Document Detail


Carboxylase overexpression effects full carboxylation but poor release and secretion of factor IX: implications for the release of vitamin K-dependent proteins.
MedLine Citation:
PMID:  12475254     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Kevin W Hallgren; Eric L Hommema; Beth A McNally; Kathleen L Berkner
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biochemistry     Volume:  41     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  2002 Dec 
Date Detail:
Created Date:  2002-12-11     Completed Date:  2003-01-30     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  15045-55     Citation Subset:  IM    
Affiliation:
Department of Molecular Cardiology, Lerner Research Institute, Cleveland, Ohio 44195, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Carbon-Carbon Ligases / biosynthesis*,  genetics*,  physiology
Cell Line
Cricetinae
Factor IX / antagonists & inhibitors,  genetics,  metabolism*,  secretion*
Genetic Vectors / metabolism,  secretion
Humans
Intracellular Fluid / metabolism,  secretion
Macromolecular Substances
Protein Binding / genetics
Protein Processing, Post-Translational / genetics
Recombinant Proteins / antagonists & inhibitors,  metabolism,  secretion
Substrate Specificity / genetics
Transfection
Vitamin K / physiology*
Grant Support
ID/Acronym/Agency:
R01 HL55666/HL/NHLBI NIH HHS
Chemical
Reg. No./Substance:
0/Macromolecular Substances; 0/Recombinant Proteins; 12001-79-5/Vitamin K; 9001-28-9/Factor IX; EC 6.4.-/Carbon-Carbon Ligases; EC 6.4.-/glutamyl carboxylase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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