From the Biozentrum, University of Basel, CH-4056 Basel, Switzerland
Received for publication, November 1, 2002, and in revised form, February 27, 2003
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ABSTRACT |
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Consensus profiles were established to screen
data bases for novel animal L-type lectins. The profiles were generated
from linear sequence motifs of the human L-type lectin-like membrane proteins ERGIC-53, ERGL, and VIP36 and by optimal alignment of the
entire carbohydrate recognition domain of these proteins. The search
revealed numerous orthologous and homologous L-type lectin-like
proteins in animals, protozoans, and yeast, as well as the sequence of
a novel family member related to VIP36, named VIPL for VIP36-like.
Sequence analysis suggests that VIPL is a ubiquitously expressed
protein and appeared earlier in evolution than VIP36. The cDNA of
VIPL was cloned and expressed in cell culture. VIPL is a high-mannose
type I membrane glycoprotein with similar domain organization as VIP36.
Unlike VIP36 and ERGIC-53 that are predominantly associated with
postendoplasmic reticulum (ER) membranes and cycle in the early
secretory pathway, VIPL is a non-cycling resident protein of the ER.
Mutagenesis experiments indicate that ER retention of VIPL involves a
RKR di-arginine signal. Overexpression of VIPL redistributed ERGIC-53
to the ER without affecting the cycling of the KDEL-receptor and
the overall morphology of the early secretory pathway. The results
suggest that VIPL may function as a regulator of ERGIC-53.
Understanding the molecular basis of secretion requires
knowledge of how secretory proteins are correctly folded and assembled in a process known as quality control, and how these itinerant proteins
are sorted from resident proteins along the secretory pathway. Evidence
is mounting that intracellular lectins play an important role in these
processes (1, 2). A majority of secretory proteins acquires
N-glycans during translocation into the
ER,1 and these modifications
are most often required for the folding of the proteins. The folding
process is assisted by the membrane lectin calnexin and the related
soluble lectin calreticulin. These two lectins recognize
monoglucosylated trimming intermediates and, in a remarkable cycle of
de- and reglucosylation, guarantee that only correctly folded
glycoproteins leave the ER (2-4).
If folding is unsuccessful, newly synthesized glycoproteins are
degraded by the proteasome after retrotranslocation to the cytosol.
Degradation appears to involve calnexin and an ER Correctly folded glycoproteins leave the ER in COPII-coated vesicles
(8) and are transported to the Golgi via the ER-Golgi intermediate
compartment (ERGIC, Ref. 9). The exit signals that direct proteins out
of the ER are poorly understood with few exceptions (10). The mannose
lectin ERGIC-53 (p58 in rat) mediates efficient ER-exit of some
secretory glycoproteins by serving as a transport receptor (9, 11-13).
ERGIC-53-assisted proteins include blood coagulation factors V and VIII
(14), cathepsin C (15), and cathepsin Z (16). ERGIC-53 is a major membrane protein of the tubulovesicular clusters of the ERGIC (17) and
efficiently cycles between ERGIC and ER (18-20). A second lectin
cycling in the early secretory pathway, VIP36 is related to ERGIC-53
(21). VIP36 is associated with ERGIC and Golgi (22) and to some extent
with the plasma membrane (23). VIP36 binds mannose (24) and GalNAc (25)
and may operate in glycoprotein transport to the apical plasma membrane
of polarized cells (26).
ERGIC-53 and VIP36 are type I membrane proteins. In their luminal
segment they carry a single domain of about 200 amino acids that
exhibits sequence similarity to the carbohydrate recognition domain
(CRD) of soluble lectins of leguminous plants (21). This domain is
known as L-type CRD (27). The L-type lectin-like domain of animal
lectins is designated LTLD in the present study. The family of plant
L-type lectins includes numerous members whereas only three L-type
lectins are known in animals: ERGIC-53, VIP36, and a recently
discovered ERGIC-53-like protein termed ERGL (28). Unlike ERGIC-53 and
VIP36 that are ubiquitously expressed proteins, the ERGL
gene is expressed in a limited number of tissues only, with highest
mRNA levels in normal and neoplastic prostate cells. The ERGL
protein remains to be characterized.
We wondered if there are additional, yet unidentified members of the
animal L-type lectin family. To identify such proteins we established
profiles characteristic for animal L-type lectins. By scanning data
bases with these profiles we identified orthologous and homologous
L-type lectin-like proteins in animals, protozoans, and yeast. A novel
member of this family displays molecular similarities with VIP36 and
was therefore dubbed VIPL (for VIP36-like). The corresponding protein
was cloned and expressed in cell culture. VIPL was found to localize to
the ER and to affect the recycling of ERGIC-53.
Reagents--
Mouse monoclonal antibodies (mAb): 9E10.2 (IgG1)
against c-Myc, 12CA5 (IgG2b) and 16B12 (IgG1, CRP) against the
hemagglutin (HA) epitope, A1/182 (IgG2a) against BAP31 (20), A1/296
(IgG2a) against CLIMP-63 (29), G1/93 (IgG1) against ERGIC-53 (17), G1/133 (IgG1) against giantin (30), A1/118 (IgG1) against GPP130 (31).
Rabbit antibodies against human KDEL receptor and rat Sec31p were
kindly provided by H.-D. Söling, University of Göttingen, and F. Gorelick, Yale University, respectively. Secondary
goat-anti-rabbit and goat-anti-mouse antibodies (either against whole
IgG (H+L) or IgG-subtypes) conjugated with AlexaFluor 488 or AlexaFluor 568 were from Molecular Probes (The Netherlands). Secondary
peroxidase-conjugated goat-anti-rabbit and goat-anti-mouse antibodies
were from Jackson ImmunoResearch Laboratories Inc. Brefeldin A (BFA)
was from Epicentre Technologies, and cell culture media and reagents
were from Invitrogen and Sigma.
Recombinant DNA--
Standard molecular biology protocols were
adapted from Refs. 32 or 33. Oligonucleotides were from Microsynth
(Switzerland). ERGIC-53 constructs containing a Myc-tag and an
artificial N-glycosylation site, termed GM, have been
described (34, 35). VIPL cDNA was obtained by RT-PCR. Total RNA of
subconfluent HepG2 cells was isolated using peqGOLD RNApure reagent
(peqLab, Biotech GmbH, Germany) with a protocol modified from Ref. 36.
Reverse transcription was done with 2 µg of total RNA using
Omniscript RT enzyme (Qiagen, Switzerland) and oligo d
(T)14V primer. Subsequent PCR was performed with
VIPL-specific primer pairs and Tgo DNA proof-reading
polymerase (Roche Molecular Biochemicals, Switzerland). The 5'-end VIPL
primer contained an additional BamHI site and maintains the
Kozak transcription initiation sequence preceding the start AUG codon
of VIPL. The 3'-end primer had a XbaI site after the stop
codon. The resulting cDNA was cloned into pcDNA 3.1 vector
(Invitrogen) via BamHI and XbaI sites. A HA
epitope (YPYDVPDYA) was introduced downstream of the signal sequence
cleavage site between amino acids 44 and 45 of full-length VIPL. This
construct was generated by PCR-based splicing (37). Selected amino
acids were substituted by oligonucleotide-directed PCR mutagenesis or
sequence-overlap-extension PCR (38), and mutant fragments were recloned
via BamHI/XbaI or
SacII/XbaI sites into VIPL constructs. For
creating chimeric GM constructs with the cytoplasmic tail of VIPL
(GM-ViTa), a BglII restriction site was introduced adjacent
to the transmembrane domain of GM by silent mutagenesis changing the
codon of the arginine 499 in the tail to AGA. This site allows
insertion of fragments via BglII and XbaI into GM
constructs. A cDNA encoding the cytoplasmic tail of VIPL was
prepared by annealing complementary oligonucleotides as described (10)
and cloned into the GM construct via BglII and
XbaI sites. Additional mutations were introduced by PCR and recloned as AccI/XbaI fragments into GM-ViTa
construct. All constructs were confirmed by sequencing using standard
methods and an ABI Prism 310 Genetic Analyser (PE Applied Biosystems, Switzerland).
