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Vol. 14, Issue 6, 2592-2602, June 2003
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* Department of Biological Sciences, University of Warwick, Coventry CV4 7AL,
United Kingdom;
Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle
Ricerche, 20133 Milano, Italy;
Centre for Plant Sciences, Leeds Institute for Plant Biotechnology and
Agriculture, School of Biology, The University of Leeds, Leeds LS2 9JT, United
Kingdom; and
¶ Unit of Immunology, Department of Oral Medicine and Pathology, Guy's Hospital,
London SE1 9RT, United Kingdom
Submitted November 27, 2002;
Revised February 19, 2003;
Accepted February 26, 2003
Monitoring Editor: Maarten J. Chrispeels
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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)
and can even produce complex, multimeric proteins in large amounts. One such
example is the expression of a decameric, secretory IgA
(Ma et al., 1995), a
molecule composed of two IgA units (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 all the IgA subunits in the endoplasmic
reticulum (ER), where complex assembly occurs with high efficiency
(Frigerio et al.,
2000). The overall production of IgA is up to 8% of protein of a
mature tobacco plant. 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
(Ma et al., 1995;
Larrick et al.,
2001). However, we have recently discovered that unlike in
mammals, not all assembled antibody is secreted by plant 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significant proportion of the molecules (at least 80%, our unpublished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 (Frigerio et al.,
2000). All IgA assembly intermediates— from the decamer to
the simplest, heterotetrameric unit— share this intracellular fate, and
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 (Frigerio et
al., 2000). Because the 
light chain is 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. This could
in turn be due either to a stress imposed on 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 on the plant ER but is the result of a cryptic signal that resides in the C-terminal domain 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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).
For protoplast transfection experiments, wild-type N. tabacum cv
Petit Havana SR1 was used. Plants were grown 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 (Ma et al.,
1994) using the PCR. The oligonucleotides
5'-ccatcgatggaatggacctgggttttt-3' and
5'-ccctctagactagtagcataggccatc-3', containing ClaI and
XbaI 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
(Denecke et 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 (glycan4 pJLH43) were introduced using the
oligonucleotides 5'-actgtagacaattccgccacctcagcctaca-3',
5'-tgtaggctgaggtggcggaattgtctacagt-3', 5'-gagcag
ctcaacagcgttttccgctcagtcag-3',
5'-ctgactgagcggaaaacgctgttgagctgctc-3',
5'-ttgcccatgaacttcgtccagaagaccatcga-3',
5'-tcgatggtcttctggacgaagttcatgggcaa-3',
5'-aaacccaccaatgtcgctgtgtctgtgatcatg-3', or
5'-catgatcacagacacagcgacattggtgggttt-3', respectively, using
pJLH38 as a template. Multiple glycan mutant 3,4 was also produced
using Quickchange in vitro mutagenesis system (Stratagene) with the
oligonucleotides 5'-aaacccaccaatgtcgctgtgtctgtgatcatg-3' and
5'-catgatcacagacacagcgacatt ggtgggttt-3' and pJLH42 as template.
The glycan mutant pJLH45, containing no glycosylation sites (1,2,3,4)
was produced by isolating the ClaI-NcoI fragment from
pJLH40, the NcoI-EcoRI fragment pJLH41, and the
EcoRI-XbaI fragment from pJLH44 and ligating them into the
pUC vector previously cut with ClaI and XbaI.
Removal of the last 18 amino acids of the / heavy chain
(C18 pJLH47) was achieved by PCR using the antisense oligonucleotide
5'-ccctctagactatttacccgacagacggtc-3' producing a stop codon
followed by an XbaI site at position 537 with the sense oligo
5'-gagcagctcaacagcgttttccgctcagtcag-3'. The resulting PCR product
was cut with EcoRI and XbaI and ligated into the expression
vector cut with ClaI and XbaI along with a
ClaI-EcoRI fragment of pJLH38.
Phaseolin expression constructs T343F and 418 are described in
Pedrazzini et al.
(1997) and Frigerio et
al. (1998a),
respectively.
The portion of the region encoding the predicted mature portion of the IgG
light chain was amplified and inserted between the blunted
KpnI site and the BamHI site of pDE300d, a pUC19-based
plasmid carrying a 35S promoter, the signal peptide of PR1b
(Denecke et al.,
1990), a polylinker, and a 3'nos polyadenylation site. This
resulted in plasmid pSK3 and encodes a secreted form of the IgG light
chain that is formed after cleavage of the PR1b signal peptide.
