The ornamental variety, Japanese striped corn, contains high anthocyanin levels and PAL specific activity: establishing the potential for development of an oral therapeutic.

Phenylalanine ammonia-lyase [PAL, EC 4.3.1.24 (formerly EC 4.3.1.5)], functions in the plant phenylpropanoid biosynthetic pathway to deaminate the amino acid L-phenylalanine forming trans-cinnamic acid and ammonia. The human inherited metabolic disorder phenylketonuria (PKU) is characterized by an inability of individuals to metabolize phenylalanine. Toward the development of a plant-PAL based therapeutic for the treatment of this disorder, a comparative analysis of PAL activities within various members of the Poaceae was undertaken. This led to the identification of a Zea mays cultivar, Japanese Striped corn with very high levels of PAL specific activity in seedling tissues. The root tissues of this corn variety contain greater levels of PAL gene transcripts and PAL activities, compared to those of the shoot tissues, and are intensely colored due to the accumulation of anthocyanin pigments. PAL activities in the root tissues of young seedlings of another corn variety that lacked root anthocyanins (Indian Blue corn) were generally 30-50% lower than those of Japanese Striped corn seedlings at equivalent growth stages. In general, various stress or hormonal treatments led to minimal changes in PAL specific activity of maize tissues, as compared to controls. The PAL enzymes of Japanese Striped corn root tissues are robust; roots retained 90% of their PAL activity after freeze-drying and >50% activity after freeze-drying and a subsequent 15-week storage at 4 degrees C. This work serves as a prelude to the formulation of a dietary supplement for treatment of PKU based on preserved edible cereal root tissues with high levels of intrinsic PAL activity.

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.