Cell Culture and Transfection--
COS-1 cells were cultured and
transfected (DEAE-dextran method) as described (35). HEK293 cells
(kindly provided by T. Meier, Myocontract, Switzerland) were cultured
in Dulbecco's minimal essential medium (4.5 g/liter
glucose) supplemented with 10% fetal calf serum and 100 IU/ml
penicillin, 100 µg/ml streptomycin, and 1 µg/ml fungizone. HEK293
cells were transfected by the calcium phosphate precipitation method
(32). HepG2 cells were cultured as described (20) and transfected by
use of FuGENE 6 transfection reagent (Roche Molecular Biochemicals,
Switzerland). For immunofluorescence experiments cells were plated in
poly-L-lysine-coated 8-well multichamber glass slides (Lab
Tek, Nalgene-Nunc Intl.). All cultures were grown at 37 °C with 5%
CO2 in humidified air, and transfection of cells was
carried out 1 day after plating.
Metabolic Labeling and Immunoprecipitation--
42 h after
transfection the cells were washed twice with phosphate-buffered
saline, starved in labeling medium (MEM without methionine,
supplemented with 10% dialyzed fetal calf serum) and pulsed with 100 µCi/ml [35S]methionine/cysteine (EasyTagTM
EXPRE35S35S Protein Labeling, PerkinElmer Life
Sciences). Cells were immediately processed or chased with complete
Dulbecco's minimal essential medium containing 10 mM
L-methionine. For immunoprecipitation the cells were washed
twice with ice-cold phosphate-buffered saline and resuspended in lysis
buffer (100 mM sodium phosphate, 1% Triton X-100, pH 8)
supplemented with 0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 0.5 µg/ml pepstatin). In some experiments 20 mM iodoacetamide or N-methylmaleimide was
included in the lysis buffer. Cells were lysed by passing them five
times through a 25-gauge needle. After 1 h on ice, the lysate was
cleared by centrifugation at 100,000 × g for 1 h.
The supernatant was added to protein A-Sepharose beads (Amersham
Biosciences) to which antibodies had been prebound. After incubation
for at least 1 h on a rotary shaker in the cold, the beads were
washed four times with lysis buffer, once with 100 mM
sodium phosphate (pH 8) and once with 10 mM sodium
phosphate (pH 8).
Endoglycosidase Digestion--
For digestion with
endoglycosidase H (endo H) the immunoprecipitates were boiled for 3 min
in 50 mM TrisCl, 1% SDS, and 0.1 M
Sodium Carbonate Extraction--
Membrane association of
proteins was tested by the sodium carbonate procedure (39). After
metabolic labeling, the cells were collected in ice-cold homogenization
buffer (20 mM Hepes/KOH, 300 mM sucrose, 0.2 mM phenylmethylsulfonyl fluoride, 20 mM NEM, pH
7.4), resuspended in 0.1 M sodium carbonate (pH 11.5),
passed 10 times through a G25 needle, and kept on ice for 30 min. The sample was then layered onto a small cushion of homogenization buffer,
and membranes were pelleted by centrifugation at 100,000 × g for 1 h and resuspended in lysis buffer (see
immunoprecipitation). The pH of the supernatant was neutralized by
adding one-fifth volume of 0.5 M potassium phosphate (pH
8), and Triton X-100 was added to 1% final concentration. All samples
were kept on ice for 30 min before centrifugation at 100,000 × g for 1 h. The supernatants were subjected to immunoprecipitation.
Subcellular Fractionation--
42 h after transfection with
VIPL-HA cDNA, HepG2 cells were incubated for 3 h with 5 µg/ml BFA or with solvent only before subcellular fractionation using
Nycodenz gradients (35). The distribution of organelle markers and
VIPL-HA was determined by Western blotting.
Gel Electrophoresis and Western Blotting--
Samples were
separated by gradient SDS-PAGE. Radioactivity was visualized by
fluorography using sodium salicylate and BioMax MR-1 films (Rochester).
Fluorograms were quantified with a ChemImagerTM and
AlphaEaseTM software (Alpha Inotech Corporation). For
Western blotting, proteins were transferred to nitrocellulose B85 (45 µm, Schleicher & Schuell, Germany) at 100 V for 1 h in the cold
using transfer buffer containing 15.6 mM Tris, 120 mM glycine, and 20% (v/v) methanol (pH 8.4). The
nitrocellulose was rinsed with 50% methanol and stained by Amido Black
(Serva, Germany). All subsequent incubations were in PBS containing 5%
nonfat dry milk and 0.05% Tween 20 (Serva, Germany): Blocking for
1 h, incubation with the first antibody for 60 min, rinsing three
times 10 min, incubation with peroxidase-coupled secondary antibody for
60 min, and rinsing 10 min. After three final 10-min washes with
phosphate-buffered saline containing 0.05% Tween 20, the
nitrocellulose was processed using enhanced chemiluminescence
(ChemiGlow ECL reagent, AlphaInotech Corp.) and exposed to BioMax MR-1
films or directly analyzed in a ChemImagerTM.
Immunofluorescence Microscopy--
The procedure has been
described (10). Specimens were examined with a Polyvar microscope or a
Leica confocal laser scanning microscope.
Biocomputing--
The GCG programs (Madison, WI) were used for
sequence analysis (40). Other software used is available at ExPASy
server (www.expasy.org/) (41). SwissProt (release 40.7) and
TrEMBL/release 19.1) data bases were searched. MEME/MotifSearch (42)
was performed with full-length sequences of human ERGIC-53, ERGL, and
VIP36 with default settings. The algorithm automatically generated a
profile, based on settings with 6 motifs of 8 amino acids in length
each with one or zero occurrence in each sequence. A ProfileSearch (43)
against SwissProt and TrEMBL data bases was done with a profile
generated of the alignment of ERGIC-53 (amino acids 44-292), VIP36
(amino acids 50-286), and ERGL (amino acids 31-271). For secondary
structure predictions we used the GCG program PeptideStructure and
PredictProtein (44). Potential sites for domains and motifs were
identified by MOTIF search in the Prosite library (45) and by PSORT
(psort.ims.u-tokyo.ac.jp). Coiled coil regions were identified with
CoilScan (46), and signal sequences with SPScan and signalP (47). For
detection of putative transmembrane domains the programs HMMTOP (48),
and TMpred were applied in combination with structure predictions. The
phylogenetic tree was constructed using the GrowTree software of the
GCG package, and distance correction was calculated according to
Jukes-Cantor.
Consensus Profiles for the Identification of Animal L-type
Lectins--
Profiles for the identification of lectins have been
established in several data bases (27). For plant L-type lectins such profiles include PS00307 and PS00308 in PROSITE (45) and PF00138 and
PF00139 in Pfam (49). We noticed, however, that these profiles do not
identify animal L-type lectins accurately in the existing data bases.
Therefore, we developed new consensus profiles specific for animal
L-type lectins.
In a first approach we used software MEME/MotifSearch of GCG (42) to
establish a consensus motif pattern from non-aligned full-length
sequences of the known human L-type lectins ERGIC-53, ERGL, and VIP36.
The algorithm generated a consensus motif pattern of 6 linear sequence
motifs of 8 amino acids each with single occurrence in a defined order.
All sequences were restricted to the LTLD. One motif encompassed part
of loop A of the CRD of L-type lectins (50) that includes a
conserved aspartate required for sugar and metal binding (Fig.
1B). Two motifs covered the
two cysteines known to form an intracellular disulfide bond in native ERGIC-53 (Ref. 51 and our own observations). The cysteines are conserved in the LTLD of all three human lectins and in most of their
orthologs assembled in Fig. 1. The other four motifs encompassed conserved regions of
A second consensus profile for proteins with a putative LTLD was
generated by a ProfileSearch method included in the GCG package (43).
