A Radioimmunoassay for Ependymins β and γ: Two Goldfish Brain Proteins Involved in Behavioral Plasticity

Abstract: A radioimmunoassay (RIA) using ¹²⁵I-labeled antigen was developed for the quantitative determination of two goldfish brain proteins (ependymins β and γ). The proteins were isolated from the cerebrospinal fluid (CSF) and cells of the ependymal zone surrounding goldfish brain ventricles. The turnover rates of β and γ were previously shown to be specifically enhanced after the animals successfully acquired a new pattern of swimming behavior. Femtomole quantities of ependymin β were measurable by the RIA. In applications of the assay, β and γ ependymins were found to have common immunological properties, since ¹²⁵I-β-antigen bound to antibody could be displaced by unlabeled ependymin γ as well as ependymin β but not by a variety of other proteins including several purified glycoproteins isolated from goldfish brain. The ependymins were shown to constitute 14% of the total protein content of the brain extracellular fluid and also to be present as a minor component of the serum proteins (0.3%). Ependymins β and γ have an immunological reactivity in these fractions that can be increased by a factor of 30 on heating. The data suggest that the antigenicity of the molecules is highly masked, and that it may require some unraveling of the quaternary structure of the proteins before maximal interaction with the antisera becomes possible.     

Molecular Characterization of an Ependymin Precursor from Goldfish Brain

Ependymins are thought to be implicated in fundamental processes involved in plasticity of the goldfish CNS. Gas-phase sequencing of purified ependymins beta and gamma revealed that they share the same N-terminal sequence. Each sequence displays microheterogeneities at several positions. Based on the protein sequences obtained, we constructed synthetic oligonucleotides and used them as hybridization probes for screening cDNA libraries of goldfish brain. In this article we describe the full-length sequence of a mRNA encoding a precursor of ependymins. A cleavable signal sequence characteristic of secretory proteins is located at the N-terminal end, followed directly by the ependymin sequence. Also, two potential N-glycosylation sites were detected. A computer search revealed that ependymins form a novel family of unique proteins.     

The role of brain extracellular proteins in neuroplasticity and learning

Double labeling studies of the pattern of protein synthesis in goldfish and mouse brain identified a class of glycoproteins (the ependymins) whose turnover rate was enhanced after training. A variety of control experiments indicated that these macromolecules have an important role in the molecular and cell biology of learning. Antisera to the ependymins when injected into the brains of trained goldfish cause amnesia of a newly acquired behavior. Isolation and localization studies by immunocytochemical methods indicate that the ependymins are released into the brain extracellular fluid by a class of neurosecretory cells. In mammalian brain ependymin-containing cells are highly concentrated in the limibic system. The ependymins are constituted from two disulfide-linked acidic polypeptide chains (M.W.37K and 31K). They contain at least 5% covalently bound carbohydrate per chain with mannose, galactose, N-acetylglucosamine and N-acetylneuraminic acid as the predominant components. The highly soluble ependymins can rapidly polymerize to form an insoluble fibrous matrix if calcium is removed from solution by the addition of a Ca2+-chelating agent or dialysis. The self-aggregation property of the ependymins can be triggered by the depletion of Ca2+ from the extracellular space. Studies of the kinetics of the aggregation phenomenon by measurements of turbidity changes indicate that the process can be terminated but not reversed by restoring Ca2+ to its normal CSF level. Immunohistochemical studies of the brains of trained goldfish show the presence of punctate statining sites in the perimeter of certain cells located in specific brain regions. This suggests that ependymin aggregation might occur in vivo during learning. A molecular hypothesis relating the aggregation properties of the ependymins to neuroplasticity and learning is proposed.     

