From the Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
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ABSTRACT |
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Chinese hamster ovary (CHO) mutants belonging to
the Lec2 complementation group are unable to translocate CMP-sialic
acid to the lumen of the Golgi apparatus. Complementation cloning in these cells has recently been used to isolate cDNAs encoding the CMP-sialic acid transporter from mouse and hamster. The present study
was carried out to determine the molecular defects leading to the
inactivation of CMP-sialic acid transport. To this end, CMP-sialic acid
transporter cDNAs derived from five independent clones of the Lec2
complementation group, were analyzed. Deletions in the coding region
were observed for three clones, and single mutants were found to
contain an insertion and a point mutation. Epitope-tagged variants of
the wild-type transporter protein and of the mutants were used to
investigate the effect of the structural changes on the expression and
subcellular targeting of the transporter proteins. Mutants derived from
deletions showed reduced protein expression and in immunofluorescence
showed a diffuse staining throughout the cytoplasm in transiently
transfected cells, while the translation product derived from the
point-mutated cDNA (G189E) was expressed at the level of the
wild-type transporter and co-localized with the Golgi marker
-mannosidase II. This mutation therefore seems to directly affect
the transport activity. Site-directed mutagenesis was used to change
glycine 189 into alanine, glutamine, and isoleucine, respectively.
While the G189A mutant was able to complement CMP-sialic acid
transport-deficient Chinese hamster ovary mutants, the exchange of
glycine 189 into glutamine or isoleucine dramatically affected the
transport activity of the CMP-sialic acid transporter.
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INTRODUCTION |
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Carbohydrates added to cell surface proteins and lipids provide major contact and communication elements for animal cells. The biosynthesis of the carbohydrate structures occurs mainly in the luminal parts of the endoplasmic reticulum (ER)1 and Golgi apparatus and therefore requires specific nucleotide sugar transport systems (1, 2). Nucleotide sugar transporters have been described for CMP-sialic acid, UDP-galactose, UDP-GlcNAc, UDP-GalNAc, GDP-fucose, UDP-xylose, GDP-mannose, UDP-glucuronic acid, and UDP-glucose (2-4). These proteins function as antiporters in an ATP- and ion-independent manner by exchanging the nucleotide sugar with the corresponding nucleoside monophosphate generated in the organellar lumen through the action of glycosyltransferases and nucleoside diphosphatases (2, 5). The high substrate specificity of the nucleotide sugar transporters, which has been demonstrated in biochemical and genetic analysis (2), makes these molecules ideal targets for the selective inhibition of glycoconjugate maturation. Increased sialylation has been described for tumor cell surfaces and has been shown to correlate positively with malignant potential (6-9). Since numerous sialyltransferases (for a review, see Ref. 10) but probably only a single CMP-sialic acid transporter (2) exist, the transporter may provide an effective target to inhibit cell surface sialylation. Accordingly, the inhibition of the UDP-galactose transporter and CMP-sialic acid transporter by somatic mutations and synthetic inhibitors resulted in strong reduction of the metastatic potential in the murine MDAY-D2 tumor cell line and in human colorectal cancer lines in nude mice (11-13).
Considerable progress in studying the transport of nucleotide sugars into the Golgi lumen has been made by the molecular cloning of nucleotide sugar transporter genes. CMP-Sia-Tr and UDP-Gal-Tr cDNAs were cloned from mammalian species (14-17), and the GDP-mannose transporter from Leishmania donovani (18, 19) and the UDP-GlcNAc- and UDP-Gal-transporters were cloned from yeast (20, 21). Related cDNAs from human, Saccharomyces cerevisiae, and Caenorhabditis elegans were also identified by homology searches in the gene data bases (16, 19, 20). Heterologous expression of the murine CMP-Sia-Tr on the zero background of S. cerevisiae was used to confirm the biological function of the protein (22). The transfected yeast cells acquired the ability to translocate CMP-sialic acid.