This approach is based on a sequence matrix profile that was generated
on the optimal alignment of the LTLDs of human ERGIC-53, ERGL, and
VIP36. The similarity of LTLDs was found to be 58% for ERGIC-53 and
ERGL, 46% for ERGIC-53 and VIP36, and 45% for VIP36 and ERGL. The
algorithm of the profile scan calculates the score (quality) of the
optimal alignment between the consensus profile and each sequence in
the scanned data bases.
The scored proteins of both scanning methods were then analyzed by
alignment and comparison to animal L-type lectins, and the presence of
residues functionally important for metal/sugar binding was
investigated. Moreover, structural features of the LTLD were examined
by hydrophilicity plots and secondary structure predictions. Finally we
analyzed the identified proteins for possible protein motifs and domains.
Identification of Orthologs and New Family Members of Animal L-type
Lectins by Profile Scanning--
Both profile scans recovered all the
known orthologs of ERGIC-53, ERGL, and of VIP36 from the data bases
(Table I and Fig. 1A). The
scores were higher for ERGIC-53 orthologs since profile characteristics
were contributed mostly by ERGIC-53 and ERGL and to a lesser extent by
VIP36. Two entries (SpTrEMBL accession numbers Q9H0V9 and Q9BQ14) also
appeared with high scores. They stand for the same sequence that
encodes a novel VIP36-like protein, we termed VIPL (VIP36-like, see
below).
With lower scores the scans also identified lectin-like proteins in
yeast. These proteins are the ERGIC-53 like Emp47p (52) and its close
relative Emp46p of Saccharomyces cerevisiae (56% similarity
to each other) and two entries of Schizosaccharomyces pombe
(SpTrEMBL O42707 and O94401). Emp47p and Emp46p are type I membrane
proteins with a putative LTLD fold and a stalk containing a coiled coil
domain preceding the transmembrane domain. Emp47p contains a
DXL(X)5N metal/sugar binding motif in
loop C of the LTLD that is found in the plant lectins UEA I and II as well as LAA I (50). By contrast, animal lectins have a metal/sugar binding site of the DXF/YXN type in loop C that
is common to most plant lectins (Fig. 1B). The Emp46p
protein does not contain any of the key residues for metal/sugar
binding although a LTLD fold is predicted. A deletion of Emp47p results
in intolerance to high Ca2+ concentration in the growth
medium, while no such phenotype was observed in an Emp46p deletion
strain (53). Nevertheless, overexpression of Emp46p can suppress the
Emp47p defect, suggesting that the two proteins functionally overlap.
Both lectin-like proteins of S. pombe contain a LTLD fold
similar to animal L-type lectins. Examination of the similarity to
animal lectins and of the domain organization suggests that entry
O42707 is related to Emp47p and Emp46p whereas entry O94401 is
presumably the ortholog of the novel VIPL (Figs. 1A and 2).
While the O94401 protein has all the hallmarks of a functional LTLD,
the O42707 protein lacks typical key residues required for metal and
sugar binding in its LTLD fold.
The scans identified two putative lectins in the protozoans
Trypanosoma cruzi (SpTrEMBL Q9GPB0) and Leishmania
major (SpTrEMBL Q9GRK5). These proteins exhibit a LTLD fold
similar to that of animal L-type lectins including the
putative intramolecular disulfide bond (Fig. 1B). Both
lectins contain predicted coiled-coil regions downstream of the LTLD
similar to ERGIC-53, but apparently no transmembrane segment,
suggesting that they are secretory proteins.
Rescanning the data bases with consensus profiles that include the
newly identified VIPL sequence gave a result similar to that of the
initial screen, with the exception that an additional protein of
S. cerevisiae was uncovered, known as Uip5 (Ulp1-interacting protein 5, SwissProt P36137). The protein is predicted to be a type I
membrane protein with a signal sequence, suggesting localization in the
secretory pathway. The LTLD fold of Uip5 resembles that of animal
L-type lectins. It comprises all key residues for metal/sugar binding
of a functional LTLD (Fig. 1B) but does not display the
conserved cysteine residues of the animal LTLD. Uip5 has been
identified in a two-hybrid screen as interacting partner of the
Smt3-specific protease Ulp1 that mediates deconjugation of
Smt3 from septin components during G2/M phase transition of the cell cycle (54). A GFP-tagged version of Uip5 was localized to the
nuclear membrane. No specific phenotype was found in the deletion strain.
VIPL, a Novel Gene Related to VIP36--
Alignment
of the SpTrEMBL entries Q9H0V9 (gene designation
DKFZP564L2423) and Q9BQ14 revealed that they encode the same
hypothetical protein although the latter lacks a defined N terminus.
The predicted protein is 43% similar (35.2% identity) to human
ERGIC-53 and 68% similar (57.8% identity) to human VIP36. Because of
its high similarity to VIP36 we named the protein VIPL for VIP36-like. The LTLD fold of VIPL is similar to that of ERGIC-53 and VIP36. It
contains all residues required for a functional LTLD (Fig. 1B) including the conserved cysteine residues required for
intramolecular disulfide bond formation in ERGIC-53.
VIPL is predicted to be a type 1 membrane protein with a cytoplasmic
tail of 12 amino acids. Unlike ERGIC-53, the LTLD of VIPL is not
followed by a stalk, and no coiled coil is predicted. Thus, VIPL
exhibits a domain organization very similar to VIP36. The VIPL protein
is expected to be translocated into the ER by a 38-amino acid long
N-terminal signal sequence. The mature protein (residues 39-348) has a
predicted mass of 35.6 kDa and a pI of 7.51. Moreover, the net charge
is almost zero in a pH range of 6.8-8.0.
Detailed examination by gap alignments and evolutionary analysis
indicates that the previously found lectins of Drosophila (SpTrEMBL Q9VCC2) and Caenorhabditis elegans (SpTrEMBL
Q22170) are orthologs of VIPL rather than VIP36, suggesting that VIP36 appeared later in evolution than VIPL. An alignment of human VIPL and
its orthologs in Drosophila (55.1% similarity), C. elegans (53.5% similarity), and the newly discovered S. pombe ortholog (O94401, 44.2% similarity) is shown in Fig.
2. The mouse VIPL (NCBI XP_129848)
exhibits 92.5% similarity to the human sequence. Sequence conservation
among VIPL orthologs is striking in the LTLD fold, particularly in the
functional loops comprising the key residues for metal and sugar
binding (Fig. 2). The cysteine residues at positions 200 and 237 of
human VIPL are also conserved.
The human gene DKFZP564L2423 (EMBL AL136617) for VIPL is located on
chromosome 2 (q11.2) and has a total length of 34.14 kb with 8 exons.
For comparison, the gene of human VIP36 with a total length of 14.9 kb
also contains 8 exons but is located on chromosome 5 (q35.5). The VIPL
gene has an ATG start codon within a proper initiation context (55) and
an in-frame termination codon. The 5'-untranslated region showed no
other open reading frame of significant length suggesting the defined
start site is correct. The correctly spliced cDNA has a total
length of 2416 bp with a coding region of 1046 bp. An expression
profile obtained from cDNA and expressed sequence tag (EST) sources
(UniGene cluster Hs. 18627 for VIPL at NCBI server, and SAGE expression
profile of VIPL gene with Ensemble ID ENSG00000114988 at EBI server)
suggests that VIPL is a ubiquitous protein expressed in many cell types.
VIPL Is an N-Glycosylated Membrane Protein of the ER--
To study
the VIPL protein the corresponding cDNA was isolated from total RNA
of HepG2 cells by RT-PCR. The cloned sequence was identical to the
hypothetical cDNA of the gene DKFZP564L2423. VIPL was tagged with a
HA tag and expressed in various cell lines. To test for membrane
association, homogenates of transfected HEK293 cells metabolically
labeled with [35S]methionine were subjected to the
carbonate/pH 11 extraction procedure. Like the integral membrane
protein ERGIC-53, VIPL-HA quantitatively distributed to the pellet
fraction, while Sec31p, a component of the COPII coat that is
peripherally associated with membranes was quantitatively recovered in
the soluble fraction as expected (Fig.
