Deletion of the GPI pre-anchor sequence in human p97—a general approach for generating the soluble form of GPI-linked proteins

Yang, Jenq-Lin; Tiong, Jacqueline; Kennard, Malcolm L.; Jefferies, Wilfred A.

doi:10.1016/j.pep.2003.09.007

Cited by 24 publications

(36 citation statements)

References 54 publications

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“…In our system, Neuritin changed the morphology of NIH 3T3 cells and supported their anchorage-independent growth but did not seem to increase proliferation. We observed morphologic changes upon expressing either a truncated, secreted, or full-length version of Neuritin in NIH 3T3 cells, suggesting that GPI-linked Neuritin might give rise to a secreted version via phospholipase cleavage as observed for many GPI-linked proteins (37). Further studies will help to clarify this question.…”

Section: Discussionmentioning

confidence: 75%

Novel Cellular Genes Essential for Transformation of Endothelial Cells by Kaposi's Sarcoma–Associated Herpesvirus

et al. 2005

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Kaposi's sarcoma-associated herpesvirus (KSHV) is involved in the development of lymphoproliferative diseases and Kaposi's sarcoma. The oncogenicity of this virus is reflected in vitro by its ability to transform B cells and endothelial cells. Infection of dermal microvascular endothelial cells (DMVEC) transforms the cells from a cobblestone-like monolayer to foci-forming spindle cells. This transformation is accompanied by dramatic changes in the cellular transcriptome. Known oncogenes, such as c-Kit, are among the KSHV-induced host genes. We previously showed that c-Kit is an essential cellular component of the KSHV-mediated transformation of DMVEC. Here, we test the hypothesis that the transformation process can be used to discover novel oncogenes. When expression of a panel of KSHV-induced cellular transcripts was inhibited with antisense oligomers, we observed inhibition of DMVEC proliferation and foci formation using antisense molecules to RDC1 and Neuritin. We further showed that transformation of KSHV-infected DMVEC was inhibited by small interfering RNA directed at RDC1 or Neuritin. Ectopic expression of Neuritin in NIH 3T3 cells resulted in changes in cell morphology and anchorage-independent growth, whereas RDC1 ectopic expression significantly increased cell proliferation. In addition, both RDC1-and Neuritin-expressing cells formed tumors in nude mice. RDC1 is an orphan G proteincoupled receptor, whereas Neuritin is a growth-promoting protein known to mediate neurite outgrowth. Neither gene has been previously implicated in tumorigenesis. Our data suggest that KSHV-mediated transformation involves exploitation of the hitherto unrealized oncogenic properties of RDC1 and Neuritin. (Cancer Res 2005; 65(12): 5084-95)

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Section: Discussionmentioning

confidence: 75%

Novel Cellular Genes Essential for Transformation of Endothelial Cells by Kaposi's Sarcoma–Associated Herpesvirus

et al. 2005

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“…However, the sequence was complete at the carboxy-terminal end, suggesting that no cleavage had occurred there. Current evidence indicates that these cleavable regions found in GPI-anchored proteins do not have conserved sequences (Yang et al 2004), so there is nothing with which the PEBP 1 sequence can be compared. Thus it would appear that DF-R/PEBP 1 lacks the typical structure characteristic of GPI-anchored proteins.…”

Section: Discussionmentioning

confidence: 99%

“…It is interesting to note that , 80% of GPI-anchored proteins can exist in multiple forms, e.g. soluble as well as anchored externally (Yang et al 2004).…”

Section: Discussionmentioning

confidence: 99%

A mouse sperm decapacitation factor receptor is phosphatidylethanolamine-binding protein 1

