©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Enhanced Folding and Processing of a Disulfide Mutant of the Human Asialoglycoprotein Receptor H2b Subunit (*)

(Received for publication, April 4, 1995; and in revised form, June 14, 1995)

Ming Huam Yuk (1) (2) Harvey F. Lodish (1) (2)(§)

From the  (1)Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142 and the (2)Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Unfolded forms of the H2b subunit of the human asialoglycoprotein receptor, a galactose-specific C-type lectin, are degraded in the endoplasmic reticulum (ER), whereas folded forms of the protein can mature to the cell surface (Wikström, L., and Lodish, H. F.(1993) J. Biol. Chem. 268, 14412-14416). There are eight cysteines in the exoplasmic domain of the protein, forming four disulfide bonds in the folded protein. We have constructed double cysteine to alanine mutants for each of the four disulfide bonds and examined the folding and metabolic fate of each of the mutants in transfected 3T3 fibroblasts. We find that mutation of the two cysteines nearest to the transmembrane region (C1) does not prevent proper folding of the protein, whereas mutations of the other three disulfides prevent proper folding of the protein and all of the mutant proteins are degraded in the ER. A normal (20%) fraction of the C1 mutant protein exits the endoplasmic reticulum and is processed in the Golgi complex, and it does so at a faster rate compared to the wild-type. Furthermore, the folded form of this mutant protein is more resistant to unfolding by dithiothreitol than the wild-type. The C1 mutant protein is expressed on the cell surface and can form a functional receptor with the H1 subunit with similar binding affinities for natural ligands as that of the wild-type receptor. The same fraction of newly made mutant and wild-type proteins (80%) remain in the ER, but the mutant protein is degraded more quickly. Thus, the presence of the C1 disulfide bond in the wild-type receptor both reduces the rate of protein folding and exit to the Golgi and slows the rate of ER degradation of the portion (80%) of the receptor that never folds properly.


INTRODUCTION

The asialoglycoprotein (ASGP) (^1)receptor, also known as the hepatic lectin, is a type II integral membrane protein that is normally expressed only on mammalian hepatocytes. This Ca-dependent lectin binds to terminal galactose residues on sugar side chains; these ligands are removed from the circulation by receptor-mediated endocytosis via coated pits and degraded in lysosomes(2, 3, 4) . The functional human ASGP receptor is a hetero-oligomer consisting of two types of subunits, H1 and H2, with a minimum stoichiometry of (H1)(3)(H2)(1)(5) . H1 and H2 are 60% homologous in amino acid sequence(6, 7) , and both subunits are required for a functional receptor(8, 9) . The polypeptide chain of each subunit consists of four main domains: a short cytosolic amino-terminal segment, a single hydrophobic transmembrane segment that also functions as an uncleaved signal anchor sequence, followed by an exoplasmic ``stalk'' domain and, at the very carboxyl terminus, the Ca-dependent carbohydrate recognition domain. There are two subtypes of H2: H2a and H2b, which differ only by the presence of five extra amino acids in H2a near the transmembrane region on the exoplasmic side that results from alternative splicing. In HepG2 human hepatoma cells, most of the H2 expressed on the cell surface is H2b(10) . In addition to the human receptor, highly homologous ASGP receptors with identical functions have also been cloned and analyzed in rat (11) and mouse(12) . The ASGP receptor belongs to a family of animal proteins known as the C-type lectins with the characteristic of requiring calcium ions for ligand binding(13) . All members of these family share sequence homology in the carbohydrate recognition domain. Within this domain of about 130 amino acids, 18 invariant and 32 conserved residues are found in all members(13) .

The important role of disulfide bonds in determining and maintaining tertiary and quaternary structures of secreted proteins and exoplasmic domains of membrane proteins has long been recognized. These proteins form disulfide bonds between cysteine residues due to the more oxidative redox state of the endoplasmic reticulum (ER) compared to the cytosol(14) . The formation of disulfide bonds in newly synthesized proteins occurs within the ER and is catalyzed by the ER-resident enzyme protein disulfide isomerase(15) . Intracellular disulfide-bonded folding intermediates of the human chorionic gonadotropin beta subunit have been identified(16) , and disulfide-bonded in vivo folding intermediates have also been reported for influenza hemagglutinin(17) , retinol-binding protein(18) , and the ASGP receptor H1 subunit(19) . Studies on the influenza hemagglutinin have shown that all the disulfide bonds are required for proper folding and protein maturation(20) . When cells are treated with a reducing agent, dithiothreitol (DTT), which disrupts disulfide bonds, newly made proteins that contain disulfide bonds, e.g. albumin and the ASGP receptor, are retained within the ER, whereas proteins that do not have disulfides are secreted normally in the presence of DTT(21) .

