Rek, an Axl/Tyro3-related receptor-class tyrosine kinase, is expressed in Müller glia and differentiating neural subpopulations in the avian retina.

Rek is a receptor-class tyrosine kinase of the Axl/Tyro3 family that has transforming capability and is expressed in the avian nervous system. To gain insight into its normal cellular function, the spatial and temporal expression of Rek was analyzed in the developing chick retina by immunoperoxidase staining and Western blotting using Rek-specific antibodies. Rek was developmentally regulated in the retina with maximal expression of the 140 kD full-length kinase at embryonic stage 34 (E8), when retinal glia and neurons begin to differentiate. Rek immunoreactivity was most prominent in the processes of developing and mature retinal Müller glia, and appeared transiently in the bodies of differentiating ganglion and amacrine neurons. In the developing optic tract Rek was found in oligodendroglial-type cells but not in ganglion cell axons. Rek antibodies also stained brain ependymal cells and some cerebellar neuronal cell types (Purkinje, basket and stellate cells). This expression pattern suggests that the Rek tyrosine kinase participates in an aspect of Müller glial function, and may also contribute to the development of restricted populations of glia and neurons in the brain and retina.

Rek, a gene expressed in retina and brain, encodes a receptor tyrosine kinase of the Axl/Tyro3 family.

Rek (retina-expressed kinase) has been identified as a putative novel receptor-type tyrosine kinase of the Axl/Tyro3 family with a potential role in neural cell development. rek clones were isolated from a chick embryonic brain cDNA library with a DNA probe obtained by reverse transcriptase-polymerase chain reaction of mRNA from Müller glia-like cells cultured from chick embryonic retina. Sequence analysis indicated that Rek is a protein of 873 amino acids with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains with eight predicted N-glycosylation sites. Two consensus src homology 2 domain binding sites are present in the cytoplasmic domain, suggesting that Rek activates several signal transduction pathways. Northern analysis of rek mRNA revealed a 5.5-kilobase transcript in chick brain, retina, and kidney and in primary cultures of retinal Müller glia-like cells. Rek protein was identified by immunoprecipitation and immunoblotting as a 140-kDa protein expressed in the chick retina at embryonic days 6-13, which corresponded to the major period of neuronal and glial differentiation. Transfection of rek cDNA into COS cells resulted in transient expression of a putative precursor of 106 kDa that autophosphorylated in immune complex protein kinase assays. Overexpression of rek cDNA in mouse NIH3T3 fibroblasts resulted in activation of the 140-kDa rek kinase and induction of morphologically transformed foci. These properties indicated that Rek has oncogenic potential when overexpressed, but its normal function is likely to be related to cell-cell recognition events governing the differentiation or proliferation of neural cells.

Binding of native kappa-neurotoxins and site-directed mutants to nicotinic acetylcholine receptors.

The kappa-neurotoxins are useful ligands for the pharmacological characterization of nicotinic acetylcholine receptors because they are potent antagonists at only a subgroup of these receptors containing either alpha 3- or alpha 4-subunits (IC50 < or = 100 nM). Four of these highly homologous, 66 amino acid peptides have been purified from the venom of Bungarus multicinctus (kappa-bungarotoxin (kappa-Bgt), kappa 2-Bgt, kappa 3-Bgt] and Bungarus flaviceps [kappa-Fvt)]. Two approaches were taken to examine the binding of these toxins to nicotinic receptors. First, venom-derived kappa-Fvt and kappa-Bgt were radioiodinated and the specific binding was measured of these toxins to overlapping synthetic peptides (16-20 amino acids in length) prepared based on the known sequence of the nicotinic receptor alpha 3-subunit. At least two main regions of interaction between the toxins and the receptor subunit were identified, both lying in the N-terminal region of the subunit that is exposed to the extracellular space. The second approach examined the importance of several sequence position in kappa-Bgt for binding to alpha 3-containing receptors in autonomic ganglia and alpha 1-containing muscle receptors. This was done using site-directed mutants of kappa-Bgt produced by an Escherichia coli expression system. Arg-34 and position 36 were important for binding to both receptor subtypes, while replacing Gln-26 with Trp-26 (an invariant in alpha-neurotoxins) increased affinity for the muscle receptor by 8-fold. The results confirm that kappa-neurotoxins bind potently to the alpha 3-subunit and bind with considerably reduced affinity (Kd approximately 10 microM) to muscle receptors. Site-directed mutagenesis of recombinant kappa-Bgt is thus an important approach for the study of structure-function relationships between kappa-Bgt and nicotinic receptors.

Facile production of native-like kappa-bungarotoxin in yeast: an enhanced system for the production of a neuronal nicotinic acetylcholine receptor probe.

