In vitro expression demonstrates impaired secretion of the gammaAsn319, Asp320 deletion variant fibrinogen
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Updated: Aug 16, 2026
High-throughput Purification of Affinity-tagged Recombinant Proteins
Published on: August 26, 2012
Satomi Kani1, Fumiko Terasawa, Susan T Lord
1Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, 3-1-1 Asahi, Matsumoto, 390-8621, Japan.
Abstract:
The hypodysfibrinogenemia Otsu is caused by the two-residue deletion, gammaAsn319 and gammaAsp320. Analysis of plasma or purified fibrinogen from the heterozygous propositus revealed that the amount of variant gamma-chain was lower than that of normal gamma-chain. In order to examine the basis for this difference, we transfected Chinese hamster ovary cells and established stable cell lines that expressed both chains, gammaDelta/gammaN, only the normal chain, gammaN, and only the variant chain, gammaDelta. We measured fibrinogen concentration of confluent cultures by ELISA. We found the ratios of the concentrations in the media to the concentrations in the cell lysates of gammaDelta, gammaDelta/gammaN, and gammaN-cells were 0.42, 0.60, and 1.00, respectively. We measured the concentrations of the gammaDelta and gammaN chains by densitometric analysis of samples following separation by SDS-PAGE and found the fraction of gammaDelta-chains in cell lysates was always greater than the fraction in the respective culture media. We examined the kinetics of fibrinogen synthesis, assembly and secretion in pulse-chase experiments, and found that the gammaDelta-chain was assembled into intact fibrinogen at a rate similar to assembly of the gammaN-chain into normal fibrinogen, but was secreted into the medium at a slightly slower rate than normal fibrinogen. Considered together, these experiments indicate secretion of the variant fibrinogen was slightly impaired. These results suggest that the reduced level of gammaDelta319,320 fibrinogen in the plasma of the Otsu patient arises from modestly impaired secretion of this variant fibrinogen.
Insights
Hypodysfibrinogenemia Otsu, caused by a gamma-chain deletion, results in lower variant fibrinogen levels. Impaired secretion of this variant fibrinogen explains its reduced presence in patients.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- Hypodysfibrinogenemia Otsu is a genetic disorder characterized by a specific two-residue deletion (gammaAsn319 and gammaAsp320) in the fibrinogen gamma-chain.
- Plasma analysis of affected individuals reveals a reduced amount of the variant gamma-chain compared to the normal gamma-chain.
Purpose of the Study:
- To investigate the underlying cause for the reduced levels of variant fibrinogen in hypodysfibrinogenemia Otsu.
- To determine if the variant gamma-chain affects fibrinogen secretion or stability.
Main Methods:
- Stable Chinese hamster ovary (CHO) cell lines were established to express normal (gammaN) and variant (gammaDelta) gamma-chains, individually or together (gammaDelta/gammaN).
- Fibrinogen concentrations in cell lysates and culture media were quantified using ELISA.
- SDS-PAGE and densitometry were used to measure gamma-chain fractions.
- Pulse-chase experiments assessed fibrinogen synthesis, assembly, and secretion kinetics.
Main Results:
- The ratio of secreted to intracellular fibrinogen was significantly lower for cells expressing the variant gammaDelta-chain (0.42) compared to normal gammaN-cells (1.00).
- The variant gammaDelta-chain was assembled into fibrinogen at a rate similar to the normal gammaN-chain.
- Secretion of the variant fibrinogen into the medium was slightly slower than that of normal fibrinogen.
Conclusions:
- The reduced level of variant gammaDelta319,320 fibrinogen in hypodysfibrinogenemia Otsu patients is attributed to a modest impairment in the secretion of this variant fibrinogen.
- These findings highlight the role of gamma-chain structure in regulating fibrinogen secretion and plasma levels.
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