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Cited by 70Hemophilia Treatment and ResearchViral Infectious Diseases and Gene Expression in InsectsVitamin K Research Studies

In vitro and in vivo functional characterization of bovine vitamin K-dependent gamma-carboxylase expressed in Chinese hamster ovary cells

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Rehemtulla, A, Roth, D A, Wasley, L C, Kuliopulos, A, Walsh, C T, Furie, B, Furie, B C, Kaufman, R J·1993·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.90.10.4611PMC PMC46562
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Abstract

Coagulation factor IX is a serine protease for which high-level expression of biologically active protein in heterologous cells is limited due to inefficient proteolytic removal of the propeptide as well as vitamin K-dependent carboxylation of multiple amino-terminal glutamic acid residues. We have overexpressed the vitamin K-dependent gamma-carboxylase cDNA and monitored its ability to improve factor IX processing in Chinese hamster ovary (CHO) cells. From amino acid sequence analysis of bovine liver vitamin K-dependent gamma-carboxylase, degenerate oligonucleotides were used to isolate a 3.5-kbp bovine cDNA that encoded a 758-residue open reading frame. Expression of the cDNA in COS-1 and CHO cells yielded 17- and 16-fold increases in the in vitro gamma-carboxylase activity of microsomal preparations, respectively. Anti-serum raised against a predicted peptide sequence reacted with a 94-kDa polypeptide in the partially purified bovine liver preparation as well as in stably transfected CHO cells. The amount of antibody reactivity correlated with the increased ability to carboxylate a peptide substrate in vitro. These results strongly support the conclusion that the cDNA encodes the vitamin K-dependent gamma-carboxylase. Transient transfection of the gamma-carboxylase expression vector into factor IX-expressing CHO cells did not improve the specific procoagulant activity of secreted factor IX. In contrast, transfection of an expression vector encoding the propeptide processing enzyme PACE (paired basic amino acid cleaving enzyme) did improve the specific activity of secreted factor IX by 3-fold. These results demonstrate that the ability of CHO cells to modify glutamic acid residues to gamma-carboxyglutamic acid in secreted factor IX is not limited by the expression of the vitamin K-dependent gamma-carboxylase alone.

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  • CHO cells have limited ability to gamma-carboxylate recombinant proteins such as clotting factors.

    “CHO cells also have limited ability to gamma-carboxylate recombinant proteins such as clotting factors (Kumar 2015), even though some improvements have been achieved through metabolic engineering work (Rehemtulla et al., 1993;Liu et al., 2014).”

    From Therapeutic glycoprotein production in mammalian cells (2017) · cites this paper as Rehemtulla et al., 1993

    The paper reports that expressing gamma-carboxylase in CHO cells increased in vitro enzyme activity, but did not improve the specific procoagulant activity of secreted factor IX. Expressing the propeptide-processing enzyme PACE improved factor IX activity threefold. Thus, the cited work supports an improvement through cell engineering, but not an improvement in gamma-carboxylation of the secreted clotting factor. (LLM, conf=0.84)

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Research works printed but not linked: 24

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Cited by

Sorted by citations

Authors
Rehemtulla, A., Roth, D. A., Wasley, L. C., Kuliopulos, A., Walsh, C. T., Furie, B., Furie, B. C., & Kaufman, R. J.
Affiliations
Genetics Institute, Cambridge, MA 02140.
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
1993-05-15
Volume
90
Issue
10
Pages
4611-4615
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.90.10.4611
PMC
PMC46562
OpenAlex
W2020338953
Cited by
70
References
0
Citations by year
2022: 2 · 2020: 1 · 2019: 1 · 2017: 1 · 2016: 2 · 2015: 3 · 2014: 2 · 2013: 2 · 2012: 1
FWCI
2.50
Type
journal article
Language
en
Open access
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health (5k11hl002574-04)
  • National Heart, Lung, and Blood Institute (HL42443, HL02574, P01HL042443)
Topics
  • Health Sciences › Medicine › Hematology › Hemophilia Treatment and Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
  • Health Sciences › Nursing › Nutrition and Dietetics › Vitamin K Research Studies
Keywords
coagulation factor IX, gamma-carboxylation, propeptide processing, PACE, bovine liver, cDNA cloning, heterologous expression, CHO cells, COS-1 cells, procoagulant activity, glutamate residues, gamma-carboxyglutamic acid
MeSH terms
Amino Acid Sequence, Animals, Cattle, Cloning, Molecular, Factor IX, Cricetinae, Ligases, Molecular Sequence Data, Oligodeoxyribonucleotides, Peptides, Protein Processing, Post-Translational, Recombinant Proteins, RNA, Messenger, Transfection, Gene Expression, Sequence Alignment, CHO Cells, Carbon-Carbon Ligases

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