The role of ependymin in the development of long lasting synaptic changes

1.) Three types of training experiments (a complex motor task, avoidance conditioning and classical conditioning) in the goldfish and one in the mouse (T-maze learning) indicate that the brain extracellular glycoprotein (ependymin) has a role in the consolidation process of long-term memory formation. 2.) Direct ELISA measures of the concentration of ependymin in the brain extracellular fluid (ECF) indicate that its level decreases after goldfish learn to associate a light stimulus (cs) with the subsequent arrival of a shock (US): paired CS-US gave changes whereas an unpaired presentation of CS-US gave no changes in comparison to unstimulated controls. 3.) Ependymin is released into ECF and CSF as mixtures of three types of disulfide-linked dimers of two acidic polypeptide chains (M. W. 37 kDa and 31 kDa). It contains 10% carbohydrate as an N-linked glycan. 4.) Ependymin has the capacity to polymerize in response to events that deplete Ca2+ from the brain extracellular environment. A molecular hypothesis relating polymerization properties to the process of formation of long-lasting synaptic changes is proposed. 5.) Investigations of the pattern of regeneration of goldfish optic nerve and the mechanisms of long-term potentiation (LTP) of rat brain hippocampal slices suggest that ependymin has a role in the formation of long-lasting synaptic changes. The E.M. data show that polymerized products which stain with anti-ependymin sera accumulate at synapses and in new spines after LTP.     

Extracellular Fluid Proteins of Goldfish Brain: Evidence for the Presence of Proteases and Esterases

Preparations of enriched fractions of extracellular fluid (ECF) proteins from goldfish brain were found to contain protease(s) and esterase(s). The N-substituted furanacryloyl (FA) peptides FA-Phe-Gly-Gly and FA-Phe-OMe were used as model substrates for determining protease and esterase activity, respectively, in a spectrophotometric assay. Studies of the profile of substrate specificity and identification of the types of compounds that were effective as inhibitors showed that these ECF enzymes have some distinctive properties. GSH, but not GSSG, and EDTA inhibited the protease(s) without influencing the esterase(s), whereas L-1-tosylamide-2-phenylethylchloromethyl ketone blocked both protease and esterase activities of ECF. Most of the protease and esterase properties of ECF could be bound to concanavalin A-Sepharose affinity chromatographic columns in association with ependymin--a brain extracellular protein. These observations indicate that ECF may contain a metalloprotease(s) and raise the possibility that the ependymins might be a substrate for these ECF enzymes.     

Major pertussis-toxin-sensitive GTP-binding protein of bovine lung. Purification, characterization and production of specific antibodies

Two GTP-binding proteins which can be ADP-ribosylated by islet-activating protein, pertussis toxin, were purified from the cholate extract of bovine lung membranes. Both proteins had the same heterotrimeric structure (alpha beta gamma), but the alpha subunits were dissociated from the beta gamma when they were purified in the presence of AlCl3, MgCl2 and NaF. The molecular mass of the alpha subunit of the major protein (designated GLu, with beta gamma) was 40 kDa and that of the minor one was 41 kDa. The results of peptide mapping analysis of alpha subunits with a limited proteolysis indicated that GLu alpha was entirely different from the alpha of brain Gi or Go, while the 41-kDa polypeptide was identical with the alpha of bovine brain Gi. The kinetics of guanosine 5'-[3-O-thio]triphosphate (GTP[gamma S]) binding to GLu was similar to that to lung Gi but quite different from that to brain Go. On the other hand, incubation of GLu alpha at 30 degrees C caused a rapid decrease of GTP[gamma S] binding, the inactivation curve being similar to that of Go alpha but different from that of Gi alpha. The alpha subunits of lung Gi and GLu did not react with the antibodies against the alpha subunit of bovine brain Go. The antibodies were raised in rabbits against GLu alpha and were purified with a GLu alpha-Sepharose column. The purified antibodies reacted not only with GLu alpha but also with the 41-kDa protein and purified brain Gi alpha. However, the antibodies adsorbed with brain Gi alpha reacted only with GLu alpha, indicating antisera raised with GLu alpha contained antibodies that recognize both Gi alpha and GLu alpha, and those specific to GLu alpha. These results further indicate that GLu is different from Gi or Go. Anti-GLu alpha antibodies reacted with the 40-kDa proteins in the membranes of bovine brain and human leukemic (HL-60) cells. The beta gamma subunits were also purified from bovine lung. The beta subunit was the doublet of 36-kDa and 35-kDa polypeptides. The lung beta gamma could elicit the ADP-ribosylation of GLu alpha by islet-activating protein, increase the GTP[gamma S] binding to GLu and protect the thermal denaturation of GLu alpha. The antibodies raised against brain beta gamma cross-reacted with lung beta but not with lung gamma.     