All nucleotide-sugar transporters cloned to date have been identified by complementation cloning in glycosylation mutants. The murine and hamster CMP-Sia-Tr were isolated by expression cloning in a clone of the Lec2 complementation group. Although the Lec2 mutation is known to inhibit translocation of CMP-sialic acid into the Golgi lumen (23), the molecular basis responsible for the asialo phenotype is still unknown. This study was undertaken to determine the molecular basis of the Lec2 phenotype. Thereby, we took advantage of the fact that several independent Lec2 mutants have been isolated in the laboratory (14). The results summarized in this study demonstrate that Lec2 cells carry defects in the CMP-Sia-Tr gene leading to aberrations in the transporter protein. The analysis of these mutants provides a useful system to gain insight into structure-function relationships of the transporter protein.
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EXPERIMENTAL PROCEDURES |
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Cell Culture--
Chinese hamster ovary (CHO) K1 wild-type cells
and Lec2 cells (24) were obtained from the American Type Culture
Collection (Rockville, MD). The CHO mutants 1E3, 6B2, 8G8, and 9D3 have
been described recently (14). All cells, except clone Lec2, were maintained in Dulbecco's modified Eagle's medium nutrient mixture F12
(Life Technologies, Inc.) supplemented with 5% fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate, penicillin
(100 units/ml), and streptomycin (100 µg/ml) at 37 °C in a 5%
CO2 incubator. Lec2 cells were grown in -minimal
essential medium with Glutamax (Life Technologies), supplemented with
10% fetal calf serum. COS cells were grown in Dulbecco's modified
Eagle's medium with Glutamax (Life Technologies) and 5% fetal calf
serum.
Northern Blot Analysis-- Total RNA was isolated from CHO cells by CsCl gradient centrifugation of guanidinium isothiocyanate lysates (25). RNA (5 µg) was electrophoresed in a 1% agarose, 1 M formaldehyde gel in 20 mM MOPS (pH 7.0), 10 mM sodium acetate, 1 mM EDTA and transferred to a nylon membrane (Qiagen). Nylon filters were hybridized overnight at 65 °C in high SDS buffer (5× SSC, 50% formamide, 50 mM sodium phosphate, 7% SDS, 1% blocking reagent (Boehringer Mannheim)) to a digoxigenin-labeled antisense RNA probe of the murine CMP-Sia-Tr cDNA (14). After hybridization, the filters were washed twice in 2× SSC, 0.1% SDS at room temperature for 5 min and twice in 0.1× SSC, 0.1% SDS at 65 °C for 20 min. Bound probes were detected by incubation with anti-digoxigenin Ig alkaline phosphate conjugate (Boehringer Mannheim) and chemiluminescence detection using disodium-3-(4-methoxyspiro(1,2-dioxetane-3,2'-(5'-chloro)tricyclo-(3.3.1.13,7)decan)-4-yl)phenylphosphate (Boehringer Mannheim) as a substrate.
Reverse Transcription Polymerase Chain Reaction
(RT-PCR)--
Polyadenylated RNA was selected from total RNA by using
oligotex beads (Qiagen) according to the manufacturer's instructions. One µg of mRNA was reverse transcribed using 200 units of
Superscript II RNase H reverse transcriptase (Life
Technologies) and the oligonucleotide 5'-TGTTTAAGCTACCATCTG-3', which
anneals to nucleotides 1202-1185 of the cloned hamster CMP-Sia-Tr
cDNA (17). The cDNA was then amplified by PCR (26) using the
sense primer 5'-CGTCTCTATGGCTGCAGGG-3', annealing to nucleotides
59-77, and the antisense primer 5'-CTGGGGTTGACTTAAGGCTC-3', annealing
to nucleotides 1187-1168. PCR conditions were 1 min at 64 °C, 1 min
at 72 °C, and 1 min at 94 °C for 35 cycles.
