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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

Purification of βγ-CAT.(A) Lyophilized frog B. maxima skin secretions (5.0 g, containing 550 mg proteins, from a stock in Kunming Institute of Zoology) was dissolved in 10 ml 50 mM Tris-HCl buffer, pH 7.3, containing 5 mM EDTA, dialyzed against the same buffer at 4°C overnight and centrifuged. The supernatant was loaded on a DEAE Sephadex A-50 (Pharmacia) column (2.6×50 cm). The elution was performed at a flow rate of 30 ml/h with a linear NaCl gradient, collecting fractions of 5 ml per tube. Hemolytic activity was mainly found in NaCl-eluted peak III. This peak was collected as indicated by a bar. (B) The peak III from DEAE Sephadex A-50 column was concentrated, then applied on a Sephadex G-100 (Pharmacia, superfine) column (2.6×100 cm) equilibrated with 50 mM Tris-HCl buffer, pH 7.8, containing 150 mM NaCl and 5 mM EDTA. Elution was achieved with the same buffer at a flow rate of 12 ml/h, collecting fractions of 2 ml per tube. The hemolytic activity was found in peak II. In A and B, The protein concentration was estimated from the absorbance at 280 nm (•). The hemolytic activity on human erythrocytes (○) was determined as described in “Methods”. (C) The peak II of Sephadex G-100 column was lyophilized and dialyzed against 25 mM Tris-HCl, pH 7.8, for 24 h at 4°C, and finally applied on an AKTA Mono-Q HR5/5 anion ion exchange column equilibrated with the same buffer. Elution was performed at a flow rate of 1.0 ml/min with the NaCl gradient, as shown in the figure. Hemolytic activity was found in peaks I, II and III. Peak I is highly purified βγ-CAT, as indicated by an arrow.
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pone-0001770-g001: Purification of βγ-CAT.(A) Lyophilized frog B. maxima skin secretions (5.0 g, containing 550 mg proteins, from a stock in Kunming Institute of Zoology) was dissolved in 10 ml 50 mM Tris-HCl buffer, pH 7.3, containing 5 mM EDTA, dialyzed against the same buffer at 4°C overnight and centrifuged. The supernatant was loaded on a DEAE Sephadex A-50 (Pharmacia) column (2.6×50 cm). The elution was performed at a flow rate of 30 ml/h with a linear NaCl gradient, collecting fractions of 5 ml per tube. Hemolytic activity was mainly found in NaCl-eluted peak III. This peak was collected as indicated by a bar. (B) The peak III from DEAE Sephadex A-50 column was concentrated, then applied on a Sephadex G-100 (Pharmacia, superfine) column (2.6×100 cm) equilibrated with 50 mM Tris-HCl buffer, pH 7.8, containing 150 mM NaCl and 5 mM EDTA. Elution was achieved with the same buffer at a flow rate of 12 ml/h, collecting fractions of 2 ml per tube. The hemolytic activity was found in peak II. In A and B, The protein concentration was estimated from the absorbance at 280 nm (•). The hemolytic activity on human erythrocytes (○) was determined as described in “Methods”. (C) The peak II of Sephadex G-100 column was lyophilized and dialyzed against 25 mM Tris-HCl, pH 7.8, for 24 h at 4°C, and finally applied on an AKTA Mono-Q HR5/5 anion ion exchange column equilibrated with the same buffer. Elution was performed at a flow rate of 1.0 ml/min with the NaCl gradient, as shown in the figure. Hemolytic activity was found in peaks I, II and III. Peak I is highly purified βγ-CAT, as indicated by an arrow.

Mentions: Each step of the purification was followed by determining hemolytic activity on human erythrocytes for each fraction. Initiated with 5.0 g of lyophilized B. maxima skin secretions (containing 550 mg proteins), DEAE Sephadex A-50 column at pH 7.3 resulted in separation of three protein peaks, in which hemolytic activity was predominantly associated with NaCl-eluted peak III (Fig. 1A). In addition to antimicrobial and mytropic activities caused by antimicrobial peptides and Bv8 analogous [18], [19], platelet activation activity was found in NaCl-free eluted peaks I and II, from which Bm-TFF2 has been purified previously [12]. Subsequent gel filtration of DEAE-Sephadex A-50 column peak III resulted in separation of three protein peaks, in which hemolytic activity was found in peak II (Fig. 1B). The peak II of Sephadex G-100 column was collected and finally applied on an AKTA Mono-Q HR5/5 anion ion exchange column. This purification step resulted in separation of several protein peaks, in which peak I is highly purified βγ-CAT associated again with hemolytic activity (Fig. 1C). Finally, about 650 µg product was obtained. Purified βγ-CAT showed lethal toxicity on mice. The LD50 values were 20 µg/kg and 400 µg/kg under intravenous and intraperitoneal injections, respectively. Purified protein showed neither proteolytic nor phospholipase A2 activity. Hemolytic activity was also determined in Peaks II and III of AKTA Mono-Q HR5/5 column, but these fractions lacked lethal toxicity on mice (LD50 value>5 mg/kg under intraperitoneal injection) and native polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE analysis revealed their heterogeneity.


