title: Human α-mannosidase produced in transgenic tobacco plants is processed in human α-mannosidosis cell lines.
abstract: Deficiency in human lysosomal α-mannosidase (MAN2B1) results in α-mannosidosis, a lysosomal storage disorder; patients present a wide range of neurological, immunological, and skeletal symptoms caused by a multisystemic accumulation of mannose-containing oligosaccharides. Here, we describe the expression of recombinant MAN2B1 both transiently in Nicotiana benthamiana leaves and in the leaves and seeds of stably transformed N. tabacum plants. After purification from tobacco leaves, the recombinant enzyme was found to be N-glycosylated and localized in vacuolar compartments. In the fresh leaves of tobacco transformants, MAN2B1 was measured at 10,200 units/kg, and the purified enzyme from these leaves had a specific activity of 32-45 U/mg. Furthermore, tobacco-produced MAN2B1 was biochemically similar to the enzyme purified from human tissues, and it was internalized and processed by α-mannosidosis fibroblast cells. These results strongly indicate that plants can be considered a promising expression system for the production of recombinant MAN2B1 for use in enzyme replacement therapy.
authors.affiliation: Institute of Plant Genetics, Italian National Council of Research (CNR), Perugia, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: De Marchis
authors.forename: Chiara
authors.initials: C
authors.lastname: Balducci
authors.forename: Andrea
authors.initials: A
authors.lastname: Pompa
authors.forename: Hilde M F
authors.initials: HM
authors.lastname: Riise Stensland
authors.forename: Marco
authors.initials: M
authors.lastname: Guaragno
authors.forename: Rita
authors.initials: R
authors.lastname: Pagiotti
authors.forename: Anna R
authors.initials: AR
authors.lastname: Menghini
authors.forename: Emanuele
authors.initials: E
authors.lastname: Persichetti
authors.forename: Tommaso
authors.initials: T
authors.lastname: Beccari
authors.forename: Michele
authors.initials: M
authors.lastname: Bellucci
mesh_terms: D002460:Cell Line; D004789:Enzyme Activation; D057075:Enzyme Assays; D005347:Fibroblasts / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D047468:Immunoprecipitation; D044904:Mannosidase Deficiency Diseases / Q000201:enzymology; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011523:Protoplasts / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D014617:Vacuoles / Q000378:metabolism; D043323:alpha-Mannosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D043323:alpha-Mannosidase