| Lectins are usually considered as non-enzyme and non-immune proteins or carbohydrate-binding proteins which can promote aggregation of cells. Lectins widely exist in animals, plants and microbes. Lectins exhibit a wide range of biological activities, such as cell adhesion, bacterial infection, pinocytosis mediated by receptor, innate immune defense, phagocytic activity, recognition and binding between sperms and eggs. Lectins have been widely applied in many research fields, for example, lectins are extensive used as mitogenic reagent and cell toxin reagent in biochemical and clinical research. At present, researches of lectins mainly focus on terrestrial plants and microbes. Lectins from marine invertebrate have been less studied, especially the interaction between marine invertebrate lectins and microorganisms. In this study, Primary structure, carbohydrate specificity, thermodynamic properties of two kind of marine invertebrate lectins(Crenomytius grayanus, CGL; Craniella australiensis, CAL) and interaction between lectins and microorganisms were investigated.The main conclusions are as follows:1. The primary structure of CGL was studied by nano-ESI-MS/MS. Amino acid sequences of six peptides were obtained, respectively FAMDFFNDNLMHK, HAAMEFLFVSPK, LQGLVSWGSGGSGTK, MYFQFDVVDER, PPNETNMVIHQDR and ALFAMDFFNDNLMHK.2. Thermodynamic properties and reaction activation energy of CGL, CAL were also studied. The agglutination activity of CGL remained stable in 60 minutes at 40℃, dropped 75% in 5 minutes at 50℃, declined rapidly at 60℃or 70℃. Reaction activation energy of CGL in this inactivation process was 61.53 kcal·mol-1. The agglutination activity of CAL dropped 50% in 5 minutes at 40 or 50℃, then remained stable, declined rapidly at 70 or 90℃. Reaction activation energy of CAL in this inactivation process was 22.3 kcal·mol-1.3. Carbohydrate specificity of CAL was studied by solid phase method. Asialo-fetuin and asialo-PSM were the most effective inhibitor. The results of enzyme-linked lectin test showed that CAL had carbohydrate and collagen binding sites. The binding constant between PSM and CAL was also investigated by solid phase method. The results showed that CAL had two PSM binding sites. The binding constants were 2.98 x 107 M-1 and 1.11 x 105 M-1. PSM only bind the high affinity site when there was a low concentration of PSM.4. Effects on yeast fermentation of CGL and CAL were studied. After adding these two lectins into the fermentation broth, the ethanol concentration of media was always higher than the negative control media. After two hours of addition of 6μg/ml CGL, ethanol concentration become twice as the negative control media. Ethanol concentration was still 15% higher than control media after adding CGL 12 hours. Also, ethanol concentration was 70% higher than control media after adding 13.8μg/ml CAL 72 hours. These results showed that both lectins can stimulate yeast to produce ethanol by glucose fermentation. CGL also exhibited positive effects on yeast cell growth, whereas CAL did not show this activity. After 12 or 24 hours of addition of CGL, yeast cells become 2×108 cfu/100ml, which was 40% or 30% higher than control media.5. Interactions between CGL, CAL and E. coli were studied. CGL agglutinated natural E. coli, and E. coli treated by methanol and trypsase. CAL agglutinated natural E. coli, and E. coli treated by acid and trypsase. CAL also inhibited the growth of E. coli K1 in media broth.6. Effects on the growth of (3-1,3-glucanase marine bacteria and enzymatic activities of two lectins were also studied. Both lectins promoted the growth ofβ-1,3-glucanase marine bacteria. More lectin added, higher growth of bacteria can be observed. Two kind of lectins also stimulated bacteria to secrete (3-1,3-glucanase. The activities ofβ-1, 3-glucanase from media broth with lectins added were higher than the control media. The highest enzyme activity can be obtained in the media broth with 6μg/ml CGL, which was 26% higher than the control media. The highest enzyme activity can be obtained in the media broth with 15μg/ml CAL, which was 35% higher than the control media.7. Interaction between CAL and HIV was investigated. CAL showed no toxicity to human C8166 lymphocyte. Cell viability of human C8166 lymphocyte remained 101.9% when the concentration of CAL was 150μg/ml. CAL exhibited promising inhibition effects on C8166 syncytial cells. Inhibition ratio was 66% when the concentration of CAL was 50 μg/ml, whereas inhibition ratio was 50% when the concentration of CAL was 16.41μg/ml. CAL did not show the activity to blockade infection and fusion of HIV. With the prolongation of adding time, inhibition effects of CAL on C8166 syncytial cells gradually reduced. CAL effectively suppressed the infection of HIV, and inhibited the formation of C8166 syncytial cells in 30 minutes with inhibition rate of C8166 syncytial cells more than 60%. This paper is funded by the National Natural Science Foundation (31071612). |
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