When Transforming Growth Factor ß family precursor proteins are ectopically expressed in Xenopus laevis embryos, they dimerize, get cleaved and are secreted into the blastocoele, which begins at the late blastula to early gastrula stage. We describe a method for aspirating cleavage products from the blastocoele cavity for immunoblot analysis.
The two arms of the Transforming Growth Factor ß (Tgfß) superfamily, represented by Tgfß/Nodal or Bone morphogenetic protein (Bmp) ligands, respectively, play essential roles in embryonic development and adult homeostasis. Members of the Tgfß family are made as inactive precursors that dimerize and fold within the endoplasmic reticulum. The precursor is subsequently cleaved into ligand and prodomain fragments. Although only the dimeric ligand can engage Tgfß receptors and activate downstream signaling, there is growing recognition that the prodomain moiety contributes to ligand activity. This article describes a protocol that can be used to identify cleavage products generated during activation of Tgfß precursor proteins. RNA encoding Tgfß precursors are first microinjected into X. laevis embryos. The following day, cleavage products are collected from the blastocoele of gastrula stage embryos and analyzed on Western blots. This protocol can be completed relatively quickly, does not require expensive reagents and provides a source of concentrated Tgfß cleavage products under physiologic conditions.
Members of the Transforming Growth Factor ß (Tgfß) superfamily are synthesized as inactive, dimerized precursor proteins. The precursors are then cleaved by members of the proprotein convertase (PC) family, either within the secretory pathway or outside of cells. This creates an active, disulfide-bonded ligand dimer and two prodomain fragments1. Although it has been known for over 30 years that the prodomain of Tgfß family precursors is required to generate an active ligand2, the understanding of how prodomains contribute to ligand function is incomplete.
Although the understanding of the process of proteolytic activation of Tgfß family members remains incomplete, there is increasing interest in understanding which PC consensus motif(s) are cleaved in vivo, whether the cleavage occurs in a specific subcellular or extracellular compartment, and whether the prodomain remains covalently or noncovalently associated with the cleaved ligand3. Several studies have shown that the prodomain not only guides ligand folding before cleavage4,5, but can also influence growth factor stability and range of action6,7,8,9, drive the formation of homodimers or heterodimers10, anchor the ligand in the extracellular matrix to maintain ligand latency11, and in some cases, function as a ligand in its own right to activate heterologous signaling12. Heterozygous point mutations within the prodomain of many members of the Tgfß family are associated with eye, bone, kidney, skeletal or other defects in humans3. These findings highlight the critical role of the prodomain in generating and maintaining an active ligand and stress the importance of identifying and deciphering the role of cleavage products developed during proteolytic maturation of Tgfß family precursors.
Here we describe a detailed protocol for aspirating cleavage products generated during maturation of Tgfß family precursors from the blastocoele of X. laevis embryos and then analyzing them on immunoblots. This protocol can be used to determine whether one or more PC consensus motif(s) in a precursor protein are cleaved in vivo10,13, identify the endogenous PC(s) that cleave each motif13,14, compare in vivo formation of Tgfß family homodimers versus heterodimers10 or analyze whether human disease-associated point mutations in Tgfß precursors impact their ability to form functional dimeric ligands.
All procedures described are approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Utah. Frogs are housed in an IACUC approved facility. Male frogs are euthanized by immersion in tricaine following by clipping the ventricle of the heart. Female frogs are housed in the laboratory for a maximum of 24 h following hormonal induction of spawning to allow for egg collection and then returned to the care facility.
1. Collection of X. laevis Testes
2. Collection and Fertilization of X. laevis Eggs
NOTE: Frogs should be handled with vinyl gloves or hands washed before and after handling the frogs. Latex gloves, lotions and detergent residues on human hands will damage the frogs' fragile skin.
