resonance (NMR) spectroscopy, gel filtration chromatography and paper chromatography. The radioprotective properties of BG isolated from the macro fungi Ganoderma lucidum was assessed by single cell gel electrophoresis (comet assay). Human lymphocytes were exposed to 0, 1, 2 and 4 Gy gamma radiation in the presence and absence of BG. RESULTS: The comet parameters were reduced by BG. The results indicate that the BG of G. lucidum possessed significant radioprotective activity with DNA repairing ability and antioxidant activity as the suggestive mechanism. CONCLUSIONS: The findings suggest the potential use of this mushroom for the prevention of radiation induced cellular damages.

Recombinant form of human wild type mannan-binding lectin (MBL/A) but not its structural variant (MBL/C) promotes phagocytosis of zymosan by activating complement.

Mannan-binding lectin (MBL) mediates innate immune responses, such as activation of the complement lectin pathway and phagocytosis, to help fight infections. In the present study, employing recombinant forms of human MBL (rMBL), the role of wild type MBL (rMBL/A) and its structural variant rMBL/C in mediating THP-1 phagocytosis of fluorescent-labeled zymosan was examined and compared to MBL purified from human plasma (pMBL/A). Flow cytometric analyses revealed that opsonization of zymosan with rMBL/A and pMBL/A (0.5-30microg/ml) resulted in a 1.9- and 2.7-fold enhancement in its uptake by THP-1 cells in the presence of serum that was depleted of both MBL and the classical pathway component, C1q (MBL/C1q Dpl serum). In contrast, no enhancement in phagocytosis was observed when zymosan was opsonized with rMBL/C. Addition of MBL monoclonal antibody, EDTA, or mannan to the opsonization reaction mixture inhibited THP-1 phagocytosis of pMBL/A opsonized zymosan. Heat inactivation of MBL/C1q Dpl serum abolished the 2-fold increase in phagocytosis and in the absence of serum the direct opsonic activity of MBL did not contribute significantly to the uptake of zymosan into THP-1 cells. Activation products of complement components C3 and C4 were deposited on zymosan opsonized with pMBL/A and rMBL/A but not rMBL/C indicating that MBL-mediated phagocytosis of zymosan requires activation of the complement lectin pathway. The findings imply that impaired MBL-mediated phagocytosis may put individuals homozygous for the mutant allele MBL/C but not wild type MBL/A at increased risk to infections such as yeast.

Human astrovirus coat protein binds C1q and MBL and inhibits the classical and lectin pathways of complement activation.

Human astroviruses (HAstVs) constitute a family of non-enveloped, RNA viruses which cause infantile gastroenteritis. We have previously demonstrated that purified HAstV coat protein (CP), multiple copies of which compose the viral capsid, bind C1q resulting in inhibition of classical complement pathway activity. The objective of this study was to further analyze the mechanism by which CP inhibits C1 activation. CP inhibited C1 activation, preventing cleavage of C1s to its active form in the presence of heat-aggregated IgG, a potent classical pathway activator. CP also inhibited generation of the potent anaphylatoxin C5a. CP dose-dependently bound to C1q, the isolated globular heads and the collagen-like regions of the C1q molecule. When CP was added to C1, C1s dissociated from C1q suggesting that CP functionally displaces the protease tetramer (C1s-C1r-C1r-C1s). Given the structural and functional relatedness of C1q and MBL, we subsequently investigated the interactions between CP and MBL. CP bound to purified MBL and was able to inhibit mannan-mediated activation of the lectin pathway. Interestingly, CP did not bind to a variant of MBL that replaces a lysine residue (Lys55) critical for binding to MASP-2, a functional homolog of C1s. Finally, CP was shown to cross the species barrier to inhibit C3 activation and MAC formation in rat serum. These findings suggest CP inhibits C1 and MBL activation via a novel mechanism of interference with the normal interaction of the recognition molecule with its cognate serine proteases.

Stringent regulation of complement lectin pathway C3/C5 convertase by C4b-binding protein (C4BP).

