Muscular dystrophies are genetic diseases characterized by progressive muscle degeneration and muscular weakening. Defects in glycosylation of α-dystroglycan (α-DG), one of the dystrophin-glycoprotein complex components, are responsible for certain congenital muscular dystrophies including Walker-Warburg syndrome (WWS) and muscle-eye-brain disease (MEB), so-called α-dystroglycanopathies. α-DG has unique glycans whose structure is Siaα2-3Galβ1-4GlcNAcβ1-2Manα1-Ser/Thr, and this glycan is required for binding to basal lamina proteins such as laminin, perlecan and agrin. Protein O-mannosyltransferases (POMT1 and POMT2) catalyze the first step in O-mannosyl glycan synthesis on α-DG when only they are co-expressed, and defects in POMT1 or POMT2 result in WWS. Next, O-mannose β-1, 2-N-acetylglucosaminyltransferase 1 (POMGnT1) catalyzes the transfer of GlcNAc to the protein O-mannosyl residue and it is responsible for MEB.
Zebrafish is a useful vertebrate model for studying genetically higher levels of biological phenomena such as organogenesis, morphogenesis, toxicology, and teratology. Muscle structure of zebrafish embryogenesis has been well studied and is an attractive model for investigation of genes involved in muscle development and degeneration.
In this study, we cloned complete cDNAs encoding zebrafish POMT1 (zPOMT1), zPOMT2 and zPOMGnT1 and examined whether they had enzymatic activity. zPOMGnT1 expressed in human embryonic kidney 293T (HEK293T) cells revealed enzymatic activity, whereas zPOMT1 and zPOMT2 showed high level of enzymatic activity only when zPOMT1 and zPOMT2 were co-expressed in HEK293T cells. Furthermore, we performed knockdown analysis using antisense morpholino oligonucleotides. As a result, while zPOMT2 morphant showed more severe phenotypes than zPOMT1 morphant, zPOMGnT1 morphant had bended body, small eyes and edematous pericardium. To investigate the glycosylation status of α-DG in morpholino-injected embryos against zPOMT1, zPOMT2 and zPOMGnT1, embryos were immunostained with the anti-glycosylated α-DG antibody (IIH6). Strong signals by IIH6 antibody were detected in the horizontal and vertical myosepta of control embryos. However, the reactivity in each morphant was almost completely lost.
