The transgenic expression of LARGE exacerbates the muscle phenotype of dystroglycanopathy mice.

Mutations in fukutin-related protein (FKRP) underlie a group of muscular dystrophies associated with the hypoglycosylation of α-dystroglycan (α-DG), a proportion of which show central nervous system involvement. Our original FKRP knock-down mouse (FKRP(KD)) replicated many of the characteristics seen in patients at the severe end of the dystroglycanopathy spectrum but died perinatally precluding its full phenotyping and use in testing potential therapies. We have now overcome this by crossing FKRP(KD) mice with those expressing Cre recombinase under the Sox1 promoter. Owing to our original targeting strategy, this has resulted in the restoration of Fkrp levels in the central nervous system but not the muscle, thereby generating a new model (FKRP(MD)) which develops a progressive muscular dystrophy resembling what is observed in limb girdle muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE) is a bifunctional glycosyltransferase previously shown to hyperglycosylate α-DG. To investigate the therapeutic potential of LARGE up-regulation, we have now crossed the FKRP(MD) line with one overexpressing LARGE and show that, contrary to expectation, this results in a worsening of the muscle pathology implying that any future strategies based upon LARGE up-regulation require careful management.

Plasmodium falciparum†SERA5 plays a non-enzymatic role in the malarial asexual blood-stage lifecycle.

The malaria parasite Plasmodium falciparum replicates in an intraerythrocytic parasitophorous vacuole (PV). The most abundant P. falciparum PV protein, called SERA5, is essential in blood stages and possesses a papain-like domain, prompting speculation that it functions as a proteolytic enzyme. Unusually however, SERA5 possesses a Ser residue (Ser596) at the position of the canonical catalytic Cys of papain-like proteases, and the function of SERA5 or whether it performs an enzymatic role is unknown. In this study, we failed to detect proteolytic activity associated with the Ser596-containing parasite-derived or recombinant protein. However, substitution of Ser596 with a Cys residue produced an active recombinant enzyme with characteristics of a cysteine protease, demonstrating that SERA5 can bind peptides. Using targeted homologous recombination in P. falciparum, we substituted Ser596 with Ala with no phenotypic consequences, proving that SERA5 does not perform an essential enzymatic role in the parasite. We could also replace an internal segment of SERA5 with an affinity-purification tag. In contrast, using almost identical targeting constructs, we could not truncate or C-terminally tag the SERA5 gene, or replace Ser596 with a bulky Arg residue. Our findings show that SERA5 plays an indispensable but non-enzymatic role in the P. falciparum blood-stage life cycle.

Prenatal muscle development in a mouse model for the secondary dystroglycanopathies.

BACKGROUND: The defective glycosylation of α-dystroglycan is associated with a group of muscular dystrophies that are collectively referred to as the secondary dystroglycanopathies. Mutations in the gene encoding fukutin-related protein (FKRP) are one of the most common causes of secondary dystroglycanopathy in the UK and are associated with a wide spectrum of disease. Whilst central nervous system involvement has a prenatal onset, no studies have addressed prenatal muscle development in any of the mouse models for this group of diseases. In view of the pivotal role of α-dystroglycan in early basement membrane formation, we sought to determine if the muscle formation was altered in a mouse model of FKRP-related dystrophy. RESULTS: Mice with a knock-down in FKRP (FKRP(KD)) showed a marked reduction in α-dystroglycan glycosylation and reduction in laminin binding by embryonic day 15.5 (E15.5), relative to wild type controls. In addition, the total number of Pax7(+) progenitor cells in the FKRP(KD) tibialis anterior at E15.5 was significantly reduced, and myotube cluster/myofibre size showed a significant reduction in size. Moreover, myoblasts isolated from the limb muscle of these mice at E15.5 showed a marked reduction in their ability to form myotubes in vitro. CONCLUSIONS: These data identify an early reduction of laminin α2, reduction of myogenicity and depletion of Pax7(+) progenitor cells which would be expected to compromise subsequent postnatal muscle growth and its ability to regenerate postnatally. These findings are of significance to the development of future therapies in this group of devastating conditions.