Depletion of Ca in the ER by treating cells with Ca pump inhibitors or Ca ionophores also blocks exit of the ASGP receptor from the ER and unfolded proteins accumulate(1, 19) . Therefore, proteins that are misfolded or cannot attain a properly folded state in the ER cannot exit this organelle to the Golgi complex and are ultimately degraded in the ER (22, 23) .

When expressed without H1 in transfected fibroblasts, about 80% of ASGP receptor H2b subunits are degraded in the ER(10, 24, 25) . The unfolded portion of the newly made H2b protein is selectively degraded within the ER, whereas the 20% that is folded matures to the cell surface(1) . Therefore, we felt it important to analyze further the relationship between folding and degradation of H2b within the ER. In this study, we determine the importance of each of the four disulfide bonds in the folding and degradation of the H2b protein by specifically mutating each pair of cysteine residues and observing the metabolic fate of the mutant proteins in transfected fibroblasts. We find that disruption of three of the disulfide bonds results in the inability of the protein to fold properly, and these cannot exit the ER and are degraded. However, mutation of the two cysteines nearest the transmembrane region (C1 mutation) actually causes the protein to fold faster than the wild-type and the mutant protein exits the ER to the Golgi complex more rapidly than does the wild-type. Furthermore, the C1 H2b mutant will form a functional receptor with H1 and bind a natural ligand with similar affinity to the wild-type receptor.


EXPERIMENTAL PROCEDURES

Materials

Materials and chemicals were purchased from Sigma or sources previously listed(9, 10, 24, 26) . Iodination reagents were purchased from Pierce. Reagents for PCR reactions and Pfu polymerase were from Stratagene. Restriction enzymes were from New England Biolabs. The Sequenase 2.0 kit for DNA sequencing was from U. S. Biochemical Corp. Cy3-labeled goat-anti-rabbit antibody was from Jackson Immunoresearch Laboratories (West Grove, PA).

Cell Surface Iodination

NIH 3T3 cells expressing wild-type H2b (2C cells) (10) were cell surface-iodinated using water-soluble Bolton-Hunter reagent (sulfo-SHPP) as described previously(10, 27) .

Cyanogen Bromide Digestion

NIH 3T3 cells expressing wild-type H2b (2C cells) that had been treated with 0.1 M iodoacetamide and cell surface-iodinated were lysed in detergent and immunoprecipitated by antiserum against the carboxyl terminus of the H2 protein. The immunoprecipitate was washed and a portion was digested for 12 h by 50 mg/ml cyanogen bromide in 70% formic acid at room temperature in the dark. Products were analyzed on SDS-PAGE under non-reducing or reducing (50 mM DTT) conditions.

Mutagenesis of ASGP Receptor H2b cDNA

Four mutations of the H2b protein were made corresponding to the four putative disulfide linkages (C1-C4, see Fig. 1), and each mutant has both cysteines of the disulfide bond mutated to alanine residues. The substitution mutations of H2b were introduced by overlap extension PCR (28) using Pfu polymerase for primer extension. The concentrations of reagents and enzymes used in the PCR reactions were according to the manufacturer's recommendations. The double mutations were made sequentially, and all mutations were verified by double-stranded dideoxy sequencing using the Sequenase 2.0 kit from U. S. Biochemical Corp.


Figure 1: Schematic diagram of putative exoplasmic disulfide bonds of the human asialoglycoprotein receptor H2b subunit. C, cysteine residues in the exoplasmic domain; M, methionine residues; Cyto, cytosolic domain; TM, transmembrane domain; Exo, exoplasmic domain; numbers above the cysteine and methionine residues refer to their position in the amino acid sequence; C1-C4 refer to the putative disulfide bonds. Asterisk indicates position of N-linked glycosylation sites. Diagram is not to scale.