Protoplast Transfection
Protoplasts prepared from axenic leaves of tobacco (Nicotiana
tabacum cv Petit Havana SRI), grown in sterile in vitro conditions under
a 12-h light-dark regime, were subjected to polyethylene glycol–
mediated transfections as described by Pedrazzini et al.
(1997). Forty micrograms of
each plasmid was used to transform 106 protoplasts in 1 ml. When
only single antibody chains were expressed, the total amount of DNA was
maintained constant among samples by adding 40 µg of empty expression
vector pDHA (Tabe and Higgins,
1998). After transfection, cells were incubated at 25°C before
metabolic labeling.
In Vivo Labeling of Protoplasts and Analysis of Expressed
Polypeptides
Pulse-chase experiments were conducted by labeling protoplasts using ProMix
(a mixture of 35S-Met and 35S-Cys; Amersham Biosciences,
Piscataway, NJ) and chasing with an excess of cold amino acids for the times
stated (Predrazzini et al. 1997). After harvesting at desired time
points, protoplasts and incubation media were frozen and homogenized by adding
two volumes of ice-cold homogenization buffer (150 mM Tris-HCl, 150 mM NaCl,
1.5 mM EDTA, and 1.5% (wt/vol) Triton X-100, pH 7.5) supplemented with
Complete protease inhibitor cocktail (Roche Products Ltd., Welwyn Garden City,
United Kingdom). Immunoprecipitation of expressed polypeptides was performed
as described previously (Frigerio et
al. 1998a) using rabbit polyclonal antisera raised against
mouse IgG (whole molecule, Sigma Chemical, St. Louis, MO), heavy
chain, light chain (Southern Biotechnology, Birmingham, AL), or bean
phaseolin (Pedrazzini et al.
1997). Digestion of immunoprecipitated proteins with
endoglycosidase H (Roche) was performed as described previously
(Ceriotti et al.,
1991).
For the analysis of the secreted free light chains by sedimentation
velocity, after radioactive labeling and homogenization of cells, homogenates
and incubation media were loaded on top of a continuous 5–25% (wt/vol)
linear sucrose gradient made in 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, and
50 mM Tris-Cl, pH 7.5. Samples were centrifuged at 39,000 rpm in a SW40 Ti
rotor (Beckman Instruments, Inc., Fullerton, CA) for 20 h at 20°C.
light chain was then immunoselected from each gradient fraction.
Immunoselected proteins were resolved by 15% (wt/vol) nonreducing or reducing
SDS-PAGE and revealed by fluorography. The relative intensity of polypeptide
bands was determined by densitometry using the Total Lab software package
(Nonlinear Dynamics, Newcastle, UK).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). We therefore decided to focus our efforts on the latter for
simplicity of analysis. Tetrameric IgA/G consists of two light chains
and two hybrid IgA/G chains. These polypeptides are schematically illustrated
in Figure 1. The hybrid heavy
chain (denominated /) consists of the IgG variable
domain and constant C1 and C2 domains from monoclonal IgG Guy's
13 (Ma et al., 1994),
fused to the constant C2 and C3 domain from a secretory IgA
(Ma et al., 1994).
The C3 domain contains the C-terminal cysteine that is responsible for
binding the J chain and contains regions necessary for contact with the
secretory component (Mestecky and McGhee,
1987). The additional C2 domain was originally added to
provide an extra-affinity tag, to facilitate purification of the antibody from
plant tissue (Ma et al.,
1994). The presence or the absence of this extra domain does not
affect the trafficking of the molecule (Ma
et al., 1994, and our unpublished observations).
