N-linked glycan recognition and processing: the molecular basis of endoplasmic reticulum quality control

Nascent polypeptides emerging into the lumen of the endoplasmic reticulum (ER) are N-glycosylated on asparagines in Asn-Xxx-Ser/Thr motifs. Processing of the core oligosaccharide eventually determines the fate of the associated polypeptide by regulating entry into and retention by the calnexin chaperone system, or extraction from the ER folding environment for disposal. Recent advances have shown that at least two N-glycans are necessary for protein access to the calnexin chaperone system and that polypeptide cycling in the system is a rather rare event, which, for folding-defective polypeptides, is activated only upon persistent misfolding. Additionally, dismantling of the polypeptide-bound N-glycan interrupts futile folding attempts, and elicits preparation of the misfolded chain for dislocation into the cytosol and degradation.     

Unglycosylation at Asn-633 made extracellular domain of E-cadherin folded incorrectly and arrested in endoplasmic reticulum, then sequentially degraded by ERAD

The human E-cadherin is a single transmembrane domain protein involved in Ca²⁺-dependent cell–cell adhesion. In a previous study, we demonstrated that all of four potential N-glycosylation sites in E-cadherin are occupied by N-glycans in human breast carcinoma cells in vivo and the elimination of N-glycan at Asn-633 dramatically affected E-cadherin expression and made it degraded. In this study we investigated the molecular mechanism of E-cadherin, which lacks N-glycosylation at Asn-633 (M4), degradation and the role of the N-glycan at Asn-633 in E-cadherin folding. We treated cells stably expressed M4 E-cadherin with MG123, DMM, respectively. Either MG132 or DMM could efficiently block degradation of M4 E-cadherin. M4 E-cadherin was recognized as the substrate of ERAD and was retro-translocated from ER lumen to cytoplasm by p97. It was observed that the ration of M4 E-cadherin binding to calnexin was significantly increased compared with that of other variants, suggesting that it was a misfolded protein, though cytoplasmic domain of M4 E-cadherin could associate with β-catenin. Furthermore, we found that N-glycans of M4 E-cadherin were modified in immature high mannose type, suggesting that it could not depart to Golgi apparatus. In conclusion, this study revealed that N-glycosylation at Asn-633 is essential for E-cadherin expression, folding and trafficking.     

Multiple Mechanism–Mediated Retention of a Defective Brassinosteroid Receptor in the Endoplasmic Reticulum of Arabidopsis

Endoplasmic reticulum–mediated quality control (ERQC) is a well-studied process in yeast and mammals that retains and disposes misfolded/unassembled polypeptides. By contrast, how plants exert quality control over their secretory proteins is less clear. Here, we report that a mutated brassinosteroid receptor, bri1-5, that carries a Cys69Tyr mutation, is retained in the ER by an overvigilant ERQC system involving three different retention mechanisms. We demonstrate that bri1-5 interacts with two ER chaperones, calnexin and binding protein (BiP), and is degraded by a proteasome-independent endoplasmic reticulum–associated degradation (ERAD). Mutations in components of the calnexin/calreticulin cycle had little effect on the fidelity of the Arabidopsis thaliana ERQC for bri1-5 retention. By contrast, overexpression of bri1-5, treatment with an ERAD inhibitor, RNA interference–mediated BiP silencing, or simultaneous mutations of Cys-69 and its partner Cys-62 can mitigate this quality control, resulting in significant suppression of the bri1-5 phenotype. Thus, bri1-5 is an excellent model protein to investigate plant ERQC/ERAD in a model organism.     

Consequences of individual N-glycan deletions and of proteasomal inhibition on secretion of active BACE

BACE is an aspartic protease involved in the production of a toxic peptide accumulating in the brain of Alzheimer's disease patients. After attainment of the native structure in the endoplasmic reticulum (ER), BACE is released into the secretory pathway. To better understand the mechanisms regulating protein biogenesis in the mammalian ER, we determined the fate of five variants of soluble BACE with 4, 3, 2, 1, or 0 N-linked glycans. The number of N-glycans displayed on BACE correlated directly with folding and secretion rates and with the yield of active BACE harvested from the cell culture media. Addition of a single N-glycan was sufficient to recruit the calnexin chaperone system and/or for oligosaccharide de-glucosylation by the ER-resident α-glucosidase II. Addition of 1–4 N-glycans progressively enhanced the dissociation rate from BiP and reduced the propensity of newly synthesized BACE to enter aberrant soluble and insoluble aggregates. Finally, inhibition of the proteasome increased the yield of active BACE. This shows that active protein normally targeted for destruction can be diverted for secretion, as if for BACE the quality control system would be acting too stringently in the ER lumen, thus causing loss of functional polypeptides.     