Cell Transfection and Culture

NIH 3T3 cells expressing wild-type H2b (2C cells) were kind gifts of Dr. G. Lederkremer (Tel Aviv University)(10) . NIH 3T3 cells expressing H2 mutants were generated by using a calcium phosphate transfection protocol(29) , using the pMEX-neo mammalian expression vector containing mutant H2 cDNAs subcloned into the BamHI and EcoRI sites in the multicloning site of the vector. Colonies resistant to G418 were subcloned and tested for expression of H2 protein by metabolic labeling. Wild-type H1 cDNA subcloned into the pCB7 expression vector was similarly transfected into 3T3 cells expressing wild-type or C1 mutant H2b and selected by hygromycin resistance. All 3T3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated calf serum.

Metabolic Labeling, Immunoprecipitation, and Enzyme Digestions

Confluent or near confluent (80%) cells in 100-mm or 60-mm diameter tissue culture dishes were labeled with [S]cysteine using techniques described previously(24, 26) . Antisera against the carboxyl terminus of the ASGP receptor H2 subunit (8) were kind gifts of Drs. L. Wikström and G. Lederkremer. Immunoprecipitation and Endo H and N-glycanase digestions of cell lysates were done as described previously(24, 26) .

Gel Electrophoresis, Fluorography, and Scanning Densitometry

Immunoprecipitates were subjected to SDS-PAGE using 0.75-mm 10% or 12% Laemmli gels and analyzed by autoradiography or fluorography using 20% 2,5-diphenyloxazole as described previously (30) . Autoradiograms and fluorograms were quantitated with a Molecular Dynamics laser microdensitometer as described previously(31) .

Immunofluorescence Microscopy

Immunofluorescence localization of H2b wild-type and cysteine mutant proteins on the cell surface of transfected 3T3 fibroblasts were done as described previously(10) . Basically, live 3T3 cells expressing wild-type or mutant proteins were reacted at 4 °C first with a primary rabbit antibody against the carboxyl terminus of the H2 protein followed by a secondary Cy3-labeled goat anti-rabbit antibody. Cells were washed with cold phosphate-buffered saline, fixed with methanol/acetone, and visualized with a Zeiss epifluorescence microscope.

Ligand Binding, Uptake, and Degradation Assays

Orosomucoid was desialylated with immobilized neuraminidase (Sigma) and radioiodinated with Iodobeads (Pierce) as described previously(9, 32, 33) . Saturation binding, uptake, and degradation assays were performed as described previously(9, 32, 33) . Binding assays were done with radioligand (specific activity = 10^7 cpm/µg) concentrations in the range of 20 ng/ml to 5 µg/ml. Nonspecific binding was measured in the presence of 100-fold excess unlabeled ligand and typically represented less than 10% of the total binding. Degradation products were measured as radioactivity remaining in the culture medium after it was precipitated with trichloroacetic acid, oxidized with potassium iodide/hydrogen peroxide, and extracted with chloroform(32) .


RESULTS

A Disulfide Bond (C1) Connects the H2 Protein between Cysand Cys

Fig. 1shows that the exoplasmic domain of the ASGP receptor H2b subunit contains eight cysteine residues (at positions 157, 171, 172, 185, 200, 275, 287, and 295) that are conserved in man, rat, and mouse. The six cysteines nearest to the carboxyl terminus are conserved in C-type lectins and are predicted to form three disulfide bonds, based on biochemical and crystallographic data obtained on homologous proteins (34, 35, 36, 37) . They are Cys/Cys, Cys/Cys, and Cys/Cys and are labeled here as C2, C3, and C4 disulfides, respectively. The two cysteines nearest to the transmembrane region, Cys and Cys, are not conserved in other C-type lectins.

