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Processing and expression of rat and human clotting factor-X-encoding cDNAs
C Stanton1, R P Ross, S Hutson
1Department of Medicine, Wake Forest University Medical Center, Winston Salem, NC 27157, USA.
Gene
|March 9, 1996
Summary
Researchers cloned rat clotting factor X (FX) cDNA, finding its propeptide cleavage site differs from human FX. This difference may explain variations in how human and rat FX are processed by proteases.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Clotting Factor X (FX) is a vitamin-K-dependent protein crucial for blood coagulation.
- Vitamin-K-dependent proteins typically have a dibasic sequence at their propeptide cleavage site.
- Human FX has a unique Thr-Arg sequence at this site, unlike the expected dibasic sequence.
Purpose of the Study:
- To clone and characterize rat clotting factor X (FX) cDNA.
- To compare the propeptide cleavage site of rat FX with that of human FX.
- To investigate the implications of sequence differences on FX processing.
Main Methods:
- Cloning of rat liver cDNA library to obtain FX encoding cDNA.
- DNA sequencing to determine the nucleotide and amino acid sequences.
- Expression of rat and human FX cDNAs in Cos-1 cells.
- Analysis of secreted Factor X forms (single- and two-chain).
Main Results:
- Rat FX cDNA encodes a protein with an Arg-Arg dibasic sequence at the propeptide cleavage site.
- Human and rat FX share 76% amino acid sequence identity but differ at the cleavage site.
- Expression studies showed both rat and human cDNAs produced single- and two-chain FX.
- Two-chain forms, lacking the propeptide, were secreted at similar rates for both species.
Conclusions:
- Rat FX processing follows the typical pattern for vitamin-K-dependent proteins.
- The unique Thr-Arg cleavage site in human FX suggests involvement of additional proteases.
- Further investigation is needed to identify proteases processing human FX at the Thr-Arg motif.