3A). The results confirm the
prediction that VIPL is an integral membrane protein.
VIPL carries a single consensus site for N-glycosylation at
position 181 (Fig. 2). To test if this site is used, VIPL-HA was immunoprecipitated from cells and treated with glycosidases. Fig. 3B shows that VIPL-HA is sensitive to both endo H and PNGase
F indicating that VIPL is a glycoprotein. The persistent sensitivity to
endo H suggests that the N-glycan of VIPL is of the
high-mannose type. This finding was confirmed by pulse-chase
experiments in which VIPL remained endo H-sensitive throughout a 3-h
chase (Fig. 3C). Similarly, VIPL-HA did not acquire Endo D
sensitivity when expressed in Lec-1, a cell line deficient in
GlcNAc-transferase (data not shown).
We next studied the localization of transiently transfected VIPL-HA by
immunofluorescence microscopy. In HepG2 cells VIPL-HA displayed a
reticular pattern typical for ER (Fig. 4,
E-H). Double immunofluorescence microscopy with the ER
marker BAP31 confirmed this finding (Fig. 4A). A similar
localization was found in COS-1, HEK293, Lec-1, and HeLa cells (Fig.
7B and not shown).
To test if VIPL cycles in the early secretory pathway, VIPL-HA
transfected cells were treated with BFA. BFA redistributes rapidly
cycling proteins of ERGIC and Golgi, such as ERGIC-53 (18), VIP36 (22),
and the KDEL receptor (56), to characteristic punctate structures in
the cytoplasm that remain separate from the ER. While BFA treatment led
to the expected punctuate pattern for the KDEL receptor (Fig. 6,
E and G) it had no effect on the localization of
VIPL-HA (Fig. 6, D, H, L). These
results suggest that VIPL-HA does not cycle between ER and post-ER compartments.
We confirmed the ER resident localization of VIPL-HA by subcellular
fractionation (Fig. 5). A postnuclear
supernatant of HepG2 cells transiently expressing VIPL-HA was separated
by Nycodenz density gradient centrifugation, and the position of
VIPL-HA and organelle markers was probed by SDS-PAGE followed by
immunoblotting. Fig. 5A shows the expected distribution of
BAP31 (ER), GPP130 (cis-Golgi), and ERGIC-53 (ER and ERGIC) (20). VIPL
co-distributed with the ER. Upon BFA-treatment, GPP130 relocalized to
ER fractions at the bottom of the gradient (Fig. 5B), while
ERGIC-53 accumulated in the ERGIC near the top of the gradient typical
for cycling proteins. The distribution of VIPL-HA was largely unchanged
by BFA treatment. The results confirm the IF data that VIPL is a non-cycling ER protein.
Overexpression of VIPL Affects the Localization of
ERGIC-53--
ERGIC-53 is localized in punctuate structures scattered
in the cytoplasm with concentration near the Golgi (17) (Figs.
4B and 6I). Surprisingly, overexpression of VIPL
led to a striking redistribution of ERGIC-53 to the ER (Figs.
4B and 6I). This effect was observed in all the
tested cell lines transiently transfected with VIPL. Interestingly, the
relocalized ERGIC-53 was insensitive to BFA. It remained in the ER
without accumulation in dots (Fig. 6K). To test if the
redistribution of ERGIC-53 to the ER required an active LTLD we
substituted the conserved asparagine 163 of VIPL by aspartate (N163D).
This mutation can be expected to disrupt VIPL's lectin function.
VIPL(N163D) still redistributed ERGIC-53 to the ER suggesting that an
active LTLD was not required (not shown). Likewise, this redistribution
does not involve an active LTLD of ERGIC-53 since the lectin-impaired
N156A mutant of ERGIC-53 was also redistributed (not shown)
To test if overexpression of VIPL affects the morphology of the early
secretory pathway we studied the localization of Sec31p (a component of
COPII coats) and GPP130 by immunofluorescence microscopy. Figs. 4 and 6
show that the distribution of these markers was unchanged suggesting
that the general morphology of the secretory pathway is unaffected by
overexpressing VIPL. Moreover, the normal distribution of the cycling
proteins KDEL-receptor (Fig. 6) and VIP36 (not shown) in
VIPL-overexpressing cells indicates that VIPL does not just arrest any
rapidly cycling protein in the ER.
ER Retention of VIPL Requires an RKR Motif in the Cytoplasmic
Segment--
The localization of VIPL to the ER is surprising since
its cytoplasmic segment possesses a putative ER-exit motif
(i.e. an aromatic amino acid in position
To test if ER retention of VIPL is caused by its luminal domain we
generated a VIPL mutant lacking the transmembrane and cytosolic domains. This soluble mutant was secreted into the cell culture medium
(not shown) suggesting that the ER localization signal is not contained
in the luminal domain. We noted a conserved RKR motif in the
cytoplasmic tail of all orthologs of VIPL that is not present in VIP36
(Fig. 7A). The motif is reminiscent of di-arginine motifs
(RR or RXR) initially characterized as ER retention motifs near the N terminus of type II membrane proteins (57, 59) and near the
cytoplasmic C terminus of multispanning membrane proteins (60). To
study the role of the RKR motif in ER retention we
substituted it by three serines. Furthermore, to prevent interference by a possible lectin activity of VIPL, we also mutated asparagine 163 to aspartate in the putative lectin domain. Like wild-type VIPL-HA,
VIPL(N163D)-HA was localized to ER in transfected COS cells (Fig.
7B, panels a and b). By contrast, VIPL
lacking the RKR motif was also found at the cell surface in addition to
intracellular membranes (Fig. 7B, panel c). This
became more obvious when the cells were not permeabilized. While hardly
any staining of VIPL-HA was detected in cells transfected with
wild-type or VIPL(N163D)-HA cDNA (Fig. 7B, panels
d and e), surface staining of VIPL-HA lacking the RKR
motif was clearly evident (Fig. 7B, panel f).
Despite its transport to the cell surface the SSS mutant of VIPL-HA
remained endo H-sensitive as revealed by pulse-chase experiments (Fig. 7C). In addition, no acquisition of Endo D sensitivity was
observed when expressing the VIPL constructs in Lec-1 cells (data not
shown). The results of this mutagenesis approach indicate that the RKR motif is required for the ER localization of VIPL.
VIPL Lacking the ER Retention Signal Does Not Pull ERGIC-53 to the
Cell Surface--
Does overexpression of VIPL lacking the RKR
retention motif result in surface localization of ERGIC-53? To test
this, HepG2 cells were transiently transfected with VIPL-HA carrying a
RKR to SSS substitution and probed surface expression of ERGIC-53 by
IF. The transfected cells showed intracellular but no apparent cell
surface staining of ERGIC-53 (Fig.
8A), while the mutant VIPL
exhibited cell surface in addition to intracellular staining, as
expected (Fig. 8B). The results were confirmed by using
non-permeabilized cells. Mutant VIPL but not ERGIC-53 was detectable at
the cell surface (Fig. 8, C and D). These results
suggest that the interaction of overexpressed VIPL with ERGIC-53 does
not persist beyond the ER.
RKR-mediated ER Retention Can Be Overridden by an ER-exit Signal
Presented in an Oligomeric Form--
We wondered if the RXR
motif was capable of retaining ERGIC-53 in the ER. To this end the
cytoplasmic tail of VIPL was appended to an ERGIC-53 variant carrying a
Myc-tag and a N-glycosylation site (GM, Fig.
9, A and B; Ref.
