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Xenopus as a model system for the study of GOLPH2/GP73 function: Xenopus GOLPH2 is required for pronephros development.

Li L, Wen L, Gong Y, Mei G, Liu J, Chen Y, Peng T - PLoS ONE (2012)

Bottom Line: The morpholino-mediated knockdown of golph2 results in edema formation.Expression patterns of the transcription factors WT1, Pax2, Pax8, Lim1, GATA3, and HNF1β are also examined in the golph2 knockdown embryos, the expression of WT1 is increased in the glomus and expanded laterally in the pronephric region.We conclude that the deletion of golph2 causes an increase in the expression of WT1, which may promote glomus formation and inhibit pronephric tubule differentiation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Respiratory Disease, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.

ABSTRACT
GOLPH2 is a highly conserved protein. It is upregulated in a number of tumors and is being considered as an emerging biomarker for related diseases. However, the function of GOLPH2 remains unknown. The Xenopus model is used to study the function of human proteins. We describe the isolation and characterization of Xenopus golph2, which dimerizes and localizes to the Golgi in a manner similar to human GOLPH2. Xenopus golph2 is expressed in the pronephros during early development. The morpholino-mediated knockdown of golph2 results in edema formation. Additionally, Nephrin expression is enhanced in the glomus, and the expression of pronephric marker genes, such as atp1b1, ClC-K, NKCC2, and NBC1, is diminished in the tubules and duct. Expression patterns of the transcription factors WT1, Pax2, Pax8, Lim1, GATA3, and HNF1β are also examined in the golph2 knockdown embryos, the expression of WT1 is increased in the glomus and expanded laterally in the pronephric region. We conclude that the deletion of golph2 causes an increase in the expression of WT1, which may promote glomus formation and inhibit pronephric tubule differentiation.

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Xenopus golph2 forms a disulfide bond-linked dimer and interacts with human GOLPH2. A.Whole lysates were prepared from stage 22 embryos of Xenopus and 293 T cell lines; the supernatant was boiled with loading buffer with (lines 1 and 3) or without (lines 2 and 4) 140 mM 2-mercaptoethanol. Samples were resolved on a 12% SDS-PAGE gel and analyzed by Western blotting with anti-golph2, 10F12, (lines 1 and 2) and anti-GOLPH2, 5B12 (lanes 3 and 4). B. The 293 T cell line was co-transfected with pCS2+-golph2 and pCR3.1-GOLPH2-FLAG (line 2) or a single construct with the empty vectors (lines 1 and 3). Cell lysates were prepared at 36 hours post-transfection (Input). The supernatant was immunoprecipitated (IP) with an anti-golph2 antibody, 10F12, and the captured proteins were immunoblotted (IB) with an anti-FLAG antibody.
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pone-0038939-g001: Xenopus golph2 forms a disulfide bond-linked dimer and interacts with human GOLPH2. A.Whole lysates were prepared from stage 22 embryos of Xenopus and 293 T cell lines; the supernatant was boiled with loading buffer with (lines 1 and 3) or without (lines 2 and 4) 140 mM 2-mercaptoethanol. Samples were resolved on a 12% SDS-PAGE gel and analyzed by Western blotting with anti-golph2, 10F12, (lines 1 and 2) and anti-GOLPH2, 5B12 (lanes 3 and 4). B. The 293 T cell line was co-transfected with pCS2+-golph2 and pCR3.1-GOLPH2-FLAG (line 2) or a single construct with the empty vectors (lines 1 and 3). Cell lysates were prepared at 36 hours post-transfection (Input). The supernatant was immunoprecipitated (IP) with an anti-golph2 antibody, 10F12, and the captured proteins were immunoblotted (IB) with an anti-FLAG antibody.

Mentions: It has been shown that human GOLPH2 is a dimer [7]. To determine whether golph2 also exists as a disulfide bond-linked dimer, endogenous golph2 from stage 22 embryos was analyzed using nonreducing SDS-PAGE and detected by Western blotting. As shown in Fig. 1A, golph2 has the same migration pattern as the human GOLPH2 protein. Furthermore, the Golgi segment of Xenopus golph2 was expressed and purified (from 117 to the C terminal of the protein). The molecular mass is around 70 KD, determined by size fractionation column (data not shown). The theoretical molecular mass for monomer is 32.4 KD. It suggests that golph2 is a dimer. Because the dimerization of GOLPH2 is mediated via an interaction between the conserved coiled-coil domains, we sought to determine whether golph2 could interact with GOLPH2 when the two proteins are co-expressed. The plasmids expressing GOLPH2-FLAG and golph2 were transiently transfected in 293 T cells. As shown in Fig. 1B, GOLPH2-FLAG co-precipitated with golph2 (compare lane 2 with lanes 1 and 3), thus indicating that golph2 is capable of forming a hetero-complex with GOLPH2.