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The Synthesis of High Amounts of IgA/G Does Not Impose Stress on the
Secretory Pathway of Mesophyll Cells
Is the observed vacuolar delivery of a proportion of SIgA/G the result of
stress imposed to the secretory system by the high expression of this
heterologous protein? Could vacuolar targeting be the effect of saturation of
secretion? To test these possibilities, protoplasts from plants expressing
SIgA/G were transiently transfected with plasmids encoding different
constructs of phaseolin, a well-characterized vacuolar storage protein from
bean. The mutated phaseolin 418 lacks the vacuolar sorting signal of
phaseolin and is efficiently secreted after traffic through the Golgi complex
when expressed in tobacco (Frigerio et
al., 1998a). If expression of the antibody were saturating
the ER capacity and imposing stress onto the system to the point of reducing
secretion and causing missorting to the vacuole, we would expect secretion of
phaseolin 418 to be inhibited as well. However,
Figure 2A shows that in the
antibody-expressing cells, the fate of phaseolin is not affected, and the
protein is still secreted with the same efficiency observed in SR1 cells where
IgA/G is not coexpressed. In the vacuole of tobacco cells, phaseolin undergoes
proteolytic fragmentation to polypeptides in the 20–25-kDa range. The
appearance of these fragments is therefore indicative of vacuolar delivery
(Pedrazzini et al.,
1997). Because no fragmentation of phaseolin 418 in
SigA/G-expressing cells occurred during the chase
(Figure 2A), we conclude that
vacuolar delivery of IgA cannot be attributed to a general partial missorting
of secretory proteins to vacuoles in these cells.
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To further explore the possibility of a spurious effect due to stress on
the secretory system, we wanted to test whether expression of IgA/G was
affecting transport of a naturally vacuolar protein. We used T343F, a form of
phaseolin that contains the normal vacuolar sorting signal of this protein and
is efficiently transported to the vacuole via the Golgi complex also in
tobacco (Pedrazzini et al.,
1997). The appearance of fragmentation products during the chase
indicated that in the IgA/G-expressing cells, phaseolin was still delivered to
vacuoles, with an efficiency comparable to wild-type cells
(Figure 2B). A proportion of
T343F molecules was also secreted in the incubation medium. We have previously
shown that this is due to saturation of the sorting machinery during transient
expression (Frigerio et al.,
1998a). In IgA/G expressing cells, there is no shift in the ratio
vacuolar/secreted phaseolin with respect to nontransgenic SR1 cells
(Figure 2B). Because sorting of
phaseolin to the vacuole is a saturable process
(Frigerio et al.,
1998a), this result indicates that expression—and vacuolar
delivery— of IgA/G is not competing with vacuolar sorting of phaseolin,
suggesting that the two proteins are sorted to the vacuole by different
mechanisms.
Intracellular Trafficking of IgA/G Is Faithfully Reproduced in
Transiently Transfected Tobacco Protoplasts
Transient expression in protoplasts has been successfully used to study the
behavior and trafficking of proteins in the plant secretory pathway (Frigerio
et al., 1998a,
1998b;
Phillipson et al.,
2001; Törmäkangas
et al., 2001), although it has never been used for
complex multimeric proteins. The transient expression system allows rapid
testing of mutant constructs and would therefore be a valuable tool for the
analysis of potential of trafficking mutants of IgA/G. To reliably use this
technique in this study we first needed to prove that the behavior of the Ig
chains was comparable in transiently and permanently transformed tobacco
cells. To achieve these aims we cloned the Ig coding sequences (heavy and
light chains, Figure 1), fused
to the CaMV35S promoter, into a CaMV35S-driven vector for transient
expression. Tobacco mesophyll protoplasts were transfected with plasmids
encoding chain and IgA/G / heavy chain. Transfected
cells were metabolically labeled for 2 h with 35S-methionine and
-cysteine and homogenized. Protoplasts from transgenic plants expressing IgA/G
were also treated similarly, for comparison. Homogenates were
immunoprecipitated with either anti- or anti- antisera.
Anti- recognizes only the constant domains in the heavy chains,
whereas anti- binds to the light chain only. Both of these antisera
were capable of coselecting both heavy and light chains, indicating assembly
of the IgA molecule after transient and permanent expression
(Figure 3A, lanes 1– 4).
Assembly is very efficient, as further confirmed by the fact that the
molecules migrate with the mobility expected for fully assembled
heterotetramers when resolved on nonreducing SDS-PAGE
(Figure 3A, lanes 5– 8)
with no free heavy chain detected in either transient of stably expressing
protoplasts. An excess of free light chain can be observed in lane 8 and will
be discussed further below. The discrepancy observed in the mobility of the
chain between transient and stable expression is due to the fact that
a different signal peptide (Denecke et
al., 1992) was used to prepare the transient expression
construct compared with the murine signal peptide used to generate transgenic
plants (Ma et al.,
1994 and see MATERIALS AND METHODS) Because the original sequence
contains additional residues between the predicted signal peptide cleavage
site and the first codon of mature chain
(Ma et al., 1994),
this probably results in a slightly different electrophoretic mobility.
