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A novel non-lens betagamma-crystallin and trefoil factor complex from amphibian skin and its functional implications.

Liu SB, He YY, Zhang Y, Lee WH, Qian JQ, Lai R, Jin Y - PLoS ONE (2008)

Bottom Line: Furthermore, betagamma-CAT showed multiple cellular effects on human umbilical vein endothelial cells.Bafilomycin A1 (a specific inhibitor of the vacuolar-type ATPase) and nocodazole (an agent of microtuble depolymerizing), while inhibited betagamma-CAT induced vacuole formation, significantly inhibited betagamma-CAT induced cell detachment, suggesting that betagamma-CAT endocytosis is important for its activities.These findings illustrate novel cellular functions of non-lens betagamma-cyrstallins and action mechanism via association with trefoil factors, serving as clues for investigating the possible occurrence of similar molecules and action mechanisms in mammals.

View Article: PubMed Central - PubMed

Affiliation: Biotoxin Units, Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming, Yunnan, China.

ABSTRACT

Background: In vertebrates, non-lens betagamma-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive.

Principal findings: A naturally existing 72-kDa complex of non-lens betagamma-crystallin (alpha-subunit) and trefoil factor (beta-subunit), named betagamma-CAT, was identified from frog Bombina maxima skin secretions. Its alpha-subunit and beta-subunit (containing three trefoil factor domains), with a non-covalently linked form of alphabeta(2), show significant sequence homology to ep37 proteins, a group of non-lens betagamma-crystallins identified in newt Cynops pyrrhogaster and mammalian trefoil factors, respectively. betagamma-CAT showed potent hemolytic activity on mammalian erythrocytes. The specific antiserum against each subunit was able to neutralize its hemolytic activity, indicating that the two subunits are functionally associated. betagamma-CAT formed membrane pores with a functional diameter about 2.0 nm, leading to K(+) efflux and colloid-osmotic hemolysis. High molecular weight SDS-stable oligomers (>240-kDa) were detected by antibodies against the alpha-subunit with Western blotting. Furthermore, betagamma-CAT showed multiple cellular effects on human umbilical vein endothelial cells. Low dosages of betagamma-CAT (25-50 pM) were able to stimulate cell migration and wound healing. At high concentrations, it induced cell detachment (EC(50) 10 nM) and apoptosis. betagamma-CAT was rapidly endocytosed via intracellular vacuole formation. Under confocal microscope, some of the vacuoles were translocated to nucleus and partially fused with nuclear membrane. Bafilomycin A1 (a specific inhibitor of the vacuolar-type ATPase) and nocodazole (an agent of microtuble depolymerizing), while inhibited betagamma-CAT induced vacuole formation, significantly inhibited betagamma-CAT induced cell detachment, suggesting that betagamma-CAT endocytosis is important for its activities.

Conclusions/significance: These findings illustrate novel cellular functions of non-lens betagamma-cyrstallins and action mechanism via association with trefoil factors, serving as clues for investigating the possible occurrence of similar molecules and action mechanisms in mammals.

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Related in: MedlinePlus

Erythrocyte membrane pore formation.(A) Osmotic protection of erythrocytes from hemolysis induced by βγ-CAT. Human erythrocytes (4×109 cells/ml) were incubated with various concentrations of βγ-CAT at 37°C for 30 min in the presence of PEGs of different molecular sizes. Hemolysis was tested as described in “Methods”. (B) Human erythrocyte K+ efflux and hemolysis induced by βγ-CAT in the absence and presence of PEG 1000. The value of the erythrocytes lysed with 1% Triton X-100 was taken as 100%. Data were expressed as means±SEM of triplicate measurements (*, p<0.05; **, p<0.01, compared with βγ-CAT only group (A–B); #, p<0.05; ##, p<0.01, compared with zero βγ-CAT (B). (C) Western blotting analysis of SDS-stable oligomers formed in erythrocyte membranes. Human erythrocytes (6×107 cells/ml) were incubated with various concentrations of βγ-CAT (0.5–3 nM) at 37°C for 30 min. Samples were treated as described in “Methods”, and loaded on a SDS-PAGE gel (linear gradient acrylamide gel of 3–15%) and blotted with rabbit polyclonal antibodies against native βγ-CAT, and each subunit, respectively. Lanes1, 6, 11, βγ-CAT control; lanes 2, 7, 12, erythrocyte control; lanes 3, 8, 13, lanes 4, 9, 14, and lanes 5, 10,15, erythrocytes treated with 0.5, 1.5, and 3 nM βγ-CAT, respectively.
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pone-0001770-g005: Erythrocyte membrane pore formation.(A) Osmotic protection of erythrocytes from hemolysis induced by βγ-CAT. Human erythrocytes (4×109 cells/ml) were incubated with various concentrations of βγ-CAT at 37°C for 30 min in the presence of PEGs of different molecular sizes. Hemolysis was tested as described in “Methods”. (B) Human erythrocyte K+ efflux and hemolysis induced by βγ-CAT in the absence and presence of PEG 1000. The value of the erythrocytes lysed with 1% Triton X-100 was taken as 100%. Data were expressed as means±SEM of triplicate measurements (*, p<0.05; **, p<0.01, compared with βγ-CAT only group (A–B); #, p<0.05; ##, p<0.01, compared with zero βγ-CAT (B). (C) Western blotting analysis of SDS-stable oligomers formed in erythrocyte membranes. Human erythrocytes (6×107 cells/ml) were incubated with various concentrations of βγ-CAT (0.5–3 nM) at 37°C for 30 min. Samples were treated as described in “Methods”, and loaded on a SDS-PAGE gel (linear gradient acrylamide gel of 3–15%) and blotted with rabbit polyclonal antibodies against native βγ-CAT, and each subunit, respectively. Lanes1, 6, 11, βγ-CAT control; lanes 2, 7, 12, erythrocyte control; lanes 3, 8, 13, lanes 4, 9, 14, and lanes 5, 10,15, erythrocytes treated with 0.5, 1.5, and 3 nM βγ-CAT, respectively.