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)

Purification of βγ-CAT.(A) Lyophilized frog B. maxima skin secretions (5.0 g, containing 550 mg proteins, from a stock in Kunming Institute of Zoology) was dissolved in 10 ml 50 mM Tris-HCl buffer, pH 7.3, containing 5 mM EDTA, dialyzed against the same buffer at 4°C overnight and centrifuged. The supernatant was loaded on a DEAE Sephadex A-50 (Pharmacia) column (2.6×50 cm). The elution was performed at a flow rate of 30 ml/h with a linear NaCl gradient, collecting fractions of 5 ml per tube. Hemolytic activity was mainly found in NaCl-eluted peak III. This peak was collected as indicated by a bar. (B) The peak III from DEAE Sephadex A-50 column was concentrated, then applied on a Sephadex G-100 (Pharmacia, superfine) column (2.6×100 cm) equilibrated with 50 mM Tris-HCl buffer, pH 7.8, containing 150 mM NaCl and 5 mM EDTA. Elution was achieved with the same buffer at a flow rate of 12 ml/h, collecting fractions of 2 ml per tube. The hemolytic activity was found in peak II. In A and B, The protein concentration was estimated from the absorbance at 280 nm (•). The hemolytic activity on human erythrocytes (○) was determined as described in “Methods”. (C) The peak II of Sephadex G-100 column was lyophilized and dialyzed against 25 mM Tris-HCl, pH 7.8, for 24 h at 4°C, and finally applied on an AKTA Mono-Q HR5/5 anion ion exchange column equilibrated with the same buffer. Elution was performed at a flow rate of 1.0 ml/min with the NaCl gradient, as shown in the figure. Hemolytic activity was found in peaks I, II and III. Peak I is highly purified βγ-CAT, as indicated by an arrow.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2262142&req=5

pone-0001770-g001: Purification of βγ-CAT.(A) Lyophilized frog B. maxima skin secretions (5.0 g, containing 550 mg proteins, from a stock in Kunming Institute of Zoology) was dissolved in 10 ml 50 mM Tris-HCl buffer, pH 7.3, containing 5 mM EDTA, dialyzed against the same buffer at 4°C overnight and centrifuged. The supernatant was loaded on a DEAE Sephadex A-50 (Pharmacia) column (2.6×50 cm). The elution was performed at a flow rate of 30 ml/h with a linear NaCl gradient, collecting fractions of 5 ml per tube. Hemolytic activity was mainly found in NaCl-eluted peak III. This peak was collected as indicated by a bar. (B) The peak III from DEAE Sephadex A-50 column was concentrated, then applied on a Sephadex G-100 (Pharmacia, superfine) column (2.6×100 cm) equilibrated with 50 mM Tris-HCl buffer, pH 7.8, containing 150 mM NaCl and 5 mM EDTA. Elution was achieved with the same buffer at a flow rate of 12 ml/h, collecting fractions of 2 ml per tube. The hemolytic activity was found in peak II. In A and B, The protein concentration was estimated from the absorbance at 280 nm (•). The hemolytic activity on human erythrocytes (○) was determined as described in “Methods”. (C) The peak II of Sephadex G-100 column was lyophilized and dialyzed against 25 mM Tris-HCl, pH 7.8, for 24 h at 4°C, and finally applied on an AKTA Mono-Q HR5/5 anion ion exchange column equilibrated with the same buffer. Elution was performed at a flow rate of 1.0 ml/min with the NaCl gradient, as shown in the figure. Hemolytic activity was found in peaks I, II and III. Peak I is highly purified βγ-CAT, as indicated by an arrow.
Mentions: Each step of the purification was followed by determining hemolytic activity on human erythrocytes for each fraction. Initiated with 5.0 g of lyophilized B. maxima skin secretions (containing 550 mg proteins), DEAE Sephadex A-50 column at pH 7.3 resulted in separation of three protein peaks, in which hemolytic activity was predominantly associated with NaCl-eluted peak III (Fig. 1A). In addition to antimicrobial and mytropic activities caused by antimicrobial peptides and Bv8 analogous [18], [19], platelet activation activity was found in NaCl-free eluted peaks I and II, from which Bm-TFF2 has been purified previously [12]. Subsequent gel filtration of DEAE-Sephadex A-50 column peak III resulted in separation of three protein peaks, in which hemolytic activity was found in peak II (Fig. 1B). The peak II of Sephadex G-100 column was collected and finally applied on an AKTA Mono-Q HR5/5 anion ion exchange column. This purification step resulted in separation of several protein peaks, in which peak I is highly purified βγ-CAT associated again with hemolytic activity (Fig. 1C). Finally, about 650 µg product was obtained. Purified βγ-CAT showed lethal toxicity on mice. The LD50 values were 20 µg/kg and 400 µg/kg under intravenous and intraperitoneal injections, respectively. Purified protein showed neither proteolytic nor phospholipase A2 activity. Hemolytic activity was also determined in Peaks II and III of AKTA Mono-Q HR5/5 column, but these fractions lacked lethal toxicity on mice (LD50 value>5 mg/kg under intraperitoneal injection) and native polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE analysis revealed their heterogeneity.

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