Alkaline phosphatase fusions of ligands or receptors as in situ probes for staining of cells, tissues, and embryos
Publisher Summary
Polypeptide ligands and their cell surface receptors bind to one another with high affinity and specificity. These biological properties can be exploited to make affinity probes to detect their cognate ligands or receptors. This approach has been applied for decades, using radiolabeled ligands as probes to detect their receptors. More recently, it has also been found that receptor ectodomains can be used as soluble probes to detect their ligands. When producing soluble receptor or ligand affinity probes, it has been common to produce the probe as a fusion protein with a tag. This can make detection and purification procedures much easier. Two tags that are widely used for this purpose are alkaline phosphatase (AP) or the immunoglobulin Fc region. Both of these tags are dimeric, and are expected to produce a fusion protein with a pair of ligand or receptor moieties facing away from the tag in the same direction. This chapter describes the production of alkaline phosphatase (AP) fusion proteins, and also describes in situ procedures in which these affinity probes are used to detect the distribution of cognate ligands or receptors in tissues or cells.
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Cited by (107)
Progranulin-derived granulin E and lysosome membrane protein CD68 interact to reciprocally regulate their protein homeostasis
2022, Journal of Biological ChemistryProgranulin (PGRN) is a glycoprotein implicated in several neurodegenerative diseases. It is highly expressed in microglia and macrophages and can be secreted or delivered to the lysosome compartment. PGRN comprises 7.5 granulin repeats and is processed into individual granulin peptides within the lysosome, but the functions of these peptides are largely unknown. Here, we identify CD68, a lysosome membrane protein mainly expressed in hematopoietic cells, as a binding partner of PGRN and PGRN-derived granulin E. Deletion analysis of CD68 showed that this interaction is mediated by the mucin–proline-rich domain of CD68. While CD68 deficiency does not affect the lysosomal localization of PGRN, it results in a specific decrease in the levels of granulin E but no other granulin peptides. On the other hand, the deficiency of PGRN, and its derivative granulin peptides, leads to a significant shift in the molecular weight of CD68, without altering CD68 localization within the cell. Our results support that granulin E and CD68 reciprocally regulate each other’s protein homeostasis.
The EphA2 receptor tyrosine kinase activates signaling pathways with different, and sometimes opposite, effects in cancer and other pathologies. Thus, highly specific and potent biased ligands that differentially control EphA2 signaling responses could be therapeutically valuable. Here, we use EphA2-specific monomeric peptides to engineer dimeric ligands with three different geometric configurations to combine a potential ability to differentially modulate EphA2 signaling responses with the high potency and prolonged receptor residence time characteristic of dimeric ligands. The different dimeric peptides readily induce EphA2 clustering, autophosphorylation and signaling, the best with sub-nanomolar potency. Yet, there are differences in two EphA2 signaling responses induced by peptides with different configurations, which exhibit distinct potency and efficacy. The peptides bias signaling when compared with the ephrinA1-Fc ligand and do so via different mechanisms. These findings provide insights into Eph receptor signaling, and proof-of-principle that different Eph signaling responses can be distinctly modulated.
Recombinant Expression and Purification of Mouse Nectin-like 4 Glycoprotein in 293ET Cell Line
2018, Chinese Medical Sciences JournalTo screen the transient and stable cell lines with high production of Nectin-like 4 (Necl-4) protein.
First, cDNA sequences encoding the extracellular domain of Necls were cloned into the modified vector pAPtag at the N terminus of alkaline phosphatase (AP) for fusion expression. Next, 293ET cells stably expressed Necls-AP fusion protein and secreted it into the culture medium which were detected by the AP activity assay and Western blot analysis. Then, by adding N-glycosylation processing inhibitor kifunensine into the medium, complex glycan was inhibited to generate. The residual glycan of purified protein was removed by endoglycosidase H. Finally, AP protein was removed by using human rhinovirus protease and size exclusion chromatography. The concentration of purified Necl-4 protein was monitored by measuring the absorbance at 280 nm and analyzed by SDS-PAGE.