Ependymins are expressed in the meninx of goldfish brain

Summary Ependymins are dimeric glycoproteins found in the extracellular fluid of goldfish brain. They were originally observed because of their enhanced turnover rates after learning. In this paper we present the first investigation concerning the expression of these secretory proteins in goldfish brain via in situ hybridization with synthetic oligonucleotides and cRNA probes. It is shown that ependymin-mRNAs are predominantly expressed in the meninx surrounding the brain and in an invaginated part of the meninx called the cavum cranii. These results have been confirmed by immunhistochemical analysis. This indicates that, in fish, the meninx synthesizes a major protein constituent of the cerebrospinal fluid; furthermore, this suggests that the functional sites of ependymins are removed from the place of their synthesis. Distribution between different compartments may be achieved via the open communication system of the perivascular spaces.     

Mapping the functional domains of the eukaryotic elongation factor 1 beta gamma

The functional domains of the eukaryotic elongation factor (EF) 1 beta gamma have been delineated with the use of limited proteolysis, protein microsequencing, gel electrophoresis under non-denaturing conditions and antibodies against EF-1 beta and EF-1 gamma. By means of limited proteolysis, it was possible to obtain large fragments of EF-1 beta. In contrast to amino-terminal fragments, those derived from the carboxy-terminal part of EF-1 beta were still active in enhancing the guanine nucleotide exchange of GDP bound to EF-1 alpha. With the same technique of limited proteolysis, it was possible to isolate a trypsin-resistant core from EF-1 beta gamma containing polypeptide chain fragments derived from both subunits. A polyvalent antiserum against EF-1 beta and two monoclonal antibodies against EF-1 gamma were used to identify the protein fragments in this core. The monoclonal antibodies were shown to recognize different epitopes, one localized on the amino-terminal and another on the carboxy-terminal half of EF-1 gamma. The antiserum against EF-1 beta and one of the monoclonal antibodies (mAb 36E5), which recognized the amino-terminal half of EF-1 gamma, reacted with this trypsin-resistant core. We conclude that the amino-terminal halves of both EF-1 beta and EF-1 gamma are firmly attached to each other, and that the carboxy-terminal part of EF-1 beta interacts with EF-1 alpha.     

Existence of two gamma subunits of the G proteins in brain

Although amino acid sequences have been determined for the alpha and beta subunits of Gs, Gi, and Go, sequences have not been reported for the gamma subunits of these G proteins. In the present paper, we determined the sequences of peptides prepared by partial proteolysis of two different forms of the gamma subunit of Gs, Gi, and Go from bovine brain. Using oligonucleotide probes based on the sequences of two of these peptides, a cDNA clone was isolated from a bovine adrenal cDNA library. This clone contained a 0.9-kilobase cDNA insert that included an open reading frame of 213 bases encoding a 71-amino acid polypeptide with an estimated Mr of 7850. The amino acid sequence predicted for the adrenal cDNA clone was identical to that determined for one form of the gamma subunit from brain. In addition, an antibody to a peptide based on the predicted amino acid sequence of this cDNA clone reacted specifically with one of the brain gamma subunits, indicating the adrenal cDNA clone encodes a gamma subunit present in both adrenal gland and brain. Also, evidence is presented, demonstrating the existence of a second, structurally distinct, form of the gamma subunit of Gs, Gi, and Go in brain.     