Construction of Epitope-tagged CMP-Sia-Tr Mutants-- Amplified CMP-Sia-Tr cDNAs of Lec2 mutants were subjected to 15 cycles of PCR using the oligonucleotides ME41 (5'-GCGGATCCATGGCTCCGGCGAGAG-3') and ME42 (5'-GCGGATCCCACACCAATGATTCTCTCTTTT-3'), which introduce BamHI restriction sites (underlined) upstream and downstream of the protein coding sequence. The PCR products were treated with BamHI, purified, and ligated into the BamHI site of the eukaryotic expression vector pEVRF0-HA (a kind gift of R. Janknecht, The Salk Institute, La Jolla, CA). The resulting plasmids direct the expression of hemagglutinin (HA)-tagged transport proteins under the control of the cytomegalovirus promoter. In these constructs, the stop codon has been replaced by the nucleotide sequence coding for the HA tag (GSYPYDVPDYASLRSGTRGAL; the epitope recognized by the anti-HA mAb 12CA5 is underlined). Plasmid pFLAG-WT, which encodes wild-type mouse CMP-Sia-Tr with an N-terminal FLAG sequence, was created by subcloning the 1.2-kilobase pair EcoRI-XbaI fragment from pME8 (14) into the EcoRI and XbaI sites of pCCF2 (pCDM8 with a sequence encoding the FLAG epitope immediately upstream of the EcoRI site; a kind gift of W. Bautsch, Medizinische Hochschule Hannover, Germany). In the final construct, the sequence MDYKDDDDKEF was placed in front of CMP-Sia-Tr (the sequence recognized by the anti-FLAG mAb M5 is underlined). C-terminal deletion mutants of the epitope-tagged murine CMP-Sia-Tr were generated by restriction digestion; to generate the construct FLAG-(1-165), the HindIII fragment of pFLAG-WT encoding the FLAG sequence and amino-terminal 165 amino acids was subcloned into the HindIII site of the expression vector pcDNA3 (Invitrogen). The construct FLAG-(1-96) was prepared by linearizing pFLAG-WT with SpeI, refilling with Klenow, and digesting with HindIII. The HindIII-SpeI fragment encoding the FLAG sequence and the amino-terminal 96 amino acids was then subcloned into the HindIII and XbaI sites of pcDNA3. The final constructs contain carboxyl-terminal extensions encoded by vector sequences (WYRARIH in FLAG-(1-165) and EGPIL in FLAG-(1-96)). All constructs were confirmed by sequencing.
Site-directed Mutagenesis-- Glycine at amino acid position 189 of the hamster CMP-Sia-Tr was exchanged to alanine, glutamine, and isoleucine, respectively, using PCR and Pfu polymerase (Stratagene). The following mutation primers were used: 5'-ATTGTGCTCTX1X2ATTTGCAGG-3' (corresponds to nucleotides 555-575 of the coding sequence of hamster CMP-Sia-Tr; X1X2 in this primer was GC to generate the mutant G189A, CA to generate the mutant G189Q, and AT to generate the mutant G189I) and primer 5'-TGCAAATX1X2AGAGCACAATAC-3' (corresponds to nucleotides 573-553; X1X2 in this sequence is GC for the mutant G189A, TG for the mutant G189Q, and AT for the mutant G189I). Oligonucleotides ME41 and ME42 (see above) served as end primers. Conditions for the first PCR were as follows: 30 s at 94 °C, 30 s at 46 °C, and 1 min at 72 °C for 25 cycles. PCR products were gel-purified and subjected to fusion PCR using primers ME41 and ME42. Conditions for the fusion PCR were as follows: 30 s at 94 °C, 30 s at 46 °C, and 1 min at 72 °C for 10 cycles. PCR products were digested with BamHI, gel-purified, and ligated into the BamHI site of pEVRF0-HA. All constructs were confirmed by DNA sequencing.
DNA Sequencing--
Nucleotide sequences were determined by the
dideoxy chain termination method (27) using -[35S]dATP
(Amersham Pharmacia Biotech) and T7 DNA polymerase (Amersham Pharmacia
Biotech). Products of RT-PCR reactions were purified on agarose gels
(Qiaquick DNA purification kit, Qiagen), denaturated by boiling, and
sequenced directly.