Xenopus as a model system for the study of GOLPH2/GP73 function: Xenopus GOLPH2 is required for pronephros development.

Li L, Wen L, Gong Y, Mei G, Liu J, Chen Y, Peng T - PLoS ONE (2012)

Xenopus golph2 forms a disulfide bond-linked dimer and interacts with human GOLPH2. A.Whole lysates were prepared from stage 22 embryos of Xenopus and 293 T cell lines; the supernatant was boiled with loading buffer with (lines 1 and 3) or without (lines 2 and 4) 140 mM 2-mercaptoethanol. Samples were resolved on a 12% SDS-PAGE gel and analyzed by Western blotting with anti-golph2, 10F12, (lines 1 and 2) and anti-GOLPH2, 5B12 (lanes 3 and 4). B. The 293 T cell line was co-transfected with pCS2+-golph2 and pCR3.1-GOLPH2-FLAG (line 2) or a single construct with the empty vectors (lines 1 and 3). Cell lysates were prepared at 36 hours post-transfection (Input). The supernatant was immunoprecipitated (IP) with an anti-golph2 antibody, 10F12, and the captured proteins were immunoblotted (IB) with an anti-FLAG antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375297&req=5

pone-0038939-g001: Xenopus golph2 forms a disulfide bond-linked dimer and interacts with human GOLPH2. A.Whole lysates were prepared from stage 22 embryos of Xenopus and 293 T cell lines; the supernatant was boiled with loading buffer with (lines 1 and 3) or without (lines 2 and 4) 140 mM 2-mercaptoethanol. Samples were resolved on a 12% SDS-PAGE gel and analyzed by Western blotting with anti-golph2, 10F12, (lines 1 and 2) and anti-GOLPH2, 5B12 (lanes 3 and 4). B. The 293 T cell line was co-transfected with pCS2+-golph2 and pCR3.1-GOLPH2-FLAG (line 2) or a single construct with the empty vectors (lines 1 and 3). Cell lysates were prepared at 36 hours post-transfection (Input). The supernatant was immunoprecipitated (IP) with an anti-golph2 antibody, 10F12, and the captured proteins were immunoblotted (IB) with an anti-FLAG antibody.
Mentions: It has been shown that human GOLPH2 is a dimer [7]. To determine whether golph2 also exists as a disulfide bond-linked dimer, endogenous golph2 from stage 22 embryos was analyzed using nonreducing SDS-PAGE and detected by Western blotting. As shown in Fig. 1A, golph2 has the same migration pattern as the human GOLPH2 protein. Furthermore, the Golgi segment of Xenopus golph2 was expressed and purified (from 117 to the C terminal of the protein). The molecular mass is around 70 KD, determined by size fractionation column (data not shown). The theoretical molecular mass for monomer is 32.4 KD. It suggests that golph2 is a dimer. Because the dimerization of GOLPH2 is mediated via an interaction between the conserved coiled-coil domains, we sought to determine whether golph2 could interact with GOLPH2 when the two proteins are co-expressed. The plasmids expressing GOLPH2-FLAG and golph2 were transiently transfected in 293 T cells. As shown in Fig. 1B, GOLPH2-FLAG co-precipitated with golph2 (compare lane 2 with lanes 1 and 3), thus indicating that golph2 is capable of forming a hetero-complex with GOLPH2.

Bottom Line: The morpholino-mediated knockdown of golph2 results in edema formation.Expression patterns of the transcription factors WT1, Pax2, Pax8, Lim1, GATA3, and HNF1β are also examined in the golph2 knockdown embryos, the expression of WT1 is increased in the glomus and expanded laterally in the pronephric region.We conclude that the deletion of golph2 causes an increase in the expression of WT1, which may promote glomus formation and inhibit pronephric tubule differentiation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Respiratory Disease, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.

ABSTRACT
GOLPH2 is a highly conserved protein. It is upregulated in a number of tumors and is being considered as an emerging biomarker for related diseases. However, the function of GOLPH2 remains unknown. The Xenopus model is used to study the function of human proteins. We describe the isolation and characterization of Xenopus golph2, which dimerizes and localizes to the Golgi in a manner similar to human GOLPH2. Xenopus golph2 is expressed in the pronephros during early development. The morpholino-mediated knockdown of golph2 results in edema formation. Additionally, Nephrin expression is enhanced in the glomus, and the expression of pronephric marker genes, such as atp1b1, ClC-K, NKCC2, and NBC1, is diminished in the tubules and duct. Expression patterns of the transcription factors WT1, Pax2, Pax8, Lim1, GATA3, and HNF1β are also examined in the golph2 knockdown embryos, the expression of WT1 is increased in the glomus and expanded laterally in the pronephric region. We conclude that the deletion of golph2 causes an increase in the expression of WT1, which may promote glomus formation and inhibit pronephric tubule differentiation.

Show MeSH
Related in: MedlinePlus