C-terminus glycans with critical functional role in the maturation of secretory glycoproteins

The N-glycans of membrane glycoproteins are mainly exposed to the extracellular space. Human tyrosinase is a transmembrane glycoprotein with six or seven bulky N-glycans exposed towards the lumen of subcellular organelles. The central active site region of human tyrosinase is modeled here within less than 2.5 Å accuracy starting from Streptomyces castaneoglobisporus tyrosinase. The model accounts for the last five C-terminus glycosylation sites of which four are occupied and indicates that these cluster in two pairs - one in close vicinity to the active site and the other on the opposite side. We have analyzed and compared the roles of all tyrosinase N-glycans during tyrosinase processing with a special focus on the proximal to the active site N-glycans, s6:N337 and s7:N371, versus s3:N161 and s4:N230 which decorate the opposite side of the domain. To this end, we have constructed mutants of human tyrosinase in which its seven N-glycosylation sites were deleted. Ablation of the s6:N337 and s7:N371 sites arrests the post-translational productive folding process resulting in terminally misfolded mutants subjected to degradation through the mannosidase driven ERAD pathway. In contrast, single mutants of the other five N-glycans located either opposite to the active site or into the N-terminus Cys1 extension of tyrosinase are temperature-sensitive mutants and recover enzymatic activity at the permissive temperature of 31°C. Sites s3 and s4 display selective calreticulin binding properties. The C-terminus sites s7 and s6 are critical for the endoplasmic reticulum retention and intracellular disposal. Results herein suggest that individual N-glycan location is critical for the stability, regional folding control and secretion of human tyrosinase and explains some tyrosinase gene missense mutations associated with oculocutaneous albinism type I.     

Protein quality control in the ER: The recognition of misfolded proteins

The mechanism, in molecular terms of protein quality control, specifically of how the cell recognizes and discriminates misfolded proteins, remains a challenge. In the secretory pathway the folding status of glycoproteins passing through the endoplasmic reticulum is marked by the composition of the N-glycan. The different glycoforms are recognized by specialized lectins. The folding sensor UGGT acts as an unusual molecular chaperone and covalently modifies the Man9 N-glycan of a misfolded protein by adding a glucose moiety and converts it to Glc1Man9 that rebinds the lectin calnexin. However, further links between the folding status of a glycoprotein and the composition of the N-glycan are unclear. There is little unequivocal evidence for other proteins in the ER recognizing the N-glycan and also acting as molecular chaperones. Nevertheless, based upon a few examples, we suggest that this function is carried out by individual proteins in several different complexes. Thus, calnexin binds the protein disulfide isomerase ERp57, that acts upon Glc1Man9 glycoproteins. In another example the protein disulfide isomerase ERdj5 binds specifically to EDEM (which is probably a mannosidase) and a lectin OS9, and reduces the disulfide bonds of bound glycoproteins destined for ERAD. Thus the glycan recognition is performed by a lectin and the chaperone function performed by a specific partner protein that can recognize misfolded proteins. We predict that this will be a common arrangement of proteins in the ER and that members of protein foldase families such as PDI and PPI will bind specifically to lectins in the ER. Molecular chaperones BiP and GRp94 will assist in the folding of proteins bound in these complexes as well as in the folding of non-glycoproteins.     

UDP-glucose:glycoprotein glucosyltransferase (UGGT1) promotes substrate solubility in the endoplasmic reticulum

Protein folding in the endoplasmic reticulum (ER) is error prone, and ER quality control (ERQC) processes ensure that only correctly folded proteins are exported from the ER. Glycoproteins can be retained in the ER by ERQC, and this retention contributes to multiple human diseases, termed ER storage diseases. UDP-glucose:glycoprotein glucosyltransferase (UGGT1) acts as a central component of glycoprotein ERQC, monoglucosylating deglucosylated N-glycans of incompletely folded glycoproteins and promoting subsequent reassociation with the lectin-like chaperones calreticulin and calnexin. The extent to which UGGT1 influences glycoprotein folding, however, has only been investigated for a few selected substrates. Using mouse embryonic fibroblasts lacking UGGT1 or those with UGGT1 complementation, we investigated the effect of monoglucosylation on the soluble/insoluble distribution of two misfolded α1-antitrypsin (AAT) variants responsible for AAT deficiency disease: null Hong Kong (NHK) and Z allele. Whereas substrate solubility increases directly with the number of N-linked glycosylation sites, our results indicate that additional solubility is conferred by UGGT1 enzymatic activity. Monoglucosylation-dependent solubility decreases both BiP association with NHK and unfolded protein response activation, and the solubility increase is blocked in cells deficient for calreticulin. These results suggest that UGGT1-dependent monoglucosylation of N-linked glycoproteins promotes substrate solubility in the ER.     