3. Microinjection of X. laevis Embryos
4. Blastocoele Extraction and Analysis of Tgfß Cleavage Products
The experiment's goal described below was to determine whether Bmp4 and Bmp7 form heterodimers (dimers composed of one Bmp4 ligand and one Bmp7 ligand), homodimers (composed of two Bmp4 or two Bmp7 ligands), or a mixture of each when they are co-expressed in X. laevis. Data shown in Figure 2 are extracted from a previously published study10. Figure 2A is a schematic showing cleavage products generated by proteolytic maturation of Bmp4 or Bmp7 homodimeric precursors or Bmp4/7 heterodimeric precursors. Bmp4 is cleaved at two sites within the prodomain to generate two prodomain fragments (dark green and yellow) and one dimeric ligand fragment (light green) that migrates at ~26 kDa on SDS-PAGE gels. Bmp7 is cleaved at a single site to generate one prodomain fragment (maroon) and one dimeric ligand fragment (pink) that migrates at ~35 kDa. Heterodimeric ligands migrate at ~30 kDa. The silver bar in the ligand dimers represents a myc-epitope tag inserted into this domain.
RNA encoding Bmp4myc or Bmp7myc precursors (200 pg), or both RNAs (100 pg of each) were injected into X. laevis embryos at the 2-4 cell stage. The embryos were cultured overnight at 16 °C until they reached the early gastrula stage. At this point, fluid was aspirated from the blastocele of 20 embryos in each group and sterile water was added to bring the volume to a total of 30 µL. Following deglycosylation, each sample was split into two tubes. Reducing sample buffer was added to one tube, and non-reducing sample buffer was added to the other. Samples were heated to 100 °C for 5 min. Proteins were then separated by SDS/PAGE and immunoblots were probed with antibodies that recognize the myc-epitope tag (Figure 2B). Under reducing conditions, a single band corresponding to cleaved Bmp4 monomers and a more slowly migrating band corresponding to cleaved BMP7 monomers were detected in lysates from embryos expressing only Bmp4 or Bmp7, respectively (Figure 2C, D, lower panel, lanes 1, 2). Both bands were detected in embryos co-expressing Bmp4 and Bmp7 (Figure 2C, D, lower panel, lane 3). Relatively equivalent amounts of Bmp4 and Bmp7 monomers were present in each of the three groups (lower panel, reducing). In this experiment, when proteins were separated under non-reducing conditions, a single mature Bmp4/7 heterodimer band of intermediate mobility was detected along with a trace amount of Bmp4 homodimer in embryos co-expressing Bmp4 and Bmp7 (Figure 2C, upper panel). From these results we conclude that if equivalent levels of Bmp4 and Bmp7 precursor proteins are expressed in Xenopus embryos, they preferentially form heterodimers, rather than either homodimer. In the experiment shown in Figure 2D, significantly more Bmp7 protein is present relative to Bmp4 (lower panel, reducing). As a result, in embryos expressing both Bmp7 and Bmp4 precursor proteins, Bmp4 homodimers are not detected and instead the available Bmp4 is present as a Bmp4/7 heterodimer, and the excess Bmp7 forms homodimers. While this experiment is not optimal, as the goal was to co-express the equivalent amount of Bmp4 and Bmp7 precursor, the results are still consistent with the conclusion that Bmp4 and Bmp7 preferentially form heterodimers over either homodimer when co-expressed.
Figure 1. Injection and aspiration needles. Photographs of representative needles that have been pulled but not clipped (A) pulled and clipped for use as an injection needle (B) or pulled and clipped for use as an aspiration needle (C) are shown. The arrow and arrowhead in (A) indicate the point at which the glass was clipped to generate a needle for injection and aspiration, respectively. Please click here to view a larger version of this figure.
