The functional role of protein O-mannosyltransferase POMT2 for mammalian development and fertilization
Final Report Abstract
Protein O-Mannosylation represents an evolutionarily conserved, essential modification. In mammals the protein O-mannosyltransferases POMT1 and POMT2 act as a heteromeric complex to initiate O-mannosylation in the endoplasmic reticulum. Mutations in human POMT1 and POMT2 cause a group of congenital muscular dystrophies due to reduced O-glycosylation of a-dystroglycan. The most severe of these autosomal recessive conditions is Walker-Warburg syndrome (WWS) with severe brain and ocular involvement. We previously showed that targeted disruption of POMT1 in mice results in embryonic lethality. POMT1 is expressed in WWS-related tissues both dunng embryogenesis and in adults. Whereas there is only a single Pomt1 transcript in adult mice, we demonstrated that there are two Pomt2 transcripts, somatic sPomt2 and testis specific tPomt2. Our analyses aimed to clarify the functional role of mammalian POMT2. The major objectives of this project were to elucidate i) the role of the testis specific POMT2 isoform (tPOMT2) and ii) the role of POMT2 for development. We demonstrated that somatic POMT2 (sP0MT2), but not tPOMT2, is prominently expressed in mouse embryos in the tissues that are most severely affected in WWS (developing muscle, eye, and brain). Correlation of POMT transcripts and protein isoforms with POMT mannosyltransferase enzyme activity demonstrates that sPOMT2-POMT1 complexes catalyze mannosyltransfer in adult somatic tissues and testis. It is suggested that the gonadal defects described in some WWS cases are associated with defects in O-mannosylation. Our data further show that whereas sPOMT2 is widely expressed, tPOMT2 is restricted to the acrosome of male germ cells and is not involved in the biosynthesis of O-mannosyl glycans in vivo. We prove that tPOMT2 is highly conserved among mammals, including humans, suggesting a crucial function that is distinct from sPOMT2. During embryogenesis, the murine POMT2 gene is prominently expressed in the neural tube, the developing eye, and the mesenchyme. These sites of expression correlate with those in which the main tissue alterations are observed in WWS patients. We have inactivated a POMT2 allele by gene targeting in embryonic stem cells and produced chimeras transmitting the defect allele to offspring. Although heterozygous mice were viable and fertile, the total absence of POMT2-/- pups in the progeny of heterozygous intercrosses indicated that this genotype is embryonic lethal. An analysis of the mutant phenotype revealed that homozygous POMT2-/- mice die around embryonic day (E) 0.5. Surprisingly, this phenotype is different from homozygous POMTV1-/- mice that suffer developmental arrest around embryonic day (E) 7.5 and die between E7.5 and E9.5. Ours data suggest a role of POMT2 during development that is independent of POMT1. We further showed that dermal fibroblasts can be applied to simplify the diagnostic analysis of dystroglycanopathy patients as well as to study the pathogenicity of POMT mutations.
Publications
- (2008) POMT2, a key enzyme in Walker-Warburg syndrome: somatic sPOMT2, but not testis-specific tPOMT2, is crucial for mannosyltransferase activity in vivo. Glycobiology 18, 615-625
Lommel M., Willer T. and Strahl S.
