Regulation of the lysosome-associated membrane protein in a sucrose model of lysosomal storage.

Lysosomal biogenesis is a complex process requiring the coordinated expression and colocalization of numerous soluble and membrane proteins. In storage disorders, lysosomal biogenesis is regulated at least partially at, or prior to, the level of mRNA. We have used the sucrosome storage model to determine the sites of regulation of LAMP-1, a major constituent of the lysosomal membrane. A six- to eightfold increase in the level of LAMP-1 mRNA and protein was observed in response to sucrose storage. The half-life of LAMP-1 mRNA was not significantly different in cells grown in the absence or presence of sucrose, implying that the increase observed in mRNA levels reflects an increase in the rate of transcription. The sixfold increase in mRNA did not translate into an increase in LAMP-1 synthesis, indicating an overall decrease in the translational yield in sucrosome cells. The elevation of LAMP-1 protein levels in storage cells was due in large part to a threefold increase in the half-life of the protein. These results are discussed in view of the current understanding of lysosomal biogenesis and how this process is altered during storage.

Altered trafficking and turnover of LAMP-1 in Pompe disease-affected cells.

The lysosome-associated membrane protein (LAMP-1) is elevated in the cells and plasma from lysosomal storage disorder-affected individuals; however, the mechanism of this elevation is not well defined. In this study we have investigated the synthesis, glycoprocessing, trafficking, and turnover of LAMP-1 in human skin fibroblasts from Pompe disease patients and control individuals. There were similar levels of LAMP-1 synthesis in both cell types, but glycoprocessing was retarded in Pompe (T1/2 = 25 min) compared to control (T1/2 = 17 min) fibroblasts. There was also a marked delay in trafficking of LAMP-1 to lysosomes of Pompe (T1/2 = 200 min) compared to control (T1/2 = 100 min) cells. A proportion of newly synthesized LAMP-1 (5.4% in Pompe and 8.5% in controls) was trafficked out of the cell (T1/2 = 3.5 h in controls) and, although significantly smaller than the lysosomal form, still had a transmembrane domain and cytoplasmic tail. In contrast, a soluble lysosomal pool of LAMP-1 had no tail sequence, suggesting that it had been clipped from the membrane. In turnover studies, LAMP-1 was more stable in Pompe (T1/2 = 4.9 days) compared to control (T1/2 = 1. 6 days) cells, implying either reduced proteolysis or lysosomal function, in Pompe cells. These results indicate altered traffic and turnover of LAMP-1 in storage disorders and identify different intracellular and extracellular pools of soluble LAMP-1, suggesting alternative trafficking pathways.

Lysosomal biogenesis in lysosomal storage disorders.

Lysosomal biogenesis is an orchestration of the structural and functional elements of the lysosome to form an integrated organelle and involves the synthesis, targeting, functional residence, and turnover of the proteins that comprise the lysosome. We have investigated lysosomal biogenesis during the formation and dissipation of storage vacuoles in two model systems. One involves the formation of sucrosomes in normal skin fibroblasts and the other utilizes storage disorder-affected skin fibroblasts; both of these systems result in an increase in the size and the number of lysosomal vacuoles. Lysosomal proteins, beta-hexosaminidase, alpha-mannosidase, N-acetylgalactosamine-4-sulfatase, acid phosphatase, and the lysosome-associated membrane protein, LAMP-1, were shown to be elevated between 2- and 28-fold above normal during lysosomal storage. Levels of mRNA for the lysosome-associated membrane proteins LAMP-1 and LAMP-2, N-acetylgalactosamine-4-sulfatase, and the 46- and 300-kDa mannose-6-phosphate receptors were also elevated 2- to 8-fold. The up-regulation of protein and mRNA lagged 2-4 days behind the formation of lysosomal storage vacuoles. Correction of storage, in both systems, resulted in the rapid decline of the mRNA to basal levels, with a slower decrease in the levels of lysosomal proteins. Lysosomal biogenesis in storage disorders is shown to be a regulated process which is partially controlled at, or prior to, the level of mRNA. Although lysosomal proteins were differentially regulated, the coordination of these events in lysosomal biogenesis would suggest that a common mechanism(s) may be in operation.