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As mentioned above, our observations in transgenic plants revealed that the
IgA/G tetramer is partially delivered to vacuoles. This results in the
appearance of degradation products after a long chase
(Frigerio et al.,
2000). To test whether this is also the case for transiently
expressed IgA/G, we compared the phenotype of the molecules after a 16-h
metabolic labeling (Figure 3B).
Subsequent immunoprecipitation with anti-IgG, which recognizes the whole IgG
molecule, reveals the presence of small polypeptides resulting from
degradation (Figure 3B, arrows)
when the IgA/G molecule is expressed transiently. The sizes of these fragments
are comparable in both transiently and stably expressed antibody chains. We
therefore conclude that fragments observed in transient expression are also
the result of vacuolar delivery. Therefore, both the assembly and
intracellular transport events of IgA/G are faithfully reproduced in transient
expression using tobacco mesophyll protoplasts.
Only Assembled IgA/G Is Found in Vacuoles
When a leaf extract is fractionated by velocity density centrifugation on
sucrose gradient, the position of the IgA/G vacuolar fragments with respect to
molecular weight markers indicates that neither of the fragments is monomeric
(Figure 4A; the positions of
fragments are indicated by dots at right; the bands that migrate at around 67
kDa on SDS-PAGE are not proteins: they are an artifact often visualized by ECL
due to 
-mercaptoethanol). Therefore all polypeptides delivered to
vacuoles possess some degree of assembly, indicating that vacuolar delivery
occurs after the antibody has assembled and is not a disposal route for orphan
chains.
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Vacuolar Delivery of IgA/G Is Mediated by the Golgi Complex
In previous studies we have shown that vacuolar delivery of IgA/G is not
the result of endocytosis of secreted proteins. This was demonstrated by the
fact that increasing the volume of the incubation medium did not result in a
decrease in the intensity of the intracellular fragmentation products
(Frigerio et al.,
2000). Likewise, incubating unlabeled protoplasts for 24 h with
secretion medium isolated from extensively labeled protoplasts and therefore
containing secreted, radioactive IgA/G did not result in the appearance of
labeled intracellular Ig fragments (Santoro and Vitale, unpublished results).
Both secretion and transport of IgA/G to the vacuole can be inhibited by the
fungal metabolite BFA (Frigerio et
al., 2000). BFA inhibits the formation of a number of
vesicles that mediate traffic through the secretory pathway; we therefore used
that evidence to conclude that IgA/G vacuolar delivery occurred through
vesicle traffic. Recently, however, it has become clear that there exist
alternative routes of traffic from the plant ER to vacuoles
(Hara-Nishimura et al.,
1998; Toyooka et al.,
2000). We have shown that one of these routes, although prone to
BFA inhibition, bypasses the Golgi complex
(Frigerio et al.,
2001). In the light of this new evidence it is clear that BFA
inhibition alone cannot be used as a sole evidence for transport through the
Golgi complex. Thus, in order to test whether IgA/G does indeed travel through
the Golgi, we have studied the state of one of its N-linked glycans.
Analysis of the IgA/G degradation products
(Figure 4) shows that one
fragment has a molecular mass around 30 kDa (arrowhead in
Figure 4A) and comigrates along
the gradient with a proportion of intact light chains, strongly suggesting
that the fragment is comprised of the variable and constant domains
associated with the light chain to yield the Fab fragment originating
from fragmentation of tetrameric IgA/G. Consistently, on protein blots, the
30-kDa fragment is not detected by anti-light () chain antibodies
(Figure 4B) or antibodies
against the heavy () chains of IgA (our unpublished results). Because
heavy chains are extensively glycosylated, we reasoned that the 30-kDa
fragment (Figure 4A) could be
used as a marker for traffic through the Golgi complex.