35). Appending different cytoplasmic tails to GM does not interfere
with its folding (10). The KK retrieval signal in GM has been
substituted to alanines to prevent cycling. GM is efficiently
transported from ER to Golgi when its C-terminal di-phenylalanine motif
is present (GMA5FF), but transport is inefficient when the
motif is replaced by two alanines (GMA7) (Refs. 10, 35, and
61 and Fig. 9, C and D). When the tail of VIPL
was appended to the GM reporter (GM-ViTa, Fig. 9, A and B), a slight but statistically significant reduction in ER
to Golgi transport was observed (Fig. 9, C and
D). When the RKR motif was mutated to serines the GM-mtViTa
chimera was transported as efficiently as the GM construct containing
the di-phenylalanine ER-export signal. The inability of the RKR motif
to fully retain GM may be due to the fact that the potential ER exit
motif "FY" of VIPL is more active in an oligomerized protein such
as ERGIC-53 (Ref. 10).2
Indeed, when we replaced the putative ER exit motif FY in the tail of
GM-ViTa (named GM-ViTa(AA) by two alanines, the construct hardly became
endo H-resistant (Fig. 9). Immunofluorescence analysis confirmed that
GM-ViTa(AA) was localized to the ER (not shown). We conclude that the
RKR motif can mediate efficient retention of VIPL despite the presence
of an ER exit motif. However, the RKR motif cannot override the ER exit
motif in a homo-oligomeric reporter construct. Furthermore, it appears
that the RKR motif does not mediate ER retrieval since the GM-ViTa
construct failed to localize to characteristic dots after BFA treatment
(not shown).
Our profile scanning revealed notable differences between animal
and plant L-type lectins. The differences include two conserved cysteines, present in the LTLD of animal but not plant lectins, that
form an intramolecular disulfide bond in ERGIC-53 and presumably other
animal lectins (Fig. 1B). Another characteristics is the unique turn of 310 helix in animal LTLDs that separates the
The legume lectin fold has evolved independently several times (62). It
is not only present in L-type lectins but also in galectins and
pentraxins. However, the position of the ligand binding site is not
conserved among the lectin families. In the animal L-type-like lectin
family, major features of the carbohydrate binding site of legume
lectins are conserved. Thus, a divergent evolution from a common
ancestor is likely. We propose that Uip5 represents a common ancestor
of plant and animal L-type lectins.
With the discovery of VIPL the family of L-type lectins in humans
appears to be complete although some uncertainty remains since the
annotation of the human genome has not been completed. The animal
L-type lectin family comprizes the 4 members ERGIC-53, ERGL, VIP36, and
VIPL. The predicted overall structure and domain organization of VIPL
most closely resembles that of VIP36. Different from ERGIC-53,
secondary structure predictions could not define an Unlike ERGIC-53 and VIP36 that cycle in the early secretory pathway,
VIPL is a resident of the ER when expressed in cultured cells. The
following considerations argue against the possibility that the ER
localization is due to misfolding. First, we have no evidence for
aggregation of transfected VIPL, both biochemically and by
immunofluorescence microscopy. Second, ER retention was saturable by
high expression. Third, ER retention was abolished by mutating the
conserved RKR motif in the cytoplasmic domain of the molecule or by
deleting both the transmembrane and cytoplasmic domain. These
observations suggest that endogenous VIPL is also localized to the ER.
ER targeting of VIPL is mediated by a conserved di-arginine motif, RKR.
VIPL is the first type I membrane protein with such a determinant.
Di-arginine ER targeting motifs (RR or RXR) have been found
in the N-terminal tail of type II membrane proteins (59) and more
recently in the C-terminal tail of multispanning membrane proteins (60,
63). Because lysines cannot substitute for the retention function of
the arginines (59, 63) and the X in RXR can be
any basic or neutral amino acid but not an acidic one (63, 64), we
conclude that only the two arginines in the RKR motif are responsible
for ER targeting of VIPL.
VIPL also possesses a C-terminal ER-exit motif (10) that appears to be
dominated by the di-arginine ER retention signal. However, RKR was
unable to retain a chimeric ERGIC-53 reporter protein carrying the same
ER-exit motif. In contrast, the RKR motif in the cytoplasmic tail of
Kir6.2, that lacks an ER exit motif, fully retains a CD4 reporter in
the ER (63). This discrepancy is most likely due to the fact that the
ERGIC-53 reporter is homo-oligomeric, while VIPL, like CD4, is
not.2 Our studies on transport motifs in ERGIC-53 revealed
that ER-exit motifs are more efficient when presented in an oligomeric
protein (Ref. 10).2 Accordingly, inactivation of the
ER-exit motif in the ERGIC-53 reporter led to ER retention of the
construct in the present study. The results indicate that the RKR motif
can indeed operate as an ER targeting signal in type I proteins.
An unexpected finding was the arrest of endogenous ERGIC-53 in the ER
upon overexpression of VIPL. The effect was selective since other
rapidly cycling proteins, such as KDEL receptor and VIP36, were not
affected by the overexpression of VIPL, and the secretory pathway was
apparently unaffected. Since ER arrest of ERGIC-53 was also observed in
cells expressing low apparent levels of VIPL, we assume that endogenous
VIPL must be expressed at rather low levels. The redistribution of
ERGIC-53 by VIPL overexpression was still observed when the lectin
function of either protein was abolished by mutagenesis. This suggests
that VIPL and ERGIC-53 interact in a lectin-independent manner, either
directly or indirectly via a third component. Since overexpression of
both proteins gave the same phenotype, the latter possibility appears
less likely to us, unless a third interacting component is present in
large excess. However, we could not detect any interaction by
co-immunoprecipitation or by chemical cross-linking. The mechanism by
which VIPL and ERGIC-53 interact remains to be elucidated. A
hydrophobic surface patch opposite to the carbohydrate binding site of
the LTLD of ERGIC-53 (51) may mediate this interaction given the fact
that VIPL is uncharged at physiological pH. We speculate that VIPL may
control the function of ERGIC-53 by modulating its exit from the
ER.
Is VIPL a lectin? The presence of key residues for metal and sugar
binding in the LTLD of VIPL would suggest lectin activity. However,
HA-tagged VIPL failed to bind to immobilized mannose or fluorescein
isothiocyanate-labeled bovine serum albumin conjugated with glucose,
mannose, or GlcNAc under conditions used to successfully establish the
mannose specificity of ERGIC-53 (12). Moreover, binding of ERGIC-53 to
immobilized mannose was not affected by the presence of VIPL. The
metal/sugar binding site of VIPL differs slightly from that of ERGIC-53
and VIP36 in loop C (Fig. 1B). VIPL contains an elongation
by one amino acid after the key binding site. A similar elongation is
found in Con A but not other plant lectins (Fig. 1B). This
variance in length correlates with different affinities of plant
lectins for different mannose/glucose derivatives (50). The slightly
elongated C loop in VIPL may explain why experiments that demonstrated
lectin activity of ERCIG-53 or VIP36 failed for VIPL. Nevertheless, we
speculate that VIPL possesses binding preference for mannose based on
findings by Sharma and Surolia (50) who reported that legume lectins
with a short D loop, such as Favin, LOL I, LSL, or Con A,
preferentially bind mannose and glucose while lectins with a long D
loop, such as EcoRL, prefer GalNAc (see Fig. 1B).
Like ERGIC-53 and VIP36, VIPL has a short D loop.
An ortholog of VIPL was recently identified in zebra fish (EMBL
Aam29497) that exhibits 71% similarity to human VIPL and is required
for early development (65). Embryos lacking VIPL are touch insensitive
and insensitive to tapping on the dish, but they are capable of
spontaneous movements. We speculate that this defect may be caused by
inefficient processing or transport of some secretory molecules in the
absence of VIPL. A case in point is combined deficiency of coagulation
factors V and VIII in humans, an inherited disease in which mutations
in ERGIC-53 lead to inefficient secretion of coagulation factors V and
VIII (14). Interestingly, some patients suffering from this disease
have entirely normal ERGIC-53 (66-68). It is conceivable that such
patients synthesize excessive levels of VIPL that would render ERGIC-53
non-functional by retaining it in the ER. The findings of the present
study provide a basis to test this notion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mannosidase-like protein, termed EDEM in mammalian cells, that enhances degradation of
misfolded glycoproteins carrying Man8(GlcNAc)2
glycans (5-7). The strict functional dependence of EDEM on a
Man8(GlcNAc)2 structure suggests it may
function as a lectin.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol (pH 6.8). An equal volume of 0.15 M
sodium citrate (pH 5.3) supplemented with protease inhibitors was
added, and digestion with 10 milliunits of endo H (Roche Molecular
Biochemicals) was carried out at 37 °C overnight. For digestion with
endoglycosidase F (PNGase F), the immunoisolates were boiled for 3 min
in 100 mM sodium phosphate, 0.1% SDS, 0.1 mM
-mercaptoethanol, and 10 mM EDTA (pH 7.2). An equal
volume of the same buffer containing 1% Triton X-100 instead of SDS
was added and the sample was incubated with 400 milliunits of PNGase F
(Roche Molecular Biochemicals, Switzerland) at 37 °C overnight.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheets 6 and 15 of ERGIC-53/p58 as well as the
310 helix turn that separates the
-sheets 1a and 1b of ERGIC-53/p58 (51).