Immunoblot Analysis-- For Western blot analysis, transiently transfected CHO cells were lysed in 20 mM Tris-HCl (pH 8), 150 mM NaCl, 5 mM EDTA, 200 units/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 2% Nonidet P-40. 100 µg of the postnuclear supernatant was used for SDS-polyacrylamide gel electrophoresis and Western blotting as described (28). Blots were probed with 2.5 µg/ml of anti-HA mAb 12CA5 (Boehringer Mannheim) and 5.4 µg/ml anti-FLAG mAb M5 (Eastman Kodak Co.), respectively. Primary antibodies were then detected using anti-mouse alkaline phosphatase conjugate (Dianova) and using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates.
Transient Transfection of Epitope-tagged Mutants of CMP-Sia-Tr-- 2 × 105 cells were seeded on glass coverslips in 35-mm cell culture dishes 24 h before transfection. For transfection, 1 µg of DNA was mixed with 6 µl of Lipofectamine (Life Technologies) in 1 ml of Opti-MEM medium (Life Technologies). Cells were washed twice with Opti-MEM medium and incubated with the DNA/Lipofectamine mixture for 6-8 h. Transfection was stopped by adding 2 ml of Dulbecco's modified Eagle's medium nutrient mixture F-12 medium containing 5% fetal calf serum, and cells were grown for another 24 h prior to Western blot or immunofluorescence analysis.
Indirect Immunofluorescence--
Cells were washed with PBS and
fixed in 2% paraformaldehyde in PBS for 15 min. To neutralize residual
paraformaldehyde, cells were washed in PBS and incubated twice for 20 min in 50 mM NH4Cl in PBS. Thereafter, the
cells were permeabilized with 0.2% saponin, 0.1% bovine serum albumin
in PBS for 15 min followed by an overnight incubation with the primary
antibodies at 4 °C. Immunodetections were carried out with the
anti-FLAG mAb M5 (5.4 µg/ml), the anti-HA mAb 12CA5 (2.5 µg/ml),
and rabbit anti--mannosidase II antiserum (29, 30) (1:2000; a kind
gift of K. Moremen, University of Georgia, Athens, GA) in 0.2%
saponin, 0.1% bovine serum albumin in PBS for 1 h. After washing
four times in 0.1% bovine serum albumin/PBS, cells were incubated with
anti-mouse Ig-fluorescein isothiocyanate (1:200; Dianova) and
anti-rabbit Ig-tetramethyl rhodamine isothiocyanate (1:200; Dianova)
for 1 h at room temperature. The incubation was stopped by three
washes in 0.1% bovine serum albumin/PBS and a final wash in PBS.
Slides were mounted in Moviol and analyzed under a Zeiss Axiophot
Epifluorescence microscope.
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RESULTS |
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Mutations in CMP-Sia-Tr-negative CHO Cells-- CHO mutants used in this study (Lec2, 1E3, 6B2, 8G8, and 9D3) have been described previously (14, 24), and a cDNA encoding the hamster CMP-Sia-Tr has been cloned (17). In order to identify the molecular defect causing the phenotype of this complementation group, we first investigated whether the mRNA is expressed in the mutants. Northern blot analysis of the mutants (Fig. 1A) revealed CMP-Sia-Tr mRNA expression at a level similar to that of wild-type cells. However, the hybridization signals of the mutants 1E3, 6B2, and Lec2 differ slightly in apparent molecular mass. These size variations were also visible when the entire coding region of the CMP-Sia-Tr was amplified by RT-PCR (see Fig. 1B). The second smaller PCR products in Fig. 1B correspond to an alternative splice variant that lacks exon 2 (i.e. nucleotides 17-194). This second splice product has been described earlier (17). Direct sequencing of the PCR products demonstrated deletions in clones Lec2 and 6B2. Loss of nucleotides 575-751 in Lec2 substitutes Gly192 to Phe251 by Val, the deletion of nucleotides 752-886 in 6B2 cells substitutes Phe251-Thr296 by Ser. Clone 1E3 exhibits a duplication of nucleotides 195-574, resulting in a frame shift after Gly192. In the mutant 8G8, the sequence 190CTAAAGAAA (nucleotides 190-198) is changed to 190CCAAAAA, resulting in a frame shift after Glu66. The mutations found in the CMP-Sia-Tr are listed in Table I. Since the boundaries of the observed deletions and duplications are in part identical, deletions are most likely caused by point mutations in splice acceptor or splice donor sites, causing exon skipping. In the mutant 9D3, a single missense mutation was found to be responsible for the inactivation of the CMP-Sia-Tr. The G to A transition at position 566 leads to the substitution of Gly189 by Glu. According to secondary structure predictions (17), Gly189 forms part of a transmembrane domain. Sequence comparison with other nucleotide-sugar transporters indicates that Gly189 is the starting point of a highly conserved stretch of 12 amino acids (see Fig. 7). In order to exclude PCR errors, all sequences have been determined twice from independent RT-PCR reactions.