Research on the mammalian central nervous system had been hindered by the limited number and meager supply of naturally occurring toxins that can be used as pharmacological reagents. The kappa-neurotoxins in particular are not found abundantly in nature and are difficult to obtain and isolate in quantities sufficient for research purposes. Here we report the expression and isolation of relatively large quantities of the kappa-neurotoxin, kappa-bungarotoxin, in an active form using a yeast, Pichia pastoris, expression system. The resultant product of the expression system has a short amino-terminal amino acid extension relative to venom-derived kappa-bungarotoxin, but is equivalent to the native toxin in physical and biological properties, as judged by the CD spectra, the ability to form dimers in solution, and the activity on chick ciliary ganglia. The yeast system produces approximately 0.2 mg from a 2 liter culture and the purification takes approximately 2 days. In contrast, E. coli, the only other available expression system for this toxin, produces one-fifth to one-half as much active material from a 5 liter high-density fermentation and the resulting protein takes over a week to purify. No high mol. wt disulfide-bonded aggregates were found in the yeast expression system product, indicating that the product is that of a biologically assisted folding process. This has significant implications not only for the efficient production of native toxin but also for the production of mutant proteins to study the structure-function relationship in these proteins.

Affinity of native kappa-bungarotoxin and site-directed mutants for the muscle nicotinic acetylcholine receptor.

kappa-Bungarotoxin (kappa-bgt) is a 66-residue peptide originally purified from snake venom that acts as an antagonist at certain acetylcholine receptors. It is one of four homologous kappa-neurotoxins that are distinguished from the structurally related alpha-neurotoxins by their ability to block the alpha 3-subunit-containing neuronal nicotinic acetylcholine receptor (nAChR). It has been reported that venom-purified kappa-bgt also displays some affinity for the alpha 1-subunit-containing muscle nAChR to which the alpha-neurotoxins bind with high affinity. Here we report the effects of particular mutations on the ability of recombinant kappa-bgt to block the binding of 125I-alpha-bgt to nAChRs found in fetal mouse muscle and chick skeletal muscle. While the replacement of a proline residue found in all kappa-neurotoxins with an alanine (P-42-A) has relatively little effect, the introduction of a lysine, which is found in 90% of active alpha-neurotoxins at the same position (P-42-K), eliminates muscle receptor affinity at the concentrations tested. In contrast, the replacement of a glutamine in kappa-bgt with a tryptophan found in all active alpha-neurotoxins (Q-32-W) increases the affinity of kappa-bgt for the muscle receptor. When the arginine residue found in all active alpha- and kappa-neurotoxins is replaced by an alanine (R-40-A), the ability of kappa-bgt to block the muscle receptor is reduced to undetectable levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Site-directed mutagenesis of kappa-bungarotoxin: implications for neuronal receptor specificity.

Postsynaptic polypeptide neurotoxins isolated from the venoms of elapid and hydrophid snakes exhibit the ability to bind selectively to and inhibit different types of receptors that function in nerve signal transmission. On the basis of their amino acid sequences and three-dimensional structures, these neurotoxins are clearly related, but nothing is yet known about the basis for their physiological receptor specificity. In this report, site-directed mutants of kappa-bungarotoxin, produced by an Escherichia coli expression system, are tested to determine the function of selected amino acid side chains in the interaction between toxin and receptor. Highly conserved residues at the bottom of the second loop (a region that has been shown to be a major point of contact with the receptor), particularly those residues at the junction between the beta-sheet and the end of the loop, were selected. The results demonstrate that a single amino acid substitution of the invariant arginine residue (Arg-40 to Ala-40) renders the toxin unable to inhibit nerve transmission in the chick ciliary ganglion up to a concentration of 10 microM. Significantly, the results also show that conversion to alanine of the nearby proline residue (Pro-42) found to be invariant in all kappa-neurotoxins, but not found in any potent alpha-neurotoxin, produces a toxin with full inhibitory capacity. However, the introduction of a lysine residue at this position (P-42-K), like that found in alpha-bungarotoxin, reduces activity significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a fast-exchange conformational process in alpha-bungarotoxin.

Anomalous behavior of the post-synaptic protein neurotoxin, alpha-bungarotoxin (alpha-bgt), has been observed during reverse-phase HPLC. Purified samples of this toxin from two distinct sources elute from reverse-phase columns as two separate peaks. The protein species represented by these two peaks are in rapid equilibrium, the relative ratio of which displays a pH dependency with a pKa of approximately 3. This equilibrium does not involve the dimerization or aggregation of the toxin and appears to be relatively unique to alpha-bungarotoxin in that similar behavior is not displayed by several other available alpha-neurotoxins. pH-dependent conformational changes have been documented for several alpha-neurotoxins whose crystal structures have been determined (alpha-bungarotoxin, alpha-cobratoxin, and erabutoxin b). One or more of these may account for the observed behavior of alpha-bungarotoxin on reverse-phase HPLC.

Purification of an active species of recombinant kappa-bungarotoxin.

The expression of kappa-bungarotoxin in Escherichia coli from a synthetic gene results in the production of multiple species of polypeptide. These include not only biologically active kappa-bungarotoxin but also a variety of inactive species, which include inactive monomers as well as disulfide-linked polymeric species. Identification of these species and their separation from the biologically active recombinant toxin is necessary for the use of the toxin in physiological and biochemical studies. This has been accomplished by a combination of ion-exchange and reverse-phase chromatography which results in a homogeneous toxin preparation. The active material produced is sufficient for many types of biological studies and for mutagenesis experiments directed at determining the structure function relationships of toxin interactions with the neuronal nicotinic acetylcholine receptor. In addition, the kappa-bungarotoxin produced in this manner has the distinct advantage over venom-purified kappa-bungarotoxin of not being contaminated with other venom components which could potentially affect experimental observations.

