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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

Co-localization of βγ-CAT positive vacuoles with endocytic marker proteins.PBS control (A1–A3, D1–D3) and βγ-CAT treated cells (10 nM, 10 min) (B1–C3, E1–F3) were first stained with specific antibodies against vacuolar type ATPase (A1–C3) and rab5 (D1–F3), respectively by indirect immunofluorescence (Cy3 labeled secondary antibodies, red channel). After washed with PBS (containing 1% BSA) for three times, the cells were stained again with FITC-labeled antibodies against βγ-CAT α-subunit (green channel). Nucleus was counter-stained by Hoechst (blue channel). Co-localization of βγ-CAT with vacuolar type ATPase (B3, C3) or rab5 (E3, F3) in intracellular vacuoles was indicated by arrows. The vacuoles reached to nuclear membrane were also observed, as indicated in C3, F3 by arrows. Scale bars equal to10 µm.
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pone-0001770-g010: Co-localization of βγ-CAT positive vacuoles with endocytic marker proteins.PBS control (A1–A3, D1–D3) and βγ-CAT treated cells (10 nM, 10 min) (B1–C3, E1–F3) were first stained with specific antibodies against vacuolar type ATPase (A1–C3) and rab5 (D1–F3), respectively by indirect immunofluorescence (Cy3 labeled secondary antibodies, red channel). After washed with PBS (containing 1% BSA) for three times, the cells were stained again with FITC-labeled antibodies against βγ-CAT α-subunit (green channel). Nucleus was counter-stained by Hoechst (blue channel). Co-localization of βγ-CAT with vacuolar type ATPase (B3, C3) or rab5 (E3, F3) in intracellular vacuoles was indicated by arrows. The vacuoles reached to nuclear membrane were also observed, as indicated in C3, F3 by arrows. Scale bars equal to10 µm.

Mentions: We next analyzed if, βγ-CAT positive vacuoles were endowed with endocytic proteins, like rab5, rab7 and vacuolar-type ATPase that are markers of different endocytic compartments involved in intracellular trafficking, with specific antibodies against rab5, rab7, vacuolar-type ATPase and βγ-CAT α-subunit. Among the endocytic proteins tested, rab5 and vacuolar-type ATPase, but not rab7, were found to be co-localized with βγ-CAT in some intracellular vacuoles, as indicated by arrows in Fig. 10-B3, C3, E3 and F3.


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)

Co-localization of βγ-CAT positive vacuoles with endocytic marker proteins.PBS control (A1–A3, D1–D3) and βγ-CAT treated cells (10 nM, 10 min) (B1–C3, E1–F3) were first stained with specific antibodies against vacuolar type ATPase (A1–C3) and rab5 (D1–F3), respectively by indirect immunofluorescence (Cy3 labeled secondary antibodies, red channel). After washed with PBS (containing 1% BSA) for three times, the cells were stained again with FITC-labeled antibodies against βγ-CAT α-subunit (green channel). Nucleus was counter-stained by Hoechst (blue channel). Co-localization of βγ-CAT with vacuolar type ATPase (B3, C3) or rab5 (E3, F3) in intracellular vacuoles was indicated by arrows. The vacuoles reached to nuclear membrane were also observed, as indicated in C3, F3 by arrows. Scale bars equal to10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001770-g010: Co-localization of βγ-CAT positive vacuoles with endocytic marker proteins.PBS control (A1–A3, D1–D3) and βγ-CAT treated cells (10 nM, 10 min) (B1–C3, E1–F3) were first stained with specific antibodies against vacuolar type ATPase (A1–C3) and rab5 (D1–F3), respectively by indirect immunofluorescence (Cy3 labeled secondary antibodies, red channel). After washed with PBS (containing 1% BSA) for three times, the cells were stained again with FITC-labeled antibodies against βγ-CAT α-subunit (green channel). Nucleus was counter-stained by Hoechst (blue channel). Co-localization of βγ-CAT with vacuolar type ATPase (B3, C3) or rab5 (E3, F3) in intracellular vacuoles was indicated by arrows. The vacuoles reached to nuclear membrane were also observed, as indicated in C3, F3 by arrows. Scale bars equal to10 µm.
Mentions: We next analyzed if, βγ-CAT positive vacuoles were endowed with endocytic proteins, like rab5, rab7 and vacuolar-type ATPase that are markers of different endocytic compartments involved in intracellular trafficking, with specific antibodies against rab5, rab7, vacuolar-type ATPase and βγ-CAT α-subunit. Among the endocytic proteins tested, rab5 and vacuolar-type ATPase, but not rab7, were found to be co-localized with βγ-CAT in some intracellular vacuoles, as indicated by arrows in Fig. 10-B3, C3, E3 and F3.

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