Synthesis of surface immunoglobulin E receptor in Xenopus oocytes by translation of mRNA from rat basophilic leukemia cells

Immunoglobulin E-binding activity was expressed in Xenopus oocytes injected with mRNA from rat basophilic leukemia cells which possess abundant immunoglobulin E (IgE) receptor. Such activity was demonstrated with intact oocytes by their binding of 125I-labeled mouse monoclonal IgE. Binding activity was specific as shown by the total inhibition of 125I-IgE binding by unlabeled IgE but not by unlabeled IgG1. The relevance of the IgE-binding activity to the IgE receptor was also supported by the absence of this activity in oocytes injected with mRNA from cells lacking surface IgE receptors. mRNA coding for the IgE-binding activity was enriched in fractions sedimenting at 13.5 S in sucrose density gradients. From oocytes injected with rat basophilic leukemia mRNA, two major polypeptides were isolated by affinity purification on IgE immunoadsorbent. One (Mr = 31,000) is equivalent in size to the previously identified "receptor-associated protein;" the other (Mr = 40,000) is speculated to be a partially glycosylated or unglycosylated form of the alpha subunit of the IgE receptor. The binding of IgE-coated fluorescent microspheres by oocytes injected with rat basophilic leukemia mRNA demonstrated the surface expression of the IgE-binding proteins.     

Assembly, glycosylation, and secretion of the oligomeric rat prostatic binding protein in Xenopus oocytes

Prostatic binding protein (PBP), a hormonally controlled oligomeric glycoprotein secreted by the rat ventral prostate, is composed of three different polypeptide chains, C1, C2, and C3. Microinjection of prostate mRNA into Xenopus laevis oocytes results in the synthesis, processing, and correct assembly of these three components, and also in the export of PBP into the medium. The glycosylation of component C3--the only glycopeptide of PBP--by the oocyte enzymes does not lead to the same result as in the native prostate tissue. The intracellular oocyte component contains an incompletely processed oligomannosyl core unit. Upon secretion this sugar core is further processed, probably at random because the carbohydrate chains attached to the exported C3 molecules are heterogeneous; they are also different from the oligosaccharide unit of authentic C3. However, tunicamycin experiments show that glycosylation is neither a prerequisite for secretion nor for the assembly of PBP, at least in oocytes.     

Synthesis and glycosylation of the MOPC-46B immunoglobulin in kappa chain in Xenopus laevis oocytes.

Polyadenylated mRNA isolated from MOPC-46B plasmacytoma, which secretes a glycosylated kappa chain, was injected into $\text{Xenopus laevis}$ oocytes. Analysis of the resulting product showed that [1-¹⁴C]mannose was incorporated into the MOPC-46B kappa chain. Light chains synthesized in oocytes injected with mRNA from MOPC-321 plasmacytoma, which secretes a nonglycosylated kappa chain, failed to incorporate label from [1-¹⁴C]mannose. Thus, protein glycosylation in the oocyte is apparently specific in that carbohydrate is incorporated only into the kappa chain synthesized as a glycoprotein by myeloma cells. It is thus evident that the general signals for glycosylation have remained stable during independent evolution of the amphibia and mammalia.     

Post-translational processing in Xenopus oocytes includes carboxyl-terminal amidation

Xenopus oocytes are versatile cells capable of carrying out many post-translational processes. Although previously reported not to be capable of C-terminal amidation, this report demonstrates that Xenopus oocytes do indeed have an amidating enzyme. The amidating activity from Xenopus ovaries is compared to the known amidating activity found in porcine pituitaries. The demonstration of C-terminal amidation by Xenopus oocytes extends their usefulness in studying post-translational events.     

Synthesis of carp proinsulin in Xenopus oocytes

Total poly(A)-containing RNA from Brockmann boides of carp (Cyprinus carpio) directs the synthesis of authentic carp proinsulin in Xenopus oocytes. Neither preproinsulin nor further processing of the proinsulin to insulin can be detected in the oocytes.     