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Fig. 1.
Animal L-type lectin homologs.
A, dendrogram constructed with GCG software GrowTree. For
protein names and corresponding accession numbers see Table I.
B, alignment of partial LTLDs of the human L-type
lectins ERGIC-53, VIP36, and VIPL with selected plant lectins (Favin,
Lol I, LSL, Con A, and ECorL), as well as the putative lectins Uip5 of
yeast (SwissProt P36137), Q9GBP0 of T. bruci and Q9GRK5 of
L. leishmania. Shown are sequences from -sheet 6-15
referring to the resolved structure of rat ERGIC-53/p58 (51). The
alignment performed with ClustalW (69) was edited based on
defined or predicted secondary structures of the lectins and formatted
by ESPript program
(prodes.toulouse.inra.fr/ESPript/cgi-bin/nph-ESPript_exe.cgi). The
secondary structures are indicated above the sequences.
Boxed arrows indicate
-sheets, cylinders indicate
-helices. Conserved cysteines are highlighted by $. Key residues for
metal and sugar binding are marked by asterisks. Addition
residues important for metal and sugar binding in plants are marked by
circles. Black boxes represent identity and
white boxes similarity.
Orthologs and homologs of animal L-type lectins
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Fig. 2.
VIPL and its orthologs. Alignment of
VIPL proteins of human (HSapiens), fly
(DMelanogaster), worm (CElegans) and yeast
(ScPombe) based on ClustalW using blossum65 matrix and
default settings. The alignment was formatted by the ESPript program.
Conserved amino acids are highlighted in black boxes.
Partially conserved amino acids are framed. Key residues for
metal and sugar binding are indicated by asterisks and
circles, and conserved cysteines by $. Predicted
transmembrane domains are underlined by a bar. Numbering of
amino acids is with respect to human VIPL.
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Fig. 3.
VIPL is an N-glycosylated
type I membrane protein. A, association of VIPL-HA with
the lipid bilayer. 42 h after transfection with VIPL-HA, HEK293
cells were labeled for 30 min with [35S]methionine and
subjected to carbonate extraction at pH 11.5 followed by
ultracentrifugation. Samples of total homogenate (T),
supernatant (S), and pellet (P) were subjected to
immunoprecipitation with antibodies against HA, ERGIC-53, or Sec31p.
Immunoprecipitates were separated by 7-10% SDS-PAGE and visualized by
fluorography. B, glycosylation of VIPL. HEK293 cells were
subjected to immunoprecipitation with mAb 12CA5 against HA 42 h
after transfection with VIPL-HA. Immunoprecipitates were digested (+)
with endo H or PGNase, or left untreated ( ), separated by 10%
SDS-PAGE, and transferred to nitrocellulose. VIPL-HA was detected with
mAb 16B12 against HA and ECL. C, newly synthesized VIPL
remains endo H-sensitive. COS-1 cells were transfected with VIPL-HA.
After 42 h the cells were pulsed for 5 min with
[35S]methionine, chased for the indicated times, and
subjected to immunoprecipitation with anti-HA. Immunoprecipitates were
digested with endo H (+) or left untreated (
), separated by 8-12%
SDS-PAGE and VIPL-HA was visualized by fluorography.
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Fig. 4.
VIPL is localized in ER. HepG2 cells
were permeabilized and processed for confocal scanning
immunofluorescence microscopy 42 h after transfection with
VIPL-HA. Double staining for VIPL-HA and organelle markers is shown.
A, BAP31 (ER); B, ERGIC-53 (ERGIC), C,
Sec31p (COPII coat); D, giantin (cis-Golgi);
E-H, VIPL-HA.
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Fig. 5.
VIPL co-distributes with ER on Nycodenz
gradients. HepG2 cells were transiently transfected VIPL-HA.
42 h after transfection, the cells were treated for 3 h with
BFA (panel B) or solvent only (panel A).
Postnuclear supernatants were fractionated by Nycodenz density gradient
centrifugation, and fractions collected from bottom (fraction
1) to top (fraction 13). The distribution of proteins
was determined by Western blotting. Blots were quantified, and total
counts in gradient were set to 100%. Closed rectangles, ER
marker BAP31; open circles, cis-Golgi marker GPP-130;
crosses, ERGIC-53; gray triangles, VIPL-HA. Shown
is a representative experiment.
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Fig. 6.
Overexpression of VIPL affects the
localization of ERGIC-53. 42 h post-transfection the effect
of overexpressing VIPL-HA on marker proteins was examined in HepG2
cells after treatment with BFA for 3 h, or without
(control). Analysis was carried out by confocal scanning
immunofluorescence microscopy. Double staining after permeabilization
is shown. A and C, GPP-130; E and
G, KDEL-receptor; I and K, ERGIC-53;
B, D, F, H, J,
L, VIPL-HA.
2, Ref. 10) but
no di-lysine ER localization signal typical for many type I ER membrane
proteins (57). ER localization is also unexpected because VIP36, having a similar C-terminal tail sequence (Fig.
7A), is localized to Golgi and
ERGIC (22), and the C-terminal four amino acids of VIP36 can mediate
efficient ER export when appended to ERGIC-53 (58).
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Fig. 7.
The RKR motif in the cytoplasmic tail of VIPL
is required for ER retention. A, cytoplasmic C-terminal
amino acid sequences of VIP36 and VIPL of different species. The
putative ER exit motif (FY) is shown in bold-italic. The
conserved RKR motif of VIPL orthologs is bold underlined.
B, substitution of the RKR motif to triple serines results
in surface localization of VIPL. COS-1 cells were processed for
immunofluorescence microscopy with anti-HA 42 h after transfection
with wt, N163D, or RKR to SSS VIPL-HA cDNA. a-c,
permeabilized cells; d-f, non-permeabilized cells.
Bar, 25 µm. C, pulse-chase experiment. COS-1
cells were transiently transfected with the indicated constructs and
subjected to pulse-chase/endo H analysis using
[35S]methionine. After chase the cells were lysed and
subjected to immunoprecipitation with anti-HA followed by endo H
treatment, 8-12% SDS-PAGE, and fluorography.
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Fig. 8.
Secreted VIPL does not relocalize ERGIC-53 to
cell surface. 42 h post-transfection the effect of
overexpressing VIPL-HA with RKR motif substitution by SSS (VIPL(RKR to
SSS)-HA) on ERGIC-53 was examined in HepG2 cells by confocal double
immunofluorescence microscopy using permeabilized (A and
B) or non-permeabilized cells (C and
D). A and C, ERGIC-53; B
and D, VIPL(RKR to SSS)-HA.
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Fig. 9.
The RKR motif mediates retention of a
homo-oligomeric reporter protein in absence of an ER exit motif.
A, schematic representation of GM constructs used to
transfect COS-1 cells. All constructs have an
N-glycosylation site (CHO) at position 61 and a c-Myc
epitope (34). The transmembrane domain is followed by the amino acid
sequence of the cytoplasmic tails in single letter code. The amino acid
sequence of the mutant cytoplasmic tails is shown in panel
B. GMA7 and GMA5FF have been described
(10, 35). In chimeric GM-ViTa constructs, the underlined
residues in cytoplasmic tail are RKR in wild-type constructs and SSS in
mutant (mt) constructs. The most extreme FY motif in GM-ViTa
(shown in italic letters in panels A and
B) has been additionally mutated to AA in GM-ViTa(AA).