Gibbons¹,

Adeoya-Osiguwa²,

Fraser³

2005

Reproduction

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Capacitation is a pivotal event for mammalian spermatozoa, involving the loss of surface proteins known as decapacitation factors (DF) and consequent acquisition of fertilizing ability. Earlier studies showed that a mouse sperm DF binds to a receptor, DF-R, whose attachment to the sperm plasma membrane appears to involve a glycosylphosphatidylinositol (GPI) anchor. In the present study, purification and subsequent sequencing of DF-R has identified this , 23 kDa protein as phosphatidylethanolamine-binding protein 1 (PEBP 1). To obtain functional evidence that supports sequence homology data, purified recombinant PEBP 1 and PEBP 2 were evaluated for biological activity. While PEBP 1 was able to remove DF activity in solution at concentrations above , 1 nmol/l, PEBP 2 was ineffective, even at 600 nmol/l; this confirmed that DF-R is PEBP 1. Anti-PEBP 1 antiserum recognized recombinant PEBP 1 and a ,23 kDa protein in both mouse and human sperm lysates. Immunolocalization studies revealed that DF-R/PEBP 1 is located on the acrosomal cap, the post-acrosomal region and the flagellum of both mouse and human spermatozoa, with epitope accessibility being capacitation state-dependent and reversible. Treatment of cells with a phospholipase able to cleave GPI anchors essentially abolished immunostaining, thus confirming the extracellular location of DF-R/PEBP 1. We suggest that DF-R/PEBP 1 plays its fundamental role in capacitation by causing alterations in the sperm plasma membrane in both head and flagellum, with functional consequences for membrane-associated proteins. Obtaining more detail about DF $ DF-R interactions could lead to useful applications in both fertility treatments and new contraceptive approaches.

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“…The GPI-deleted MTf was employed because deletion of the GPI anchor has been shown to allow production of the soluble form of MTf. 20 In addition, a bacteriophage T4 fibritin trimerization domain was used to connect the two binding domains, as fiber exists as trimer in Ad5 virions and trimerized sCAR is expected to bind fiber sCAR-MTf-mediated Ad5 transcytosis across the BBB Y Tang et al better than sCAR monomer. Two His 6 epitopes (in the middle and at the C-terminal end) were included to ensure protein purification.…”

Section: Generation Of Bi-specific Adaptor Protein Scar-mtfmentioning

confidence: 99%

Directing adenovirus across the blood–brain barrier via melanotransferrin (P97) transcytosis pathway in an in vitro model

et al. 2006

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Adenovirus serotype 5 (Ad5) is widely used in the development of gene therapy protocols. However, current gene therapy strategies involving brain are mostly based on intracranial injection. A major obstacle for systemically administered vectors to infect brain tissue is the blood-brain barrier (BBB). One strategy to cross the BBB is transcytosis, a transcellular transport process that shuttles a molecule from one side of the cell to the other side. Recently, melanotransferrin (MTf)/P97 was found to be able to cross the BBB and accumulate in brain. We thus hypothesize that re-directing Ad5 vectors to the MTf transcytosis pathway may facilitate Ad5 vectors to cross the BBB. To test this hypothesis, we constructed a bi-specific adaptor protein containing the extracellular domain of the coxsackie-adenovirus receptor (CAR) and the full-length melanotransferrin (sCAR-MTf), and investigated its ability to re-direct Ad5 vectors to the MTf transcytosis pathway. We found this adaptor protein could redirect Ad5 to the MTf transcytosis pathway in an in vitro BBB model, and the transcytosed Ad5 viral particles retained their native infectivity. The sCAR-MTf-mediated Ad5 transcytosis was temperature-and dose dependent. In addition, we examined the directionality of sCAR-MTf-mediated Ad5 transcytosis, and found the efficiency of apical-to-basal transcytosis was much higher than that of basal-to-apical direction, supporting a role of this strategy in transporting Ad5 vectors towards the brain. Taken together, our study demonstrated that re-directing Ad5 to the MTf transcytosis pathway could facilitate gene delivery across the BBB.

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Deletion of the GPI pre-anchor sequence in human p97—a general approach for generating the soluble form of GPI-linked proteins

Cited by 24 publications

References 54 publications

Novel Cellular Genes Essential for Transformation of Endothelial Cells by Kaposi's Sarcoma–Associated Herpesvirus

Novel Cellular Genes Essential for Transformation of Endothelial Cells by Kaposi's Sarcoma–Associated Herpesvirus

A mouse sperm decapacitation factor receptor is phosphatidylethanolamine-binding protein 1

Directing adenovirus across the blood–brain barrier via melanotransferrin (P97) transcytosis pathway in an in vitro model

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