SummaryGaucher's disease, a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy using recombinant GCD (Cerezyme ® ) expressed in Chinese hamster ovary (CHO) cells. As complex glycans in mammalian cells do not terminate in mannose residues, which are essential for the biological uptake of GCD via macrophage mannose receptors in human patients with Gaucher's disease, an in vitro glycan modification is required in order to expose the mannose residues on the glycans of Cerezyme ® . In this report, the production of a recombinant human GCD in a carrot cell suspension culture is described. The recombinant plant-derived GCD (prGCD) is targeted to the storage vacuoles, using a plant-specific C-terminal sorting signal. Notably, the recombinant human GCD expressed in the carrot cells naturally contains terminal mannose residues on its complex glycans, apparently as a result of the activity of a special vacuolar enzyme that modifies complex glycans. Hence, the plant-produced recombinant human GCD does not require exposure of mannose residues in vitro , which is a requirement for the production of Cerezyme

Glutamate decarboxylase (GAD) catalyzes the decarboxylation of glutamate to CO2 and gamma‐aminobutyrate (GABA). GAD is ubiquitous in prokaryotes and eukaryotes, but only plant GAD has been shown to bind calmodulin (CaM). Here, we assess the role of the GAD CaM‐binding domain in vivo. Transgenic tobacco plants expressing a mutant petunia GAD lacking the CaM‐binding domain (GADdeltaC plants) exhibit severe morphological abnormalities, such as short stems, in which cortex parenchyma cells fail to elongate, associated with extremely high GABA and low glutamate levels. The morphology of transgenic plants expressing the full‐length GAD (GAD plants) is indistinguishable from that of wild‐type (WT) plants. In WT and GAD plant extracts, GAD activity is inhibited by EGTA and by the CaM antagonist trifluoperazine, and is associated with a CaM‐containing protein complex of approximately 500 kDa. In contrast, GADdeltaC plants lack normal GAD complexes, and GAD activity in their extracts is not affected by EGTA and trifluoperazine. We conclude that CaM binding to GAD is essential for the regulation of GABA and glutamate metabolism, and that regulation of GAD activity is necessary for normal plant development. This study is the first to demonstrate an in vivo function for CaM binding to a target protein in plants.

Blue light regulates plant growth and development, and three photoreceptors, CRY1, CRY2, and NPH1, have been identified. The transduction pathways of these receptors are poorly understood. Transgenic plants containing aequorin have been used to dissect the involvement of these three receptors in the regulation of intracellular Ca 2؉ . Pulses of blue light induce cytosolic Ca 2؉ transients lasting about 80 s in Arabidopsis and tobacco seedlings. Use of organelle-targeted aequorins shows that Ca 2؉ increases are limited to the cytoplasm. Blue light treatment of cry1, cry2, and nph1 mutants showed that NPH1, which regulates phototropism, is largely responsible for the Ca 2؉ transient. The spectral response of the Ca 2؉ transient is similar to that of phototropism, supporting NPH1 involvement. Furthermore, known interactions between red and blue light and between successive blue light pulses on phototropic sensitivity are mirrored in the blue light control of cytosolic Ca 2؉ in these seedlings. Our observations raise the possibility that physiological responses regulated by NPH1, such as phototropism, may be transduced through cytosolic Ca 2؉ . L ight is one of the most important signals controlling plant growth and development. Separate photomorphogenic effects of red light (R) and blue light (B) (which also interact with each other) are well established. B signaling controls important plant processes such as phototropism, suppression of stem extension, chloroplast movement, circadian timing, and expression of numerous genes. Three photoreceptors absorbing in the B region of the spectrum have been identified thus far. The Arabidopsis HY4 gene encodes a protein, CRY1, for cryptochrome (1). hy4 mutants are impaired in several extension growth responses and in the expression of genes concerned with flavonoid biosynthesis in B (2, 3). CRY1 is thought to possess FAD and pterin chromophores (4, 5). A similar protein, CRY2, controls B-induced cotyledon expansion and is involved in the regulation of flowering (6, 7). A third B photoreceptor, NPH1, was discovered as a result of the isolation of mutants with impaired phototropic sensitivity (8). Although there was some doubt initially as to whether NPH1 was a receptor or a downstream component, more recent evidence shows that heterologously expressed NPH1 has an FMN chromophore and is autophosphorylated as a result of B irradiation (9, 10). Ahmad et al. (11) have reported recently that CRY1 and CRY2 may interact with NPH1 in the regulation of phototropism. This interaction implies crosstalk between the signal transduction pathways for these three receptors.The nature of the B signal transduction pathways remains uncertain, but protein phosphorylation or a kinase cascade is likely for NPH1. Furthermore, the effect of B is to change the redox status of B receptors, and redox processes have been predicted to be involved in B signal transduction (12, 13). However, despite the facts that intracellular Ca 2ϩ is involved in more processes in plant cells than any other second mess...

To date, only plants have been shown to possess a form of glutamate decarboxylase (GAD) that binds calmodulin. In the present study, a recombinant calmodulin-binding 58-kDa petunia GAD produced in Escherichia coli was purified to homogeneity using calmodulin-affinity chromatography, and its responsiveness to calcium and calmodulin was examined in vitro. At pH 7.0 -7.5, the purified recombinant enzyme was essentially inactive in the absence of calcium and calmodulin, but it could be stimulated to high levels of activity (V max ‫؍‬ 30 mol of CO 2 min ؊1 mg of protein ؊1 ) by the addition of exogenous calmodulin (K 0.5 ‫؍‬ 15 nM) in the presence of calcium (K 0.5 ‫؍‬ 0.8 M). Neither calcium nor calmodulin alone had any effect on GAD activity. Recombinant GAD displayed hyperbolic kinetics at pH 7.3 (K m ‫؍‬ 8.2 mM). A monoclonal antibody directed against the carboxyl-terminal region, which contains the calmodulin-binding domain of GAD, was able to fully activate GAD in a dose-dependent manner in the absence of calcium and calmodulin, whereas an antibody recognizing an epitope outside of this region was unable to activate GAD. This study provides the first evidence that the activity of the purified 58-kDa GAD polypeptide is essentially calcium/calmodulin-dependent at physiological pH. Furthermore, activation of GAD by two different proteins that interact with the calmodulinbinding domain, a monoclonal antibody or calcium/ calmodulin, suggests that this domain plays a major role in the regulation of plant GAD activity.