Enzyme replacement therapy from birth in a feline model of mucopolysaccharidosis type VI.

We report evidence of a dose responsive effect of enzyme replacement therapy in mucopolysaccharidosis type VI cats from birth, at the clinical, biochemical, and histopathological level. Cats treated with weekly, intravenous recombinant human N-acetylgalactosamine-4-sulfatase at 1 and 5 mg/kg, were heavier, more flexible, had greatly reduced or no spinal cord compression, and had almost normal urinary glycosaminoglycan levels. There was near normalization or complete reversal of lysosomal storage in heart valve, aorta, skin, dura, liver, and brain perivascular cells. No reduction in lysosomal vacuolation was observed in cartilage or cornea; however, articular cartilage was thinner and external ear pinnae were larger in some treated cats. Degenerative joint changes were not obviously delayed in treated cats. Skeletal pathology was reduced, with more normalized bone dimensions and with more uniform bone density and trabecular pattern clearly visible on radiographs by 5 to 6 mo; however, differences between 1 and 5 mg/kg dose rates were not clearly distinguishable. At a dose of 0.2 mg/kg, disease was not significantly altered in the majority of parameters examined. Lysosomal storage was present in all tissues examined in the midterm mucopolysaccharidosis type VI fetus and increased rapidly in extent and severity from birth.

Enzyme replacement therapy in a feline model of Maroteaux-Lamy syndrome.

We report studies that suggest enzyme replacement therapy will result in a significant reduction in disease progression and tissue pathology in patients with Maroteaux-Lamy syndrome (Mucopolysaccharidosis type VI, MPS VI). A feline model for MPS VI was used to evaluate tissue distribution and clinical efficacy of three forms of recombinant human N-acetylgalactosamine-4-sulfatase (rh4S, EC 3.1.6.1). Intravenously administered rh4S was rapidly cleared from circulation. The majority of rh4S was distributed to liver, but was also detected in most other tissues. Tissue half-life was approximately 2-4 d. Three MPS VI cats given regular intravenous infusions of rh4S for up to 20 mo showed variable reduction of storage vacuoles in Kupffer cells and connective tissues, however cartilage chondrocytes remained vacuolated. Vertebral bone mineral volume was improved in two MPS VI cats in which therapy was initiated before skeletal maturity, and increased bone volume appeared to correlate with earlier age of onset of therapy. One cat showed greater mobility in response to therapy.

Tumour promoting phorbol esters activate phospholipase D in mouse skin.

Phospholipase D catalyses a transphosphatidylation reaction in the presence of primary alcohols, resulting in the formation of phosphatidylalcohol derivatives. In the present work we show that application of phorbol esters and butanol to mouse skin causes the rapid accumulation of phosphatidylbutanol (PBol), indicating the activation of phospholipase D. A similar accumulation of PBol was observed when the skin was treated with phorbol esters in vivo and skin pieces incubated with butanol in vitro. PBol formation was stimulated by the active tumour promoters 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein and phorbol-12,13-didecanoate (PDD) but not by the inactive promoter 4 alpha-PDD. Accumulation of PBol was not observed 24 h after application of TPA, a treatment which has been shown to deplete epidermal protein kinase C activity.

Carbon dioxide-induced hypoxia causes selective loss of rat brain protein kinase C-gamma.

Protein kinase C purified from the brains of rats killed by decapitation contained isozymes -alpha, beta and -gamma, whereas brain from animals killed by CO2-anoxia contained only the -alpha and -beta forms. Immunoblotting experiments established that exposure to CO2 resulted in a selective loss of protein kinase C-gamma protein.