To determine if a disulfide is formed between these two cysteines (referred to as the C1 disulfide), we take advantage of the methionine residue at position 159, between these two cysteines, that is only one of two methionines in the whole molecule, excluding the translation initiation residue site. Met is also the only methionine in the exoplasmic domain (Fig. 1). The mature complex glycosylated H2b protein is 50 kDa, with a core protein mass of 35 kDa and three N-linked sugar side chains adding 5 kDa each to the total mass. In the experiment leading to Fig. 2, proteins in H2b-transfected fibroblasts were reacted with iodoacetamide to block free sulfhydryl groups and then radioiodinated with the membrane-impermeable Bolton-Hunter reagent (sulfo-SHPP). The cells were then dissolved in detergent and immunoprecipitated with an antiserum against the carboxyl terminus of the H2 protein. We chose to analyze only the mature, cell surface form of the H2b protein to avoid ambiguities that can arise in the intracellular forms, which may not be fully folded or are misfolded with non-native disulfides. If there is a C1 disulfide bond, on digestion of the immunoprecipitated mature cell surface H2b protein by cyanogen bromide, which specifically cleaves after methionine residues, a 27-kDa fragment (with two N-linked sugar side chains) and a 17-kDa fragment (with one N-linked sugar side chain), should be detected only in reducing conditions but not under non-reducing conditions. Fig. 2shows the 50-kDa full-length complex glycosylated H2b expressed on the cell surface of stably transfected 3T3 fibroblasts analyzed under non-reducing (lane1) and reducing (lane3) conditions. After digestion with cyanogen bromide, a portion of the 50-kDa protein is decreased in size to about 44 kDa due to cleavage at Met but no smaller fragments were observed under non-reducing conditions (lane2). However, under reducing conditions, a 27-kDa fragment (which corresponds to the carboxyl-terminal fragment with two N-linked sugar side chains) and a 17-kDa fragment (which corresponds to the amino-terminal fragment with one N-linked sugar side chain) were observed (lane4). These were not observed under non-reducing conditions (lane2). These observations strongly suggest that there is a reducible intrachain disulfide linking Cys and Cys. It is possible that the C1 disulfide bond is actually between Cys and Cys, whereas the C2 disulfide is between Cys and Cys, but the main conclusion is that Cys is disulfide-bonded. In summary, the intrachain disulfides of the H2b protein are presumed to be: Cys/Cys (C1), Cys/Cys (C2), Cys/Cys (C3), and Cys/Cys (C4).


Figure 2: Cyanogen bromide digestion of H2b protein. Fibroblasts expressing wild-type H2b were treated with 0.1 M iodoacetamide in phosphate-buffered saline on ice for 5 min, after which cell surface proteins were radioiodinated with the water-soluble Bolton-Hunter reagent as described under ``Experimental Procedures.'' Cells were lysed and immunoprecipitated with an antiserum against the carboxyl terminus of the H2 protein, and the immunoprecipitated H2b protein was reacted in 70% formic acid with (lanes2 and 4) or without (lanes1 and 3) 50 mg/ml cyanogen bromide for 12 h. After the reaction, the proteins were subjected to SDS-PAGE under non-reducing (lanes1 and 2) or after reduction with 50 mM DTT (lanes3 and 4).



Mutant H2b Proteins Lacking the C1 Disulfide Bond Show Enhanced Rate of Folding in the ER and Golgi Processing

To examine the importance of each of the four intrachain disulfides in the H2b protein, we constructed double cysteine to alanine mutants, which individually disrupted each of the disulfide bonds (C1-C4 mutants) and studied the metabolic fate and folding of each of the mutants in stably transfected 3T3 fibroblasts. Fig. 3(lanes1, 7, 13, 19, and 25) show that on pulse labeling with [S]cysteine, the wild-type and C1-C4 mutants of H2b are all synthesized as a 43-kDa precursor (openarrow), which can be deglycosylated to a 35-kDa core protein with endoglycosidase H (data not shown). Lanes2-6 show that in the subsequent chase period, a fraction of the wild-type precursor is converted to a 50-kDa mature form (solidarrow), which is resistant to Endo H (data not shown). Densitometric scans of this pulse-chase experiment (Fig. 4a) shows that about 20% of the wild-type precursor becomes complex glycosylated (i.e. Endo H-resistant). This Golgi processing proceeds with a half-time of about 90 min with most of the mature forms appearing by 3 h. After an initial lag of 60 min, 80% of the Endo H-sensitive forms of H2b are degraded with a half-life about 60 min, consistent with previous reports(1, 10) . Fig. 3(lanes 8-12) shows that a fraction of the C1 mutant precursor is also converted to the 50-kDa mature form that is Endo H-resistant (data not shown). Furthermore, densitometric scans (Fig. 4b) shows that although the fraction of the C1 mutant precursor that attains complex glycosylation is similar to the wild-type (20%), the Golgi-processed forms appear much faster than the wild-type. Substantial amounts of the complex glycosylated form of the C1 mutant protein has already appeared by 30 min, and the maximum level is reached by 60 min. The Endo H-sensitive form of the C1 mutant also disappears much faster than that of the wild-type, with a half-life of less than 30 min.