The complement lectin pathway, an essential component of the innate immune system, is geared for rapid recognition of infections as each C4b deposited via this pathway is capable of forming a C3/C5 convertase. In the present study, role of C4b-binding protein (C4BP) in regulating the lectin pathway C3/C5 convertase assembled on zymosan and sheep erythrocytes coated with mannan (E(Man)) was examined. While the C4BP concentration for inhibiting 50% (IC(50)) formation of surface-bound C3 convertase on the two surfaces was similar to that obtained for the soluble C3 convertase (1.05nM), approximately 3- and 41-fold more was required to inhibit assembly of the C5 convertase on zymosan (2.81nM) and E(Man) (42.66nM). No difference in binding interactions between C4BP and surface-bound C4b alone or in complex with C3b was observed. Increasing the C4b density on zymosan (14,000-431,000 C4b/Zym) increased the number of C4b bound per C4BP from 2.87 to 8.23 indicating that at high C4b density all seven alpha-chains of C4BP are engaged in C4b-binding. In contrast, the number of C4b bound per C4BP remained constant (3.79+/-0.60) when the C4b density on E(Man) was increased. The data also show that C4BP regulates assembly and decay of the lectin pathway C3/C5 convertase more stringently than the classical pathway C3/C5 convertase because of a approximately 7- to 13-fold greater affinity for C4b deposited via the lectin pathway than the classical pathway. C4BP thus regulates efficiently the four times greater potential of the lectin pathway than the classical pathway in generating the C3/C5 convertase and hence production of pro-inflammatory products, which are required to fight infections but occasionally cause pathological inflammatory reactions.

New insights on the structural/functional properties of recombinant human mannan-binding lectin and its variants.

Inefficient activation of complement lectin pathway in individuals with variant mannan-binding lectin (MBL) genotypes has been attributed to poor formation of higher order oligomers by MBL. But recent studies have shown the presence of large oligomers of MBL (approximately 450 kDa) in serum of individuals with variant MBL alleles. The recombinant forms of MBL (rMBL) variants except MBL/B that assemble into higher order oligomers have not yet been reported. In the present study, structural/functional properties of recombinant forms of wild type MBL (rMBL/A) and its three structural variants, rMBL/B, C, and D generated in insect cells were examined. Western blot analysis indicated covalently linked monomers to hexamers while gel filtration chromatography exhibited non-covalently linked higher order oligomers in addition to prevalent low oligomeric forms. Mannan binding determined by ELISA showed rMBL/A but not the structural variants bind to mannan. Apparent avidity of monoclonal antibody used was found to be about 18- to 52-fold weaker for rMBL structural variants than rMBL/A. Complement activation varied with maximum impairment apparent in rMBL/C followed by rMBL/B, but rMBL/D was functional to the same extent as rMBL/A. Comparison of rMBL/A to MBL purified from plasma (pMBL/A) indicated 8- and 24-fold weaker binding to mannan by BIAcore analysis and ELISA and about 5-fold lesser efficiency in activating complement. The findings provide new insights on the structural/functional properties of rMBL variants and imply that lectin pathway activation may be impaired in individuals, homozygous for the mutant alleles, MBL/C and to a lesser extent MBL/B but not MBL/D.

Activation of complement component C5: comparison of C5 convertases of the lectin pathway and the classical pathway of complement.

Although the initiating complex of lectin pathway (called M1 in this study) generates C3/C5 convertases similar to those assembled by the initiating complex (C1) of the classical pathway, activation of complement component C5 via the lectin pathway has not been examined. In the present study kinetic analysis of lectin pathway C3/C5 convertases assembled on two surfaces (zymosan and sheep erythrocytes coated with mannan (E(Man))) revealed that the convertases (ZymM1,C4b,C2a and E(Man)M1,C4b,C2a) exhibited a similar but weak affinity for the substrate, C5 indicated by a high K(m) (2.73-6.88 microm). Very high affinity C5 convertases were generated when the low affinity C3/C5 convertases were allowed to deposit C3b by cleaving native C3. These C3b-containing convertases exhibited K(m) (0.0086-0.0075 microm) well below the normal concentration of C5 in blood (0.37 microm). Although kinetic parameters, K(m) and k(cat), of the lectin pathway C3/C5 convertases were similar to those reported for classical pathway C3/C5 convertases, studies on the ability of C4b to bind C2 indicated that every C4b deposited on zymosan or E(Man) was capable of forming a convertase. These findings differ from those reported for the classical pathway C3/C5 convertase, where only one of four C4b molecules deposited formed a convertase. The potential for four times more amplification via the lectin pathway than the classical pathway in the generation of C3/C5 convertases and production of pro-inflammatory products, such as C3a, C4a, and C5a, implies that activation of complement via the lectin pathway might be a more prominent contributor to the pathology of inflammatory reactions.