Expression and characterization of Flag-epitope- and hexahistidine-tagged derivatives of saxiphilin for use in detection and assay of saxitoxin.

Saxiphilin is a plasma protein from the bullfrog (Rana catesbiana) that binds saxitoxin (STX), a causative agent of paralytic shellfish poisoning. Saxiphilin is homologous to transferrin and consists of two internally homologous domains called the N-lobe and the C-lobe. STX binds to a single site in the C-lobe of saxiphilin. In this study, cloned genes coding for recombinant saxiphilin and C-lobe saxiphilin were modified to contain two tandemly located affinity tags, Flag epitope (DYKDDDDK) and His(6) (HHHHHH), at the protein C-terminus and were expressed in cultured insect cells using baculovirus vectors. Both tagged proteins are readily detected on immunoblots by anti-Flag monoclonal antibody. Flag-His(6)-tagged saxiphilin was purified to homogeneity using Ni(2+)-chelate affinity chromatography and Heparin Sepharose chromatography. Equilibrium analysis of [3H]STX binding to tagged saxiphilin and tagged C-lobe saxiphilin gave K(D) values of 0.75 and 2.7 nM, respectively. Flag-His(6)-tagged saxiphilin was also utilized in a microtiter well solid-phase assay with Reacti-bind metal chelate plates to measure [3H]STX binding and binding competition by unlabeled STX. Such Flag-His(6)-tagged derivatives of saxiphilin have many possible applications in the assay of STX and related toxinological research.

The divergent homeobox gene PBX1 is expressed in the postnatal subventricular zone and interneurons of the olfactory bulb.

In the mammalian brain, an important phase of neurogenesis occurs postnatally in the subventricular zone (SVZ). This region consists of a heterogeneous population of cells, some mitotically active, others postmitotic. A subset of mitotically active SVZ precursor cells gives rise to a population of neurons that migrates over a long distance to their final destination, the olfactory bulb. Other SVZ precursor cells continue to proliferate or undergo cell death. The combination of genes that regulates proliferation and cell fate determination of SVZ precursor cells remains to be identified. We have used the rat homolog of the human homeobox gene PBX1 in Northern analysis and in situ hybridization studies to determine the temporal and regional localization of PBX1 expression during embryonic and postnatal rat brain development. PBX1 is expressed embryonically in the telencephalon. In addition, it is expressed at high levels postnatally in the SVZ, in the migratory pathway to the olfactory bulb, and in the layers of the olfactory bulb that are the targets of these migratory neurons. Combining in situ hybridization for PBX1 with immunostaining for markers of cell proliferation (PCNA), postmitotic neurons (class III beta-tubulin), and glia (GFAP), we show that SVZ proliferating cells and their neuronal progeny express rat PBX1 mRNA, whereas glial cells do not express detectable levels of PBX1. The expression of PBX1 in SVZ precursor cells and postmitotic neurons suggests a role for PBX1 in the generation of olfactory bulb interneurons and in mammalian neurogenesis.

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)

Molecular cloning of bullfrog saxiphilin: a unique relative of the transferrin family that binds saxitoxin.

Plasma and tissue of certain vertebrates contain a protein called saxiphilin that specifically binds the neurotoxin saxitoxin with nanomolar affinity. We describe the isolation of a cDNA clone of saxiphilin from liver of the North American bullfrog (Rana catesbeiana). The cDNA sequence encodes a protein that is evolutionarily related to members of the transferrin family of Fe(3+)-binding proteins. Pairwise sequence alignment of saxiphilin with various transferrins reveals amino acid identity as high as 51% and predicts 14 disulfide bonds that are highly conserved. The larger size of saxiphilin (91 kDa) versus serum transferrin (approximately 78 kDa) is primarily due to a unique insertion of 144 residues. This insertion contains a 49-residue domain classified as a type 1 repetitive element of thyroglobulin, which is shared by a variety of membrane, secreted, and extracellular matrix proteins. Saxiphilin also differs from transferrins in 9 of 10 highly conserved amino acids in the two homologous Fe3+/HCO3-binding sites of transferrin. Identification of saxiphilin implies that transferrin-like proteins comprise a diverse superfamily with functions other than iron binding.

Shared nuclear protein binding sites in the upstream region of the rat opsin gene.

DNase I protection and gel retardation assays have identified two sequences 5' to the rat opsin gene that interact with nuclear proteins from retina but not from a number of neuronal and non-neuronal tissues. These sites, Ret 2 and Ret 3, are over 1200 base pairs apart but seem to interact with the same protein(s). Synthetic oligonucleotides corresponding to each site were able to inhibit complex formation in a gel retardation assay using an oligonucleotide corresponding to the other site. The proteins binding to the Ret 2 and Ret 3 sites co-eluted in both ion exchange and gel filtration chromatography. The protein(s) were also present at adult levels at birth, suggesting that they may represent differentiation products expressed in the proliferating retinal epithelium.

Characterization of developmentally regulated and retina-specific nuclear protein binding to a site in the upstream region of the rat opsin gene.

DNase I protection and gel retardation assays have identified a sequence 5' to the transcription start site of the rat opsin gene that interacts with nuclear proteins from mammalian retinas but not from a variety of other neural and non-neural tissues. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to nitrocellulose the protein(s) responsible for this binding were identified with an oligonucleotide probe and were found to migrate with an apparent molecular size of 40 kilodaltons. The binding complex eluted from fast protein liquid chromatography gel filtration as a peak centered at 100 kilodaltons, suggesting the presence of more than one subunit. Binding activity could be detected in postnatal day 1 retinal extracts and increased over the next 2 weeks of development, a time course coincident with opsin gene expression and maturation of rod photoreceptors. Synthetic oligonucleotides with altered sequences showed that the binding was dependent upon residues in a CTAAT motif and was facilitated by surrounding GGCCCC sequences. The specificity of the binding interaction was measured by inhibition of complex formation in a gel retardation assay. The unaltered sequence was over 2 orders of magnitude more effective at inhibiting complex formation than either an unrelated DNA sequence or a concensus sequence corresponding to a known CCAAT box binding protein NF1.

Opsin expression in the rat retina is developmentally regulated by transcriptional activation.

The gene for rhodopsin, the primary light sensor of the visual system, is specifically expressed in the rod photoreceptor cells of the retina. We show here that in the rat, opsin RNA first accumulates to detectable levels at postnatal day 2 (PN2) and that nascent transcripts can be detected at PN1; this is the time when peak numbers of photoreceptor cells are generated by the final division of their neuroepithelial precursors. Accumulated opsin RNA then increases to reach the adult level, 0.06% of total retinal RNA, at about PN10. The transcription rate of the opsin gene increases to a similar extent over the same time course between PN3 and adulthood, suggesting that transcriptional activation is responsible for the increase in opsin expression. We used the antibody RET-P1 to show that rhodopsin protein is also detectable at PN2 and that the number of cells expressing the protein increases with time in a central-to-peripheral gradient in the retina. This increase in the number of differentiating photoreceptors in the tissue appears to account for much of the increase in opsin gene transcription and RNA accumulation. In situ hybridization to opsin RNA shows that it is restricted to the photoreceptor layer from the time it can first be detected, at PN7. Later in development, when RET-P1 staining shifts to the photoreceptor outer segments, opsin RNA becomes localized to the inner segments, suggesting that the distributions of opsin protein and RNA are related.