Figure 2. Analysis of cleaved Bmp ligands extracted from X. laevis blastocele fluid. (A) Cleavage products generated from Bmp4 and Bmp7 homodimeric or heterodimeric precursor proteins and position of myc epitope tag (silver bar) is shown schematically (B) Schematic diagram showing the timing of injection and blastocoele fluid extraction. (C-D) Xenopus embryos were injected with 200 pg of Bmp7 or Bmp4 RNA, or with Bmp4 and Bmp7 RNA mixed together (100 pg each). At the gastrula stage, fluid was extracted from the blastocoele of the same number of embryos in each experimental group. Proteins present in the blastocoele fluid were deglycosylated and separated by SDS-PAGE. Antibodies recognizing the myc-epitope tag were used to probe immunoblots. The relative position of immunoreactive ligand monomers and dimers is shown schematically to the right of each gel. Black line in (C) indicates removal of an intervening lane on the blot. Data shown are extracted from a previously published study10. Please click here to view a larger version of this figure.
Solution | Composition |
0.2% Tricaine | 20 g of Tricaine dissolved in deionized water, adjust pH to 7.4 by addition of sodium bicarbonate |
10x MBS | 880 mM NaCl, 10 mM KCl, 25 mM NaHCO3, 100 mM HEPES (pH 7.5), 10 mM MgSO4, 0.14 mM Ca(NO3)2, 0.41 mM CaCl2; Adjust to pH 7.5 with NaOH. The 10x MBS stock can be stored at 4 °C and diluted as needed. |
2% cysteine solution | 2 g of cysteine per 100 mL of deionized water, adjust pH to 7.8-8.0 with sodium hydroxide. Make fresh each day. |
20x DeBoer's pond water | 100 mM NaCl, 1.3 mM KCl, 0.44 mM CaCl2; Adjust to pH 7.4 with NaHCO3. 20x stock of DeBoer’s pond water can be stored at 4 °C and diluted as needed. |
4x non-reducing sample buffer | 200 mM Tris pH 6.8, 8% SDS, 0.4% bromophenol blue, 40% glycerol. |
4x reducing sample buffer | 200 mM Tris pH 6.8, 8% SDS, 0.4% bromophenol blue, 40% glycerol, 14.4 M ß-mercaptoethanol |
5% Ficoll Solution | 25 g of Ficoll in 500 mL 0.1x MBS, adjust pH to 7.5 with sodium hydroxide. Store at 4 °C. |
Embryo lysate buffer | 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 2.5 % NP-40. Store at 4 °C. |
Human chorionic gonadotropin | Using a 3 mL syringe attached to a 19 G needle, inject 2.5 mL of sterile water through the rubber stopper of a vial of human chorionic gonadotropin (10,000 IU ). Store at 4 °C . |
Testis buffer | 10% fetal bovine serum, 1% pen/strep (100U/mL penicillin, 100 mg/mL streptomycin in 1x MBS |
Table 1.
The main advantages to the protocol described here are that it can be completed relatively quickly, does not require expensive reagents and provides a source of concentrated Tgfß cleavage products under physiologic conditions. Another advantage is that it allows one to analyze epitope-tagged proteins and thus circumvent the shortage of commercially available antibodies that recognize most Tgfß family prodomain. Although it is also possible to analyze the cleavage of epitope-tagged Tgfß precursor proteins in transfected cultured mammalian cells, there are several advantages to using X. laevis. Early X. laevis embryos express all four members of the PC family that are candidates for endogenous Tgfß convertases (Furin, Pcsk5, Pcsk6 and Pcsk7)13,14,16. Some mammalian cell lines do not express one or more PC, requiring the ectopic expression of both the precursor protein and its convertase to examine cleavage17. A more important consideration is that very high levels of precursor proteins generated by transient transfect of transformed cells can lead to artifacts, such as the secretion of misfolded cleavage products, which can occur if precursor levels exceed the capacity for quality control in the ER18. This can mask the effect of deleterious mutations on precursor dimerization and cleavage since the misfolded cleavage products appear in the media and misfolding would not be detected unless the activity is assayed. By contrast, experiments utilizing X. laevis embryos readily detect defects in folding, leading either to loss of precursor due to the misfolded protein response in the ER or aberrant migration of cleavage products under non-reducing conditions10,18.