The transient and stable cell lines with high production of Necl-4 protein were screened by the color reaction with the AP-tag in the recombinant vector. The soluble and active form of purified Necl-4 protein was obtained after deglycosylation of native N-glycan protein with an expression level of 4 mg/L culture and purity of 95%.
By using modified AP mammalian protein expression system, we can easily screen the high productive stable cell lines by using AP activity assay. By adding mannosidase inhibitor kifunensine into the medium and cutting purified protein by using endoglycosidase H, we can obtain deglycosylated Necl-4 protein in milligram quantities. Our method might throw a light on the expression and purification of glycoprotein for structural and functional studies.
The C-terminal region of Reelin is necessary for proper positioning of a subset of Purkinje cells in the postnatal cerebellum
2016, NeuroscienceIn the normal cerebellum, Purkinje cells (PCs) are generated in a zone along the ventricular surface, migrate radially, and align to form a single-cell layer. However, in mice lacking the secreted protein Reelin or its downstream adaptor protein Dab1, the majority of PCs are located ectopically in the deep cerebellar mass. Nonetheless, how Reelin regulates migration and alignment of PCs remains incompletely understood. Reelin has a highly-conserved C-terminal region (CTR), which is required for its full activity. Here, we report an abnormality of the cerebellum in Reelin CTR-lacking knock-in (ΔC-KI) mice. In the ΔC-KI mice, cerebellar formation was largely normal, but some PCs in selected regions were found to be located ectopically and to frequently form clusters. Ectopic PCs contained a higher amount of Dab1 protein and functional Reelin receptors, including mainly very low-density lipoprotein receptor than correctly-aligned PCs. Decreasing Dab1 gene dosage exacerbated mislocalization of PCs and the cerebellar structure in Reelin ΔC-KI mice. These results indicate that ectopic PCs in ΔC-KI mice failed to receive sufficient Reelin signaling en route to their final destinations. Further, we also found that Reelin protein with intact CTR binds preferentially to PCs. Thus, it was suggested that the extent or quality of Reelin/Dab1 signaling that PCs require for correct positioning vary and that Reelin with intact CTR is required for that of a certain subset of PCs.
During the development of forebrain connectivity, ascending thalamocortical and descending corticofugal axons first intermingle at the pallial-subpallial boundary to form the internal capsule (IC). However, the identity of molecular cues that guide these axons remains largely unknown. Here, we show that the transmembrane protein Linx is robustly expressed in the prethalamus and lateral ganglionic eminence-derived corridor and on corticofugal axons, but not on thalamocortical axons, and that mice with a null mutation of Linx exhibit a complete absence of the IC. Moreover, regional inactivation of Linx either in the prethalamus and LGE or in the neocortex leads to a failure of IC formation. Furthermore, Linx binds to thalamocortical projections, and it promotes outgrowth of thalamic axons. Thus, Linx guides the extension of thalamocortical axons in the ventral forebrain, and subsequently, it mediates reciprocal interactions between thalamocortical and corticofugal axons to form the IC.
XAn extracellular interactome of immunoglobulin and LRR proteins reveals receptor-ligand networks
2013, CellExtracellular domains of cell surface receptors and ligands mediate cell-cell communication, adhesion, and initiation of signaling events, but most existing protein-protein “interactome” data sets lack information for extracellular interactions. We probed interactions between receptor extracellular domains, focusing on a set of 202 proteins composed of the Drosophila melanogaster immunoglobulin superfamily (IgSF), fibronectin type III (FnIII), and leucine-rich repeat (LRR) families, which are known to be important in neuronal and developmental functions. Out of 20,503 candidate protein pairs tested, we observed 106 interactions, 83 of which were previously unknown. We “deorphanized” the 20 member subfamily of defective-in-proboscis-response IgSF proteins, showing that they selectively interact with an 11 member subfamily of previously uncharacterized IgSF proteins. Both subfamilies interact with a single common “orphan” LRR protein. We also observed interactions between Hedgehog and EGFR pathway components. Several of these interactions could be visualized in live-dissected embryos, demonstrating that this approach can identify physiologically relevant receptor-ligand pairs.