From the Institut für Klinische Chemie und
Biochemie, Virchow-Klinikum, Humboldt-Universität zu Berlin,
D-13353 Berlin, Germany and ¶ Institut für Molekularbiologie
und Biochemie, Freie Universität Berlin,
D-14195 Berlin, Germany
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ABSTRACT |
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Primary rat hepatocytes and two hepatoma cell lines have been used to study whether high mannose-type N-glycans of plasma membrane glycoproteins may be modified by the removal of mannose residues even after transport to the cell surface. To examine glycan remodeling of cell surface glycoproteins, high mannose-type glycoforms were generated by adding the reversible mannosidase I inhibitor deoxymannojirimycin during metabolic labeling with [3H]mannose, thereby preventing further processing of high mannose-type N-glycans to complex structures. Upon transport to the cell surface, glycoproteins were additionally labeled with sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate. This strategy allowed us to follow selectively the fate of cell surface glycoproteins. Postbiosynthetic demannosylation was monitored by determining the conversion of Man8-9GlcNAc2 to smaller structures during reculture of cells in the absence of deoxymannojirimycin. The results show that high mannose-type N-glycans of selected cell surface glycoproteins are trimmed from Man8-9GlcNAc2 to Man5GlcNAc2 with Man7GlcNAc2 and Man6GlcNAc2 formed as intermediates. It could be clearly shown in MH 7777 as well as in HepG2 cells that demannosylation affects plasma membrane glycoproteins after they are routed to the cell surface. As was determined for total cell surface glycoproteins in HepG2 cells, this process occurs with a half-time of 6.7 h. By analyzing the size of high mannose-type glycans of glycoproteins isolated from the cell surface at the end of the reculture period, i.e. after trimming had occurred, we were able to demonstrate that glycoproteins carrying trimmed high mannose glycans become exposed at the cell surface. From these data we conclude that cell surface glycoproteins can be trimmed by mannosidases at sites peripheral to N-acetylglucosaminyltransferase I without further processing of their glycans to the complex form. This glycan remodeling may occur at the cell surface or during endocytosis and recycling back to the cell surface.
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INTRODUCTION |
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During their maturation, N-linked glycans of secretory
and membrane glycoproteins undergo extensive processing by specific glycosidases and glycosyltransferases in the
ER,1 the Golgi complex, and
the TGN (for reviews, see Refs. 1-4). The sequence of processing
events includes trimming of the precursor oligosaccharide,
Glc3Man9GlcNAc2, by glucosidases I
and II and by distinct 1,2-mannosidases to form
Man5GlcNAc2. Several of the processing
mannosidases have been described (for reviews, see Refs. 4 and 5) such
as ER mannosidases and Golgi mannosidase IA/IB. Following the action of
N-acetylglucosaminyltransferase I and Golgi mannosidase II,
the transfer of N-acetyl-D-glucosamine, D-galactose, L-fucose, and sialic acids by an
array of glycosyltransferases generates the wide variety of
oligosaccharide structures found on mature glycoproteins.
Several lines of evidence suggest that oligosaccharide processing of
cell surface glycoproteins is not restricted to biosynthesis but may
also occur after the initial passage through the compartments of the
secretory pathway to the cell surface (for a review, see Ref. 6).
First, measurements of the turnover rates of the different sugar
residues of glycoproteins isolated from rat liver plasma membranes have
shown that these turnover kinetics are distinctly influenced by the
position of each sugar within the N-linked oligosaccharides (7-10). The half-lives of the terminal or penultimate sugars, L-fucose, sialic acid, and D-galactose, are
only to
as long as that of the protein backbone.
From these studies it has been proposed that terminal sugar residues
may be removed from the nonreducing end of the N-glycans of
plasma membrane glycoproteins. In distinct plasma membrane
glycoproteins, even mannose residues were lost from the glycoproteins
(11). Second, studies designed to examine the return of surface
receptors to compartments of the secretory pathway have demonstrated
that selected cell surface glycoproteins may also acquire terminal
sugars, L-fucose, and sialic acid when recycling to
fucosyl- and sialyltransferases in the medial/trans-Golgi
and in the TGN (12-19). In Chinese hamster ovary cells, the
cation-independent mannose 6-phosphate/insulin-like growth factor-II
receptor has been reported to recycle even to galactosyltransferases in
the trans-Golgi region (19). It has been proposed that
reglycosylation might serve as a repair mechanism for surface
glycoproteins trimmed by glycosidases encountered on the cell surface
or during endocytosis and recycling and that cell surface glycoproteins
may pass several rounds of de- and reglycosylation (15, 18, 20).
However, as compared with recycling to glycosyltransferases, far less
is known about postbiosynthetic trimming of cell surface glycoproteins
by glycosidases. In an important study, Snider and Rogers (21)
demonstrated that TfR and glycoproteins from the total cellular protein
pool may return to mannosidase I in the early Golgi region in K 562 cells. This was shown in that cells were metabolically labeled with
[3H]mannose in the presence of the reversible mannosidase
I inhibitor 1-deoxymannojirimycin (dMM). Glycoproteins synthesized
under these conditions retained immature oligomannosidic
N-glycans during their initial transport through the Golgi
complex. A return to early Golgi mannosidase I and a subsequent passage
through peripheral Golgi elements was noticed by trimming of the
immature oligomannosidic N-glycans and conversion to
complex-type structures during reculture of cells in the absence of
dMM. Employing this experimental strategy, a return to early Golgi
mannosidase I was also shown for the cation-dependent and
the cation-independent mannose 6-phosphate receptor in BW 5147 mouse
lymphoma cells (12). It was calculated that glycoproteins recycle to
mannosidase I at very low rates with half-times of 12 h for the
total glycoprotein pool in K562 cells (19) and ~20 h for both mannose
6-phosphate receptors in BW 5147 cells (12). In none of these studies,
however, was a return to Golgi mannosidase I examined by a sample of
glycoproteins that had been covalently labeled on the cell surface
beforehand. Hence, it could not be distinguished whether glycoproteins
trimmed by early Golgi mannosidase I recycled from the cell surface or
from other post-Golgi locations such as the TGN, secretory vesicles,
endosomes, or lysosomes or even represented, in the case of the total
glycoprotein pool, at least partly glycoproteins resident in the Golgi
complex. Moreover, it remained unknown whether glycoproteins return to
the cell surface after reentering the early Golgi. A recent study
designed to examine the transport of TfR and DPPIV from the cell
surface to compartments of the secretory pathway in HepG2 cells showed
that oligomannosidic N-glycans of these two glycoproteins
were not converted to complex structures during recycling (18). In
accordance with this finding, Neefjes et al. (22) failed to
detect conversion of oligomannosidic to complex-type glycans on
recycling glycoproteins including TfR and HLA class II antigens in
different cell lines, indicating that these proteins do not encounter
mannosidase I in the early Golgi (i.e. at sites proximal to
N-acetylglucosaminyltransferase I). This enzyme is localized
in medial Golgi elements (23) and initiates the further processing of
the oligomannosidic trimming intermediate
Man5GlcNAc2 to complex-type oligosaccharides.