Mentions: K+ efflux was determined in human erythrocytes exposed to βγ-CAT. In βγ-CAT (3 nM) treated human erythrocytes (4×109 cells/ml), about 90% of intracellular K+ leaked (p<0.01, compared with zero βγ-CAT), whereas the hemolytic rate only reached to 25% (p<0.05) (Fig. 5B). The erythrocyte lysis induced by βγ-CAT was also assayed in the presence of polyethylene glycols (PEGs) with different hydrodynamic diameters. The results showed that the hemolysis induced by βγ-CAT was not affected by the addition of PEGs 200, but partially inhibited by PEGs 400 and 600, and entirely suppressed by PEGs 1000 and 2000 (p<0.01, compared with the βγ-CAT only group) (Fig. 5A). In the presence of PEGs 1000, the hemolysis was suppressed, however, the intracellular K+ releasing was not obviously blocked (Fig. 5B). The hydrodynamic diameter of PEGs 1000 was estimated to be 2.0 nm [24]. These results suggest that βγ-CAT formed membrane pores with a functional diameter about 2.0 nm, leading intracellular K+ efflux, causing the colloid osmotic burst of erythrocytes and finally inducing erythrocyte lysis.


A novel non-lens betagamma-crystallin and trefoil factor complex from amphibian skin and its functional implications.

Liu SB, He YY, Zhang Y, Lee WH, Qian JQ, Lai R, Jin Y - PLoS ONE (2008)

Erythrocyte membrane pore formation.(A) Osmotic protection of erythrocytes from hemolysis induced by βγ-CAT. Human erythrocytes (4×109 cells/ml) were incubated with various concentrations of βγ-CAT at 37°C for 30 min in the presence of PEGs of different molecular sizes. Hemolysis was tested as described in “Methods”. (B) Human erythrocyte K+ efflux and hemolysis induced by βγ-CAT in the absence and presence of PEG 1000. The value of the erythrocytes lysed with 1% Triton X-100 was taken as 100%. Data were expressed as means±SEM of triplicate measurements (*, p<0.05; **, p<0.01, compared with βγ-CAT only group (A–B); #, p<0.05; ##, p<0.01, compared with zero βγ-CAT (B). (C) Western blotting analysis of SDS-stable oligomers formed in erythrocyte membranes. Human erythrocytes (6×107 cells/ml) were incubated with various concentrations of βγ-CAT (0.5–3 nM) at 37°C for 30 min. Samples were treated as described in “Methods”, and loaded on a SDS-PAGE gel (linear gradient acrylamide gel of 3–15%) and blotted with rabbit polyclonal antibodies against native βγ-CAT, and each subunit, respectively. Lanes1, 6, 11, βγ-CAT control; lanes 2, 7, 12, erythrocyte control; lanes 3, 8, 13, lanes 4, 9, 14, and lanes 5, 10,15, erythrocytes treated with 0.5, 1.5, and 3 nM βγ-CAT, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2262142&req=5