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Expression of Epitope-tagged Transporter Proteins--
Epitope
tags were introduced to visualize translation products predicted from
the cloned cDNA sequences. The cDNAs of the wild-type and
mutant CMP-Sia-Tr were subcloned into the vector pEVRF0-HA directing
the expression of proteins with carboxyl-terminal hemagglutinin (HA)
tags. The following constructs were made: (192-251)HA from Lec2
cDNA,
(251-296)HA from 6B2 cDNA,
(7-66)HA from 8G8 cDNA, and G189E-HA from 9D3 cDNA. In addition, two CMP-Sia-Tr variants with an N-terminal FLAG sequence that lack the C-terminal 171 and 240 amino acids, respectively, were generated. All constructs are
summarized in Fig. 2. The constructs were
transiently expressed in CHO and COS cells and the expression was
analyzed by immunoblotting using the anti-HA mAb 12CA5 and the
anti-FLAG mAb M5. The results obtained in COS cells are shown in Fig.
3. The apparent molecular masses of the
fusion proteins are in good correlation with the calculated molecular
masses (see Fig. 2). However, for the deletion mutants the expression
levels are reduced compared with the wild-type and the point-mutated
protein. This reduction in the protein level probably reflects a
decreased stability of these proteins. Detection of the translation
products with the mAb 12CA5 displayed double bands (see Fig.
3A). Since the faster migrating band was not detectable when
N-terminally Flag-tagged transporter variants were analyzed (Fig.
3B and data not shown) the doublets can be explained as N-terminally truncated proteins or by the use of a second downstream ATG.
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Subcellular Localization of Mutant Transporters--
CMP-sialic
acid transport activity is strongly associated with Golgi vesicle
membranes (3), and epitope-tagged variants of mouse and hamster
CMP-Sia-Tr are targeted to the Golgi apparatus (14, 17). To investigate
if the mutations observed affect subcellular targeting of the
CMP-Sia-Tr, the HA- and FLAG-tagged constructs were transiently
expressed in CHO cells. 30 h after transfection, cells were fixed
in paraformaldehyde, permeabilized with saponin, and the localization
of each construct was determined by indirect immunofluorescence using
the anti-HA mAb 12CA5 and the anti-FLAG mAb M5. Simultaneously, the
cells were stained with an antiserum directed against -mannosidase
II, a marker for the Golgi apparatus. As has been demonstrated earlier
(14, 17), the HA-tagged wild-type CMP-Sia-Tr is effectively targeted to the Golgi apparatus (Fig. 4, A
and B). The same strong co-localization with
-mannosidase
II was found for the mutant G189E-HA (Fig. 4, I and
K), indicating Golgi targeting of the mutated protein. Loss
of functional activity in this mutant therefore is not due to
inappropriate subcellular localization. In contrast, the deletion mutants
(192-251)HA (Fig. 4, C and D),
(251-296)HA (Fig. 4, E and F), and
(7-66)HA (Fig. 4, G and H) showed a diffuse
staining throughout the cytoplasm. Co-localization of the transporter
mutants and
-mannosidase II was not obtained. The same diffuse
staining was found for the C-terminally deleted mutants FLAG (1-165)
and FLAG (1-96) (Fig. 5, E
and G). The FLAG-tagged wild-type transporter again
co-localized with
-mannosidase II (Fig. 5, C and
D). These data demonstrate that deletion mutants are
retained in a pre-Golgi compartment, most probably the ER.