In a recent immunohistochemical study, however, mannosidase I was found
to be less compartmentalized than previously assumed and was also
detected in the medial and the trans-Golgi and, in some cell
types, even in the TGN, in secretory vesicles, and in the plasma
membrane (24). Hence, it became conceivable that cell surface
glycoproteins could return to mannosidase I also at sites peripheral to
N-acetylglucosaminyltransferase I, resulting in trimming of
oligomannosidic glycans without further processing to complex
N-glycans. In that case, membrane glycoproteins retaining
trimmed oligomannosidic glycans might return to the plasma membrane and
become exposed on the cell surface.
In the present paper, this assumption was examined in two hepatoma cell lines and primary rat hepatocytes by a sample of cell surface glycoproteins of well defined function, i.e. the TfR, the serine peptidase DPPIV, the cell adhesion molecule gp110/cell-CAM105 (25) (a member of the Ig superfamily), and LI-cadherin (26) (a member of the cadherin family of cell adhesion molecules).
Using a strategy based on the experimental design initially described by Snider and Rogers (21), in conjunction with covalent labeling of cell surface glycoproteins with NHS-SS-biotin, it was determined whether plasma membrane glycoproteins might be trimmed by mannosidases after transport to the cell surface. Moreover, to examine whether subsequently glycoproteins carrying trimmed oligomannosidic N-glycans are exposed on the cell surface, glycoproteins were allowed to encounter mannosidases and were, thereafter, isolated selectively from the cell surface. In this report, we present evidence that N-glycans of selected cell surface glycoproteins are postbiosynthetically trimmed from Man8-9GlcNAc2 to Man5-7GlcNAc2. Following demannosylation, glycoproteins carrying trimmed oligomannosidic structures become exposed on the cell surface, indicating that demannosylation occurs at sites peripheral to N-acetylglucosaminyltransferase I, either at the cell surface or during endocytosis and recycling back to the cell surface.
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EXPERIMENTAL PROCEDURES |
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Materials-- Materials were obtained from the following sources. Culture media were from Biochrom (Berlin, Germany), and other materials for tissue culture were from Falcon (Heidelberg, Germany) or Nunc (Wiesbaden, Germany). Tran35S-label containing L-[35S]methionine and L-[35S]cysteine (specific radioactivity 40.29 TBq/mmol) was from ICN (Meckenheim, Germany). D-[2,6-3H]Mannose (specific radioactivity, 2 TBq/mmol) was from Amersham Buchler (Braunschweig, Germany). Protein A-Sepharose and Sepharose-4B were from Pharmacia (Freiburg, Germany). dMM was a gift of Dr. Schüller (Bayer AG, Wuppertal, Germany). Decanoyl-N-methylglucamide was from Calbiochem (Frankfurt, Germany). All glycosidases were purchased from Boehringer Mannheim (Mannheim, Germany). Sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (NHS-SS-biotin) and streptavidin-agarose were from Pierce (Oud Beijerland, The Netherlands). Dowex AG50W-X12 (hydrogen form) and Dowex AG3-X4 (free base form) were from Bio-Rad (München, Germany). Unless otherwise stated, all other chemicals and reagents were either from Sigma (Deisenhofen, Germany) or from Serva (Heidelberg, Germany). Centricon-10 microconcentrator tubes were from Amicon (Witten, Germany). The oligosaccharide alditols Man5-9GlcNAcOH, metabolically labeled with D-[2-3H]mannose, were a gift of Dr. R. Geyer (Justus-Liebig-Universität Gießen, Germany). The authentic oligosaccharides Man5-9GlcNAc2 were from Oxford Glycosystems (Krefeld, Germany). The monoclonal antibodies De 13.4, directed against rat DPPIV, and Lo 47.2, directed against rat LI-cadherin, have been described previously (27, 28). The monoclonal antibody 188 A2, recognizing rat TfR, was a gift of Dr. D. C. Hixson (Brown University). The rabbit polyclonal antibody directed against rat gp110/cell-CAM105 was that described previously (25). Ascites fluid containing monoclonal antibody HBB3/775 (29) directed against human DPPIV was kindly provided by Dr. H. P. Hauri (Biocenter of the University of Basel, Switzerland).
Cell Culture-- The rat hepatoma cell line MH 7777 derived from Morris hepatoma 7777 was described previously (30). Primary rat hepatocytes were isolated according to the procedure of Seglen (31) at a minimum viability of 80-90% as determined by trypan blue exclusion. Cells were seeded on collagen I-coated plastic dishes. Hepatocytes and hepatoma cells were maintained in DMEM supplemented with penicillin (50 units/ml), streptomycin (50 µg/ml), insulin (0.08 milliunits/ml), dexamethasone (1 µM), and 10% (v/v) complement-inactivated horse serum in a humidified atmosphere with 5% CO2 at 37 °C as described (32). HepG2 cells (33) obtained from ATCC (Rockville, MD) were cultured as described (18).