Synthesis and processing of the mouse MOPC-321 kappa chain in Xenopus laevis oocytes.

Polyadenylated mRNA isolated from mineral oil-induced plasmacytoma (MOPC)-321 was injected into Xenopus laevis oocytes that were incubated in ³H-labeled amino acids. The MOPC-321 k chain was purified from an oocyte homogenate by immunoprecipitation, followed by preparative gel electrophoresis. To determine whether the precursor segment had been properly and precisely cleaved in the oocyte, the amino acid sequence of the NH₂terminal end of the purified k chain was investigated. The NH₂-terminal sequence obtained was identical to that of the mature, secreted form of the protein. Thus the specificity of the enzyme performing the cleavage of precursor to mature chain is similar in frog oocytes and in mammalian cells. Therefore, the enzymatic specificity has been highly conserved during evolution and evidently performs an essential role in cellular metabolism.     

Hepatic lipase. Synthesis, processing, and secretion by isolated rat hepatocytes

Hepatic lipase, a glycoprotein synthesized and secreted by the hepatocyte, binds to sinusoidal endothelium where it is involved in metabolism of lipoprotein phospholipid and triglyceride. To better understand the regulation of hepatic lipase, we investigated the synthesis, post-translational processing, and secretion of the enzyme by isolated rat hepatocytes. Metabolically labeled [35S]methionine hepatic lipase protein, produced by the collagenase-dispersed hepatocytes, was immunoisolated from detergent-solubilized cells and incubation medium at designated times, using a polyclonal rabbit anti-rat hepatic lipase antibody raised against hepatic lipase purified to homogeneity from rat liver post-heparin perfusates. Following polyacrylamide gel electrophoresis and fluorography, radiolabeled hepatic lipase was quantitated by densitometry. Newly synthesized hepatic lipase was rapidly secreted and accumulated in the medium as a 59,000-dalton protein in a manner consistent with a constitutive process. An intracellular 53,000-dalton precursor of the mature 59,000-dalton hepatic lipase was identified by immunoprecipitation. The 53,000-dalton form could also be generated by endoglycosidase digestion of the secreted 59,000-dalton protein. In pulse-chase experiments, the 53,000-dalton protein was converted into the 59,000-dalton form. A 47,000-dalton form of hepatic lipase was immunoisolated from cell lysates only after tunicamycin treatment and could be generated from the secreted 59,000-dalton enzyme by prolonged endoglycosidase digestion. These data show that hepatic lipase is synthesized and rapidly secreted by isolated rat hepatocytes. Further, an intracellular 47,000-dalton precursor peptide can be identified after tunicamycin treatment, which may represent the hepatic lipase polypeptide, presumably after removal of its signal sequence; a 53,000-dalton partially glycosylated peptide exists as a major precursor form in the cell; and the mature 59,000-dalton hepatic lipase is present in the hepatocyte, but it is rapidly secreted.     

Synthesis of human Placental Lactogen in Xenopus oocytes

Oocytes from Xenopus laevis were injected with polysomes from normal human term placenta.Synthesis of the protein hormone Human Placental Lactogen (HPL) in the oocytes was demonstrated by specific immunoprecipitation with anti-HPL serum.Analysis of the immunoprecipitates on SDS-polyacrylamide gel revealed one peak, with a migration distance corresponding exactly to that of [¹⁴C]-Radioacetylated HPL added as a marker.Two days after injection the mRNA was still able to direct the synthesis of HPL.     