G-protein beta gamma forms: identity of beta and diversity of gamma subunits

Signal-transducing G-proteins are heterotrimers composed of GTP-binding alpha subunits in association with a tightly bound complex of beta and gamma subunits. While the alpha subunits are recognized as a family of diverse structures, beta and gamma subunits have also been found as heterogeneous isoforms. To investigate the diversity and tissue specificity of the beta gamma complexes, we have examined homogeneous oligomeric G-proteins from a variety of sources. The beta and gamma subunits isolated from the major-abundance G-proteins from bovine brain, bovine retina, rabbit liver, human placenta, and human platelets were purified and subjected to biochemical and immunological analysis. Protease mapping and immune recognition revealed an identical profile for each of the two distinctly migrating beta isoforms (beta 36 and beta 35) regardless of tissue or G-protein origin. Digestion with V8 protease revealed four distinct, clearly resolved terminal fragments for beta 36 and two for beta 35. Trypsin and chymotrypsin digestion yielded numerous bands, but again each form had a unique profile with no tissue specificity. Tryptic digestion was found to be conformationally specific with the most resistant structure being the native beta gamma complex. With increasing trypsin, the complex was digested but in a pattern distinct from that for denatured beta. In contrast to the two highly homologous beta structures, examination of this set of proteins revealed at least six distinct gamma peptides. Two unique gamma peptides were found in bovine retinal Gt and three gamma peptides in samples of bovine brain derived Go/Gi. Human placental and platelet Gi samples each contained a unique gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

The tryptic and chymotryptic fragments of the beta-subunit of guanine nucleotide binding proteins in brain are identical to those of retinal transducin

The 35-kDa beta-subunit of transducin purified from rod outer segment membranes is cleaved into 2 major fragments by trypsin, and 7 major fragments by chymotrypsin. Identical fragments are visualized by immunoblotting with transducin-beta specific antisera after proteolysis of rod outer segment membranes, purified brain guanine nucleotide binding proteins, and brain membranes. The results indicate that the beta-subunits of transducin and of brain guanine nucleotide binding proteins are not only similar structurally, but are also similarly oriented in membranes with respect to accessibility to proteolytic enzymes.     

Identification of exposed and buried determinants of the membrane-bound acetylcholine receptor from Torpedo californica

The ability of five rabbit anti-acetylcholine receptor antisera to recognize the membrane-bound receptor from Torpedo californica has been investigated. Two antisera, raised against affinity-purified native receptor, react extensively with purified receptor-rich membrane vesicles. Since the membrane vesicles are impermeable to macromolecules and are oriented right side out, these two antisera recognize predominantly extracellular determinants. Two antisera against sodium dodecyl sulfate denatured receptor and one against purified delta subunit react poorly with the membrane-bound receptor. Only 10-20% of the determinants recognized by these antisera are accessible to antibodies when the receptor is membrane bound. Many of the latent sites can be exposed by permeabilizing the vesicles with saponin, by alkaline extraction of the membranes to remove peripheral proteins, or by a combination of these two treatments. These treatments neither solubilize the receptors nor interfere with their ability to undergo agonist-induced affinity changes. Subunit analysis of the sites on the membrane-bound receptor that are accessible to antibodies indicates that the alpha, beta, and delta chains possess extracellular determinants. Buried sites are present on all four of the subunits. Saponin permeabilization makes latent sites accessible on alpha and delta while alkaline extraction uncovers determinants on alpha, gamma, and delta. Treatment of membranes by both procedures reveals sites on beta, gamma, and delta that are not uncovered by either treatment alone. This study, in conjunction with results from other laboratories demonstrating that the gamma chain is extracellularly exposed, suggests that all four subunits are transmembrane proteins.     

Virus-coded origin of a 32,000-dalton protein from avian retrovirus cores: structural relatedness of p32 and the beta polypeptide of the avian retrovirus DNA polymerase.