Metabolic Labeling of Cells-- Confluent layers of cells were trypsinized and seeded on collagen I-coated dishes (50-mm diameter). Cells were allowed to adhere overnight. For labeling in the polypeptide moiety, the monolayers were washed and preincubated for 60 min in DMEM without L-methionine/L-cysteine. The cells were pulse-labeled for 4 h with L-[35S]methionine/L-[35S]cysteine (5.5 Mbq/3 × 106 cells) and then chased for 3 h in DMEM with 1 mM unlabeled L-methionine/L-cysteine. When used, 3 mM dMM was present during the preincubation, pulse, and chase periods. For labeling of glycoproteins in the oligosaccharide moiety, cells were washed, preincubated for 60 min in the presence of 3 mM dMM, and then labeled for 8 h with D-[2,6-3H]mannose (14.8 Mbq/6 × 106 cells) in glucose-free DMEM supplemented with 3 mM dMM, 5 mM D-galactose, and 10 mM pyruvate. Cells were then washed and chased for 3 h in the presence of 3 mM dMM.
Labeling of Cell Surface Proteins with NHS-SS-biotin-- Cell surface proteins were labeled with NHS-SS-biotin essentially as described (18). After cooling on ice, cells were washed four times with ice-cold PBS/Ca2+/Mg2+ (PBS containing 0.9 mM CaCl2 and 0.5 mM MgCl2) and incubated with a freshly prepared solution of NHS-SS-biotin (1 mg/ml) in PBS/Ca2+/Mg2+ for 20 min at 4 °C. Cells were then washed twice with PBS/Ca2+/Mg2+ containing 0.1% (w/v) bovine serum albumin and twice with PBS/Ca2+/Mg2+ and were then either recultured or harvested for further analysis.
Immunoaffinity Absorption and SDS-Polyacrylamide Gel Electrophoresis-- The following steps were carried out at 4 °C. After washing with PBS/Ca2+/Mg2+, cells were solubilized for 2 h in lysis buffer A (150 mM NaCl, 10 mM Tris/HCl, pH 8.0, 1 mM CaCl2, 1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 1% (v/v) Nonidet P-40). Detergent-insoluble material was removed by centrifugation (100,000 × g, 30 min). The supernatants were precleared by incubation with 100 mg of Sepharose-4B for 2 h. The Sepharose was pelleted by centrifugation and discarded. For immunoaffinity absorption, 20 µg of mouse monoclonal antibody (De 13.4, Lo 47.2, or 188 A2) coupled to 8 mg of protein A-Sepharose was added to the supernatant and rotated end-over-end for 4 h. Immunocomplexes bound to protein A-Sepharose were pelleted by centrifugation and washed twice with washing buffer B (500 mM NaCl, 10 mM Tris/HCl, pH 8.0, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40), twice with washing buffer C (150 mM NaCl, 50 mM Tris/HCl, pH 7.8, 0.1% (w/v) SDS, 0.1% (w/v) sodium deoxycholate, 0.5% Nonidet P-40), and finally with PBS. Immunoaffinity absorption of gp110/cell-CAM105 with a polyclonal antibody was performed as described previously (25). Immunocomplexes bound to protein A-Sepharose were eluted by heating (95 °C) for 3 min with 50 µl of SDS electrophoresis sample buffer (4% SDS, 28.6% (v/v) glycerol, 5% (v/v) mercaptoethanol, 50 mM Tris/HCl, pH 6.8, 0.01% bromphenol blue). If not otherwise stated, electrophoresis was performed in 7.5% polyacrylamide gels in the presence of 0.1% SDS as described by Laemmli (34). Gels with 35S-labeled samples were processed and fluorographed as described (35) using Kodak XAR-5 x-ray film.
Isolation of Biotinylated Proteins--
Cells were extracted as
detailed above with lysis buffer A, which additionally contained 10 mM L-lysine (lysis buffer B). A cellular
glycoprotein fraction was prepared from detergent extracts by binding
to ConA-Sepharose. For this purpose, ConA-Sepharose (0.5 g of
ConA-Sepharose/5 mg of protein) was added to the detergent extract and
slowly shaken at 4 °C for 12 h. ConA-Sepharose was then
pelleted by centrifugation and washed four times in lysis buffer B. Bound glycoproteins were eluted with 200 mM
-methylmannopyranoside in lysis buffer B. Biotinylated cell surface
glycoproteins were then isolated from this cellular glycoprotein
fraction by binding to streptavidin-agarose. Streptavidin-agarose (100 µg/mg of protein) was added, and the mixture was shaken at 4 °C
for 4 h. After washing five times in lysis buffer A, biotinylated
proteins were eluted by boiling for 3 min in 100 µl of 0.4% SDS, 5%
mercaptoethanol. Biotinylated DPPIV was isolated as described
previously (18). Briefly, the protein was immunoadsorbed from detergent
extracts and eluted from protein A-Sepharose with 3 M KSCN,
0.5% Nonidet P-40. Eluates were diluted 1:10 in dilution buffer (10 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40), and streptavidin-agarose was
added. The suspension was shaken at 4 °C for 4 h; washed four
times in 50 mM Tris/HCl, pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.3 M KSCN; washed
twice in the same buffer without KSCN; and washed once with PBS.
Biotinylated DPPIV bound to streptavidin-agarose was eluted by boiling
for 3 min in 100 µl of 0.4% SDS, 5% mercaptoethanol.
Treatment of Glycoproteins with Endo H and PNGase F-- Immunoabsorbed glycoproteins were eluted from protein A-Sepharose by boiling for 3 min in 0.4% SDS, 5% mercaptoethanol, and 10 mM EDTA. Biotinylated proteins bound to streptavidin-agarose were eluted with the same buffer (100 µl per 100 µl of streptavidin-agarose). Before the addition of the glycosidases, all samples were diluted 4-fold in the buffer recommended by the manufacturer. The diluted samples were cleared by centrifugation followed by the addition of a mixture of proteinase inhibitors (leupeptin, chymostatin, antipain, pepstatin) each at a final concentration of 20 µg/ml. Treatment with 10 milliunits of Endo H from Streptomyces plicatus (EC 3.2.1.96) was performed in 50 mM sodium phosphate, pH 6.0, 0.1% Nonidet P-40 for 20 h at 37 °C. Treatment with PNGase F (EC 3.2.2.18) from Flavobacterium meningosepticum was performed in 50 mM sodium phosphate, pH 8.0, containing 1% (v/v) decanoyl-N-methylglucamide for 20 h at 37 °C.