Expression of rat jejunal cystine carrier in Xenopus oocytes

Functional interactive cystine-lysine carriers have been expressed in Xenopus oocytes following the injection of RNA extracted from rat intestinal mucosa. Lysine-inhibitable cystine uptake was able to be measured 16 h after oocyte injection with RNA. The longer the oocytes were maintained after injection, the more cystine transport capability was induced. Uninjected or water-injected oocytes showed virtually no lysine-inhibitable cystine uptake, and no system developed after the oocytes had been isolated and maintained in vitro. The cystine uptake expressed after RNA injection was selectively inhibited by dibasic amino acids and phenylalanine but not by other amino acids or alpha-methyl-D-glucoside. Expression of the interactive cystine-lysine system was induced only by RNA isolated from intestinal tissue and not by RNA from rat liver. The Km for cystine uptake in RNA-injected oocytes was 0.01 mM and appears identical to the single system found in the RNA source tissue.     

Opposite effects of Ni2+ on Xenopus and rat ENaCs expressed in Xenopus oocytes

Opposite effects of Ni²⁺ on Xenopus and rat ENaCs expressed in Xenopus oocytes. Am J Physiol Cell Physiol 289: C946–C958, 2005. First published June 8, 2005; .—The epithelial Na⁺ channel (ENaC) is modulated by various extracellular factors, including Na⁺, organic or inorganic cations, and serine proteases. To identify the effect of the divalent Ni²⁺ cation on ENaCs, we compared the Na⁺ permeability and amiloride kinetics of Xenopus ENaCs (xENaCs) and rat ENaCs (rENaCs) heterologously expressed in Xenopus oocytes. We found that the channel cloned from the kidney of the clawed toad Xenopus laevis [wild-type (WT) xENaC] was stimulated by external Ni²⁺, whereas the divalent cation inhibited the channel cloned from the rat colon (WT rENaC). The kinetics of amiloride binding were determined using noise analysis of blocker-induced fluctuation in current adapted for the transoocyte voltage-clamp method, and Na⁺ conductance was assessed using the dual electrode voltage-clamp (TEVC) technique. The inhibitory effect of Ni²⁺ on amiloride binding is not species dependent, because Ni²⁺ decreased the affinity (mainly reducing the association rate constant) of the blocker in both species in competition with Na⁺. Importantly, using the TEVC method, we found a prominent difference in channel conductance at hyperpolarizing voltage pulses. In WT xENaCs, the initial ohmic current response was stimulated by Ni²⁺, whereas the secondary voltage-activated current component remained unaffected. In WT rENaCs, only a voltage-dependent block by Ni²⁺ was obtained. To further study the origin of the xENaC stimulation by Ni²⁺, and based on the rationale of the well-known high affinity of Ni²⁺ for histidine residues, we designed -subunit mutants of xENaCs by substituting histidines that were expressed in oocytes, together with WT - and -subunits. Changing His²¹⁵ to Asp in one putative amiloride-binding domain (WYRFHY) in the extracellular loop between Na⁺ channel membrane segments M1 and M2 had no influence on the stimulatory effect of Ni²⁺, and neither did complete deletion of this segment. Next, we mutated His⁴¹⁶ flanked by His⁴¹¹ and Cys⁴¹⁷, a unique site for possible heavy metal ion chelation, and, with this quality, most proximal (100 amino acids upstream of the second putative amiloride binding site at the pore entrance), was found localized at M2. Replacing His⁴¹⁶ with arginine, aspartate, tyrosine, and alanine clearly affected amiloride binding in all cases, as well as Na⁺ conductance, as expressed in the xENaC current-voltage relationship, especially with regard to aspartate and tyrosine. However, similarly to those obtained with the WYRFHY stretch, none of these mutations could either abolish the stimulating effect of Ni²⁺ or reverse it to an inhibitory type. epithelia; divalent cations; amiloride; Na⁺; voltage clamp     

