Expression of recombinant human plasminogen in mammalian cells is augmented by suppression of plasmin activity

Busby, Sharon; Mulvihill, Eileen R.; Rao, D. E. C. S.; Kumar, Ashok; Lioubin, Pamela J.; Heipel, Mark; Sprecher, Cindy A.; Halfpap, Laurel; Prunkard, Donna; Gambee, Jay E.

doi:10.1016/s0021-9258(18)98614-x

Cited by 48 publications

(3 citation statements)

References 30 publications

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“…BHK cell lines that secrete low (i.e., 10 pmol per day per 10 6 cells) or high (200 pmol per day per 10 6 cells) levels of fIX were used in these studies. The lowproducing fIX BHK cells were generated by transfecting BHK cells with a human fIX cDNA (18) in the ZEM228 expression vector, which contains a cassette conferring resistance to G418 (19), as previously described (20). The high-producing fIX BHK cells were generated by transfecting BHK cells with the fIX cDNA in the ZEM229 expression vector.…”

Section: Methodsmentioning

confidence: 99%

r-VKORC1 Expression in Factor IX BHK Cells Increases the Extent of Factor IX Carboxylation but Is Limited by Saturation of Another Carboxylation Component or by a Shift in the Rate-Limiting Step

et al. 2006

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Carboxylation of vitamin K-dependent (VKD) proteins is required for their activity and depends upon reduced vitamin K generated by vitamin K oxidoreductase (VKOR) and a redox protein that regenerates VKOR activity. VKD protein carboxylation is inefficient in mammalian cells, and to understand why carboxylation becomes saturated we developed an approach that directly measures intracellular VKD protein carboxylation. Analysis of factor IX (fIX)-expressing BHK cells indicated that slow fIX egress from the endoplasmic reticulum and preferential secretion of the carboxylated form contribute to secreted fIX being more fully-carboxylated. The analysis also revealed the first reported in vivo VKD protein turnover, which was 14-fold faster than occurs in vitro, suggesting facilitation of this process in vivo. r-VKORC1 expression increased the rate of fIX carboxylation and extent of carboxylated fIX ~2-fold, which shows that carboxylation is the rate-limiting step in fIX turnover and which was surprising because turnover in vitro is limited by release of carboxylated fIX. Interestingly, the increases were significantly less than the amount of VKOR overexpression (15-fold). However, when cell extracts were tested in single turnover experiments in vitro, where redox protein is functionally substituted by dithiothreitol, VKOR overexpression increased the fIX carboxylation rate 14-fold, showing r-VKORC1 is functional for supporting fIX carboxylation. These data indicate that the effect of VKOR overexpression is limited in vivo, possibly because a carboxylation component like the redox protein becomes saturated or because another step is now rate-limiting. The studies illustrate the complexity of carboxylation and potential importance of component stoichiometry to overall efficiency.

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Section: Methodsmentioning

confidence: 99%

r-VKORC1 Expression in Factor IX BHK Cells Increases the Extent of Factor IX Carboxylation but Is Limited by Saturation of Another Carboxylation Component or by a Shift in the Rate-Limiting Step

et al. 2006

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“…In ViVo Labeling and Immunoprecipitation. 293 cells stably expressing r-fIX and r-carboxylase (r-carb) were generated by cotransfection with fIX/ZEM228, which contains the full-length fIX cDNA in an expression cassette also encoding a phosphotransferase gene conferring resistance to G418 (17), and carboxylase/ZEM228, which contains a fulllength carboxylase cDNA in the same vector. Lysates from clonal isolates were screened for fIX expression by western analysis and for carboxylase expression by peptide activity (18).…”

Section: Methodsmentioning

confidence: 99%

Carboxylase Overexpression Effects Full Carboxylation but Poor Release and Secretion of Factor IX: Implications for the Release of Vitamin K-Dependent Proteins