Preparation of High Mannose-type Oligosaccharide Alditols-- High mannose-type oligosaccharides were prepared from immunoadsorbed glycoproteins or from biotinylated proteins eluted from streptavidin-agarose as described previously (36, 37). Briefly, oligosaccharides were released from glycoproteins by incubation with Endo H and then separated from proteins by ultrafiltration through Centricon-10 microconcentrators. The filtrates were desalted by mixed bed ion exchange chromatography on a column (0.6 × 10 cm) containing 500 µl of Dowex AG50W-X12 and 500 µl of Dowex AG3-X4. After washing the column with four bed volumes of water, the combined filtrates were dried by evaporation. The oligosaccharides were converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride in 0.2 M sodium borate, pH 9.2, for 6 h at 30 °C. The reaction was stopped by the dropwise addition of 1 M acetic acid. The solution was adjusted to pH 5.0 and passed through a cation exchange column containing 4 ml of AG 50W-X12. The column was washed with five bed volumes of water, and the combined filtrates were evaporated to dryness at 30 °C. Boric acid was completely removed by 5-fold evaporation with 1 ml of methanol, and traces of acetic acid were removed by drying the oligosaccharides over sodium hydroxide in a desiccator.
HPLC Separation of Oligosaccharide Alditols-- HPLC separation of the oligosaccharide alditols was performed as described previously (37) using a Bio-Rad model 700 chromatography workstation equipped with two Bischoff (Leonberg, Germany) model 2200 pumps, a Knauer (Berlin, Germany) dynamic mixing chamber, and a Shimadzu fluorescence HPLC monitor RF-535. Briefly, oligosaccharide alditols were separated on two Spherisorb-NH2 columns (4.6 × 250 mm, 5 µm; Bischoff) equilibrated with a mixture containing 65% acetonitrile and 35% 15 mM sodium dihydrogenphosphate, pH 5.2, at a flow rate of 1.5 ml/min and were eluted by decreasing the proportion of acetonitrile to 45% within 100 min. Fractions of 1 ml were collected and assayed for radioactivity by liquid scintillation counting using a Tri-Carb 1900 CA liquid scintillation analyzer (Canberra Packard). A mixture of glucose oligomers (n = 1-20), fluorescence-labeled by reductive amination with 8-amino-2-naphthol, was used as an internal standard. Columns were calibrated with authentic oligosaccharide alditols Man5-9GlcNAcOH, prepared from HA2 subunits of influenza virus hemagglutinin by Endo H treatment and NaBH4 reduction after metabolic labeling with D-[2-3H]mannose (38).
High Performance Anion Exchange (HPAE) Separation of Oligosaccharides-- In some experiments, oligosaccharides were released from glycoproteins with PNGase F according to Anumula and Taylor (39) with some modifications and separated by HPAE chromatography. Briefly, biotinylated proteins were eluted from streptavidin-agarose with 0.4% SDS, 5% mercaptoethanol by boiling for 3 min. Eluted proteins were concentrated in a Centricon-10 microconcentrator and suspended in 0.5% NH4HCO3, pH 8.0. Trypsin (2% by mass) was added to the samples from a fresh stock solution of 20 mg/ml in 0.5% NH4HCO3 and incubated at 37 °C for 16 h. Trypsin was inactivated by heating at 100 °C for 5 min. After cooling, the pH was readjusted to 8.5, and PNGase F was added and incubated at 37 °C for 16 h in a shaker. Oligosaccharides were purified by passing the samples through a column containing AG3-X4 in the bottom layer and AG50W-X12 in the top layer. After washing with two bed volumes of water, the filtrates were dried by evaporation. Oligosaccharides were separated using a Dionex (Sunnyvale, CA) DX-300 system and a CarboPac PA-100 (4 × 250 mm) in series with a CarboPac PA-100 guard column as described (18). Columns were calibrated with authentic oligosaccharides Man5-9GlcNAc2.
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RESULTS |
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To examine whether cell surface glycoproteins lose mannose residues from their oligomannosidic N-glycans during their life span, the following protocol was employed. Cells were metabolically labeled with either L-[35S]methionine/L-[35S]cysteine or D-[2,6-3H]mannose in the presence of the Golgi-mannosidase I inhibitor dMM. As a consequence, the N-glycans of newly synthesized glycoproteins normally processed to the complex type retain high mannose-type structures. After a chase period sufficient to allow the passage of the newly synthesized glycoproteins through the Golgi region to the cell surface, the inhibition of mannosidase I was reversed by washout of the inhibitor. Cells were then recultured for different times, and the plasma membrane glycoproteins DPPIV, LI-cadherin, TfR, and gp110/cell-CAM105 were isolated and analyzed for trimming of their oligomannosidic glycans.
Characterization of the N-Glycosylation of LI-cadherin, DPPIV, gp110/cell-CAM105, and TfR-- To characterize the N-glycans of LI-cadherin, DPPIV, gp110/cell-CAM105, and TfR generated either in the absence or in the presence of dMM, MH 7777 cells were pulse-labeled with L-[35S]methionine/L-[35S]cysteine and chased for 3 h. When used, dMM was present during the pulse and the chase period. DPPIV, LI-cadherin, TfR, and gp110/cell-CAM105 were then isolated by immunoadsorption and were analyzed by SDS-PAGE and radiofluorography. LI-cadherin and DPPIV immunoadsorbed from dMM-treated cells had a molecular mass of approximately 110 and 100 kDa, respectively, as assessed from migration on 7.5% SDS-polyacrylamide gels (Fig. 1, lanes 4 and 9). GP110/cell-CAM105 and TfR from these cells migrated as doublets with a molecular mass of 84/78 kDa (lane 14) and 92/88 kDa (lane 19), respectively. As has been shown previously, the doublet observed for gp110/cell-CAM105 represents two variants generated by alternative splicing that differ in the size of their C-terminal cytoplasmic domains (40). The doublet observed for the TfR has been reported for a wide variety of cell types and most likely represents the phosphorylated and the nonphosphorylated form of the receptor (41). Glycoproteins synthesized in the absence of dMM had a molecular mass of approximately 120 kDa (LI-cadherin, lane 1), 110 kDa (DPPIV, lane 6), 110 kDa (gp 110/cell-CAM105, lane 11), and 94/90 kDa (TfR, lane 16), in accordance with previous reports (25, 26, 32). For characterization of their N-linked glycans, the immunoadsorbed glycoproteins were digested with Endo H or PNGase F. Digestion of the glycoproteins synthesized in the absence of dMM with Endo H either did not reduce the molecular mass or reduced it only slightly (LI-cadherin (lane 2), DPPIV (lane 7), gp110/cell-CAM105 (lane 12), TfR (lane 17)), indicating that the majority of the N-linked glycans of these glycoproteins is of the complex type. By contrast, digestion with Endo H reduced the molecular mass of the four glycoproteins obtained from dMM-treated cells from approximately 110 to 100 kDa (LI-cadherin, lane 5), from 100 to 88 kDa (DPPIV, lane 10), from 84/78 to 58/48 kDa (gp110/cell-CAM105, lane 15), and from 92/88 to 84/80 kDa (TfR, lane 20), respectively. Polypeptides of the same size were obtained when the glycoproteins generated in the absence of dMM were digested with PNGase F (lanes 3, 8, 13, and 18). These results show that the N-linked glycans of the four glycoproteins when synthesized in the presence of dMM are at least predominantly of the oligomannosidic type.