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A Missense Mutation Affecting CMP-sialic Acid Transport-- While no sialic acid or polysialic acid surface expression was observed after overexpression of transporter mutants derived from sequence deletions and insertions (data not shown), the overexpression of the construct G189E-HA led to a very weak polysialic acid expression. The polysialic acid (PSA) signal was best detectable by immunocytochemistry but was too faint to be displayed in Western blot (see Fig. 6). Starting from this observation, the importance of the glycine residue in position 189 was further investigated by substituting this position by amino acids with different chemical properties. Via site-directed mutagenesis HA-tagged constructs were generated in which Gly189 is exchanged to alanine (G189A), glutamine (G189Q), or isoleucine (G189I). Transient transfection of G189A into 8G8 cells resulted in full complementation, as can be seen by the reexpression of polysialic acid (Fig. 6). In contrast, the exchange of glycine to glutamine and isoleucine resulted in phenotypes that were very close to that of the G189E mutant. Western blot analysis using the anti-HA mAb 12CA5 confirmed equal protein expression for these mutants.
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DISCUSSION |
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Cells of the Lec2 complementation group are defective in the transport of CMP-NeuAc into the lumen of the Golgi apparatus (23). Clones exhibiting the lec2 defect have been isolated by lectin resistance (24, 31) or immunoselection (14). The lec2 mutation causes the expression of asialo cell surfaces. Among the carbohydrate epitopes missing is PSA. Reexpression of PSA was therefore used to isolate cDNAs encoding the murine and hamster CMP-Sia-Tr cDNAs via complementation cloning (14, 17). The present study was carried out to determine the lec2 mutation at the molecular level. Four independent mutants (1E3, 6B2, 8G8, and 9D3), which arose from chemical mutation experiments (32), together with the clone Lec2 isolated via lectin resistance (24), were analyzed by Northern blotting and RT-PCR. Deletions, insertions, and point mutations in the CMP-Sia-Tr coding region were found that demonstrated that the gene defective in these cells encodes the CMP-Sia-Tr. The high frequency by which these mutants occur after chemical mutagenesis (32, 33) makes this approach an attractive way to identify functionally important primary sequence elements and to investigate structure-function relationships of nucleotide-sugar transporters.
Deletions observed in clones 6B2, 8G8, and Lec2, are likely to result
from mutations in splice acceptor or donor sites. Support for this
assumption comes from the observation that the deleted sequence
sections share common boundaries. The extended sequence changes
associated with the mutations lec2, 6b2, and
8g8, cause mistargeting of the translation products. While
co-localization of the wild-type CMP-Sia-Tr with -mannosidase II
indicated transport to the Golgi apparatus (Ref. 14 and Figs. 4 and 5),
all internally deleted or C-terminally truncated transporter mutants
were not in the
-mannosidase II compartment but produced a diffuse
staining throughout the cytoplasma. The staining pattern observed for
the mutants might be explained by retention of the proteins in the ER.
The extended primary sequence changes caused by the deletions or
truncations most probably lead to misfolded proteins, which do not
escape the process of "ER quality control" (34). Consistent with
this, reduced expression levels were observed for the mutant proteins.