To determine whether the fragment was actually glycosylated, all
glycosylation sites of the hybrid heavy chain were inactivated by
site-specific mutagenesis (1,2,3,4 in
Figure 5A), and the mutated
polypeptide was transiently coexpressed with chain in tobacco
protoplasts. Protoplasts were labeled with 35S-labeled cysteine and
-methionine for 16 h and immunoprecipitated with anti-IgG antibodies. The
30-kDa fragment was no longer detectable
(Figure 5A, lane 3). The
observed disappearance of the 30-kDa fragment upon mutation of the
glycosylation sites is likely due to the fact that its mobility becomes very
similar to that of the chain, resulting in comigration of the two
polypeptides. The fact that the mobility shift is only detectable in the
mutant / chain where all four putative glycosylation sites have
been removed (Figure 5A, lane
3) but not in a mutant chain carrying mutations in the two sites located in
the domain (3,4, Figure
5A, lane 2) provides further evidence that the 30-kDa fragment
originates from the domain of the hybrid / heavy
chain.
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Thus, the 30-kDa fragmentation product, derives from assembled IgA/G, and
is N-glycosylated. As fragmentation occurs in the vacuolar compartment
(Frigerio et al.,
2000), we expect that the glycan present on the 30-kDa fragment
product be of the complex (i.e., Golgi-modified) type, if the molecule has
traveled through the Golgi complex en route to the vacuole. If this is the
case, this glycan should be resistant to in vitro endoglycosidase H (endo H)
digestion, because this enzyme removes high-mannose, N-linked glycans that are
attached to glycoproteins in the ER but not glycans modified by Golgi enzymes.
Indeed, treatment with endo H after transient expression of heavy and light
chains, metabolic labeling, and subsequent immunoprecipitation does not affect
the mobility of the 30-kDa fragment (Figure
5B, compare lanes 2 and 4), indicating that this glycan has
acquired endo H resistance. It is, however, clear that the enzyme is active,
because the mobility of the heavy chain is increased by endo H treatment at
the end of the pulse (Figure
5B, compare lanes 1 and 3). This is expected because, shortly
after synthesis, while still in the ER, the heavy chain glycans are in the
high-mannose, endo H–sensitive form. The same results were obtained when
the experiment was performed on protoplasts isolated from transgenic plants
expressing IgA/G (our unpublished results).
On the basis of the results shown, we therefore conclude that transport of IgA/G to the vacuole occurs through the Golgi complex.
The C-terminal Domain of Heavy Chain Determines Vacuolar
Delivery
The work presented so far shows that the secretory pathway in plants
expressing IgA/G is not overloaded and that the IgA/G tetrameric molecule is
transported to the vacuole via the Golgi complex where it is subjected to
proteolysis. The most likely explanation for IgA/G delivery to the vacuole is
therefore the existence of positive, albeit cryptic, sorting information
within the molecule. Clearly, this signal must be absent from the parent IgG
as this is secreted efficiently (Frigerio
et al., 2000). Therefore, it must be contained within the
additional constant domains of the / heavy chain
(Figure 1). We speculated that
the signal must be exposed and therefore compared the C-terminal regions of
the two heavy chains. Sequence alignment of the IgA/G C3 constant
domain with the constant C3 domain of its parent Guy's 13 IgG heavy
chain (Ma et al.,
1994) reveals that the C3 domain is 18 residues longer than
C3 (Figure 6A). This
extension constitutes the tailpiece containing the cysteine residue required
for J chain binding (Mattu et
al., 1998). Given the heterogeneous nature of C-terminal
vacuolar sorting signals (ctVSS; Vitale
and Raikhel, 1999), the tailpiece might be recognized as a ctVSS
within plant cells. To test this hypothesis, we deleted the 18-residue,
C-terminal tail to produce a truncated heavy / chain,
denominated C18. We cotransfected protoplasts with plasmids encoding
the light chain and /, , or C18 heavy
chains, respectively, and subjected them to pulse-chase analysis. When
coexpressed with light chain, C18 assembled correctly into tetramers
(our unpublished results) and was secreted very efficiently
(Figure 6, B and C). Given that
free, unassembled light chains are secreted (see below), but unassembled heavy
chains are completely retained in the endoplasmic reticulum
(Nuttall et al.,
2002), the amount of heavy chains recovered from the medium
represents the actual levels of tetramer secretion. Note that although the
extracellular amount of IgA/G heavy chains increases very slowly, to 
30%
of the initial labeled protein after a 12-h chase, the amount of secreted
IgA/G containing C18 heavy chains increases much more rapidly and
reaches 90% of the synthesized chains during the chase
(Figure 6C, and compare lanes
5– 8 with lanes 21–24 in Figure
6B). This rate and efficiency of secretion is comparable or even
higher than that of IgG (Figure 6, B and
C). The increased secretion of C18 with respect to
wild-type IgA/G was paralleled by the almost complete disappearance of
vacuolar degradation fragments. This is visible in
Figure 6B (compare lanes
2– 4, arrows, with lanes 18–20) and was further confirmed by
overexposing gels of immunoprecipitates after a 16-h labeling
(Figure 6D). This shows that
deletion of the C-terminal 18 amino acids of the IgA/G heavy chain virtually
abolishes vacuolar targeting and results in increased secretion of the
assembled tetramers. Therefore, we conclude that the C-terminal tail of the
hybrid / chain is responsible for vacuolar delivery of
IgA/G.