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The Molecular Mass of the High Mannose-type Glycoforms of DPPIV and gp110/cell-CAM105 Decreases during Reculture-- Loss of mannose residues from the nonreducing end of oligomannosidic N-glycans during the life span of a glycoprotein should result in a decrease in the molecular mass. This was studied by monitoring the molecular mass of DPPIV, LI-cadherin, TfR, and gp110/cell-CAM105 during a reculture period of up to 100 h (DPPIV, LI-cadherin, and gp110/cell-CAM105) or 72 h (TfR). MH 7777 cells were radiolabeled for 4 h with L-[35S]methionine/L-[35S]cysteine in the presence of dMM and were further chased for 3 h in the presence of the inhibitor to generate the high mannose glycoforms of the four glycoproteins as shown in Fig. 1. After washout of the inhibitor, cells were recultured in the absence of dMM. DPPIV, gp110/cell-CAM105, LI-cadherin, and TfR were immunoadsorbed at different times and were analyzed by SDS-PAGE and radiofluorography (Fig. 2A). A distinct decrease in the molecular mass was noted for DPPIV and gp110/cell-CAM105. DPPIV obtained immediately after the chase (0 h) had a molecular mass of approximately 100 kDa, which shifted to 95 kDa during reculture. The decrease in the molecular mass of gp110/cell-CAM105 was more prominent, probably due to its high carbohydrate content of approximately 50% (25). No decrease in the molecular mass was detectable for the high mannose-type glycoforms of LI-cadherin and TfR. Immunoprecipitates of DPPIV obtained during reculture contained an additional faint polypeptide band with a molecular mass of approximately 110 kDa that corresponded to the molecular mass of mature complex-type DPPIV and might represent some DPPIV processed to the complex-type glycoform. For comparison, the behavior of the complex-type glycoforms of the four glycoproteins, labeled and chased in the absence of dMM, was analyzed. In contrast to the high mannose-type glycoforms of DPPIV and gp110/cell-CAM105, none of the four glycoproteins with complex-type N-glycans exhibited a decrease in the molecular mass during reculture (shown for DPPIV and gp110/cell-CAM105 in Fig. 3, lanes 9 and 10). To exclude the possibility that the observed reduction in the molecular mass of the high mannose-type glycoforms of DPPIV and gp110/cell-CAM105 is a particular feature of transformed MH 7777 cells, the same experiments were performed with primary cultured rat hepatocytes. In accordance with the results obtained in MH 7777 cells, the molecular mass of the high mannose glycoforms of DPPIV and gp110/cell-CAM105 decreased after 70 h of reculture (Fig. 3, lanes 5 and 6), and no decrease in the molecular mass was observed for the complex-type glycoforms of the two glycoproteins (Fig. 3, lanes 13 and 14). Whereas in MH 7777 cells the two isoforms of the oligomannosidic gp110/cell-CAM105 were labeled to a similar extent, in hepatocytes the 78-kDa isoform prevailed (Fig. 3, gp110, lanes 1 and 5). The 84-kDa isoform was only faintly detectable and could not be assessed. In accord with the results shown in Fig. 1 (lanes 11 and 12) the complex-type glycoforms of the two isoforms of gp110/cell-CAM105 generated in the absence of dMM migrated as a very broad band and could not be discriminated (Fig. 3, gp 110, lanes 9-16). Taken together, these results indicate that the high mannose glycoforms of DPPIV and of gp110/cell-CAM105 undergo a trimming process most likely by loss of mannose residues from the nonreducing end of the oligomannosidic N-glycans.
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The Decrease in the Molecular Mass of the High Mannose Glycoforms of DPPIV and gp110/cell-CAM105 Is Due to Glycan Trimming and Not to Limited Proteolysis-- To rule out that the decrease in the molecular mass of the high mannose-type glycoforms of DPPIV and gp110/cell-CAM105 during reculture is caused by limited preoteolysis of the polypeptide backbone, the sizes of the deglycosylated polypeptides were compared immediately after the chase and after 70 h of reculture. MH 7777 cells and hepatocytes were radiolabeled with L-[35S]methionine/L-[35S]cysteine and chased, both in the presence of dMM, and were recultured after washout of the inhibitor. DPPIV and gp110/cell-CAM105 immunoadsorbed immediately after the chase (Fig. 3, lanes 1 and 5), and after 70 h of reculture (Fig. 3, lanes 2 and 6) they were digested with Endo H. Digestion with the endoglycosidase converted both forms of the glycoproteins (DPPIV: 0 h, 100 kDa; 70 h, 95 kDa; gp110/cell-CAM105: 0 h, 84/78 kDa; 70 h, 78/72 kDa) to polypeptides exhibiting the same molecular mass of approximately 88 kDa as for DPPIV and 58/48 kDa as for gp110/cell-CAM105 (Fig. 3, lanes 3 and 4 (MH 7777 cells) and lanes 7 and 8 (hepatocytes)). The additional polypeptide band of 110 kDa present in immunoprecipitates of DPPIV obtained from MH 7777 cells after reculture (Fig. 3, DPPIV, lanes 2 and 4) was resistant to Endo H and hence most likely reflects reprocessing of some DPPIV to the complex-type glycoform as mentioned above. These experiments demonstrate that the decrease in the molecular mass does not result from limited proteolysis but reflects trimming of the oligomannosidic N-glycans.