Actin in Xenopus oocytes

It has been found that a high-speed supernatant fraction from Xenopus oocytes extracted in the cold will form a clear, solid gel upon warming. Gel formation occurs within 60 min at 18 degrees-40 degrees C, and is, at least initially, temperature reversible. Gelation is strictly dependent upon the addition of sucrose to the extraction medium. When isolated in the presence of ATP, the gel consists principally of a 43,000-dalton protein which co-migrates with Xenopus skeletal muscle actin on SDS-polyacrylamide gels, and a prominent high molecular weight component of approx. 250,000 daltons. At least two minor components of intermediate molecular weight are also found associated with the gel in variable quantities. Actin has been identified as the major consituent of the gel by ultrastructural and immunological techniques, and comprises roughly 47% of protein in the complex. With time, the gel spontaneously contracts to form a small dense aggregate. Contraction requires ATP. In the absence of exogenous ATP, a polypeptide which co-migrates with the heavy chain of Xenopus skeletal muscle myosin becomes a prominent component of the gel. This polypeptide is virtually absent from gels which have contracted in ATP-containing extracts. It has also been found that Ca++ is required for gelation in oocyte extracts. At both low and high concentrations of Ca++ (defined as a ratio of Ca++/EGTA in the extraction medium), gelation is inhibited.     

Expression of rat liver canalicular sulfate carrier in Xenopus laevis oocytes

Poly(A)+ RNA (mRNA)extracted from rat liver was injected into Xenopus laevis oocytes and the expression of sulfate transport was determined by measuring [35S] sulfate uptake. Compared to water-injected oocytes, which exhibited virtually no sulfate uptake, injection of rat liver mRNA resulted in a time- and dose-dependent increase in uptake of sulfate. Depending on the method used for the isolation of the mRNA, sulfate uptake was stimulated after injection (40 ng after 6 days) between 8- and 72-fold compared to water-injected oocytes. Sulfate uptake of oocytes injected with mRNA was found to be sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (IC50 less than 20 microM) and could also be inhibited by thiosulfate. Sulfate uptake of injected oocytes showed Michaelis-Menten kinetics (apparent Km, 0.31 mM) which is similar to the Km of the sulfate/bicarbonate antiporter of rat liver canalicular plasma membranes. After fractionation by a sucrose density gradient, the mRNA encoding for the expressed rat liver sulfate carrier was found in fractions containing messages of 3.5-4.0 kilobases in length.     

Expression of rat liver glutamine transporters in Xenopus laevis oocytes.

As a first step in attempting to isolate the Na(+)-dependent System N transporter from rat liver we have investigated the use of prophase-arrested oocytes from Xenopus laevis for the functional expression of rat liver glutamine transporters. Individual oocytes, defolliculated by collagenase treatment, were injected with 50 nl of a 1 mg.ml-1 solution of poly(A)+ RNA (mRNA) isolated from rat liver. 50 microM L-[3H]glutamine uptake was measured 1-5 days post-injection: after 48 h, poly(A)+ RNA-injected oocytes showed a 60 +/- 12% increase in Na(+)-dependent glutamine uptake compared to controls. This increased uptake showed characteristic features of hepatic System N: that is, it tolerated Li(+)-for-Na+ substitution and was inhibited by the System N substrate L-histidine (5 mM) in Li medium, unlike endogenous Na(+)-dependent glutamine transport. In subsequent experiments rat liver poly(A)+ RNA, size-fractionated by density gradient fractionation, was injected into oocytes. Injection of poly(A)+ RNA of 1.9-2.8 kilobases (kb) in size resulted in a significant stimulation of Na(+)-dependent glutamine transport to 0.362 +/- 0.080 pmol.min-1/oocyte from 0.178 +/- 0.060 pmol.min-1/oocyte in vehicle-injected oocytes (p less than 0.01). A lighter fraction, with poly(A)+ RNA of less than 1.9 kilobases size resulted in a similar increase in Na(+)-dependent glutamine uptake which was largely Li(+)-tolerant: Li(+)-stimulated glutamine uptake in oocytes injected with this fraction increased to 0.230 +/- 0.070 pmol.min-1/oocyte from 0.098 +/- 0.029 pmol.min-1/oocyte in controls (p less than 0.05). This enhanced rate of Li(+)-stimulated glutamine uptake was inhibited 28 and 70%, respectively, by 1 and 5 mM L-histidine. Na(+)-independent uptake of glutamine rose by 72 +/- 12% in oocytes injected with poly(A)+ RNA of 2.8-3.6 kb (p less than 0.001). These results demonstrate that glutamine transporters, with characteristics associated with hepatic Systems N, L, and A (or ASC), can be expressed in X. laevis oocytes injected with specific size fractions of rat liver mRNA.     

