POMT1-associated walker-warburg syndrome: a disorder of dendritic development of neocortical neurons.

We have analyzed the morphology and dendritic development of neocortical neurons in a 2.5-month-old infant with Walker-Warburg syndrome homozygotic for a novel POMT1 gene mutation, by Golgi methods. We found that pyramidal neurons frequently displayed abnormal (oblique, horizontal, or inverted) orientation. A novel finding of this study is that members of the same population of pyramidal neurons display different stages of development of their dendritic arborizations: some neurons had poorly developed dendrites and thus resembled pyramidal neurons of the late fetal cortex; for some neurons, the level of differentiation corresponded to that in the newborn cortex; finally, some neurons had quite elaborate dendritic trees as expected for the cortex of 2.5-month-old infant. In addition, apical dendrites of many pyramidal neurons were conspiciously bent to one side, irrespective to the general orientation of the pyramidal neuron. These findings suggest that Walker-Warburg lissencephaly is characterized by two hitherto unnoticed pathogenetic changes in the cerebral cortex: (a) heterochronic decoupling of dendritic maturation within the same neuronal population (with some members significantly lagging behind the normal maturational schedule) and (b) anisotropically distorted shaping of dendritic trees, probably caused by patchy displacement of molecular guidance cues for dendrites in the malformed cortex.

Correlation of enzyme activity and clinical phenotype in POMT1-associated dystroglycanopathies.

BACKGROUND: Mutations in protein O-mannosyltransferases (POMTs) cause a heterogeneous group of muscular dystrophies with abnormal glycosylation of alpha-dystroglycan (dystroglycanopathies). The wide spectrum of clinical severities ranges from Walker-Warburg syndrome (WWS), associated with brain and eye abnormalities, to mild forms of limb girdle muscular dystrophy (LGMD). OBJECTIVE: The aim of this study was to elucidate the impact of mutations in POMT1 on the clinical phenotype. METHODS: We examined 2 patients with POMT1-associated alpha-dystroglycanopathy, 1 displaying a LGMD2K and 1 with a WWS phenotype. Using dermal fibroblasts, we analyzed the influence of the POMT1 mutations on the glycosylation status of alpha-dystroglycan, protein O-mannosyltransferase activity, and the stability of the mutant POMT1 protein. RESULTS: We report on novel compound heterozygous mutations in POMT1 (p.L171A and p.A589VfsX38) that result in LGMD2K. We further demonstrate that a homozygous splice site mutation of a recently identified WWS patient results in POMT1 p.del77-93. Using dermal fibroblasts, we show that mannosyltransferase activity is reduced in the patients and that stability of POMT1 mutant proteins p.A589VfsX38 and p.del77-93 is significantly decreased. CONCLUSIONS: Our results suggest that dermal fibroblasts can be applied to facilitate the diagnostic analysis of dystroglycanopathy patients as well as to study the pathogenic mechanism of POMT mutations. Characterization of the POMT1 substrate protein alpha-dystroglycan and POMT in vitro mannosyltransferase activity shows that the severity of the clinical phenotype of the patients analyzed is inversely correlated with POMT activity.

Mouse T cell membrane proteins Rt6-1 and Rt6-2 are arginine/protein mono(ADPribosyl)transferases and share secondary structure motifs with ADP-ribosylating bacterial toxins.

Mono ADP-ribosylation is a posttranslational protein modification that has been implicated in the regulation of key biological functions in bacteria as well as in animals. Recently, the first cDNAs for eucaryotic mono(ADPribosyl)transferases were cloned and found to exhibit significant sequence similarity only to one other known protein, the T cell differentiation antigen Rt6. In this paper we describe secondary structure analyses of Rt6 and related proteins and show conserved structure motifs and amino acid residues consistent with a common ancestry of these eucaryotic proteins and bacterial ADP-ribosyltransferases. Moreover, we have expressed soluble mouse Rt6-1 and Rt6-2 gene products in which C-terminal tags (FLAG-His6) replace the native glycosylphosphatidylinositol anchor signal sequences. Purified recombinant Rt6-2, but not Rt6-1, shows NAD+ glycohydrolase activity, which is inhibited by the arginine analogue agmatine. Immunoprecipitation of recombinant Rt6-1 and Rt6-2 with anti-FLAG M2 antibody followed by incubation with [32P]NAD+ leads to rapid and covalent incorporation of radioactivity into the light chain of the M2 antibody. The bound label is resistant to treatment with HgCl2 but sensitive to NH2OH, characteristic of arginine-linked ADP-ribosylation. These results demonstrate that Rt6-1 and RT6-2 possess the enzymatic activities typical for NAD+-dependent arginine/protein mono(ADPribosyl)transferases (EC 2.4.2.31). They are the first such enzymes to be molecularly characterized in the immune system.