Human α-mannosidase produced in transgenic tobacco plants is processed in human α-mannosidosis cell lines.

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.

Intracellular transport of human lysosomal alpha-mannosidase and alpha-mannosidosis-related mutants.

Human LAMAN (lysosomal a-mannosidase) was synthesized as a 120 kDa precursor in transfected COS cells [African-green-monkey kidney cells], which was partly secreted as a single-chain form and partly sorted to the lysosomes being subsequently cleaved into three peptides of 70, 40 and 15 kDa respectively. Both the secreted and the lysosomal forms contained endo H (endoglucosidase H)-resistant glycans, suggesting a common pathway through the trans-Golgi network. A fraction of LAMAN was retained intracellularly as a single-chain endo H-sensitive form, probably in the ER (endoplasmic reticulum). The inherited lack of LAMAN causes the autosomal recessive storage disease a-mannosidosis. To understand the biochemical consequences of the disease-causing mutations, 11 missense mutations and two in-frame deletions were introduced into human LAMAN cDNA by in vitro mutagenesis and the resulting proteins were expressed in COS cells. Some selected mutants were also expressed in Chinese-hamster ovary cells. T355P (Thr355Pro), P356R, W714R, R750W and L809P LAMANs as well as both deletion mutants were misfolded and arrested in the ER as inactive single-chain forms. Six of the mutants were transported to the lysosomes, either with less than 5% of normal specific activity (H72L, D196E/N and R220H LAMANs) or with more than 30% of normal specific activity (E402K LAMAN). F320L LAMAN resulted in much lower activity in Chinese-hamster ovary cells when compared with COS cells. Modelling into the three-dimensional structure revealed that the mutants with highly reduced specific activities contained substitutions of amino acids involved in the catalysis, either co-ordinating Zn2+ (His72 and Asp196), stabilizing the active-site nucleophile (Arg220) or positioning the active-site residue Asp319 (Phe320).