C, effect of RKR motif on GM transport. COS-1 cells were
transfected with the indicated constructs (panel B) and
subjected to pulse-chase/endo H analysis using
[35S]methionine. 60 min after chase the cells were lysed
and GM constructs were immunoprecipitated with anti-Myc.
Immunoprecipitates were digested with endo H (+) or left untreated
( ), separated by 7-10% SDS-PAGE and analyzed by fluorography. The
upper band represents the endo H-resistant and the
lower band the endo H-sensitive form of the GM constructs.
D, quantification of fluorograms including that shown in
C. Numbers on y-axis refer to
constructs in panel B. Mean values ± S.E. of at least
three independent experiments. Single and double
asterisk symbols indicate statistically significant differences in
transport between GM-ViTa and GMA5FF or GM-mtViTa,
respectively (p < 0.05, Student's t
test).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheets 1a and 1b (51). Further differences became obvious when the (partial) LTLDs of plant and animal L-type lectins were aligned (Fig.
1B). Animal lectins contain a shorter B loop but an
elongated loop between the two
-sheets after loop C. The elongated
loop forms an
-helix in ERGIC-53 (51) and is predicted to be also present in ERGL, VIP36, and the newly identified protozoan lectins, as
well as in the putative yeast lectin Uip5. Uip5 has an additional predicted
-helix in loop B not present in animal and plant lectins (Fig. 1B). Yet another difference between plant and animal
LTLDs concerns the residues mediating metal/sugar binding in loop C. While plant lectins possess a glutamate and an aspartate that are
conserved and responsible for Mn2+ binding, the animal
lectins lack these residues (Fig. 1B). Accordingly, lectin
activity of ERGIC-53 and VIP36 does not require Mn2+ (12,
25). Whether or not the protozoan lectins and the putative lectin Uip5
of yeast require Mn2+ for lectin activity is difficult to
predict since they lack the glutamate although the aspartate is
conserved. Finally, the general domain organization also differs
between plant and animal L-type lectins (including the orthologs and
homologs in lower organisms, such as yeast). Animal lectins are type I
membrane proteins as opposed to plant lectins that are soluble proteins.
-helix between
the
-sheets after loop C in the VIPL LTLD, although, similar to
ERGIC-53, the corresponding region is longer than in the LTLD of plant
lectins (Fig. 1B). Interestingly, orthologs of VIPL are
found in mouse, fly, worm, and yeast (S. pombe) but not
S. cerevisiae whereas VIP36 is restricted to higher
organisms (Fig. 1A). It appears, therefore, that VIP36 has
evolved from VIPL, presumably by gene duplication.
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ACKNOWLEDGEMENTS |
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We thank Käthy Bucher for excellent technical assistance and Hans-Dieter Söling and Fred Gorelick for providing antibodies.
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FOOTNOTES |
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* This work was supported by the University of Basel and the Swiss National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Biozentrum, University
of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. Tel.:
41-61-267-2222; Fax: 41-61-267-2208; E-mail:
Hans-Peter.Hauri@unibas.ch.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M211199200
2 O. Nufer and H.-P. Hauri, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; CRD, carbohydrate recognition domain; HA, hemagglutinin; BFA, brefeldin A; ERGIC, ER-Golgi intermediate compartment; VIPL, VIP36-like; LTLD, L-type lectin-like domain of animal lectins.
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1. | Hauri, H., Appenzeller, C., Kuhn, F., and Nufer, O. (2000) FEBS Lett. 476, 32-37[CrossRef][Medline] [Order article via Infotrieve] |
2. | Ellgaard, L., and Helenius, A. (2001) Curr. Opin. Cell. Biol. 13, 431-437[CrossRef][Medline] [Order article via Infotrieve] |
3. | Parodi, A. J. (2000) Annu. Rev. Biochem. 69, 69-93[CrossRef][Medline] [Order article via Infotrieve] |
4. | Chevet, E., Cameron, P. H., Pelletier, M. F., Thomas, D. Y., and Bergeron, J. J. (2001) Curr. Opin. Struct. Biol. 11, 120-124[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Nakatsukasa, K.,
Nishikawa, S.,
Hosokawa, N.,
Nagata, K.,
and Endo, T.
(2001)
J. Biol. Chem.
276,
8635-8638 |
6. |
Hosokawa, N.,
Wada, I.,
Hasegawa, K.,
Yorihuzi, T.,
Tremblay, L. O.,
Herscovics, A.,
and Nagata, K.
(2001)
EMBO Rep.
2,
415-422 |
7. |
Jakob, C. A.,
Bodmer, D.,
Spirig, U.,
Battig, P.,
Marcil, A.,
Dignard, D.,
Bergeron, J. J.,
Thomas, D. Y.,
and Aebi, M.
(2001)
EMBO Rep.
2,
423-430 |
8. | Antonny, B., and Schekman, R. (2001) Curr. Opin. Cell Biol. 13, 438-443[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Hauri, H. P.,
Kappeler, F.,
Andersson, H.,
and Appenzeller, C.
(2000)
J. Cell Sci.
113,
587-596 |
10. |
Nufer, O.,
Guldbrandsen, S.,
Degen, M.,
Kappeler, F.,
Paccaud, J. P.,
Tani, K.,
and Hauri, H. P.
(2002)
J. Cell Sci.
115,
619-628 |
11. |
Arar, C.,
Carpentier, V.,
Le Caer, J. P.,
Monsigny, M.,
Legrand, A.,
and Roche, A. C.
(1995)
J. Biol. Chem.
270,
3551-3553 |
12. | Itin, C., Roche, A. C., Monsigny, M., and Hauri, H. P. (1996) Mol. Biol. Cell 7, 483-493[Abstract] |
13. |
Lahtinen, U.,
Hellman, U.,
Wernstedt, C.,
Saraste, J.,
and Pettersson, R. F.
(1996)
J. Biol. Chem.
271,
4031-4037 |
14. | Nichols, W. C., Seligsohn, U., Zivelin, A., Terry, V. H., Hertel, C. E., Wheatley, M. A., Moussalli, M. J., Hauri, H. P., Ciavarella, N., Kaufman, R. J., and Ginsburg, D. (1998) Cell 93, 61-70[Medline] [Order article via Infotrieve] |
15. |
Vollenweider, F.,
Kappeler, F.,
Itin, C.,
and Hauri, H. P.
(1998)
J. Cell Biol.
142,
377-389 |
16. | Appenzeller, C., Andersson, H., Kappeler, F., and Hauri, H. P. (1999) Nat. Cell Biol. 1, 330-334[CrossRef][Medline] [Order article via Infotrieve] |
17. | Schweizer, A., Fransen, J. A., Bachi, T., Ginsel, L., and Hauri, H. P. (1988) J. Cell Biol. 107, 1643-1653[Abstract] |
18. | Lippincott-Schwartz, J., Donaldson, J. G., Schweizer, A., Berger, E. G., Hauri, H. P., Yuan, L. C., and Klausner, R. D. (1990) Cell 60, 821-836[Medline] [Order article via Infotrieve] |
19. | Aridor, M., Bannykh, S. I., Rowe, T., and Balch, W. E. (1995) J. Cell Biol. 131, 875-893[Abstract] |
20. |
Klumperman, J.,
Schweizer, A.,
Clausen, H.,
Tang, B. L.,
Hong, W.,
Oorschot, V.,
and Hauri, H. P.
(1998)
J. Cell Sci.
111,
3411-3425 |
21. | Fiedler, K., and Simons, K. (1994) Cell 77, 625-626[Medline] [Order article via Infotrieve] |
22. |
Fullekrug, J.,
Scheiffele, P.,
and Simons, K.