Glycoprotein folding and quality-control mechanisms in protein-folding diseases

Biosynthesis of proteins – from translation to folding to export – encompasses a complex set of events that are exquisitely regulated and scrutinized to ensure the functional quality of the end products. Cells have evolved to capitalize on multiple post-translational modifications in addition to primary structure to indicate the folding status of nascent polypeptides to the chaperones and other proteins that assist in their folding and export. These modifications can also, in the case of irreversibly misfolded candidates, signal the need for dislocation and degradation. The current Review focuses on the glycoprotein quality-control (GQC) system that utilizes protein N-glycosylation and N-glycan trimming to direct nascent glycopolypeptides through the folding, export and dislocation pathways in the endoplasmic reticulum (ER). A diverse set of pathological conditions rooted in defective as well as over-vigilant ER quality-control systems have been identified, underlining its importance in human health and disease. We describe the GQC pathways and highlight disease and animal models that have been instrumental in clarifying our current understanding of these processes.KEY WORDS: N-glycosylation, Glycoprotein folding, ER quality control, ER-associated degradation, ER export     

N-glycan branching requirement in neuronal and postnatal viability

The structural variations among extracellular N-glycans reflect the activity of glycosyltransferases and glycosidases that operate in the Golgi apparatus. More than other types of vertebrate glycans, N-glycans are highly branched oligosaccharides with multiple antennae linked to an underlying mannose core structure. The branching patterns of N-glycans consist of three types, termed high-mannose, hybrid, and complex. Though most extracellular mammalian N-glycans are of the complex type, some cells variably express hybrid and high-mannose forms. Nevertheless, a requirement for hybrid and complex N-glycan branching exists in embryonic development and postnatal function among mice and humans inheriting defective Mgat1 or Mgat2 alleles. The resulting defects in formation N-glycan branching patterns cause multiple abnormalities, including neurologic defects, and have inferred the presence of distinct functions for hybrid and complex N-glycan branches among different cell lineages. We have further explored N-glycan structure-function relationships in vivo by using Cre-loxP conditional mutagenesis to abolish hybrid and complex N-glycan branching specifically among neuronal cells. Our findings show that hybrid N-glycan branching is an essential posttranslational modification among neurons. Loss of Mgat1 resulted in a unique pattern of neuronal glycoprotein deficiency concurrent with caspase 3 activation and apoptosis. Such animals exhibited severe locomotor deficits, tremors, paralysis, and early postnatal death. Unexpectedly, neuronal Mgat2 deletion resulting in the loss of complex but not hybrid N-glycan branching was well tolerated without phenotypic markers of neuronal or locomotor dysfunction. Structural features associated with hybrid N-glycan branching comprise a requisite posttranslational modification to neuronal glycoproteins that permits normal cellular function and viability.     

Substrate-specific requirements for UGT1-dependent release from calnexin

Newly synthesized glycoproteins displaying monoglucosylated N-glycans bind to the endoplasmic reticulum (ER) chaperone calnexin, and their maturation is catalyzed by the calnexin-associated oxidoreductase ERp57. Folding substrates are eventually released from calnexin, and terminal glucoses are removed from N-glycans. The UDP-glucose:glycoprotein glucosyltransferase (UGT1, UGGT, GT) monitors the folding state of polypeptides released from calnexin and adds back a glucose residue on N-glycans of nonnative polypeptides, thereby prolonging retention in the calnexin chaperone system for additional folding attempts. Here we show that for certain newly synthesized glycoproteins UGT1 deletion has no effect on binding to calnexin. These proteins must normally complete their folding program in one binding event. Other proteins normally undergo multiple binding events, and UGT1 deletion results in their premature release from calnexin. For other proteins, UGT1 deletion substantially delays release from calnexin, unexpectedly showing that UGT1 activity might be required for a structural maturation needed for substrate dissociation from calnexin and export from the ER.     