Crystallization of kappa-bungarotoxin: preliminary X-ray data obtained from the venom-derived protein.

kappa-Bungarotoxin is a 66 residue polypeptide found in the venom of the Taiwanese banded krait, Bungarus multicinctus. It binds tightly to neuronal nicotinic acetylcholine receptors and inhibits nerve transmission mediated by these postsynaptic receptors. It is related, by similarity in amino acid sequence, to alpha-bungarotoxin and other alpha-neurotoxins, but differs sharply in physiologic action. The alpha-neurotoxins inhibit nerve transmission in nicotinic acetylcholine receptors associated with vertebrate skeletal muscle and fish electric organs. The kappa-neurotoxins inhibit nerve transmission in neuronal nicotinic acetylcholine receptors such as those found in chick ciliary ganglia. The kappa-neurotoxins display a low level of interaction with receptors that are strongly affected by alpha-neurotoxins, but alpha-neurotoxins are completely without effect on receptors that are affected by kappa-bungarotoxin. The structural basis for this physiologic differentiation is not known. Crystals of kappa-bungarotoxin have now been obtained that diffract to at least 2.3 A. These crystals are hexagonal, space group P6, and have dimensions of a = b = 80.2 A, c = 39.6 A, and angles of alpha = beta = 90 degrees and gamma = 120 degrees. Each unit cell contains 12 molecules of the 66 residue protein or two molecules per asymmetric unit. Comparison of the structure of kappa-bungarotoxin, which will result from further diffraction analysis of these crystals, with the structures of the alpha-neurotoxins that have been determined may provide information on the structural basis of physiologic action in these acetylcholine receptor antagonists.

Synthesis and expression in Escherichia coli of a gene for kappa-bungarotoxin.

A gene which codes for the 66-residue polypeptide of kappa-bungarotoxin has been chemically synthesized by linking together 3 synthetic double-stranded oligonucleotides in a bacterial plasmid. The synthesis incorporated six unique silent restriction sites spaced throughout the gene for use in cassette mutagenesis. Direct expression of the kappa-bungarotoxin polypeptide by itself in Escherichia coli failed to result in a stable product. The toxin polypeptide was stabilized and expressed in E. coli as part of a fusion protein with rat intestinal fatty acid binding protein under control of the nalidixic acid inducible recA promoter. Two fusion protein constructs were prepared that differed only in the cleavage site between the fatty acid binding protein and the toxin polypeptide. One contained a factor Xa cleavage site, and the other, since the toxin itself is devoid of methionine, contained a methionyl residue that served as a cyanogen bromide cleavage site. The fusion proteins were isolated by ion-exchange chromatography and reverse-phase HPLC. The construct containing the factor Xa cleavage site could not be cleaved under nondenaturing conditions. On the other hand, kappa-bungarotoxin was efficiently cleaved from the methionyl fusion protein with CNBr. The toxin polypeptide was isolated by reverse-phase HPLC and ion-exchange chromatography and produced a complete and specific blockade of neuronal nicotinic acetylcholine receptors in chick ciliary ganglia which was indistinguishable from that produced by a comparable amount of venom-purified kappa-bungarotoxin.

Comparison of superoxide dismutase, thiopental, and nimodipine for maintenance of somatosensory evoked responses during aortic cross-clamping and declamping in dogs.

Paraplegia is a potential complication of aortic cross-clamping. The occurrence of this devastating sequela has caused increased interest in the use of somatosensory evoked responses (SER) to monitor spinal cord ischemia during aortic cross-clamping. This study was designed to examine changes in SERs during clamping and declamping of the canine aorta after injection of superoxide dismutase (SOD), thiopental (T), and nimodipine (N). In the control group, cross-clamping the aorta produced an increase in latency and a decrease in amplitude of the SER starting at two minutes. Isoelectric SERs were obtained after 16 minutes of aortic cross-clamping, but recovered with cross-clamp removal. When the aorta was clamped for more than 16 minutes in the control group, the isoelectric SERs obtained were irreversible. After the injection of SOD and T, SER latencies and amplitudes changed to a smaller degree with aortic cross-clamping and did not become isoelectric even after 20 minutes of clamping. During aortic cross-clamp removal in the control group, SERs initially improved and then showed signs of reperfusion ischemia, which disappeared after eight minutes. There were no significant SER changes due to reperfusion when SOD or T or the combination was given prior to aortic cross-clamping. There was no difference in SER changes from the control group during aortic cross-clamping and after release of cross-clamping when N was given. Nimodipine did not alter SER changes from aortic cross-clamping alone. In summary, SOD and T, alone or in combination, protect the spinal cord against ischemia during aortic cross-clamping and declamping.