(1999)
J. Cell Sci.
112,
2813-2821 |
23. | Yamashita, K., Hara-Kuge, S., and Ohkura, T. (1999) Biochim. Biophys. Acta 1473, 147-160[Medline] [Order article via Infotrieve] |
24. |
Hara-Kuge, S.,
Ohkura, T.,
Seko, A.,
and Yamashita, K.
(1999)
Glycobiology
9,
833-839 |
25. |
Fiedler, K.,
and Simons, K.
(1996)
J. Cell Sci.
109,
271-276 |
26. |
Hara-Kuge, S.,
Ohkura, T.,
Ideo, H.,
Shimada, O.,
Atsumi, S.,
and Yamashita, K.
(2002)
J. Biol. Chem.
277,
16332-16339 |
27. |
Dodd, R. B.,
and Drickamer, K.
(2001)
Glycobiology
11,
71R-9R |
28. | Yerushalmi, N., Keppler-Hafkemeyer, A., Vasmatzis, G., Liu, X. F., Olsson, P., Bera, T. K., Duray, P., Lee, B., and Pastan, I. (2001) Gene (Amst.) 265, 55-60[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Schweizer, A.,
Ericsson, M.,
Bachi, T.,
Griffiths, G.,
and Hauri, H. P.
(1993)
J. Cell Sci.
104,
671-683 |
30. | Linstedt, A. D., and Hauri, H. P. (1993) Mol. Biol. Cell 4, 679-693[Abstract] |
31. | Linstedt, A. D., Mehta, A., Suhan, J., Reggio, H., and Hauri, H. P. (1997) Mol. Biol. Cell 8, 1073-1087[Abstract] |
32. | Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1997) Current protocols in molecular biology , Wiley, New York |
33. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning: A laboratory manual , 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
34. | Itin, C., Schindler, R., and Hauri, H. P. (1995) J. Cell Biol. 131, 57-67[Abstract] |
35. |
Kappeler, F.,
Klopfenstein, D. R.,
Foguet, M.,
Paccaud, J. P.,
and Hauri, H. P.
(1997)
J. Biol. Chem.
272,
31801-31808 |
36. | Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve] |
37. | Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 61-68[CrossRef][Medline] [Order article via Infotrieve] |
38. | Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59[CrossRef][Medline] [Order article via Infotrieve] |
39. | Fujiki, Y., Hubbard, A. L., Fowler, S., and Lazarow, P. B. (1982) J. Cell Biol. 93, 97-102[Abstract] |
40. | Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395[Abstract] |
41. | Appel, R. D., Bairoch, A., and Hochstrasser, D. F. (1994) Trends Biochem. Sci 19, 258-260[CrossRef][Medline] [Order article via Infotrieve] |
42. | Bailey, T. L., and Gribskov, M. (1998) J. Comput. Biol. 5, 211-221[Medline] [Order article via Infotrieve] |
43. | Gribskov, M., McLachlan, A. D., and Eisenberg, D. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 4355-4358[Abstract] |
44. | Rost, B. (1996) Methods Enzymol. 266, 525-539[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Hofmann, K.,
Bucher, P.,
Falquet, L.,
and Bairoch, A.
(1999)
Nucleic Acids Res.
27,
215-219 |
46. | Lupas, A. (1996) Methods Enzymol. 266, 513-525[Medline] [Order article via Infotrieve] |
47. | Nielsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G. (1997) Int. J. Neural Syst. 8, 581-599[Medline] [Order article via Infotrieve] |
48. | Tusnady, G. E., and Simon, I. (1998) J. Mol. Biol. 283, 489-506[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Bateman, A.,
Birney, E.,
Cerruti, L.,
Durbin, R.,
Etwiller, L.,
Eddy, S. R.,
Griffiths-Jones, S.,
Howe, K. L.,
Marshall, M.,
and Sonnhammer, E. L.
(2002)
Nucleic Acids Res.
30,
276-280 |
50. | Sharma, V., and Surolia, A. (1997) J. Mol. Biol. 267, 433-445[CrossRef][Medline] [Order article via Infotrieve] |
51. |
Velloso, L. M.,
Svensson, K.,
Schneider, G.,
Pettersson, R. F.,
and Lindqvist, Y.
(2002)
J. Biol. Chem.
277,
15979-15984 |
52. | Schroder, S., Schimmoller, F., Singer-Kruger, B., and Riezman, H. (1995) J. Cell Biol. 131, 895-912[Abstract] |
53. |
Sato, K.,
and Nakano, A.
(2002)
Mol. Biol. Cell
13,
2518-2532 |
54. | Takahashi, Y., Mizoi, J., Toh, E. A., and Kikuchi, Y. (2000) J. Biochem. (Tokyo) 128, 723-725[Abstract] |
55. | Kozak, M. (1987) J. Mol. Biol. 196, 947-950[Medline] [Order article via Infotrieve] |
56. | Tang, B. L., Low, S. H., Hauri, H. P., and Hong, W. (1995) Eur J. Cell Biol. 68, 398-410[Medline] [Order article via Infotrieve] |
57. | Teasdale, R. D., and Jackson, M. R. (1996) Annu. Rev. Cell Dev. Biol. 12, 27-54[CrossRef][Medline] [Order article via Infotrieve] |
58. | Itin, C., Kappeler, F., Linstedt, A. D., and Hauri, H. P. (1995) EMBO J. 14, 2250-2256[Abstract] |
59. | Schutze, M. P., Peterson, P. A., and Jackson, M. R. (1994) EMBO J. 13, 1696-1705[Abstract] |
60. | Ma, D., and Jan, L. Y. (2002) Curr. Opin. Neurobiol. 12, 287-292[CrossRef][Medline] [Order article via Infotrieve] |
61. |
Andersson, H.,
Kappeler, F.,
and Hauri, H. P.
(1999)
J. Biol. Chem.
274,
15080-15084 |
62. | Loris, R. (2002) Biochim. Biophys. Acta 1572, 198[Medline] [Order article via Infotrieve] |
63. | Zerangue, N., Schwappach, B., Jan, Y. N., and Jan, L. Y. (1999) Neuron 22, 537-548[Medline] [Order article via Infotrieve] |
64. |
Zerangue, N.,
Malan, M. J.,
Fried, S. R.,
Dazin, P. F.,
Jan, Y. N.,
Jan, L. Y.,
and Schwappach, B.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
2431-2436 |
65. | Golling, G., Amsterdam, A., Sun, Z., Antonelli, M., Maldonado, E., Chen, W., Burgess, S., Haldi, M., Artzt, K., Farrington, S., Lin, S. Y., Nissen, R. M., and Hopkins, N. (2002) Nat. Genet. 31, 135-140[CrossRef][Medline] [Order article via Infotrieve] |
66. |
Nichols, W. C.,
Terry, V. H.,
Wheatley, M. A.,
Yang, A.,
Zivelin, A.,
Ciavarella, N.,
Stefanile, C.,
Matsushita, T.,
Saito, H.,
de Bosch, N. B.,
Ruiz-Saez, A.,
Torres, A.,
Thompson, A. R.,
Feinstein, D. I.,
White, G. C.,
Negrier, C.,
Vinciguerra, C.,
Aktan, M.,
Kaufman, R. J.,
Ginsburg, D.,
and Seligsohn, U.
(1999)
Blood
93,
2261-2266 |
67. |
Neerman-Arbez, M.,
Johnson, K. M.,
Morris, M. A.,
McVey, J. H.,
Peyvandi, F.,
Nichols, W. C.,
Ginsburg, D.,
Rossier, C.,
Antonarakis, S. E.,
and Tuddenham, E. G.
(1999)
Blood
93,
2253-2260 |
68. | Dansako, H., Ishimaru, F., Takai, Y., Tomoda, J., Nakase, K., Fujii, K., Ogama, Y., Kozuka, T., Sezaki, N., Honda, K., and Harada, M. (2001) Ann. Hematol. 80, 292-294[CrossRef][Medline] [Order article via Infotrieve] |
69. | Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-4680[Abstract] |