et al. 2002

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Vitamin K-dependent (VKD) proteins are modified by the VKD carboxylase as they transit through the endoplasmic reticulum. In a reaction required for their activity, clusters of Glu's are converted to Gla's, and fully carboxylated VKD proteins are normally secreted. In mammalian cell lines expressing high levels of r-VKD proteins, however, under- and uncarboxylated VKD forms are observed. Overexpression of r-carboxylase does not improve carboxylation, but the lack of effect is not understood, and the intracellular events that occur during VKD protein carboxylation have not been investigated. We analyzed carboxylation in 293- and BHK cell lines expressing r-factor IX (fIX) and endogenous carboxylase or overexpressed r-carboxylase. The fIX secreted from the four cell lines was highly carboxylated, indicating fIX-carboxylase engagement during intracellular trafficking. The r-carboxylase was functional for carboxylation: overexpression resulted in a proportional increase in fIX-carboxylase complexes that yielded full fIX carboxylation. Interestingly, the carboxylated fIX product was not efficiently released from the carboxylase in r-fIX/r-carboxylase cells, resulting in decreased fIX secretion. r-Carboxylase overexpression changed the ratios of intracellular fIX to carboxylase, and we therefore developed an in vitro assay to test whether fIX levels affect release. FIX-carboxylase complexes were in vitro carboxylated with or without excess VKD substrate or propeptide. These analyses are the first to dissect the rates of release versus carboxylation and showed that release was much slower than carboxylation. In the absence of excess VKD substrate/propeptide, fIX in the fIX-carboxylase complex was fully carboxylated by 10 min, but 95% was still complexed with carboxylase after 30 min. The presence of excess VKD substrate/propeptide, however, led to a significant increase in VKD product release, possibly through a second propeptide binding site in the carboxylase. The intracellular analyses also showed that the fIX carboxylation rate was slow in vivo and was similar in r-fIX versus r-fIX/r-carboxylase cells, despite the large differences in carboxylase levels. The results suggest that the vitamin K cofactor may be limiting for carboxylation in the cell lines.

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“…To express r-carboxylase in mammalian cells, a BamH I fragment containing the carboxylase with a C-terminal FLAG epitope was isolated from r-carboxylase-FLAG/BacPAK 12 and subcloned into the 229 vector 13 encoding methotrexate resistance. The plasmid was stably transfected in baby hamster kidney (BHK) cells by selection in media containing 1 μM methotrexate.…”

Section: ■ Experimental Section Expression and Purification Of Recomb...mentioning

confidence: 99%

Methylation of γ-Carboxylated Glu (Gla) Allows Detection by Liquid Chromatography–Mass Spectrometry and the Identification of Gla Residues in the γ-Glutamyl Carboxylase

Hallgren¹,

Zhang²,

Kinter

et al. 2013

J. Proteome Res.

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Gamma-carboxylated Glu (Gla) is a post-translational modification required for the activity of vitamin K-dependent (VKD) proteins that has been difficult to study by mass spectrometry due to the properties of this negatively-charged residue. Gla is generated by a single enzyme, the gamma-glutamyl carboxylase, which has broad biological impact because VKD proteins have diverse functions that include hemostasis, apoptosis, and growth control. The carboxylase also contains Glas, of unknown function, and is an integral membrane protein with poor sequence coverage. To locate these Glas, we first established methods that resulted in high coverage (92%) of uncarboxylated carboxylase. Subsequent analysis of carboxylated carboxylase identified a Gla-peptide (729-758) and a missing region (625-647) that was detected in uncarboxylated carboxylase. We therefore developed an approach to methylate Gla, which efficiently neutralized Gla and improved mass spectrometric analysis. Methylation eliminated CO2 loss from Gla, increased the ionization of Gla-containing peptide, and appeared to facilitate trypsin digestion. Methylation of a carboxylated carboxylase tryptic digest identified Glas in the 625-647 peptide. These studies provide valuable information for testing the function of carboxylase carboxylation. The methylation approach for studying Gla by mass spectrometry is an important advance that will be broadly applicable to analyzing other VKD proteins.

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Expression of recombinant human plasminogen in mammalian cells is augmented by suppression of plasmin activity

Cited by 48 publications

References 30 publications

r-VKORC1 Expression in Factor IX BHK Cells Increases the Extent of Factor IX Carboxylation but Is Limited by Saturation of Another Carboxylation Component or by a Shift in the Rate-Limiting Step

r-VKORC1 Expression in Factor IX BHK Cells Increases the Extent of Factor IX Carboxylation but Is Limited by Saturation of Another Carboxylation Component or by a Shift in the Rate-Limiting Step

Carboxylase Overexpression Effects Full Carboxylation but Poor Release and Secretion of Factor IX: Implications for the Release of Vitamin K-Dependent Proteins

Methylation of γ-Carboxylated Glu (Gla) Allows Detection by Liquid Chromatography–Mass Spectrometry and the Identification of Gla Residues in the γ-Glutamyl Carboxylase

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