| The fruit of Lycium barbarum L. is a well-known traditional Chinese medicine and it has been employed clinically as a tonic in the treatment of diseases for thousands of years in China. The main effect of it was described as "nourishing the blood and improving the eyesight" in traditional Chinese medicine. The crude polysaccharides were extracted from it and studied in our laboratory since the 1990's. It was found that Lycium barbarum polysaccharides could significantly improve the immune function in many kinds of immunodeficient model animals such as cyclophosphamide-treated, thymectomized, tumor-borne, D-galactose- treated, naturally aged mice and senescence accelerated mice (SAM) both given in vivo and in vitro. Further study demonstrated that the polysaccharides elevated the levels of intracellular calcium and cAMP and induced the expressions of c-fos and c-myc genes of the splenocytes when them were used in vitro.Recently, the polysaccharides were further purified and three glycoconjugates, LbGp1, LbGp3 and LbGp4, were obtained and identified consequently. LbGp1-OL, LbGp3-OL and LbGp4-OL are the glycochains dissociated from the corresponding glycoconjugates, respectively. In evaluation of their activities, it was found that in vitro application of LbGp4-OL (180.8 KDa) significantly promoted the proliferation and lgG production of mouse splenocytes dose-dependently, but it showed no stimulating effect on mouse thymocytes and had no effect on CD25 expression of T lymphocytes. Further studies demonstrated that it significantly potentiated the proliferation ofsplenocytes from nude mice. These results suggested that B lymphocyte are the main target of LbGp4-OL and there may exist binding site for LbGp4-OL on B cells. In this research, we studied the binding site of LbGp4-OL on B cells.1. Binding of 3H-LbGp4 and 3H-LbGp4-OL to B lymphocytesRadio-ligand assay showed that when 5.82 pM 3H-LbGp4 was reached, the specific binding of 3H-LbGp4 to B lymphocytes tended to saturation. The balance dissociation constant Kd was 2.973 pM and the affinity constant K was 0.3364 pM"1, indicating a strong binding'affinity. Bmax was 14.50 fM. Binding kinetic study showed that the binding saturation concentration Rleq was 12.25 fM, Correlation coefficient R 0.8668, availability speed constant 0.1464 fM/min, and tV2 4.734 min in combination time-binding curve. Correlation coefficient R was 0.9249 and t\/2 50.58 min in dissociation time-binding curve.The specific binding of 3H-LbGp4-OL to B lymphocytes was also saturable. Balance dissociation constant Kd was 2.4131 pM, affinity constant K 0.4144 pM"1 and Bmax 89.82 fM, indicating a strong binding affinity. The parameters of Rleq was 21.4891 fM, correlation coefficient 0.9341, availability speed constant 0.0224 fM/min and t\/2 30.94 min in combination time-binding curve. The correlation coefficient R was 0.9594 and tV2 30.13 min in dissociation time-binding curve.The specific binding of 3H-LbGp4-OL to B cells showed typicaldisplacement curve. Unlabeled LbGp4-OL replaced the binding ofH-LbGp4-OL to B cell in a concentration-dependent manner and almostcomplete replacement was achieved at a concentration of 760 times higher thanthat of 3H-LbGp4-OL. LPS, PWM and Con A could not replace the binding ofH-LbGp4-OL even at a high concentration, 63 times higher than that ofunlabeled LbGp4-OL, supporting that the binding of 3H-LbGp4-OL to B cellsis specific.2. Effects of LbGp4 and LbGp4-OL-composing onosaccharides on specific binding of 3H-LbGp4 and 3H-LbGp4-OL to B lymphocytesThe hydrocarbon content of LbGp4 was 85.6%, which was consisted of four monosaccharides, arabinose (Ar), galactose (Gal), rhamnegin (Rha) and xylose (Xyl). The composing ratio was 1.5: 2.5: 0.43: 0.23, respectively. When Ar, Gal, Rha and Xyl, or Ar, Gal, and Rha were used together, the specific binding of 3H-LbGp4 to B cells was blocked, but any other combining or single uses of the monosaccharides showed no blocking effectsLbGp4-OL was composed of Ar, Gal and Rha. The results showed that combining use of Ar, Gal and Rha almost completely blocked the specific binding of 3H-LbGp4-OL to B lymphocytes and the other combining or single uses of the monosaccharides showed no blocking effect.3. Effects of the concentrations of monosaccharides on the specific binding of 3H-LbGp4-OL on B cellsThe effects of combining use of Ar, Gal and Rha at the different concentrations, 1, 0.8, 0.6, 0.4 and 0.2 as the same times as those of normal concentration, on the specific binding of 3H-LbGp4-OL to B cells were observed. The results showed that the specific binding of 3H-LbGp4-OL to B cells was blocked by the combining use of Ar, Gal and Rha concentration-dependently.4. Binding of FITC labeled LbGp4-OL to B lymphocytesLbGp4-OL was labeled by fluoresceiniso-thiocyanate (FITC) and the binding of FITC-LbGp4-OL to the resting B cells was observed. The results showed that the binding was saturable. Scatchard plot showed an inverse correlation. The balance dissociation constant was 232.56 pM, Bmax 162.70 fM and K=0.0043 pM"1, indicating that the binding characteristics was similar to that of 3H-LbGp4-OL but the binding affinity was lower than 3H-LbGp4-OL.5. The bindings of 3H-LbGp4-OL on T lymphocytes, testis leydig cells and hippocampal and hypothalamic neural cellsThe results indicated that the bindings showed no saturable tendency and the total binding curve was almost overlapped with non-specific binding curve, suggesting that 3H-LbGp4-OL did not bind to T lymphocytes, testis eydig cells and hippocampal and hypothalamic neural cells.6. Study of the binding site of LbGp4-OL on cells with laser confocal microscopeLbGp4-OL was labeled with FITC and FITC-LbGp4-OL cultured with the resting B cells. Then, the labeled B cells were observed and sectioned with laser confocal microscope. The results showed that the binding site of FITC-LbGp4-OL was on membrane of B cells.7. Effects of LbGp4 and LbGp4-OL on the activity of protein kinase C in B cellsLbGp4 (2.32, 1.16, 0.58 nM) and LbGp4-OL (2.76, 1.38, 0.69 nM) were incubated with the resting B lymphocytes and the activity of protein kinase C was then determinated. The results indicated that LbGp4 and LbGp4-OL had no effect on the activity of protein kinase C in the resting B cells.8. Effects of LbGp4 and LbGp4-OL on the activity of protein tyrosine kinase in B cellsLbGp4 (2.32, 1.16, 0.58 nM) and LbGp4-OL (2.76, 1.38, 0.69 nM) were incubated with resting B cells and the activity of protein tyrosine kinase was then determined. The results showed that both LbGp4 and LbGp4-OL significantly intensified the activity of protein tyrosine kinase in resting B cells in concentration-dependent manners.9. Effects of LbGp4 and LbGp4-OL on nuclear transcription factor NF-kB and B cell specific activator protein in B cellsThe results showed that LbGp4 and LbGp4-OL significantly up-regulated the expressions of the nuclear transcription factor NF-kB and B cell specific activator protein (BSAP).
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