During the blastocoele aspiration procedure, it is essential to try to extract an approximately equivalent amount of blastocoele fluid from each embryo, particularly if, for example, the goal was to compare cleavage products generated from wild type and mutant precursors. This is an inexact science but aspirating for an approximately equal amount of time from each embryo helps to normalize the volume. In addition, aspirating fluid from a greater number of embryos in each group helps to normalize any differences in volume between individual embryos.
In vitro cleavage reactions are an alternative procedure that can be used to determine whether a particular PC motif present in a Tgfß precursor protein is cleaved and to identify and/or rule out potential PCs as endogenous convertases for a given substrate. Existing protocols describe a simple assay in which radiolabeled precursor proteins are generated in vitro, using rabbit reticulocytes, for example, or in vivo, using X. laevis oocytes. Substrates are then incubated in vitro with candidate recombinant PCs and cleavage products analyzed by electrophoresis and autoradiography19. While this protocol provides a fast and easy way to determine whether a protein is a PC substrate and to identify which potential cleavage sites are utilized in vivo, the precursor proteins are unlikely to be adequately folded or dimerized and thus cleavage information obtained from these experiments may not reflect the in vivo situation.
One shortcoming of the protocol we describe here is that it is impossible to visualize properly folded, dimerized precursor protein in X. laevis embryos. The same is true in other ectopic expression systems, such as transiently transfected mammalian cells, since uncleaved monomeric precursor protein accumulates within the ER at much higher levels than post-ER dimerized and folded precursors. The dimerized precursor is rapidly cleaved and secreted once it leaves the ER. If it is essential to analyze dimerization and folding of precursor proteins, a useful alternative procedure is to examine the trafficking and cleavage of radiolabeled precursors in X. laevis oocytes20. The high sensitivity of autoradiography, coupled with the fact that proteins move through the secretory system more slowly in oocytes than they do in cultured mammalian cells or embryos allows one to detect dimerized precursor proteins within oocytes and to test whether the proteins are properly folded and have left the ER by examining their glycosylation status18,21.
In summary, this protocol provides a rapid and straightforward method to analyze cleavage products generated by the maturation of Tgfß family precursor proteins.
The authors have nothing to disclose.
We thank Mary Sanchez for excellent animal care. The authors' research is supported by the National Institute of Child Health and Human Development of the National Institutes of Health (NIH/NICHD) grants R01HD067473-08 and R21 HD102668-01 and by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK/NIH) grant R01DK128068-01.
ß-mercaptoethanol | Fisher | AC125470100 | |
Bromophenol Blue | Fisher | B392-5 | |
Calcium Chloride | Fisher | C79-500 | |
Calcium Nitrate | Fisher | C109-500 | |
Disposable Pellet Pestle/Tissue Grinder | Fisher | 12-141-364 | |
Dumont #5 forceps | Fine Science tools | 11251-10 | |
Fetal Bovine Serum | Atlanta Biologicals | S11150H | |
Ficoll 400 | Sigma Aldrich | F9378-500G | |
Glass capillary, 1 X 90 mm | Narshige | G-1 | |
Glycerol | Fisher | G33-4 | |
HEPES | Fisher | BP310-500 | |
Human chorionic gonadotropin | Sigma Aldrich | CG10-10VL | |
Injection Syringe, 1 mL | Fisher | 8881501368 | |
L-Cysteine | Sigma Aldrich | C7352 | |
Magnesium Sulfate | Fisher | M63-500 | |
Needle, 26 G | Fisher | 305111 | |
Penicillin/Streptomycin | Gibco | 15140148 | |
Picoliter Microinjector | Warner Instruments | PLI-100A | |
Pipette Puller | Narashige | PC-100 | |
Potassium Chloride | Fisher | P217-500 | |
PVDF Membrane | Sigma Aldrich | IPVH00010 | |
Sodium Bicarbonate | Fisher | S233-500 | |
Sodium Chloride | Fisher | S271-10 | |
Sodium Dodecyl Sulfate | Fisher | BP166-500 | |
Sodium Hydroxide | Fisher | S318-500 | |
Tricaine-S | Pentair | TRS5 | |
Tris | Fisher | BP152-5 |