Carbohydrate-binding domain of the POMGnT1 stem region modulates O-mannosylation sites of α-dystroglycan.

The dystrophin glycoprotein complex, which connects the cell membrane to the basement membrane, is essential for a variety of biological events, including maintenance of muscle integrity. An O-mannose-type GalNAc-ß1,3-GlcNAc-ß1,4-(phosphate-6)-Man structure of α-dystroglycan (α-DG), a subunit of the complex that is anchored to the cell membrane, interacts directly with laminin in the basement membrane. Reduced glycosylation of α-DG is linked to some types of inherited muscular dystrophy; consistent with this relationship, many disease-related mutations have been detected in genes involved in O-mannosyl glycan synthesis. Defects in protein O-linked mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGnT1), a glycosyltransferase that participates in the formation of GlcNAc-ß1,2-Man glycan, are causally related to muscle-eye-brain disease (MEB), a congenital muscular dystrophy, although the role of POMGnT1 in postphosphoryl modification of GalNAc-ß1,3-GlcNAc-ß1,4-(phosphate-6)-Man glycan remains elusive. Our crystal structures of POMGnT1 agreed with our previous results showing that the catalytic domain recognizes substrate O-mannosylated proteins via hydrophobic interactions with little sequence specificity. Unexpectedly, we found that the stem domain recognizes the ß-linked GlcNAc of O-mannosyl glycan, an enzymatic product of POMGnT1. This interaction may recruit POMGnT1 to a specific site of α-DG to promote GlcNAc-ß1,2-Man clustering and also may recruit other enzymes that interact with POMGnT1, e.g., fukutin, which is required for further modification of the GalNAc-ß1,3-GlcNAc-ß1,4-(phosphate-6)-Man glycan. On the basis of our findings, we propose a mechanism for the deficiency in postphosphoryl modification of the glycan observed in POMGnT1-KO mice and MEB patients.

Mutations in POMGNT1 cause non-syndromic retinitis pigmentosa.

A growing number of human diseases have been linked to defects in protein glycosylation that affects a wide range of organs. Among them, O-mannosylation is an unusual type of protein glycosylation that is largely restricted to the muscular and nerve system. Consistently, mutations in genes involved in the O-mannosylation pathway result in infantile-onset, severe developmental defects involving skeleton muscle, brain and eye, such as the muscle-eye-brain disease (MIM no. 253280). However, the functional importance of O-mannosylation in these tissues at later stages remains largely unknown. In our study, we have identified recessive mutations in POMGNT1, which encodes an essential component in O-mannosylation pathway, in three unrelated families with autosomal recessive retinitis pigmentosa (RP), but without extraocular involvement. Enzymatic assay of these mutant alleles demonstrate that they greatly reduce the POMGNT1 enzymatic activity and are likely to be hypomorphic. Immunohistochemistry shows that POMGNT1 is specifically expressed in photoreceptor basal body. Taken together, our work identifies a novel disease-causing gene for RP and indicates that proper protein O-mannosylation is not only essential for early organ development, but also important for maintaining survival and function of the highly specialized retinal cells at later stages.

Implication of Akt-dependent Prp19 alpha/14-3-3beta/Cdc5L complex formation in neuronal differentiation.

PRP19alpha and CDC5L are major components of the active spliceosome. However, their association process is still unknown. Here, we demonstrated that PRP19 alpha/14-3-3beta/CDC5L complex formation is regulated by Akt during nerve growth factor (NGF)-induced neuronal differentiation of PC12 cells. Analysis of PRP19 alpha mutants revealed that the phosphorylation of PRP19 alpha at Thr 193 by Akt was critical for its binding with 14-3-3beta to translocate into the nuclei and for PRP19 alpha/14-3-3beta/CDC5L complex formation in neuronal differentiation. Forced expression of either sense PRP19 alpha or sense 14-3-3beta RNAs promoted NGF-induced neuronal differentiation, whereas down-regulation of these mRNAs showed a suppressive effect. The nonphosphorylation mutant PRP19 alpha T193A lost its binding ability with 14-3-3beta and acted as a dominant-negative mutant in neuronal differentiation. These results imply that Akt-dependent phosphorylation of PRP19 alpha at Thr193 triggers PRP19 alpha/14-3-3beta/CDC5L complex formation in the nuclei, likely to assemble the active spliceosome against neurogenic pre-mRNAs.

Involvement of crosstalk between Oct4 and Meis1a in neural cell fate decision.

Oct4 plays a critical role both in maintaining pluripotency and the cell fate decision of embryonic stem (ES) cells. Nonetheless, in the determination of the neuroectoderm (NE) from ES cells, the detailed regulation mechanism of the Oct4 gene expression is poorly understood. Here, we report that crosstalk between Oct4 and Meis1a, a Pbx-related homeobox protein, is required for neural differentiation of mouse P19 embryonic carcinoma (EC) cells induced by retinoic acid (RA). During neural differentiation, Oct4 expression was transiently enhanced during 6-12 h of RA addition and subsequently disappeared within 48 h. Coinciding with up-regulation of Oct4 expression, the induction of Meis1a expression was initiated and reached a plateau at 48 h, suggesting that transiently induced Oct4 activates Meis1a expression and the up-regulated Meis1a then suppresses Oct4 expression. Chromatin immunoprecipitation (ChIP) and luciferase reporter analysis showed that Oct4 enhanced Meis1a expression via direct binding to the Meis1 promoter accompanying histone H3 acetylation and appearance of 5-hydoxymethylcytosine (5hmC), while Meis1a suppressed Oct4 expression via direct association with the Oct4 promoter together with histone deacetylase 1 (HDAC1). Furthermore, ectopic Meis1a expression promoted neural differentiation via formation of large neurospheres that expressed Nestin, GLAST, BLBP and Sox1 as neural stem cell (NSC)/neural progenitor markers, whereas its down-regulation generated small neurospheres and repressed neural differentiation. Thus, these results imply that crosstalk between Oct4 and Meis1a on mutual gene expressions is essential for the determination of NE from EC cells.

The U-box-type ubiquitin ligase PRP19ß regulates astrocyte differentiation via ubiquitination of PTP1B.

U-box protein PRP19ß, a splicing variant of PRP19α, suppresses neuronal differentiation and conversely promotes astrocyte differentiation as a neuron/glia switch molecule. However, the mechanistic basis of PRP19ß in astrocyte differentiation is not well understood. Here, we demonstrated that PRP19ß regulates the stability of protein tyrosine phosphatase 1B (PTP1B) via ubiquitination during N(6),2'-O-dibutyryl cyclic AMP (cAMP)-induced astrocyte differentiation of C6 cells. Only overexpression of PRP19ß conferred astrocyte properties at a certain level, and induced more astrocyte markers, glial fibrillary acidic protein (GFAP) and S100ß, in the presence of cAMP, whereas its down-regulation by antisense RNA showed a suppressive effect. In addition, ectopic expression of PRP19ß led to robust phosphorylation of signal transducer and activator of transcription 3 (STAT3) accompanying the reduction in PTP1B stability during astrocyte differentiation. Immunological analysis revealed that PRP19ß interacted with PTP1B and ubiquitinated PTP1B via its U-box region. Forced expression of the U-box deletion mutant of PRP19ß resulted in inhibition of astrocyte differentiation. Moreover, down-regulation of PTP1B by short hairpin (sh)RNA enhanced astrocyte differentiation, while forced expression of PTP1B showed an inhibitory effect. Thus, these results indicate that PRP19ß activates the gp130/Janus kinase (JAK)/STAT signaling pathway during astrocyte differentiation of C6 cells via PTP1B ubiquitination.