title: Protein O-mannosylation is necessary for normal embryonic development in zebrafish.
abstract: Two distinct cDNAs corresponding to two zebrafish protein O-mannosyltransferase genes, zPOMT1 and zPOMT2, were cloned from early developmental embryos. Gene expression analysis revealed that zPOMT1 and zPOMT2 were expressed in similar patterns during early embryonic development and in all adult tissues. To study the regulation of zPOMT1 and zPOMT2 mRNA distribution during zebrafish embryogenesis, we injected enhanced green fluorescent protein (EGFP) mRNA fused to the 3'untranslated regions of each zPOMT gene. The distribution of EGFP resulting from the two constructs was similar. Injection of antisense morpholino oligonucleotides of zPOMT1 and zPOMT2 resulted in several severe phenotypes-including bended body, edematous pericaridium and abnormal eye pigmentation. Immunohistochemistry using anti-glycosylated alpha-dystroglycan antibody (IIH6) and morphological analysis revealed that the phenotypes of zPOMT2 knockdown were more severe than those of zPOMT1 knockdown, even though the IIH6 reactivity was lost in both zPOMT1 and zPOMT2 morphants. Finally, only when both zPOMT1 and zPOMT2 were expressed in human embryonic kidney 293T cells were high levels of protein O-mannosyltransferase activity detected, indicating that both zPOMT1 and zPOMT2 were required for full enzymatic activity. Moreover, either heterologous combination, zPOMT1 and human POMT2 (hPOMT2) or hPOMT1 and zPOMT2, resulted in enzymatic activity in cultured cells. These results indicate that the protein O-mannosyltransferase machinery in zebrafish and humans is conserved and suggest that zebrafish may be useful for functional studies of protein O-mannosylation.
authors.affiliation: Department of Life Science, Mie University Graduate School of Bioresources, 1577 Kurimamachiya, Tsu, Mie 514-8507, Japan.
authors.forename: Eriko
authors.initials: E
authors.lastname: Avsar-Ban
authors.forename: Hisayoshi
authors.initials: H
authors.lastname: Ishikawa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Manya
authors.forename: Masatoki
authors.initials: M
authors.lastname: Watanabe
authors.forename: Shinichi
authors.initials: S
authors.lastname: Akiyama
authors.forename: Hideo
authors.initials: H
authors.lastname: Miyake
authors.forename: Tamao
authors.initials: T
authors.lastname: Endo
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Tamaru
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D003001:Cloning, Molecular; D004625:Embryo, Nonmammalian; D047108:Embryonic Development / Q000235:genetics / Q000502:physiology*; D006031:Glycosylation; D008358:Mannose / Q000378:metabolism*; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000502:physiology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics / Q000378:metabolism*
chemical_list: D008364:Mannosyltransferases; C082696:protein O-mannosyltransferase; D008358:Mannose