Analysis of the CMP-Sia-Tr mRNA from clone 9D3 revealed a single
missense mutation, resulting in exchange of glycine at position 189 for
glutamic acid. Like the other mutants, the steady-state level of
mRNA expression in this mutant was comparable with that of
wild-type cells (Fig. 1), and Western blot analysis of cells transiently transfected with the epitope-tagged G189E cDNA
indicated that this protein was expressed at the same level as the
epitope-tagged wild-type protein. In contrast to the mutants described
above, this protein co-localizes with -mannosidase II, indicating
correct targeting to the Golgi apparatus. Therefore, the G189E mutation seems to directly affect the transport activity of the CMP-Sia-Tr. Changing glycine 189 to alanine did not influence the activity of
CMP-Sia-Tr, measured as polysialic acid expression in clones of the
Lec2 complementation group. In contrast, the activities of G189Q and
G189I mutants were drastically decreased and resembled that of the
G189E mutant. These results suggest that not the charge repulsion
between glutamine and CMP-sialic acid, but rather the size of the amino
acid at position 189, is a critical factor for the transport activity.
Large amino acids at this position may lead to steric hindrance of a
hydrophilic "channel" required to translocate CMP-sialic acid
through the membrane. This hypothesis is in good agreement with the
transporter model proposed recently (17), where Gly189 is
part of a transmembrane helix, closely located to the cytosolic face of
the membrane. Another explanation would be that the mutation destroys a
potential site involved in the binding of cofactors or in protein
dimerization. So far, however, there are no experimental data
supporting the idea.
The G189E mutation identified a functionally important region of CMP-Sia-Tr. In the mutant 9D3, sialic acid is undetectable by either Western blotting or immunocytochemistry. Overexpression of the mutant G189E in 9D3 cells, however, restored transport activity at a very low level. Due to the high sensitivity of the anti-PSA mAb 735, a very faint PSA signal was visible in immunocytochemistry and Western blot after transient transfection of cells. The signal intensity is however too low to be reproduced in Fig. 6, and sialic acid reexpression was not detectable with Maackia amurensis lectin. Thus, the mutant transporter from 9D3 cells is not completely inactive, but the endogenous expression of the mutated protein in 9D3 cells is insufficient to translocate CMP-sialic acid at a rate necessary for detectable amounts of (poly)sialic acid at the cell surface. Transient overexpression of the deletion mutants in CHO cells of the Lec2 cells did not lead to a detectable complementation. Together with the above results, this strongly suggests that these mutants are completely inactive.
The change Gly189 to Glu occurs in a region that is conserved among CMP-Sia-Tr and UDP-Gal-Tr isolated from mammals and the yeast Schizosacchaccharomyces pombe. Furthermore, this sequence is found in a putative nucleotide-sugar transporter of C. elegans. All transporter sequences containing this motif are listed in Fig. 7. The high conservation strongly suggests that this amino acid stretch is essential for a functionally active transporter. On the other hand, the appearance of this domain in transporters of different specificity argues against a direct involvement in nucleotide-sugar binding or recognition. Additional studies are required to define the functional role of this sequence motif.
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An important aspect of this study consists in the fact that a CMP-Sia-Tr mutant exhibiting residual transport activity could be isolated after chemical mutation of CHO cells. Isolation and functional analysis of such mutants provides a powerful way to gain further insight into structure-function relationships for this structurally ambitious group of molecules.
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ACKNOWLEDGEMENTS |
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We thank Michael Cahill for critical remarks on the manuscript, K. Moremen for kindly providing the mannosidase II antiserum, and D. Bitter-Suermann for continuous support.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft and Boehringer Mannheim.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.
A postdoctoral fellow of the Deutsche Forschungsgemeinschaft.
§ To whom correspondence should be addressed: Institut für Medizinische Mikrobiologie, MHH, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Tel.: 49-511-532-4359; Fax: 49-511-532-4366; E-mail: rgs{at}mikrobio.h.shuttle.de.
The abbreviations used are: ER, endoplasmic reticulum; CHO, Chinese hamster ovary; CMP-Sia-Tr, CMP-sialic acid transporter; HA, hemagglutinin; PCR, polymerase chain reaction; RT-PCR, reverse transcription PCR; UDP-Gal-Tr, UDP-galactose transporter; MOPS, 4-morpholinepropanesulfonic acid; mAb, monoclonal antibody; PBS, phosphate-buffered saline; PSA, polysialic acid.
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REFERENCES |
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