pone-0001770-g005: Erythrocyte membrane pore formation.(A) Osmotic protection of erythrocytes from hemolysis induced by βγ-CAT. Human erythrocytes (4×109 cells/ml) were incubated with various concentrations of βγ-CAT at 37°C for 30 min in the presence of PEGs of different molecular sizes. Hemolysis was tested as described in “Methods”. (B) Human erythrocyte K+ efflux and hemolysis induced by βγ-CAT in the absence and presence of PEG 1000. The value of the erythrocytes lysed with 1% Triton X-100 was taken as 100%. Data were expressed as means±SEM of triplicate measurements (*, p<0.05; **, p<0.01, compared with βγ-CAT only group (A–B); #, p<0.05; ##, p<0.01, compared with zero βγ-CAT (B). (C) Western blotting analysis of SDS-stable oligomers formed in erythrocyte membranes. Human erythrocytes (6×107 cells/ml) were incubated with various concentrations of βγ-CAT (0.5–3 nM) at 37°C for 30 min. Samples were treated as described in “Methods”, and loaded on a SDS-PAGE gel (linear gradient acrylamide gel of 3–15%) and blotted with rabbit polyclonal antibodies against native βγ-CAT, and each subunit, respectively. Lanes1, 6, 11, βγ-CAT control; lanes 2, 7, 12, erythrocyte control; lanes 3, 8, 13, lanes 4, 9, 14, and lanes 5, 10,15, erythrocytes treated with 0.5, 1.5, and 3 nM βγ-CAT, respectively.
Mentions: K+ efflux was determined in human erythrocytes exposed to βγ-CAT. In βγ-CAT (3 nM) treated human erythrocytes (4×109 cells/ml), about 90% of intracellular K+ leaked (p<0.01, compared with zero βγ-CAT), whereas the hemolytic rate only reached to 25% (p<0.05) (Fig. 5B). The erythrocyte lysis induced by βγ-CAT was also assayed in the presence of polyethylene glycols (PEGs) with different hydrodynamic diameters. The results showed that the hemolysis induced by βγ-CAT was not affected by the addition of PEGs 200, but partially inhibited by PEGs 400 and 600, and entirely suppressed by PEGs 1000 and 2000 (p<0.01, compared with the βγ-CAT only group) (Fig. 5A). In the presence of PEGs 1000, the hemolysis was suppressed, however, the intracellular K+ releasing was not obviously blocked (Fig. 5B). The hydrodynamic diameter of PEGs 1000 was estimated to be 2.0 nm [24]. These results suggest that βγ-CAT formed membrane pores with a functional diameter about 2.0 nm, leading intracellular K+ efflux, causing the colloid osmotic burst of erythrocytes and finally inducing erythrocyte lysis.

Bottom Line: Furthermore, betagamma-CAT showed multiple cellular effects on human umbilical vein endothelial cells.Bafilomycin A1 (a specific inhibitor of the vacuolar-type ATPase) and nocodazole (an agent of microtuble depolymerizing), while inhibited betagamma-CAT induced vacuole formation, significantly inhibited betagamma-CAT induced cell detachment, suggesting that betagamma-CAT endocytosis is important for its activities.These findings illustrate novel cellular functions of non-lens betagamma-cyrstallins and action mechanism via association with trefoil factors, serving as clues for investigating the possible occurrence of similar molecules and action mechanisms in mammals.

View Article: PubMed Central - PubMed

Affiliation: Biotoxin Units, Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming, Yunnan, China.

ABSTRACT

Background: In vertebrates, non-lens betagamma-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive.

Principal findings: A naturally existing 72-kDa complex of non-lens betagamma-crystallin (alpha-subunit) and trefoil factor (beta-subunit), named betagamma-CAT, was identified from frog Bombina maxima skin secretions. Its alpha-subunit and beta-subunit (containing three trefoil factor domains), with a non-covalently linked form of alphabeta(2), show significant sequence homology to ep37 proteins, a group of non-lens betagamma-crystallins identified in newt Cynops pyrrhogaster and mammalian trefoil factors, respectively. betagamma-CAT showed potent hemolytic activity on mammalian erythrocytes. The specific antiserum against each subunit was able to neutralize its hemolytic activity, indicating that the two subunits are functionally associated. betagamma-CAT formed membrane pores with a functional diameter about 2.0 nm, leading to K(+) efflux and colloid-osmotic hemolysis. High molecular weight SDS-stable oligomers (>240-kDa) were detected by antibodies against the alpha-subunit with Western blotting. Furthermore, betagamma-CAT showed multiple cellular effects on human umbilical vein endothelial cells. Low dosages of betagamma-CAT (25-50 pM) were able to stimulate cell migration and wound healing. At high concentrations, it induced cell detachment (EC(50) 10 nM) and apoptosis. betagamma-CAT was rapidly endocytosed via intracellular vacuole formation. Under confocal microscope, some of the vacuoles were translocated to nucleus and partially fused with nuclear membrane. Bafilomycin A1 (a specific inhibitor of the vacuolar-type ATPase) and nocodazole (an agent of microtuble depolymerizing), while inhibited betagamma-CAT induced vacuole formation, significantly inhibited betagamma-CAT induced cell detachment, suggesting that betagamma-CAT endocytosis is important for its activities.

Conclusions/significance: These findings illustrate novel cellular functions of non-lens betagamma-cyrstallins and action mechanism via association with trefoil factors, serving as clues for investigating the possible occurrence of similar molecules and action mechanisms in mammals.

Show MeSH
Related in: MedlinePlus