High Mannose-type N-Glycans of Surface Glycoproteins Are Trimmed
from Man8-9GlcNAc2 to
Man5-7GlcNAc2 after Transport to the Cell
Surface--
To characterize the mannose trimming of the high mannose
N-glycans of DPPIV and gp110/cell-CAM105, in particular to
determine the end product of the trimming, oligosaccharides were
analyzed immediately after washout of dMM, i.e. after the
chase period, and after reculture using the following protocol. MH 7777 cells were radiolabeled with
D-[2,6-3H]mannose and then chased, both in
the presence of dMM, and recultured in the absence of dMM. From the
glycoproteins exhibiting either a decrease in the molecular mass during
reculture (DPPIV and gp110/cell-CAM105) or not (TfR and LI-cadherin),
DPPIV and TfR were analyzed. The glycoproteins were immunoadsorbed
immediately after the chase (0 h) and after 85 h (DPPIV) or
70 h (TfR) of reculture. The radiolabeled oligosaccharides were
released from the glycoproteins by Endo H and then converted into their
corresponding oligosaccharide alditols by reduction with
NaBH4 and separated by HPLC (Fig.
4). Columns were calibrated with
authentic oligosaccharide alditols (Man5-9GlcNAcOH), and
samples were mixed with fluorescence-labeled glucose oligomers as
internal standards. From DPPIV and TfR isolated immediately after the
chase (0 h), Man9GlcNAcOH and Man8GlcNAcOH were
obtained as the major components (Fig. 4, A and
D), in line with the inhibitory effect of dMM on processing
mannosidases (for reviews, see Refs. 4 and 5). After reculture of
cells, TfR and DPPIV exhibited a different pattern in the sizes of
oligomannosidic N-glycans. In the case of DPPIV,
Man5GlcNAcOH was the major component, while
Man6-9GlcNAcOH were present as minor components (Fig.
4B). These results demonstrate that high mannose-type
N-glycans of DPPIV are trimmed from
Man8-9GlcNAc2 to
Man5GlcNAc2 during reculture with
Man6-7GlcNAc2, representing trimming
intermediates. In contrast to DPPIV, no decrease in the size of the
high mannose-type N-glycans was detectable for the TfR
during reculture of cells (Fig. 4E). To examine whether
DPPIV being trimmed from Man8-9GlcNAc2 to
Man5GlcNAc2 derives in fact from the plasma
membrane, demannosylation of DPPIV was examined after covalent labeling
with NHS-SS-biotin at the cell surface, as schematized in Fig.
5A. This approach allows us to
unambiguously discriminate between cell surface proteins and proteins
localized in intracellular compartments. Cells were radiolabeled and
chased as above and then surface-labeled at 4 °C with NHS-SS-biotin
prior to reculture. Biotin-labeled surface DPPIV was isolated by
immunoadsorption in conjunction with affinity chromatography on
streptavidin-agarose. HPAE-separation of the high mannose-type
oligosaccharides released from biotinylated DPPIV by PNGase F revealed
the same shift from Man8-9GlcNAc2 to
Man5-7GlcNAc2 structures (Fig.
6, A and B) that
was observed for total cellular DPPIV (Fig. 4, A and
B). This clearly demonstrates that trimming of high
mannose-type N-glycans affects DPPIV molecules that have
exited the secretory pathway and were exposed at the cell surface.
Demannosylation was also observed for the bulk of cell surface
glycoproteins in MH7777 cells (Fig. 6, C and D),
showing that this process is not restricted to DPPIV. In an attempt to
find out whether a class I or class II mannosidase is involved that is
known to be inhibited by dMM or swainsonine, respectively (for reviews,
see Refs. 4 and 5), reculture was performed in the presence of either
one of the inhibitors. When cells were recultured in the presence of
dMM, trimming of DPPIV was completely blocked (Fig. 4C),
which is in agreement with previous reports (12, 21). The inhibitory
effect of dMM on demannosylation could also be demonstrated by the
finding that the molecular mass of DPPIV and gp110/cell-CAM105 as
analyzed by SDS-PAGE did not decrease during reculture in the presence
of the inhibitor (Fig. 2B). In contrast, in the presence of
swainsonine (3 µg/ml) the high mannose-type N-glycans of
DPPIV were trimmed to the same extent as in the absence of the
inhibitor (not shown). In summary, these results show that high
mannose-type glycans of cell surface glycoproteins are
postbiosynthetically trimmed by a dMM-sensitive, swainsonine-resistant
-mannosidase.
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|
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Kinetics of Postbiosynthetic Mannose Trimming--
A quantitative
analysis of the kinetics of postbiosynthetic demannosylation was
performed for total cell surface proteins in HepG2 cells. In these
experiments, high mannose-type glycans of cell surface-labeled
glycoproteins were analyzed after different times of reculture. Data
from HPAE fractionations (shown in Fig.
8A) were quantitated by
totaling the radioactivity in each peak and by correcting for the
number of mannose residues. In Fig. 8B, the radioactivity of
each oligosaccharide species, expressed as a percentage of the total
oligosaccharides recovered, is plotted versus time of
reculture. As can be seen from the hydrolysis curves,
Man9GlcNAc2 is converted to
Man5GlcNAc2 with
Man6GlcNAc2,
Man7GlcNAc2, and
Man8GlcNAc2 formed as trimming intermediates.
