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The role of beta‐hydroxyaspartate and adjacent carboxylate residues in the first EGF domain of human factor IX.
Author(s) -
Rees D. J.,
Jones I. M.,
Handford P. A.,
Walter S. J.,
Esnouf M. P.,
Smith K. J.,
Brownlee G. G.
Publication year - 1988
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1988.tb03045.x
Subject(s) - classics , theology , philosophy , history
beta‐Hydroxyaspartic acid is a post‐translationally modified amino acid found in a number of plasma proteins in a domain homologous to epidermal growth factor. Its presence can be correlated with a high affinity Ca2+ binding site, with a dissociation constant of 10‐100 microM. We describe a system for the expression of human coagulation factor IX in dog kidney cells in tissue culture, in which the post‐translational modifications and the biochemical activity are indistinguishable from factor IX synthesized in vivo. This system has been used to express eight different point mutations of human factor IX in the first epidermal growth factor domain in order to study the role of beta‐hydroxyaspartate at residue 64, and the adjacent carboxylate residues at positions 47, 49 and 78. We conclude that this domain is essential for factor IX function and suggest that Ca2+ binds to carboxylate ions in this domain and stabilizes a conformation necessary for the interaction of factor IXa with factor X, factor VIII and phospholipid in the next step of the clotting cascade.