Figure 3: Metabolic fate of H2b wild-type and cysteine mutant proteins in stably transfected 3T3 fibroblasts. 3T3 cells expressing wild-type (lanes 1-6) or mutant H2b proteins (C1 mutant, lanes 7-12; C2 mutant, lanes 13-18; C3 mutant, lanes 19-24; C4 mutant, lanes 25-30) were pulse-labeled with 0.3 mCi/ml [S]Cys for 15 min (lanes1, 7, 13, 19, and 25) and then chased in unlabeled medium for up to 4 h (lanes 2-6, 8-12, 14-18, 20-24, and 26-30). Cell lysates from various times of chase were immunoprecipitated with an antiserum against the carboxyl terminus of the H2 protein and then subjected to SDS-PAGE under reducing conditions. Openarrow indicates the high mannose precursor form of the protein. Solidarrow indicates the complex glycosylated form of the protein.




Figure 4: Kinetics of ER degradation and Golgi processing of H2b wild-type and cysteine mutant proteins in transfected 3T3 fibroblasts. The 43-kDa high mannose precursors and 50-kDa complex glycosylated forms of the H2b wild-type or mutant proteins in the fluorograms from Fig. 2were quantitated by scanning densitometry, normalized to amount of precursor after each pulse, and plotted against time of chase. Panel a, rate of loss of high mannose forms and appearance of complex glycosylated forms of wild-type protein. Panel b, rate of loss of high mannose forms and appearance of complex glycosylated forms of C1 mutant protein. Panel c, rate of loss of high mannose forms of C4 mutant protein.



Fig. 3(lanes 14-18, 20-24, and 26-30) shows that the C2, C3 and C4 mutants do not form any detectable complex glycosylated mature forms. All the pulse-labeled proteins remain as 43-kDa forms (Endo H-sensitive, data not shown). A 55-kDa polypeptide that appears in all lanes is unlikely to be a processed form of the mutant H2b receptor, since it is present in the pulse-labeled samples and its mobility is not affected by N-glycanase digestion (data not shown). We do not know the identity of this co-immunoprecipitated protein. The immunoprecipitated 35-kDa polypeptide that appears in the chase periods comprises the carboxyl terminus of the H2b protein; it is produced by a proteolytic cleavage in the exoplasmic domain near the membrane-spanning region (24, 25) . We consistently observe that more of this fragment is produced in cells expressing the C2, C3, and C4 mutants, consistent with the finding that no detectable fraction of these proteins matures out of the ER. Fig. 4c shows that the kinetics of degradation of the Endo H-sensitive form of the C4 mutant is similar to that of the wild-type. Similar densitometric scans show that the C2 and C3 mutants also have similar kinetics of degradation compared to the wild-type H2b (data not shown).

On non-reducing SDS-PAGE, less compact forms of a protein should experience more hydrodynamic resistance, and thereby migrate more slowly than more compact forms of the same protein. Formation of disulfide bonds in a protein should allow it to attain more compact forms and migrate faster under non-reducing conditions on SDS-PAGE than non-compact forms without disulfide bonds. Previous studies on folding of the ASGP receptor subunits showed folding intermediates that were separable by non-reducing SDS-PAGE(1, 19) . Fig. 5(lanes1 and 2) shows that, as judged by their differences in mobility on non-reducing SDS-PAGE, pulse-labeled H2b wild-type and C1 mutant proteins have a less compact and presumably unfolded structure (shadedarrow) but that they attain a more compact structure (lanes6 and 7, stripedarrow) after a 30-min chase period. However, if the cells are treated with 5 mM DTT for another 5 min at the end of the 30-min chase, a fraction of the wild-type H2b protein is ``unfolded'' to a less compact form (lane11), while almost all of the C1 mutant protein remains as the more compact species (lane12). Lanes 3-5 show that the C2, C3, and C4 mutants are also synthesized as less compact unfolded forms, similar to the wild-type (lane1, shadedarrow). After 30 min, the C2 and C4 mutants (lanes8 and 10) also attain a more compact or more folded form (stripedarrow) similar to the wild-type (lane6). However, most of the C3 mutant remains as the less compact unfolded form after 30 min (lane9). On treatment with 5 mM DTT, only a fraction of the wild-type is unfolded to the less compact form (lane11) but all of the C2, C3 and C4 proteins are unfolded to the less compact form (lanes 13-15). On reducing SDS-PAGE, all the bands migrate to the same positions (data not shown) and therefore results under non-reducing conditions reflect differential oxidative isoforms of the proteins. Experiments in which longer chase times were done (up to 3 h) showed identical results concerning the relative mobilities and DTT sensitivities of the wild-type and mutant proteins (data not shown). We conclude that the C2, C3, and C4 disulfides are required for proper folding of the protein. If any of these disulfides are missing, the protein cannot attain its properly folded form and cannot exit the ER and is all degraded within the ER. However, the absence of the C1 disulfide bond enhances protein folding and processing.