The influence of topology and glycosylation on the fate of heterologous secretory proteins made in Xenopus oocytes

Secretory proteins made in Xenopus laevis oocytes under the direction of heterologous messenger RNA are modified, topologically segregated and exported. Thus the oocyte may serve as a useful surrogate secretory system and we have studied some of the factors governing access to the export pathway. Unglycosylated chicken ovalbumin, synthesized and trapped in the cytosol, is not secreted but glycosylated ovalbumin, found sequestered within vesicles, is exported from oocytes. However, ovalbumin, which is transferred across the endoplasmic reticulum in the presence of tunicamycin and which is indistinguishable by immunoprecipitation, by two-dimensional gel electrophoresis and by concanavalin-A--Sepharose binding from the cytosolic form, is still secreted. Guinea-pig milk proteins and human interferon are also exported from tunicamycin-treated frog cells. These observations demonstrate that access to the endoplasmic reticulum but not glycosylation is a mandatory intermediate step in secretion, and emphasize the advantages of the oocyte as a surrogate system for the study of the later events in the gene expression pathway.     

Cytoskeleton in Xenopus oocytes and eggs

The Xenopus egg is a huge cell divided into compartments with distinct characteristics. The organization of the cytoskeleton reflects both the size of the egg and its regional differences. We review the information concerning the deployment and function of cytoskeletal elements during the changes in cellular organization accompanying oogenesis, oocyte maturation, and following fertilization.     

Biosynthesis, processing and secretion of mucus glycoprotein in the rat stomach

For the study of the biosynthesis, processing and secretion of mucus glycoproteins in rat gastric mucous cells, antibodies were raised against purified gastric mucus glycoproteins and against deglycosylated gastric mucus glycoproteins. Indirect immunofluorescence analysis of gastric mucosa sections revealed that both antibodies specifically labelled the mucus glycoprotein-synthesizing cells in the gastric mucosa. Stomach segments were pulse-labelled with [35S]cysteine and chased for various times. The radioactively labelled (glyco)proteins were quantitatively immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophoresis. Less than 3% of the total radioactivity incorporated in protein was found to be present in mucus glycoproteins. Antibodies raised against native mucus glycoproteins recognized only high-molecular-weight mucus glycoproteins, while the antibodies against deglycosylated glycoproteins also bound to probable precursor forms. The synthesis of mature mucus glycoproteins (Mr greater than 300 000) required about 90 min. After 3 h of chase, only a small portion of the pulse-labelled mucus glycoproteins had been secreted; the majority of the radioactive glycoproteins at that time was still associated with the tissue. Immature (glyco)proteins were not secreted into the medium.     

Single-channel currents of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes

The neuronal nicotinic acetylcholine receptor subunits alpha 2, alpha 3, and alpha 4 form functional receptors with the beta 2 subunit. Each of these subunit combinations shows two distinct open states (referred to as primary and secondary). The primary open states of alpha 2 beta 2, alpha 3 beta 2, and alpha 4 beta 2 receptors were 33.6 +/- 1.8 pS, 15.4 +/- 0.8 pS, and 13.3 +/- 1.5 pS, respectively. The open times of the alpha 3 beta 2 primary open state were significantly longer than the open times of the other primary conductance states. The secondary open states of alpha 2 beta 2 and alpha 3 beta 2 were 15.5 +/- 1.3 pS and 5.1 +/- 0.4 pS, respectively. Secondary open states were seen infrequently with alpha 4 beta 2. Oocytes injected with alpha 2 RNA and a 9-fold excess of beta 2 RNA showed an enhanced expression of the secondary open state.