A 32,000-dalton protein (p32) located in avian retrovirus cores was immunoprecipitated from [35S]methionine-labeled avian myeloblastosis virus (AMV) propagated in cultured chicken embryo fibroblast cells by an antiserum preparation (sarc III) derived from tumor-bearing hamsters injected with cloned and passaged cells from an avian sarcoma virus-induced primary hamster tumor. Since sarc III serum apparently contained antibodies only to virus-coded proteins and not to chicken cellular proteins, the immunoprecipitation of p32 from AMV by sarc III serum strongly suggested that p32 is virus coded. The origin of p32 was more definitively established by demonstrating the existence of a structural relationship between p32 and the AMV DNA polymerase. AMV p32 cross-reacted with the beta polypeptide of AMV alphabeta DNA polymerase in radioimmunoprecipitation and radioimmunoprecipitation inhibition assays, indicating that p32 and beta share common antigenic determinants. This relationship was clarified by sodium do-decyl sulfate-polyacrylamide gel electrophoretic analysis of the peptides generated by limited proteolysis of 125I-labeled AMV DNA polymerase polypeptides and of 125I-labeled AMV p32 by chymotrypsin or Staphylococcus aureus V-8 protease. The peptides which appeared during proteolytic digestion of p32 were a subset of those produced by digestion of the beta polypeptide; however, p32 had no discernible peptides in common with the alpha polypeptide. Further, all of the peptides produced by limited proteolysis of beta were present in the digests of either p32 or alpha. Our findings suggest that p32 is apparently derived by cleavage of the beta polypeptide of AMV DNA polymerase, presumably at a site near or identical to that at which alpha is generated from beta by proteolytic cleavage.     

Structural relationships between human erythrocyte sialoglycoproteins beta and gamma and abnormal sialoglycoproteins found in certain rare human erythrocyte variants lacking the Gerbich blood-group antigen(s).

The human erythrocyte membrane sialoglycoproteins beta and gamma are important for the maintenance of the discoid shape of the normal erythrocyte. In this paper we show that the human erythrocyte sialoglycoproteins beta and gamma (hereafter called beta and gamma) are structurally related. Rabbit antisera produced against purified beta and beta 1 and rendered specific to the cytoplasmic portion of these proteins also react with the cytoplasmic portion of gamma. Some human anti-Gerbich (Ge) sera react with the extracellular portion of both beta and gamma. This reactivity is shown to be directed towards a common epitope on beta and gamma. However, most anti-Ge sera do not react with beta, but react with an extracellular epitope only present on gamma. All individuals who lack the Ge antigens lack beta and gamma. In some cases abnormal sialoglycoproteins are present in the erythrocytes, and these are shown to be structurally related to beta and gamma. Rabbit antisera raised against the purified abnormal sialoglycoprotein from a Ge-negative erythrocyte type reacted with the cytoplasmic portion of both beta and gamma. Unlike normal beta and gamma, the abnormal sialoglycoproteins found in Ge-negative erythrocytes migrate as a diffuse band on SDS/polyacrylamide-gel electrophoresis. Studies using endoglycosidases suggest that the diffuse nature of these bands results from carbohydrate heterogeneity and that the abnormal sialoglycoproteins contain N-glycosidically linked oligosaccharides with repeating lactosamine units. Such polylactosamine chains are not present on normal beta or gamma.     

Evidence of Native-like Substructure(s) in Polypeptide Chains of Carbonic Anhydrase Deposited into Insoluble Aggregates During Thermal Unfolding

A non-specific protease, subtilisin, was used to probe the existence of folded structure in thermal aggregates of bovine carbonic anhydrase-II (BCA). BCA aggregates and native BCA were subjected to proteolysis and electrophoretic analyses which revealed the accumulation of polypeptide fragments of similar size, indicating survival of similar sections of folded structure burying peptide bonds away from scission in the two samples. N-terminal sequencing revealed that the termini of size-matched fragments from the two samples were either identical, or located very close to each other, and predominantly on the surface of the 3-dimensional structure of native BCA. The susceptibility to proteolysis of very nearly the same sites in the two samples suggests that native-like elements of structure survive within BCA aggregates. The finding that thermal aggregation can involve interactions among molecules retaining elements of native-like structure, suggests that complete chain unfolding may not be a necessary prerequisite for all aggregation.     