Figure 5: Folding of H2b wild-type and cysteine mutant proteins in transfected 3T3 fibroblasts. 3T3 cells expressing wild-type (lanes1, 6, and 11) or mutant H2b proteins (C1 mutant, lanes2, 7, and 12; C2 mutant, lanes3, 8, and 13; C3 mutant, lanes4, 9, and 14; C4 mutant, lanes5, 10, and 15) were pulse-labeled with 0.3 mCi/ml [S]Cys for 10 min (lanes 1-5) and then chased in unlabeled medium for 30 min (lanes 6-15). Where indicated, 5 mM DTT was added to the medium at the end of the chase period for 5 min (lanes 11-15). All samples were treated with 0.1 M iodoacetamide before the cells were lysed. Cell lysates were immunoprecipitated with an antiserum against the carboxyl terminus of the H2 protein, treated with N-glycanase to remove the sugar side chains, and then subjected to SDS-PAGE under non-reducing conditions. Shadedarrow indicates the less compact form of the protein. Stripedarrow indicates the more compact form of the protein.



The H2b C1 Mutant Is Expressed on the Cell Surface and, Together with the H1 Subunit, Can Form a Functional Receptor

The data from Fig. 3show that a similar fraction of the wild-type and C1 mutant H2b proteins becomes Golgi-processed. Previous studies showed that wild-type H2b protein is expressed on the cell surface of transfected fibroblasts(10) . This is confirmed in Fig. 6a, which shows binding of an antibody against the carboxyl terminus of the H2 protein to the surface of transfected fibroblasts expressing wild-type H2b protein. A similar immunolabeling experiment (Fig. 6b) shows that the C1 mutant protein is also detected on the cell surface of transfected fibroblasts. Fig. 6c shows that none of the C4 mutant protein can be detected on the cell surface of transfected fibroblasts. These results are consistent with the pulse-chase experiments in which no complex glycosylated forms of the C4 protein could be detected. Similar negative results were obtained for cells expressing the C2 and C3 mutants (data not shown).


Figure 6: Immunofluorescence localization of H2b wild-type and cysteine mutant proteins on the surface of transfected 3T3 fibroblasts. Live 3T3 cells expressing wild-type (a), C1 mutant (b), or C4 mutant (c) H2b protein were reacted at 4 °C first with a primary rabbit antibody against the carboxyl terminus of the H2 protein followed by a secondary Cy3-labeled goat anti-rabbit antibody. Cells were washed, fixed, and visualized by fluorescence microscopy.



A functional ASGP receptor requires both the H1 and H2 subunits, and it is possible that the C1 disulfide bond is required to form the functional receptor complex with H1. To determine if the H2b C1 mutant can form a functional receptor, we stably transfected cDNA encoding the H1 subunit into 3T3 fibroblasts expressing the wild-type or C1 mutant. Both cell lines showed specific and calcium-dependent binding to a well studied ligand for the ASGP receptor, human asialo-orosomucoid (data not shown), whereas cell lines expressing only the H2b wild-type or C1 mutant protein did not show any specific binding (data not shown). Fig. 7a shows the Scatchard plot of a binding study at 4 °C, employing cells expressing H1 and wild-type H2b, and radioiodinated asialo-orosomucoid. The results indicate a dissociation constant of 11 nM and about 600,000 surface receptors/cell. Fig. 7b shows the Scatchard plot of a similar binding study employing cells expressing H1 and C1 mutant H2b. The results indicate a binding constant of 9 nM and about 120,000 surface receptors/cell. These levels of expression are within the range of that exhibited by human hepatoma HepG2 cells (32) . The different levels of surface expression may be accounted for by different levels of H2 protein expression in the two cell lines used here. Pulse-labeling studies with the two cell lines show that they express equal levels of the H1 protein but that the wild-type H2b is expressed at about a 5-fold higher level than the C1 mutant (data not shown). However, both cell lines show specific ligand binding and similar binding affinities; the values are similar to those of wild-type receptors naturally expressed in cultured HepG2 cells(32) .