Allele-specific suppression of a defective brassinosteroid receptor reveals a physiological role of UGGT in ER quality control

UDP-glucose:glycoprotein glucosyltransferase (UGGT) is a presumed folding sensor of protein quality control in the endoplasmic reticulum (ER). Previous biochemical studies with nonphysiological substrates revealed that UGGT can glucosylate nonnative glycoproteins by recognizing subtle folding defects; however, its physiological function remains undefined. Here, we show that mutations in the Arabidopsis EBS1 gene suppressed the growth defects of a brassinosteroid (BR) receptor mutant, bri1-9, in an allele-specific manner by restoring its BR sensitivity. Using a map-based cloning strategy, we discovered that EBS1 encodes the Arabidopsis homolog of UGGT. We demonstrated that bri1-9 is retained in the ER through interactions with several ER chaperones and that ebs1 mutations significantly reduce the stringency of the retention-based ER quality control, allowing export of the structurally imperfect yet biochemically competent bri1-9 to the cell surface for BR perception. Thus, our discovery provides genetic support for a physiological role of UGGT in high-fidelity ER quality control.     

The Arabidopsis homolog of the mammalian OS-9 protein plays a key role in the endoplasmic reticulum-associated degradation of misfolded receptor-like kinases

The endoplasmic reticulum-associated degradation (ERAD) is a highly conserved mechanism to remove misfolded membrane/secretory proteins from the endoplasmic reticulum (ER). While many of the individual components of the ERAD machinery are well characterized in yeast and mammals, our knowledge of a plant ERAD process is rather limited. Here, we report a functional study of an Arabidopsis homolog (AtOS9) of an ER luminal lectin Yos9 (OS-9 in mammals) that recognizes a unique asparagine-linked glycan on misfolded proteins. We discovered that AtOS9 is an ER-localized glycoprotein that is co-expressed with many known/predicted ER chaperones. A T-DNA insertional atos9-t mutation blocks the degradation of a structurally imperfect yet biochemically competent brassinosteroid (BR) receptor bri1-9, causing its increased accumulation in the ER and its consequent leakage to the cell surface responsible for restoring the BR sensitivity and suppressing the dwarfism of the bri1-9 mutant. In addition, we identified a missense mutation in AtOS9 in a recently discovered ERAD mutant ems-mutagenized bri1 suppressor 6 (ebs6-1). Moreover, we showed that atos9-t also inhibits the ERAD of bri1-5, another ER-retained BR receptor, and a misfolded EFR, a BRI1-like receptor for the bacterial translation elongation factor EF-Tu. Furthermore, we found that AtOS9 interacted biochemically and genetically with EBS5, an Arabidopsis homolog of the yeast Hrd3/mammalian Sel1L known to collaborate with Yos9/OS-9 to select ERAD clients. Taken together, our results demonstrated a functional role of AtOS9 in a plant ERAD process that degrades misfolded receptor-like kinases.     

EDEM1 regulates ER-associated degradation by accelerating de-mannosylation of folding-defective polypeptides and by inhibiting their covalent aggregation

Proteins expressed in the endoplasmic reticulum (ER) are covalently modified by co-translational addition of pre-assembled core glycans (glucose(3)-mannose(9)-N-acetylglucosamine(2)) to asparagines in Asn-X-Ser/Thr motifs. N-Glycan processing is essential for protein quality control in the ER. Cleavages and re-additions of the innermost glucose residue prolong folding attempts in the calnexin cycle. Progressive loss of mannoses is a symptom of long retention in the ER and elicits preparation of terminally misfolded polypeptides for dislocation into the cytosol and proteasome-mediated degradation. The ER stress-induced protein EDEM1 regulates disposal of folding-defective glycoproteins and has been described as a mannose-binding lectin. Here we show that elevation of the intralumenal concentration of EDEM1 accelerates ER-associated degradation (ERAD) by accelerating de-mannosylation of terminally misfolded glycoproteins and by inhibiting formation of covalent aggregates upon release of terminally misfolded ERAD candidates from calnexin. Acceleration of Man(9) or Man(5)N-glycans dismantling upon overexpression was fully blocked by substitution in EDEM1 of one catalytic residue conserved amongst alpha1,2-mannosidases, thus suggesting that EDEM1 is an active mannosidase. This mutation did not affect the chaperone function of EDEM1.     

