Traffic of Human α-Mannosidase in Plant Cells Suggests the Presence of a New Endoplasmic Reticulum-to-Vacuole Pathway without Involving the Golgi Complex  

Abstract

The transport of secretory proteins from the endoplasmic reticulum to the vacuole requires sorting signals as well as specific transport mechanisms. This work is focused on the transport in transgenic tobacco (Nicotiana tabacum) plants of a human a-mannosidase, MAN2B1, which is a lysosomal enzyme involved in the turnover of N-linked glycoproteins and can be used in enzyme replacement therapy. Although ubiquitously expressed, a-mannosidases are targeted to lysosomes or vacuoles through different mechanisms according to the organisms in which these proteins are produced. In tobacco cells, MAN2B1 reaches the vacuole even in the absence of mannose-6-phosphate receptors, which are responsible for its transport in animal cells. We report that MAN2B1 is targeted to the vacuole without passing through the Golgi complex. In addition, a vacuolar targeting signal that is recognized in plant cells is located in the MAN2B1 amino-terminal region. Indeed, when this amino-terminal domain is removed, the protein is retained in the endoplasmic reticulum. Moreover, when this domain is added to a plantsecreted protein, the resulting fusion protein is partially redirected to the vacuole. These results strongly suggest the existence in plants of a new type of vacuolar traffic that can be used by leaf cells to transport vacuolar proteins.

Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana

“…Retention of Abs in the endoplasmic reticulum (ER) frequently results in increased yields (De Muynck et al ., ). Although the plant central vacuole is considered a hostile environment for foreign protein accumulation, it has been shown that some proteins, such as human alpha‐mannosidase (De Marchis et al ., ), human complement factor C5a, (Nausch et al ., ), collagen (Stein et al ., ) and human transglutaminase 2 (Marin Viegas et al ., ), accumulate in this organelle. In contrast, in carrot suspension cell cultures, secretory versions of human IgG1 and G4 have higher yields than ER and vacuolar variants (Shaaltiel et al ., ).…”

Unconventional pathways of secretory plant proteins from the endoplasmic reticulum to the vacuole bypassing the Golgi complex

“…Observing the behavior of a human lysosomal enzyme (α-mannosidase) expressed in plant cells, a new Golgi-independent vacuolar deliver of a soluble protein has been revealed. 8 In this paper it has been demonstrated that this glycoprotein is completely transported to the vacuole in a BFA-insensitive manner. Moreover, the processed α-mannosidase vacuolar fragments are not modified by Golgi enzymes, suggesting that this protein uses an alternative route the glycosylated proteins present in the Golgi.…”

“…Moreover, the processed α-mannosidase vacuolar fragments are not modified by Golgi enzymes, suggesting that this protein uses an alternative route the glycosylated proteins present in the Golgi. 8 In conclusion, even if the determinants that regulate this unconventional route are not yet well characterized, we think that its importance for the regulation of cell functionality in eukaryotes is becoming more and more evident.…”

“…However, the classical Golgi pathway turns out to be only one route of a complex system of pathways which deliver proteins to the vacuoles, thus it is not the predominant mechanism for vacuolar protein transport. [6][7][8] Here we want to give a brief overview of the direct ER-vacuole transport of secretory proteins.…”

Lysosomal and vacuolar sorting: not so different after all!

“…Plants in particular seem to represent a promising system thanks to the cost-effective and easy production of lysosomal proteins with already successful attempts of expressing human hydrolases such as mannosidases and glucocerebrosidase [61,62]. Most importantly, plant-produced (carrot) recombinant human glucocerebrosidase has been shown to naturally possess terminal mannose residues, a step which has to be additionally performed in vitro for Chinese hamster ovary (CHO)-derived glucocerebrosidase [63].…”