Figure 7: Saturation binding of I-asialo-orosomucoid to transfected 3T3 fibroblasts expressing H1 and wild-type H2b proteins (a) and H1 and C1 mutant H2b proteins (b). Cells were incubated with various concentrations of radiolabeled ligand for 2 h at 4 °C with or without excess unlabeled ligand. Total and nonspecific binding were measured as described under ``Experimental Procedures,'' and their difference, the specific binding, was analyzed by Scatchard plots. B, specific bound ligand; B/F, ratio of specific bound ligand to free ligand. K, dissociation constant.



Fig. 8(a and b) shows that both the cell lines expressing H1 and wild-type H2b and the cell lines expressing H1 and C1 mutant H2b are capable of endocytosis and degradation of asialo-orosomucoid. With both cell lines, cell-associated radioactivity plateaus after about 30 min of incubation at 37 °C and degradation products appear in the medium within 30 min. The rates of total ligand uptake and degradation are similar in both cell lines: 3.5 pg of ligand/min/µg of cell protein over 4 h at 37 °C. It is interesting that although the cell line expressing H1 and H2b C1 receptors has about 5-fold fewer surface receptors than one expressing H1 and wild-type H2b (as determined by the saturation binding experiments), they show a similar rate of ligand endocytosis and degradation. This may indicate that the turnover rate of the endocytic cycle of the mutant receptor is higher than that of the wild-type receptor.


Figure 8: Uptake and degradation of I-asialo-orosomucoid by transfected 3T3 fibroblasts expressing H1 and wild-type H2b proteins (a) and H1 and C1 mutant H2b proteins (b). Cells were incubated with 2 µg/ml radiolabeled ligand for various times at 37 °C. At each time point, the medium was analyzed for I degradation products and the cell-associated I was also determined as described under ``Experimental Procedures.''



We conclude that the absence of the C1 disulfide bond in the H2b protein does not prevent its transport to the cell surface; it can form a functional receptor when co-expressed with the H1 subunit. This receptor binds to a natural ligand, asialo-orosomucoid, with an affinity similar to that of the wild-type receptor. The mutant receptor can also perform its natural functions of receptor-mediated endocytosis, ligand degradation, and presumably receptor recycling. Therefore, it is unlikely that the these two cysteines are required for the formation of a functional receptor complex.


DISCUSSION

A large fraction of the ASGP receptor H2b subunit expressed in transfected fibroblasts in the absence of the H1 subunit is degraded in the ER. Depletion of calcium ions in the ER causes all of H2b to be retained in a misfolded state in the ER and eventually is degraded there(1) . When cells expressing the H1 subunit are depleted of calcium ions or treated with DTT, all of the H1 protein is also retained in and degraded in the ER(19) . Studies on the T-cell receptor subunits also showed that the ER degradation process is enhanced in the presence of DTT (38) or depletion of calcium ions(39) , conditions that prevent proper folding of proteins. Therefore, proper folding and formation of disulfide bonds in secretory or membrane proteins are normally required for transport out of the ER.

As we expected, three of the disulfide bonds (C2, C3, and C4) in the H2b protein are essential for proper folding of the protein as absence of any one of them prevents transport of the protein out of the ER to the Golgi and all of the newly made protein is degraded in a pre-medial Golgi compartment. As judged by non-reducing SDS-PAGE and sensitivity to DTT, the C2, C3, and C4 mutants cannot fold as well as the wild-type protein, as only the wild-type protein can attain resistance to DTT unfolding (although only a fraction of it can do so). These observations confirm the previous deduction that only folded forms of the H2b protein can exit the ER to the cell surface(1) . Presumably, a folded form of the H2b protein that is resistant to DTT unfolding, a conformation attained by a fraction (20%) of the wild-type protein, is the prerequisite for ER to Golgi transport and appearance on the plasma membrane. An interesting result from these assays is that the C3 mutant cannot attain any compact structure after a 30-min chase period, whereas the C2 and C4 mutants can attain some compact structure(s), although these are all sensitive to DTT unfolding (Fig. 5). This suggests that the C3 disulfide bond plays a more important role in attaining or maintaining a folded structure of the H2b protein than do the C2 and C4 disulfide bonds, although the C3 disulfide bond is formed by two cysteines that are very close in amino acid sequence positions (Cys and Cys) relative to the C2 and C4 disulfide bonds.

