Recombinant human alpha-mannosidase: molecular analysis of its traffic to the vacuole in the plant cell and biochemical characterization of the purified enzyme

We are carrying on with our studies on the optimization of the production of human ?-mannosidase (MAN2B1) in tobacco plants grown in greenhouse and, in the meantime, we are also try to understand which are the targeting signals that direct the human enzyme to the vacuole. Traffic of ?-mannosidases to the lysosomal or vacuolar compartment follows alternative routes in different kingdoms. In animal cells, these hydrolases are transported to lysosomes by the mannose 6-phosphate pathway, whereas in plants vacuolar ?-mannosidase is targeted to its final destination via the classic secretory pathway involving the ER-Golgi system. MAN2B1 in tobacco tissues is localised in vacuolar compartments. Due to the absence of a plant counterpart of the mannose 6-phosphate, we tried to understand what kind of mechanism uses this human protein in plants to reach the vacuole. Studies performed on transgenic leaf protoplasts using different drugs which inhibit protein traffic and glycosylation indicate that traffic to the vacuole of the precursor MAN2B1 polypeptide is not dependent on Golgi-mediated delivery. In order to understand which structural part of the protein acts as a vacuolar targeting signal, a comparative analysis of various ?-mannosidase sequences from different organisms was carried out. It was therefore decided to focus our attention in the first two hundred amino acids that were removed from the original sequence of MAN2B1 and added to the N-terminal sequence of a secreted form of phaseolin (named ?418). What has been observed is that MAN2B1 without the N-terminal domain no longer reaches the vacuole and is localized in the endoplasmic reticulum, conversely the fusion protein that we have constructed (called Man?418) is directed to the vacuole instead of being secreted. We have also shown that Man?418 is transported to the vacuole without passing through the Golgi complex. Taken together these results indicate that in plant cells exists a sorting mechanism that target soluble secretory proteins from ER directly to the vacuole. A functional human ?-mannosidase was previously expressed in stably transformed tobacco plants to develop an enzyme replacement therapy for alpha-mannosidosis which is a rare lysosomal storage disease. Recent analyses on tobacco lines expressing the recombinant enzyme show that in young leaves the 110-kD precursor MAN2B1 polypeptide is prevalently found, conversely in the oldest leaf there are only the post-translationally processed forms. In seeds the MAN2B1 polypeptide is much more stable than in leaves and there are not differences in the pattern (i.e. presence of precursor and processed forms) of accumulation among different stages of seed development. As regards the ?-mannosidase specific activity, in leaves the activity is higher than in seeds and in the leaves the activity increases with the leaf age. DEAE elution profiles of recombinant MAN2B1 revealed the separation of the total activity into two major peaks, A and B, which do not have the same profiles in different tobacco tissues. We are currently investigating these differences and their meaning for MAN2B1 purification and functional activity.