Pieces of eight: bioactive fragments of extracellular proteins as regulators of angiogenesis

Specific stages of angiogenesis are regulated by extracellular proteins. This review discusses the endogenous proteolysis of eight of these proteins and the release of polypeptide fragments that have biological activities different from those of the native, parent protein. The generation of natural cleavage products could provide a precise mechanism for the regulation of angiogenesis. Although further experimental confirmation of this mechanism is needed, this leitmotif offers an attractive explanation, in part, for the complex role of proteolysis in vascular morphogenesis.     

Processing of structural polypeptides of infectious flacherie virus of the silkworm, Bombyx mori: VP1 and VP4 are derived from VP0.

Infectious flacherie virus of the silkworm, Bombyx mori, contains five major polypeptides termed VP0, VP1, VP2, VP3, and VP4 in the order of descending molecular weight. Immunoblot analysis with specific antisera against each of these structural polypeptides showed that antisera against VP1 and VP4 unequivocally reacted with VP0 as well as their homologous structural polypeptides. Limited proteolysis of VP0 and VP1 by Staphylococcus aureus V8 protease gave several common polypeptide fragments. Amino acid sequence analysis showed that VP0 and VP4 shared a common NH2-terminal amino acid sequence. These results indicate that VP1 and VP4 are generated from VP0 and that VP4 occupies the NH2-terminal portion of VP0.     

Characterization of transducin from bovine retinal rod outer segments. Participation of the amino-terminal region of T alpha in subunit interaction

The GTP-induced dissociation of T alpha from T beta gamma initiates the release of transducin from photolyzed rhodopsin and the subsequent activation of the cGMP phosphodiesterase. In this study, site-specific proteolysis and immunoprecipitation were used to map the domain of T alpha that interacts with T beta gamma. We found that Staphylococcus aureus V8 protease rapidly removes a small fragment from T alpha under native conditions, resulting in the formation of a single 38-kDa polypeptide (T alpha'). Under the same conditions, T beta gamma remains intact. A 4.5-fold decrease in the rate of T alpha cleavage by S. aureus protease was observed in the presence of T beta gamma, suggesting T beta gamma binding blocks the protease-sensitive site on T alpha. Amino acid sequence analysis indicated that T alpha' is derived from the cleavage of T alpha at Glu-21. The ability of T alpha' to interact with and activate the retinal phosphodiesterase is not diminished. However, T alpha' is unable to participate in T beta gamma-dependent activities such as the light-stimulated binding of guanine nucleotides, binding to photoexcited rhodopsin, and ADP-ribosylation catalyzed by pertussis toxin. Moreover, the anti-T alpha monoclonal antibody TF16 was able to precipitate T beta gamma in the presence of T alpha, but not with either T alpha' or T alpha-guanosine 5'-O-(3-thiotriphosphate). We conclude that the amino-terminal region of T alpha participates in T beta gamma interaction and discuss our results with respect to the known structure and function of transducin.     

Ionic-strength-dependent changes in the structure of the major protein of the human erythrocyte membrane.

The effect of ionic strength on the proteolysis by trypsin of the major membrane-penetrating protein (polypeptide 3) in the erythrocyte membrane was studied. Both the intracellular and extracellular regions of the protein are susceptible to trypsin proteolysis under hypo-osmotic conditions, whereas under iso-osmotic conditions the extracellular region of the protein is resistant to trypsin, and the intracellular region yields only two cleavage products with trypsin. Studies of the fragments obtained from polypeptide 3 by trypsin digestion under iso-osmotic conditions of 'ghosts' radioiodinated with lactoperoxidase confirmed our earlier conclusions that the polypeptide chain of polypeptide 3 traverses the membrane twice. Ionic-strength-dependent changes were also observed in the incorporation of iodine by lactoperoxidase into the individual extracellular tyrosine sites of the protein. These results show that polypeptide 3 undergoes ionic-strength-dependent changes in structure.