The extent of demannosylation at each time point, defined as the
conversion of Man8-9GlcNAc2 to
Man5-7GlcNAc2, was calculated based on the
amount of Man8-9GlcNAc2 obtained immediately
after the chase and plotted versus time. As can be seen in
Fig. 8C, the extent of demannosylation increased during the
first 24 h and then reached a plateau. With the assumption of
first order kinetics, demannosylation followed the equation
Dt = Dt
·(1
e
kt), where Dt is
the extent of demannosylation at the time t = x. The constant k can be determined from this
equation as the negative slope of a plot of
ln(Dt
Dt) over the time (Fig. 8D). For total
cell surface glycoproteins of HepG2 cells, a half-time of 6.7 h
was calculated for demannosylation according to the equation
t1/2 = ln 2/k.
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Trimmed Glycoproteins Can Be Isolated from the Cell Surface after Postbiosynthetic Demannosylation-- Trimming of plasma membrane glycoproteins after their transport to the cell surface could occur either at the cell surface or after endocytosis in intracellular compartments. In the latter case, it could not be distinguished on the basis of the previous experiments whether trimmed glycoproteins remain in intracellular compartments or return to the cell surface. To examine whether trimmed glycoproteins are exposed at the cell surface, mannose trimming was analyzed for glycoproteins isolated selectively from the cell surface at the end of the reculture period. To do so, MH 7777 cells were radiolabeled and recultured as in the experiments shown in Fig. 4. After reculture, proteins exposed at the cell surface were labeled with biotin immediately prior to harvesting and solubilization of cells and were then analyzed for mannose trimming, as schematized in Fig. 5B. HPLC separation of the high mannose-type glycans released from the biotinylated glycoproteins revealed that surface glycoproteins isolated after reculture carried mainly Man5GlcNAc2 and minor amounts of Man6-9GlcNAc2 (Fig. 9B). In accordance with the results shown in Figs. 4 and 6, glycoproteins isolated immediately after the chase carried mainly Man8-9GlcNAc2 (Fig. 9A). These results demonstrate that after demannosylation had occurred, trimmed glycoproteins were exposed at the cell surface. This provides additional evidence that demannosylation can occur without further processing to complex structures.
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DISCUSSION |
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Two major conclusions can be drawn from the results of the present
study. First, after exit from the secretory pathway and transport to
the cell surface, selected cell surface glycoproteins undergo trimming
of their oligomannosidic N-glycans by -mannosidase(s)
sensitive to dMM. The observed demannosylation of cell surface
glycoproteins results in the conversion of
Man8-9GlcNAc2 species to
Man5-7GlcNAc2 with
Man5GlcNAc2 being the major product. Second,
subsequent to demannosylation, trimmed glycoproteins are exposed at
least partly at the cell surface. Hence, it is likely that
demannosylation of glycoproteins occurs at sites peripheral to
N-acetylglucosaminyltransferase I either at the cell surface
or during endocytosis and recycling back to the cell surface. The data
clearly demonstrate that modification of plasma membrane glycoproteins
by trimming of their oligomannosidic N-glycans is not
restricted to biosynthesis. These conclusions are based on the
following evidence.
Analysis of the size of high mannose-type N-glycans of cell surface glycoproteins demonstrated that during reculture of MH 7777 cells and HepG2 cells Man8-9GlcNAc2 species were trimmed to Man5GlcNAc2 with Man6GlcNAc2 and Man7GlcNAc2 formed as trimming intermediates. Mannose trimming has also been observed for the cation-dependent and the cation-independent mannose 6-phosphate receptors in BW 5147 mouse lymphoma cells (12) and for TfR and for the total cellular glycoprotein pool in K562 cells (21). However, in both of these studies, mannose trimming was not examined by a sample of glycoproteins after previous labeling on the cell surface. Therefore, it remained unknown whether glycoproteins modified by mannosidase I were derived from the plasma membrane or from intracellular compartments. In comparison, by analyzing membrane glycoproteins that had been labeled with biotin at the cell surface prior to reculture of cells (schematized in Fig. 5A), we were able to examine selectively the fate of cell surface glycoproteins. The results of these experiments unequivocally demonstrate that demannosylation of oligomannosidic N-glycans affects glycoproteins after they have been delivered to the cell surface. Moreover, previous studies did not address the question of whether trimmed glycoproteins become exposed at the cell surface. By analyzing glycoproteins that were allowed to encounter mannosidases and were thereafter isolated selectively from the cell surface (schematized in Fig. 5B), it is clearly shown in the present study that subsequent to demannosylation trimmed glycoproteins are present at the cell surface.
Which -mannosidase is involved in the postbiosynthetic mannose
trimming remains to be established. Based on the finding that
postbiosynthetic demannosylation could be inhibited by dMM, and since
the oligomannosidic N-glycans were converted from
Man8-9GlcNAc2 species to
Man5GlcNAc2, it is likely that class I
-mannosidases of the ER and the Golgi complex are involved. These
enzymes are known to cleave up to four mannose residues from
Man9GlcNAc2 to yield
Man5GlcNAc2 during the maturation of
N-linked oligosaccharides. In contrast to class I
-mannosidases of the ER and the Golgi complex, the class II
-mannosidases (Golgi
-mannosidase II, ER/cytosolic
-mannosidase, and the lysosomal mannosidase (for review, see Refs. 4
and 5)) cannot account for the observed trimming reactions. These
enzymes are dMM-resistant and inhibited to some extent by swainsonine,
a mannose analogue that had no effect on demannosylation. In addition,
class II
-mannosidases do not have the specificities required to
account for the trimming from Man9GlcNAc2 to
Man5GlcNAc2 and therefore cannot be involved in
postbiosynthetic demannosylation. Several as yet unclassified
1,2/1,3/1,6-mannosidases have been described, purified from rat
brain (42), rat sperm (43), and rat liver microsomes (44), that share
many common characteristics. Swainsonine and dMM are only weakly
inhibitory or not inhibitory to these enzymes. For example, rat liver
1,2/1,3/1,6-mannosidase is inhibited by dMM at concentrations more
than 100-fold higher than that reported for purified Golgi mannosidase
I (44). These
1,2/1,3/1,6-mannosidases cleave
Man4-9GlcNAc substrates to Man3GlcNAc, an
oligosaccharide that could not be isolated from cell surface
glycoproteins in the present study. Taken together, it is unlikely that
these enzymes are involved in postbiosynthetic mannose trimming of cell
surface glycoproteins. It should, however, be noted that the substrate
specificities of several mannosidases were determined using
ManXGlcNAc oligosaccharides as substrates and that the enzymes
may yield other products with ManXGlcNAc2 or
glycopeptides as substrates as has been shown for the pig liver
Man9-mannosidase (45) and the neutral
-mannosidase from
Japanese quail oviduct (46). Apart from the known
-mannosidases
sensitive to dMM the possibility cannot be excluded that another as yet
unidentified dMM-sensitive
-mannosidase might be involved.
