Synthesis of enzymatically active human alpha-L-iduronidase in Arabidopsis cgl (complex glycan-deficient) seeds.
As an initial step to develop plants as systems to produce enzymes for the treatment of lysosomal storage disorders, Arabidopsis thaliana wild-type (Col-0) plants were transformed with a construct to express human alpha-l-iduronidase (IDUA; EC 3.2.1.76) in seeds using the promoter and other regulatory sequences of the Phaseolus vulgaris arcelin 5-I gene. IDUA protein was easily detected on Western blots of extracts from the T(2) seeds, and extracts contained IDUA activity as high as 2.9 nmol 4-methylumbelliferone (4 MU)/min/mg total soluble protein (TSP), corresponding to approximately 0.06 microg IDUA/mg TSP. The purified protein reacted with an antibody specific for xylose-containing plant complex glycans, indicating its transit through the Golgi complex. In an attempt to avoid maturation of the N-linked glycans of IDUA, the same IDUA transgene was introduced into the Arabidopsis cgl background, which is deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of complex glycan biosynthesis. IDUA activity and protein levels were significantly higher in transgenic cgl vs. wild-type seeds (e.g. maximum levels were 820 nmol 4 MU/min/mg TSP, or 18 microg IDUA/mg TSP). Affinity-purified IDUA derived from cgl mutant seeds showed a markedly reduced reaction with the antibody specific for plant complex glycans, despite transit of the protein to the apoplast. Furthermore, gel mobility changes indicated that a greater proportion of its N-linked glycans were susceptible to digestion by Streptomyces endoglycosidase H, as compared to IDUA derived from seeds of wild-type Arabidopsis plants. The combined results indicate that IDUA produced in cgl mutant seeds contains glycans primarily in the high-mannose form. This work clearly supports the viability of using plants for the production of human therapeutics with high-mannose glycans.
Downing
Willa L
WL
Department of Biological Sciences, Simon Fraser University, 8888 University Dr., Burnaby, BC, Canada V5A 1S6.
Galpin
Jason D
JD
Clemens
Sabine
S
Lauzon
Shauna M
SM
Samuels
A Lacey
AL
Pidkowich
Mark S
MS
Clarke
Lorne A
LA
Kermode
Allison R
AR
D017360:Arabidopsis / Q000235:genetics*; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D007068:Iduronidase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000201:enzymology*; D011134:Polysaccharides / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012639:Seeds / Q000201:enzymology / Q000235:genetics; D014170:Transformation, Genetic
D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I; D007068:Iduronidase; D008358:Mannose