Calnexin mediates the maturation of GPI-anchors through ER retention

The protein folding and lipid moiety status of glycosylphosphatidylinositol-anchored proteins (GPI-APs) are monitored in the endoplasmic reticulum (ER), with calnexin playing dual roles in the maturation of GPI-APs. In the present study, we investigated the functions of calnexin in the quality control and lipid remodeling of GPI-APs in the ER. By directly binding the N-glycan on proteins, calnexin was observed to efficiently retain GPI-APs in the ER until they were correctly folded. In addition, sufficient ER retention time was crucial for GPI-inositol deacylation, which is mediated by post-GPI attachment protein 1 (PGAP1). Once the calnexin/calreticulin cycle was disrupted, misfolded and inositol-acylated GPI-APs could not be retained in the ER and were exposed on the plasma membrane. In calnexin/calreticulin-deficient cells, endogenous GPI-anchored alkaline phosphatase was expressed on the cell surface, but its activity was significantly decreased. ER stress induced surface expression of misfolded GPI-APs, but proper GPI-inositol deacylation occurred due to the extended time that they were retained in the ER. Our results indicate that calnexin-mediated ER quality control systems for GPI-APs are necessary for both protein folding and GPI-inositol deacylation.     

A conserved basic residue cluster is essential for the protein quality control function of the Arabidopsis calreticulin 3

Calreticulin (CRT) is a highly conserved chaperone-like lectin that regulates Ca²⁺ homeostasis and participates in protein quality control in the endoplasmic reticulum (ER). Most of our CRT knowledge came from mammalian studies, but our understanding of plant CRTs is limited. Many plants contain more than two CRTs that form two distinct groups: CRT1/CRT2 and CRT3. Previous studies on plant CRTs were focused on their Ca²⁺-binding function, but recent studies revealed a crucial role for the Arabidopsis CRT3 in ER retention of a mutant brassinosteroid receptor, brassinosteroid-insensitive 1-9 (bri1-9) and in complete folding of a plant immunity receptor EF-Tu Receptor (EFR). However, little is known about the molecular basis of the functional specification of the CRTs. We have recently shown that the C-terminal domain of CRT3, which is rich in basic residues, is essential for retaining bri1-9 in the ER; however, its role in assisting EFR folding has not been studied. Here, we used an insertional mutant of CRT3, ebs2-8 (EMS mutagenized bri1 suppressor 2-8), in the bri1-9 background as a genetic system to investigate the functional importance of two basic residue clusters in the CRT3′s C-terminal domain. Complementation experiments of ebs2-8 bri1-9 with mutant CRT3^M transgenes showed that a highly conserved basic tetrapeptide Arg³⁹²Arg³⁹³Arg³⁹⁴Lys³⁹⁵ is essential but a less conserved basic tetrapeptide Arg⁴⁰¹Arg⁴⁰²Arg⁴⁰³Arg⁴⁰⁴ is dispensable for the quality control function of CRT3 that retains bri1-9 in the ER and facilitates the complete folding of EFR.     

Pharmacologic rescue of conformationally-defective proteins: implications for the treatment of human disease

The process of quality control in the endoplasmic reticulum involves a variety of mechanisms which ensure that only correctly folded proteins enter the secretory pathway. Among these are conformation-screening mechanisms performed by molecular chaperones that assist in protein folding and prevent non-native (or misfolded) proteins from interacting with other misfolded proteins. Chaperones play a central role in the triage of newly formed proteins prior to their entry into the secretion, retention, and degradation pathways. Despite this stringent quality control mechanism, gain- or loss-of-function mutations that affect protein folding in the endoplasmic reticulum can manifest themselves as profound effects on the health of an organism. Understanding the molecular, cellular, and energetic mechanisms of protein routing could prevent or correct the structural abnormalities associated with disease-causing misfolded proteins. Rescue of misfolded, "trafficking-defective", but otherwise functional, proteins is achieved by a variety of physical, chemical, genetic, and pharmacological approaches. Pharmacologic chaperones (or "pharmacoperones") are template molecules that may potentially arrest or reverse diseases by inducing mutant proteins to adopt native-type-like conformations instead of improperly folded ones. Such restructuring leads to a normal pattern of cellular localization and function. This review focuses on protein misfolding and misrouting related to various disease states and describes promising approaches to overcoming such defects. Special attention is paid to the gonadotropin-releasing hormone receptor, since there is a great deal of information about this receptor, which has recently emerged as a particularly instructive model.     

