Sequence of the rat factor VIII cDNA

Summary

FactorVIII acts as an essential compound of the tenase complex of the coagulation system. Herein we report the cDNA of the rat factor VIII. The rat cDNA comprises 6777 nucleotides and encodes a protein of 2258 amino acids, 61 amino acids less than mouse and 92 amino acids less than human factor VIII. The overall identity compared to human cDNA is 61% on the cDNA and 51% on the amino acid level. In cDNA, highest levels of sequence identity can be observed in the A and C domains (ranging between 68% and 73%), whereas B domain and the small acidic regions are more divergent (34%-49%). Compared to mouse and human most sites for posttranslational modifications such as sulfatation and glycosylation as well as thrombin and protein C cleavage sites are conserved in rat. Alternative transcripts lacking exon 17 and/or comprising additional 26 bp due to alternative splicing of exon 20 were found. Furthermore, 13 polymorphisms (seven in exon 14, one in exon 20, 23, 24, and 25, two in the 3’UTR) three of which lead to an amino acid exchange could be detected. Our findings might provide new insights into the structure-function analysis of the factor VIII protein and might prove useful for future animal models addressing the function of factor VIII.

Keywords

Factor VIII - rat - cDNA - alternative splicing - exon skipping

• References

• 1 Fay PJ, Anderson MT, Chavin SI. et al.. The size of human factor VIII heterodimers and the effects produced by thrombin. Biochim Biophys Acta 1986; 871 (03) 268-78.
• 2 Thompson AR. Structure and Function of the Factor VIII Gene and Protein. Semin Thromb Hemost 2003; 29 (01) 11-22.
• 3 Toole JJ, Knopf JL, Wozney JM. et al.. Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature 1984; 312 5992 342-7.
• 4 Gitschier J, Wood WI, Goralka TM. et al.. Characterization of the human factor VIII gene. Nature 1984; 312 5992 326-30.
• 5 Elder B, Lakich D, Gitschier J. Sequence of the murine factor VIII cDNA. Genomics 1993; 16 (02) 374-9.
• 6 Healey JF, Lubin IM, Lollar P. The cDNA and derived amino acid sequence of porcine factor VIII. Blood 1996; 88 (11) 4209-14.
• 7 Cameron C, Notley C, Hoyle S. et al.. The canine factor VIII cDNA and 5’ flanking sequence. Thromb Haemost 1998; 79 (02) 317-22.
• 8 Davidson CJ, Hirt RP, Lal K. et al.. Molecular evolution of the vertebrate blood coagulation network. Thromb Haemost 2003; 89 (03) 420-8.
• 9 Nielsen H, Engelbrecht J, Brunak S. et al.. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 1997; 10 (01) 1-6.
• 10 Pittman DD, Wang JH, Kaufman RJ. Identification and functional importance of tyrosine sulfate residues within recombinant factor VIII. Biochemistry 1992; 31 (13) 3315-25.
• 11 Monigatti F, Gasteiger E, Bairoch A. et al.. The Sulfinator: predicting tyrosine sulfation sites in protein sequences. Bioinformatics 2002; 18 (05) 769-70.
• 12 Medzihradszky KF, Besman MJ, Burlingame AL. Structural characterization of site-specific N-glycosylation of recombinant human factor VIII by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry. Anal Chem 1997; 69 (19) 3986-94.
• 13 McMullen BA, Fujikawa K, Davie EW. et al.. Locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII (antihemophilic factor A). Protein Sci 1995; 04 (04) 740-6.
• 14 Tagliavacca L, Moon N, Dunham WR. et al.. Identification and functional requirement of Cu(I) and its ligands within coagulation factor VIII. J Biol Chem 1997; 272 (43) 27428-34.
• 15 Eaton D, Rodriguez H, Vehar GA. Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry 1986; 25 (02) 505-12.
• 16 Kozak M. Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem 1991; 266 (30) 19867-70.
• 17 Saenko EL, Scandella D. The acidic region of the factor VIII light chain and the C2 domain together form the high affinity binding site for von Willebrand factor. J Biol Chem 1997; 272 (29) 18007-14.
• 18 Fuentes-Prior P, Fujikawa K, Pratt KP. New insights into binding interfaces of coagulation factors V and VIII and their homologues lessons from high resolution crystal structures. Curr Protein Pept Sci 2002; 03 (03) 313-39.
• 19 Maugard C, Tuffery S, Aguilar-Martinez P. et al.. Protein truncation test: detection of severe haemophilia a mutation and analysis of factor VIII transcripts. Hum Mutat 1998; 11 (01) 18-22.
• 20 David D, Santos IMA, Johnson K. et al.. Analysis of the consequences of premature termination codons within the factor VIII coding sequences. J Thromb Haemost 2003; 01 (01) 139-46.
• 21 Culbertson MR, Leeds PF. Looking at mRNA decay pathways through the window of molecular evolution. Curr Opin Genet Dev 2003; 13 (02) 207-14.
• 22 Lewis BP, Green RE, Brenner SE. Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans. Proc Natl Acad Sci U S A 2003; 100 (01) 189-92.
• 23 Wang J, Chang YF, Hamilton JI. et al.. Nonsense-associated altered splicing: a framedependent response distinct from nonsensemediated decay. Mol Cell 2002; 10 (04) 951-7.
• 24 Kemball-Cook G, Tuddenham EG, Wacey AI. The factor VIII Structure and Mutation Resource Site: HAMSTeRS version 4. Nucleic Acids Res 1998; 26 (01) 216-9.
• 25 Higuchi M, Antonarakis SE, Kasch L. et al.. Molecular characterization of mild-to-moderate hemophilia A: detection of the mutation in 25 of 29 patients by denaturing gradient gel electrophoresis. Proc Natl Acad Sci USA 1991; 88 (19) 8307-11.