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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

Sequence comparison of βγ-CAT α- and β-subunits.(A) Sequence comparison of βγ-CAT α-subunit with EP37L1 from newt C. pyrrhogaster [2]. The characteristic βγ-crystallin domains were marked. (B) Sequence comparison of βγ-CAT α-subunit C-terminal part (residues 173–287) with an internal fragment (residues 118–209) of ETX from bacterial C. perfringens [21]. (C) Sequence comparison of βγ-CAT β-subunit with human TFF3 [22], Bm-TFF2 [12] and human TFF2 [23]. The characteristic cysteine residues of TFF domains are boxed. In (A) and (C), Peptides corresponding to determined amino acid sequences by Edman degradation are underlined. In (A), (B) and (C), identical residues are shown by asterisks. Gaps have been introduced to optimize the sequence homology.
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pone-0001770-g003: Sequence comparison of βγ-CAT α- and β-subunits.(A) Sequence comparison of βγ-CAT α-subunit with EP37L1 from newt C. pyrrhogaster [2]. The characteristic βγ-crystallin domains were marked. (B) Sequence comparison of βγ-CAT α-subunit C-terminal part (residues 173–287) with an internal fragment (residues 118–209) of ETX from bacterial C. perfringens [21]. (C) Sequence comparison of βγ-CAT β-subunit with human TFF3 [22], Bm-TFF2 [12] and human TFF2 [23]. The characteristic cysteine residues of TFF domains are boxed. In (A) and (C), Peptides corresponding to determined amino acid sequences by Edman degradation are underlined. In (A), (B) and (C), identical residues are shown by asterisks. Gaps have been introduced to optimize the sequence homology.

Mentions: The full protein sequence of the α-subunit, as deduced from its coding cDNA (data deposited in GenBank, EU003881), is composed of 336 amino acids. The determined internal amino acid sequences were found exactly in the deduced sequence (Fig. 3A, underlined), thereby unequivocally confirming the identity of the obtained cDNA clone. A BLAST search in databases revealed that the α-subunit is homologous to ep37 proteins, which are non-lens βγ-crystallin members identified in various tissues of newt C. pyrrhogaster [2]–[4], [20]. Its sequence shows 32% identity with that of EP37L1 (also called EDSP, epidermic differentiation specific protein). In addition, the sequence identity between its βγ-crystallin domains and human AIM1 βγ-crystallin domains 5–6 is 28%. The sequence of the α-subunit actually consists of two distinct parts (Fig. 3A and B). Similar to EP37L1, it contains four consecutive Greek key motifs organized into two βγ-crystallin domains in its N-terminal part (residues 1–170). The C-terminal part (residues 173–287) shows the most homology (sequence identity 24%) to an internal fragment (residues 118–209) of epsilon toxin (ETX) from bacterial Clostridium perfringens [21] (Fig. 3B).


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)

Sequence comparison of βγ-CAT α- and β-subunits.(A) Sequence comparison of βγ-CAT α-subunit with EP37L1 from newt C. pyrrhogaster [2]. The characteristic βγ-crystallin domains were marked. (B) Sequence comparison of βγ-CAT α-subunit C-terminal part (residues 173–287) with an internal fragment (residues 118–209) of ETX from bacterial C. perfringens [21]. (C) Sequence comparison of βγ-CAT β-subunit with human TFF3 [22], Bm-TFF2 [12] and human TFF2 [23]. The characteristic cysteine residues of TFF domains are boxed. In (A) and (C), Peptides corresponding to determined amino acid sequences by Edman degradation are underlined. In (A), (B) and (C), identical residues are shown by asterisks. Gaps have been introduced to optimize the sequence homology.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001770-g003: Sequence comparison of βγ-CAT α- and β-subunits.(A) Sequence comparison of βγ-CAT α-subunit with EP37L1 from newt C. pyrrhogaster [2]. The characteristic βγ-crystallin domains were marked. (B) Sequence comparison of βγ-CAT α-subunit C-terminal part (residues 173–287) with an internal fragment (residues 118–209) of ETX from bacterial C. perfringens [21]. (C) Sequence comparison of βγ-CAT β-subunit with human TFF3 [22], Bm-TFF2 [12] and human TFF2 [23]. The characteristic cysteine residues of TFF domains are boxed. In (A) and (C), Peptides corresponding to determined amino acid sequences by Edman degradation are underlined. In (A), (B) and (C), identical residues are shown by asterisks. Gaps have been introduced to optimize the sequence homology.
Mentions: The full protein sequence of the α-subunit, as deduced from its coding cDNA (data deposited in GenBank, EU003881), is composed of 336 amino acids. The determined internal amino acid sequences were found exactly in the deduced sequence (Fig. 3A, underlined), thereby unequivocally confirming the identity of the obtained cDNA clone. A BLAST search in databases revealed that the α-subunit is homologous to ep37 proteins, which are non-lens βγ-crystallin members identified in various tissues of newt C. pyrrhogaster [2]–[4], [20]. Its sequence shows 32% identity with that of EP37L1 (also called EDSP, epidermic differentiation specific protein). In addition, the sequence identity between its βγ-crystallin domains and human AIM1 βγ-crystallin domains 5–6 is 28%. The sequence of the α-subunit actually consists of two distinct parts (Fig. 3A and B). Similar to EP37L1, it contains four consecutive Greek key motifs organized into two βγ-crystallin domains in its N-terminal part (residues 1–170). The C-terminal part (residues 173–287) shows the most homology (sequence identity 24%) to an internal fragment (residues 118–209) of epsilon toxin (ETX) from bacterial Clostridium perfringens [21] (Fig. 3B).

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