Mutations of an α1,6 Mannosyltransferase Inhibit Endoplasmic Reticulum–Associated Degradation of Defective Brassinosteroid Receptors in Arabidopsis

Asn-linked glycans, or the glycan code, carry crucial information for protein folding, transport, sorting, and degradation. The biochemical pathway for generating such a code is highly conserved in eukaryotic organisms and consists of ordered assembly of a lipid-linked tetradeccasaccharide. Most of our current knowledge on glycan biosynthesis was obtained from studies of yeast asparagine-linked glycosylation (alg) mutants. By contrast, little is known about biosynthesis and biological functions of N-glycans in plants. Here, we show that loss-of-function mutations in the Arabidopsis thaliana homolog of the yeast ALG12 result in transfer of incompletely assembled glycans to polypeptides. This metabolic defect significantly compromises the endoplasmic reticulum–associated degradation of bri1-9 and bri1-5, two defective transmembrane receptors for brassinosteroids. Consequently, overaccumulated bri1-9 or bri1-5 proteins saturate the quality control systems that retain the two mutated receptors in the endoplasmic reticulum and can thus leak out of the folding compartment, resulting in phenotypic suppression of the two bri1 mutants. Our results strongly suggest that the complete assembly of the lipid-linked glycans is essential for successful quality control of defective glycoproteins in Arabidopsis.     

Quality control in the secretory pathway: retention of a misfolded viral membrane glycoprotein involves cycling between the ER, intermediate compartment, and Golgi apparatus

Proteins synthesized in the ER are generally transported to the Golgi complex and beyond only when they have reached a fully folded and assembled conformation. To analyze how the selective retention of misfolded proteins works, we monitored the long-term fate of a membrane glycoprotein with a temperature-dependent folding defect, the G protein of tsO45 vesicular stomatitis virus. We used indirect immunofluorescence, immunoelectron microscopy, and a novel Nycodenz gradient centrifugation procedure for separating the ER, the intermediate compartment, and the Golgi complex. We also employed the folding and recycling inhibitors dithiothreitol and AIF4-, and coimmunoprecipitation with calnexin antibodies. The results showed that the misfolded G protein is not retained in the ER alone; it can move to the intermediate compartment and to the cis-Golgi network but is then recycled back to the ER. In the ER it is associated with calnexin and BiP/GRP78. Of these two chaperones, only BiP/GRP78 seems to accompany it through the recycling circuit. Thus, the retention of this misfolded glycoprotein is the result of multiple mechanisms including calnexin binding in the ER and selective retrieval from the intermediate compartment and the cis-Golgi network.     

A KDEL-tagged monoclonal antibody is efficiently retained in the endoplasmic reticulum in leaves, but is both partially secreted and sorted to protein storage vacuoles in seeds

Transgenic plants are attractive biological systems for the large-scale production of pharmaceutical proteins. In particular, seeds offer special advantages, such as ease of handling and long-term stable storage. Nevertheless, most of the studies of the expression of antibodies in plants have been performed in leaves. We report the expression of a secreted (sec-Ab) or KDEL-tagged (Ab-KDEL) mutant of the 14D9 monoclonal antibody in transgenic tobacco leaves and seeds. Although the KDEL sequence has little effect on the accumulation of the antibody in leaves, it leads to a higher antibody yield in seeds. sec-Ab(Leaf) purified from leaf contains complex N-glycans, including Lewis(a) epitopes, as typically found in extracellular glycoproteins. In contrast, Ab-KDEL(Leaf) bears only high-mannose-type oligosaccharides (mostly Man 7 and 8) consistent with an efficient endoplasmic reticulum (ER) retention/cis-Golgi retrieval of the antibody. sec-Ab and Ab-KDEL gamma chains purified from seeds are cleaved by proteases and contain complex N-glycans indicating maturation in the late Golgi compartments. Consistent with glycosylation of the protein, Ab-KDEL(Seed) was partially secreted and sorted to protein storage vacuoles (PSVs) in seeds and not found in the ER. This dual targeting may be due to KDEL-mediated targeting to the PSV and to a partial saturation of the vacuolar sorting machinery. Taken together, our results reveal important differences in the ER retention and vacuolar sorting machinery between leaves and seeds. In addition, we demonstrate that a plant-made antibody with triantennary high-mannose-type N-glycans has similar Fab functionality to its counterpart with biantennary complex N-glycans, but the former antibody interacts with protein A in a stronger manner and is more immunogenic than the latter. Such differences could be related to a variable immunoglobulin G (IgG)-Fc folding that would depend on the size of the N-glycan.