The subcellular site of postbiosynthetic mannose trimming is unknown.
Demannosylation of glycoproteins could occur at the cell surface,
during passage through endocytic compartments, or even after return to
compartments of the secretory pathway. Trimming of high mannose-type
N-glycans at the cell surface seems feasible, since in a
recent immunohistochemical study Golgi -mannosidase I has been
detected at the cell surface of enterocytes, pancreatic acinar cells,
and goblet cells (24). However, it is unknown whether the mannosidase
is enzymatically active at this location. Moreover, a rat sperm
1,2/1,3/1,6-mannosidase has been reported to be an intrinsic plasma
membrane component that is enzymatically active when assayed in sperm
plasma membranes and intact spermatozoa, respectively (43). As a second
possibility, postbiosynthetic mannose trimming could occur in endocytic
compartments during endocytosis and recycling of glycoproteins back to
the cell surface. It has been shown that rat liver
1,2/1,3/1,6-mannosidase activity is enriched in endosomal fractions
(47). Although, as discussed above, this enzyme is probably not
involved with respect to its substrate specificity, other mannosidases
might also be present in endosomes. Third, since several plasma
membrane glycoproteins have been shown to return to the Golgi complex
and the TGN (12-21), demannosylation could also occur after the return
of cell surface glycoproteins to these compartments. From our data, the
possibility cannot be excluded that some glycoproteins return to the
processing mannosidases in the cis-Golgi, since a small
fraction of the oligomannosidic glycoform of DPPIV was processed to the
complex form during reculture in MH 7777 cells (Fig. 2A),
whereas no reconversion to the complex glycoform could be detected for
surface DPPIV in HepG2 cells (18). From these observations, combined
with the fact that surface DPPIV is trimmed from
Man8-9GlcNAc2 species to
Man5-7GlcNAc2 in MH 7777 cells as well as in
HepG2 cells, we conclude that most of the DPPIV is trimmed without
further processing to the complex glycoform. This explanation is
supported by the observation that trimmed glycoproteins can be found on
the cell surface and is consistent with the recent finding of Velasco
et al. (24) that Golgi mannosidase I previously assumed to
reside specifically in the cis-Golgi (48) is less
compartmentalized. In this study, the enzyme was primarily detected in
the medial- and trans-Golgi cisternae, and in some cell
types it was also localized in the TGN and even in secretory vesicles.
Therefore, in case cell surface glycoproteins return to the Golgi at
sites peripheral to N-acetylglucosaminyltransferase I, an
enzyme that is localized in medial Golgi elements (23) and initiates
the synthesis of hybrid and complex oligosaccharides, it seems feasible
that high mannose-type N-glycans of recycling glycoproteins
might be trimmed by Golgi mannosidase I without being further processed
to complex structures.
Comparison of DPPIV, TfR, gp110/cell-CAM105, and LI-cadherin showed that postbiosynthetic trimming did not affect each of the four glycoproteins. This may be due to differences in the kinetics or routes of internalization and recycling or due to a different susceptibility of the oligomannosidic glycans to trimming mannosidases. Which of these different mechanisms is responsible remains to be established. Although distinct proteins escape demannosylation, this process seems to affect a large number of cell surface glycoproteins, since it could be demonstrated for the bulk of plasma membrane glycoproteins in MH 7777 cells as well as in HepG2 cells. A quantitative analysis of the time course of demannosylation revealed that this process obeyed first order kinetics with a calculated half-time of 6.7 h as determined for total cell surface glycoproteins in HepG2 cells. The process of demannosylation occurs distinctly faster than degradation of [35S]methionine-labeled total membrane proteins in HepG2 cells (t1/2 = 65 h2). This indicates that cell surface glycoproteins may encounter trimming mannosidase(s) several times during their life span. The physiological role of postbiosynthetic demannosylation is unknown. Trimming of oligomannosidic N-glycans could reflect the occasional removal of mannose residues from surface glycoproteins by a mannosidase present at the cell surface or encountered during endocytosis and recycling. Alternatively, postbiosynthetic processing could provide a means by which cells can modify N-glycans of cell surface glycoproteins. With respect to the role of cell surface glycoproteins in cell-substratum and cell-cell recognition processes, this remodeling of cell surface glycoproteins may be of relevance for cell surface functions.
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ACKNOWLEDGEMENTS |
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We are indebted to Prof. Dr. R. Geyer (Biochemisches Institut, Universität Gießen, Germany) for the generous gift of the authentic glycan standards and to Dr. D. Hixson (Brown University) and Dr. H. P. Hauri (Biocenter of the University of Basel, Switzerland) for the gift of monoclonal antibodies.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (Re 523/4-1 (to W. R.) and Sonderforschungsbereich 312 (to R. T.)), and by the Sonnenfeld-Stiftung.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.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: Institut für
Klinische Chemie und Pathobiochemie, Universitätsklinikum
Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany. Tel.:
49-30-8445-2555; Fax: 49-30-8445-4152.
1
The abbreviations used are: ER, endoplasmic
reticulum; ConA, concanavalin A; dMM, 1-deoxymannojirimycin; DMEM,
Dulbecco's modified Eagle's medium; DPPIV, dipeptidyl peptidase IV;
Endo H, endo--N-acetylglucosaminidase H; HPAE, high
performance anion exchange; HPLC, high performance liquid
chromatography; PBS, phosphate-buffered saline; NHS-SS-biotin,
sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate; PNGase F,
peptide
N4-(N-acetyl-
-glucosaminyl)
asparagine amidase F; TfR, transferrin receptor; TGN,
trans-Golgi network; PAGE, polyacrylamide gel
electrophoresis.
2 G. Orberger and R. Tauber, unpublished results.
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REFERENCES |
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