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Unassembled Light Chains Are Efficiently Secreted as Monomers
When protoplasts from transgenic plants expressing IgA/G are subjected to
pulse-chase labeling and IgA/G are analyzed by SDS-PAGE under nonreducing
conditions, it can be observed that at the end of the 1-h pulse only a very
minor proportion of heavy chains is recovered as unassembled polypeptides,
indicating very rapid and efficient assembly
(Figure 7A). Conversely, a
relevant proportion of light chains is still monomeric, indicating that in
these plants light chains are produced in excess with respect to heavy chains.
Analysis of the protoplast incubation medium shows that, besides the
heterotetramers, unassembled light chains are also secreted
(Figure 7A). To test this, we
transiently transformed tobacco protoplasts with plasmid encoding the
chain, the / heavy chain, or both chains together and subjected
them to metabolic labeling for 16 h. Protoplasts were then homogenized and
immunoprecipitated with anti-IgG. When both chains are coexpressed,
immunoprecipitation with anti-IgG antiserum reveals the presence of a larger
amount of light chains compared with heavy chain in both the cells and
the incubation medium (Figure
7B, lanes 3 and 6). When the samples are resolved on nonreducing
SDS-PAGE (Figure 7B, lanes
7–12), it appears evident that the excess light chain observed
is free light chain not associated with heavy chain. In mammalian
cells there is evidence that free unassembled light chain can be secreted as
covalent or noncovalent dimer (Leitzgen
et al., 1997). From the experiment in nonreducing
conditions (Figure 7B, lanes 10
and 12), it is clear that there are no disulfide-linked dimers of .
When the chain is expressed alone, it is mainly recovered from the
incubation medium (Figure 7B, lanes 1 and 4). Conversely, the / chain remains mainly
intracellular when chain is not coexpressed
(Figure 7B, lanes 2 and 5;
Nuttall et al.,
2002). To determine if noncovalent dimerization occurs before
secretion of chain in plants, we transfected protoplasts with
chain and metabolically labeled them for 16 h. We then loaded cell homogenates
and incubation medium onto linear 5–25% sucrose gradients and subjected
them to sedimentation velocity centrifugation, along with molecular mass
markers. Gradient fractions were then immunoprecipitated with anti-IgG
antiserum (Figure 7C). Clearly,
the vast majority of secreted light chain is retrieved in fractions
corresponding to a mass around 30 kDa, a size compatible with the monomeric
form. We conclude that is efficiently secreted as a monomer in tobacco
mesophyll protoplasts. This is in agreement with the finding that vacuolar
delivery of assembled IgA/G depends on the presence of the C-terminal portion
of heavy chains.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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, and here). In this respect, the plant ER is a very
versatile and efficient folding compartment for foreign proteins, as shown by
other studies (Frigerio et al.,
1998b; Crofts et al.,
1999; Frigerio et
al., 2001). Production of biologically active SIgA/G in
transgenic tobacco leaves reaches 8% of total soluble protein
(Ma et al., 1995).
