Abstract
Human Goodpasture antigen-binding protein (GPBP) is an atypical protein kinase that phosphorylates the Goodpasture auto-antigen, the alpha3 chain of collagen IV. The COL4A3BP gene is alternatively spliced producing two protein isoforms: GPBP and GPBPDelta26. The latter lacks a serine-rich domain composed of 26 amino acid residues. Both isoforms also function as ceramide transfer proteins (CERT). Here, we explored the function of Gpbp and GpbpDelta26/CERT during embryogenesis in zebrafish. We cloned both splice variants of the zebrafish gene and found that they are differentially expressed during development. We used antisense oligonucleotide-mediated loss-of-function and synthetic mRNA-based gain-of-function approaches. Our results show that the loss-of-function phenotype is linked to cell death, evident primarily in the muscle of the somites, extensive loss of myelinated tracks, and brain edema. These results indicate that disruption of the nonvesicular ceramide transport is detrimental to normal embryonic development of somites and brain because of increased apoptosis. Moreover, this phenotype is mediated by Gpbp but not GpbpDelta26/CERT, suggesting that Gpbp is an important factor for normal skeletal muscle and brain development.
Highlights
Our results suggest that the observed phenotype is a consequence of depletion of the full-length Gpbp splice isoform, selectively inducing apoptosis in muscle and brain during early development
Stockdale for the F59 antibody, and Mercedes Montero-Balaguer for assistance in the initial stages of the project
Summary
Materials—The preparation of monoclonal antibody against human GPBP (Mab14) was previously described (1). Using the pRCX vectors as templates and the primers EcoMFLAGF and Eco2R, the isoforms were reamplified and digested with EcoRI Both PCR products were inserted in the EcoRI site of pcDNA3.1 expression vector (Invitrogen). After extensive washes in PTT, the embryos were incubated with biotinylated secondary antibodies (Vector) at 1:200 dilution in blocking solution for 1 h at room temperature. After extensive washes with PBT (0.1% Tween 20 in PBS), the embryos were incubated with secondary anti-mouse Alexa 488, and Alexa 555 IgG antibodies at 1:400 dilution (Molecular Probes). Rehydrated embryos were permeabilized by proteinase K digestion, washed in PBT, and assayed by TUNEL using the in situ cell death detection kit POD (Roche Applied Science) according to the manufacturer’s instructions. A stock solution of 5 mg/ml in egg water was diluted 300 times in egg water, and dechorionated live embryos were bathed in this solution for 20 min in the dark, extensively washed in egg water, analyzed under a fluorescence microscope, and photographed
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