Expression of saxiphilin in insect cells and localization of the saxitoxin-binding site to the C-terminal domain homologous to the C-lobe of transferrins.
Saxiphilin is a plasma protein from the bullfrog (Rana catesbeiana) that is homologous to transferrin. Most known transferrins contain two binding sites for Fe3+/HCO3-, one in each of two homologous domains called the N-lobe and C-lobe. However, native saxiphilin does not bind Fe3+ but stoichiometrically binds one molecule of the neurotoxin saxitoxin (STX) with a dissociation constant (KD) of approximately 0.2 nM. To pursue structural analysis of the STX binding sites, cDNA encoding saxiphilin was used to construct a baculovirus expression vector that directs synthesis and secretion of a approximately 92-kDa recombinant saxiphilin protein (R-sax) in cultured insect cells. Culture medium harvested from infected cells contained 25-67 pmol of [3H]STX binding sites/mL with a KD of 0.22 nM. The kinetics and pH dependence (pK0.5 = 5.4) of [3H]STX binding to R-sax are similar to native saxiphilin, implying proper folding and functional activity. Another baculovirus expression vector was constructed to encode a deletion mutant of saxiphilin consisting of the first 20 N-terminal residues containing the secretory signal sequence spliced to the C-terminal, 361-residue fragment homologous to the C-lobe domain of transferrins. This vector directed the secretion of a approximately 38-kDa derivative of saxiphilin (C-sax) that was recognized by antisaxiphilin antibody. C-sax also exhibited [3H]STX binding activity with a lower affinity KD of approximately 0.9 nM, a 4-fold faster dissociation rate for [3H]STX than native saxiphilin, and a pH dependence (pK0.5 = 5.7) similar to R-sax (pK0.5 = 5.4).(ABSTRACT TRUNCATED AT 250 WORDS)
Morabito
M A
MA
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066, USA.
Llewellyn
L E
LE
Moczydlowski
E G
EG
D029845:Amphibian Proteins; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D011892:Rana catesbeiana; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012530:Saxitoxin / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D014168:Transferrin / Q000737:chemistry
D029845:Amphibian Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D014168:Transferrin; C027525:saxitoxin-binding protein, Rana catesbeiana; D012530:Saxitoxin