However, we have previously shown that part of the protein is directed to the
vacuole instead of being secreted into the apoplast
(Frigerio et al.,
2000). Vacuolar SIgA/G is not an irrelevant proportion, it is
detectable at the electron microscope level and accumulates as proteolytic
fragments (Frigerio et al.,
2000). We do not know whether fragmentation is then followed by
full degradation, but the fragments are unlikely to have the same activity as
the intact molecules. Understanding the mechanism that leads to vacuolar
delivery of these immunoglobulins would therefore increase our knowledge on
how plants handle foreign secretory proteins and more in general on plant
vacuolar delivery mechanisms, but would also allow us to plan strategies to
increase the production of SIgA/G.
The Capacity of the Plant Secretory Pathway Is Very High
By transiently expressing vacuolar or secreted forms of phaseolin we have
shown that the secretory pathway of IgA/G plants still has spare
translocational and traffic activity. Therefore, delivery of IgA/G to the
vacuole is not due to overloading of the endomembrane system. This is
consistent with the current view that in plants, secretion is the default
pathway for proteins introduced into the secretory pathway and lacking sorting
signals (Denecke et al.,
1990). We have also shown here that at least one of the heavy
chain fragments present in the vacuole has N-linked oligosaccharide chains
that, similarly to secreted intact IgA/G, have undergone modifications in the
Golgi complex. This implies that the Golgi complex mediates vacuolar delivery
of the heterologous protein and that most probably the sorting occurs within
or at the exit of this compartment.
Defective proteins introduced into the secretory pathway are often
dislocated from the ER into the cytosol using the Sec61 translocation channel
in reverse and are finally degraded by the proteasome
(Chevet et al., 2001).
It has however been shown both in plants and yeast that structurally defective
or foreign nonvacuolar proteins can also be directed to vacuoles, in processes
that could be alternative quality control mechanism with respect to
dislocation to the cytosol (Coleman et
al., 1996; Hong et
al., 1996; Bagga et
al., 1997). In plants the mechanism could involve direct
autophagy of portions of the ER by the vacuole
(Coleman et al., 1996;
Bagga et al., 1997).
We have also shown that overaccumulation of a protein in the plant ER due to
addition of the ER localization signal KDEL can lead to vacuolar delivery
without Golgi complex involvement
(Frigerio et al.,
2001). A role of such mechanisms in IgA/G vacuolar delivery is
however ruled out by our observation that IgA/G fragments have Golgi-modified
oligosaccharide chains. Quality control vacuolar delivery in yeast can instead
be mediated by the Golgi complex and has shown to be dependent on the vacuolar
sorting receptor Vps10p. This receptor also mediates sorting of a number of
yeast natural vacuolar enzymes through clathrin-coated vesicle delivery from
the Golgi complex to prevacuolar endosomes
(Hong et al., 1996).
The receptor would therefore be involved both in normal vacuolar sorting and
in quality control, possibly through distinct recognition activities
(Hong et al., 1996).
We therefore cannot rule out that SIgA/G is also partially directed to the
vacuole by a form of Golgi-mediated quality control, although, as explained
below, we favor the hypothesis of the presence of a cryptic vacuolar sorting
signal.
A Signal for Vacuolar Delivery in the IgA Heavy Chains
We have deleted the last 18 amino acids from the hybrid heavy chain. When
this truncated chain was coexpressed with light chain we observed efficient
secretion of the assembled immunoglobulin and could not detect fragmentation.
The deleted segment is part of the C3 constant domain of IgA and is an
extension with respect to the IgG heavy chain. Therefore, the segment contains
a signal that determines vacuolar delivery of Ig tetramers. We do not know if
this signal is also sufficient. We intend to test this by fusing it to
reporter secretory proteins although probably many random C-terminal sequences
can lead to vacuolar delivery in plant cells
(Dombrowski et al.,
1993; Neuhaus et al.,
1994). Therefore, even if the C3 tailpiece should prove
sufficient to deliver reporter proteins to the plant vacuole, this would not
mean that the signal is the result of a selective pressure for the delivery of
IgA to a similar hydrolytic compartment in animal cells.
The deleted fragment contains the cysteine residue that forms a disulfide
bridge with the J chain (Mattu et
al., 1998). This residue is therefore necessary for IgA
dimerization and must be maintained in an engineered heavy chain. The free
cysteine, however, is not responsible for vacuolar delivery because dimers of
IgA/G, where the cysteine is engaged in J chain interaction, have the same
intracellular fate as the individual units
(Frigerio et al.,
2000). Therefore, it could be possible to mutagenize the C termini
of the heavy chain to inhibit vacuolar sorting without affecting dimerization.
