== Permanently and transiently expressed IgA/G chains have the same fate in tobacco protoplasts

== Permanently and transiently expressed IgA/G chains have the same fate in tobacco protoplasts. IgA models (2 heavy and 2 light chains), a joining J chain, and a secretory component. It has been shown that transgenic tobacco cells are able to translocate Rabbit Polyclonal to TOP2A (phospho-Ser1106) all the IgA subunits in the endoplasmic reticulum (ER), where complex assembly occurs with high efficiency (Frigerioet al.,2000). The overall production of IgA is usually up to 8% of protein of a mature tobacco herb. This very high accumulation makes plants the system of choice for the expression of these molecules, which at present are produced only to minimal levels in transfected mammalian cells (Maet al.,1995;Larricket al.,2001). However, we have recently discovered that unlike in mammals, not all assembled antibody is usually secreted by herb cells. Rather, a significant proportion of the molecules (at least 80%, our unpublished observation) are retained intracellularly and eventually delivered to vacuoles, where they are ultimately fragmented and probably degraded. We have shown that vacuolar delivery is not the result of endocytosis of secreted polypeptides (Frigerioet al.,2000). All IgA assembly intermediates from the decamer to the simplest, heterotetrameric unit share this intracellular fate, and Tadalafil this transport and subsequent degradation in the vacuolar compartment can be inhibited by treatment with the fungal metabolite brefeldin A (BFA). In contrast, when the parent IgG molecule was expressed, it was efficiently and quantitatively secreted (Frigerioet al.,2000). Because the light chain is usually common to both IgG and IgA/G molecules, this led us to speculate that features of the IgA/G heavy chain might be responsible for its intracellular diversion. Tadalafil This could in turn be due either to a stress imposed around the ER, with subsequent mis-sorting or quality control delivery to the vacuole or to the presence of cryptic signals for vacuolar sorting. In the present report we have tested these hypotheses. We have analyzed the fate of IgA molecules in transgenic tobacco plants or transiently transfected tobacco protoplasts. We demonstrate that assembled IgA molecules travel through the Golgi complex before reaching the vacuole. IgA/G transport to the vacuole is not due to stress imposed around the herb ER but is the result of a cryptic signal that resides in the C-terminal domain name of the IgA/G heavy chains. In addition, we demonstrate that antibody light chains are expressed in excess of the heavy chains and that free light chains are secreted in their monomeric form. == MATERIALS AND METHODS == == Transgenic Plants == TransgenicNicotiana tabacumcv Xanthi herb lines expressing assembled IgG and IgA/G under the control of the cauliflower mosaic computer virus 35S-promoter have Tadalafil previously been described (Maet al.,1994). For protoplast transfection experiments, wild-typeN. tabacumcv Petit Havana SR1 was used. Plants were produced in axenic conditions under a 12-h light-dark regime. == Recombinant DNA == All DNA manipulations were performed using established procedures. The full length IgA/G / heavy chain was amplified from the binary vector pMON530 (Maet Tadalafil al.,1994) using the PCR. The oligonucleotides 5-ccatcgatggaatggacctgggttttt-3 and 5-ccctctagactagtagcataggccatc-3, containingClaI andXbaI restriction sites before the start codon and after the stop codon for cloning purposes, were used. The digested PCR products were ligated into a pUC-based vector downstream of the CaMV35S-promoter (Deneckeet al.,1992). The resulting plasmid was designated pJLH38. Glycosylation site mutations were produced using the Quickchange in vitro mutagenesis system (Stratagene, La Jolla, CA). Potential glycan sites mutations Ser 76 to Ala (glycan1 pJLH40), Thr 289 to Val (glycan2 pJLH41), Thr 526 to Ala (glycan3 pJLH42), and Ser 541 to Ala.