A number of natural propeptide vacuolar sorting signals have been identified,
both for storage and vegetative vacuoles (see
Vitale and Raikhel, 1999 for a
review). A comparison with the C-terminal segment of IgA shows that the IgA
pentapeptide VSVSV is a repetition of dipeptides (VS or SV) that are present
three times in the C-terminal propeptides of tobacco -glucanase (one SV
and two VS) and once in potato PT20 (VS;
Koide et al., 1999;
Vitale and Raikhel, 1999). It
may therefore be possible that just by chance the IgA sequence is recognized
by one of the plant vacuolar sorting mechanisms. The fact that vacuolar
delivery of IgA/G is only partial could be explained considering that IgAs
have clearly not evolved as vacuolar proteins: the fortuitous signal would
therefore be inefficient. It has been shown that mutagenesis of natural plant
sorting signals often results in decreased but not abolished vacuolar sorting,
whereas full deletion causes efficient secretion
(Dombrowski et al.,
1993; Neuhaus et al.,
1994).
These observations are not in favor of vacuolar delivery of IgA/G because of quality control. It seems more likely that the protein is misrecognized as a natural vacuolar protein. This would be a more favorable situation for site-directed mutagenesis aimed to obtain complete secretion.
The Light Chains Do Not Have the Same Behavior in Mammalian
and Plant Cells
We have shown that tobacco protoplasts efficiently secrete unassembled
chains. Light chains are also secreted when expressed in mammalian
cells in the absence of heavy chains, and their secretion requires
dimerization (Leitzgen et al.,
1997). These homodimers do not form when heavy chains are also
present, but the variable and first constant domain are similar enough in the
two types of chains to allow this assembly to occur in the absence of
competing heavy chains. Tobacco protoplasts instead secrete monomeric light
chains. This was demonstrated both by transient expression in the absence of
heavy chains and in the transgenic IgA/G plants we have analyzed, where light
chains are synthesized in excess with respect to their partners. Secretion of
monomeric or homodimeric heavy chains seems negligible. In mammalian cells,
before assembly with the light chains, heavy chains are quantitatively
associated with BiP and retained in the ER. Indeed, we have recently shown
that this is also the case in plant cells
(Nuttall et al.,
2002). However, the behavior of light chains is clearly different
in plant and mammalian cells. It has been reported that an unusual mAb,
comprised exclusively of light chains, is recovered both as monomers and
dimers from hybridoma cell cultures (Masat
et al., 1994). It has been suggested, however, that the
detection of monomers would be the result of partial dissociation of dimers
during purification (Leitzgen et
al., 1997). We do not think that this explains our results,
because we never observed any dimer formation in our experiments. It has been
hypothesized that monomers of light chains are retained in the ER until
dimerization because they would interact with the ER chaperone machinery
(Leitzgen et al.,
1997). Some features of the mammalian and plant ER folding
machineries may therefore be different, contrary to previous indications from
a number of studies (Vitale and Denecke,
1999).
Biotechnological Perspectives
The ultimate goal of secretory antibody expression in plants is to produce
large amounts of protein in a way that allows for easy purification. Protein
secretion by the roots is one promising approach
(Borisjuk et al.,
1999). Maximizing SIgA/G secretion is therefore an attractive
goal. We have identified the signal that prevents a vast proportion of the
immunoglobulin molecules from being secreted. Deletion of this signal leads to
hybrid IgA/G secretion at levels comparable to IgG secretion, indicating that
no further intracellular processes exist to prevent exocytosis. This is the
first essential step toward the production of a fully secreted complex
immunoglobulin. We are aware that complete deletion of the C-terminal tail,
which also contains the C-terminal cysteine involved in J chain binding, is
too drastic as it also prevents assembly of the decameric molecule. We are
currently aiming to develop a set of heavy chain mutants that are devoid of
vacuolar sorting information but are still able to allow decamer assembly.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Both authors contributed equally to this work. 
|| Corresponding authors. E-mail addresses: vitale{at}ibba.cnr.it; l.frigerio{at}warwick.ac.uk.
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REFERENCES |
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