Surprisingly, mutation of the two cysteines nearest to the transmembrane region to alanine residues does not prevent maturation of the protein to the cell surface. In contrast, the mutant protein is folded more rapidly than the wild-type, as all of the mutant protein that attains a compact structure (as judged by mobility on non-reducing SDS-PAGE) within 30 min is resistant to unfolding by DTT, whereas less than half of the compact form of the wild-type protein is resistant to DTT unfolding. Furthermore, the mutant protein is processed more rapidly than the wild-type in that it is transported to the Golgi complex more quickly. However, the proportion of the mutant protein that becomes Golgi-processed (20%) is about the same as that of the wild-type. The portion of the mutant H2b protein that remains in the ER is degraded at a higher rate than the wild-type.

We speculate that, when present, the C1 cysteines cause the protein to be misfolded and to be bound by chaperones in the ER such as protein disulfide isomerase or BiP (heavy chain binding protein). The binding would cause the protein to remain in the ER for longer periods, during which a fraction of them may refold to form the native disulfide bonds after which the protein is exported to the Golgi complex. As a result, ER to Golgi transport of the protein is delayed and degradation in the ER is also slowed down. In the C1 mutant, these cysteines are absent. The misfolded proteins would not be formed, and the protein would not bind chaperones. Therefore, the C1 mutant would fold faster and exit the ER quicker, while the fraction that is unable to fold would remain in the ER and would be quickly degraded. This hypothesis indicates that binding of H2 to certain chaperones might delay both folding and ER degradation. It is consistent with our observation that the C2, C3, and C4 mutant proteins are degraded at the same rate as the fraction of the wild-type that remains in the ER, as all these proteins would be misfolded and bound to chaperones. We have no direct evidence for this hypothesis, as we have not been able to detect binding of specific chaperones to the wild-type or mutant H2b proteins.

We have demonstrated previously that degradation of the H2 subunit in the ER occurs via two pathways, one which involves an initial cleavage of the protein (most likely by signal peptidase) to form a 35-kDa carboxyl-terminal fragment as a degradation intermediate, and another pathway which does not go through this intermediate. The pathway that is not dependent on the formation of the 35-kDa intermediate is inhibited by the protease inhibitors TLCK or TPCK and is probably the major pathway(25) . In the presence of TLCK or TPCK, unfolded forms of the H2b protein accumulate in the ER, therefore suggesting that it is the unfolded forms of H2b that are degraded in the ER(1) . The degradation of the C1 mutant protein in the ER is also inhibited in the presence of TLCK or TPCK(^2); this is consistent with the notion that the unfolded fraction of the C1 mutant protein that remains in the ER is degraded via the major TLCK and TPCK-sensitive degradation pathway. Relative to wild-type H2b, more of the 35-kDa carboxyl-terminal fragment was produced from the C2, C3 and C4 mutants. Its higher abundance may indicate that a larger fraction of each of these mutants is degraded via the pathway that goes through this intermediate rather than the pathway that does not involve cleavage to the 35-kDa fragment.

We find that the H2b C1 mutant protein is capable of forming a functional receptor when co-expressed in fibroblasts with the wild-type H1 subunit. The H1/H2b C1 mutant receptor binds a natural ligand with an affinity similar to that of the wild-type receptor and is capable of catalyzing ligand endocytosis and degradation. The rate of endocytosis and recycling of the mutant receptor actually appears to be slightly higher than the wild-type. This may indicate that the mutant protein exhibits a higher rate of turnover in the secretory/endocytic pathway. Another possibility is that access to the coated-pits is the rate-limiting step for rate of endocytosis and that this process is saturated at low numbers of receptors. Therefore, the rate of endocytosis and recycling would be similar for the wild-type and the C1 mutant even though they have a 5-fold difference in the number of cell-surface receptors. Importantly, the two C1 cysteines in the H2 protein are not required for proper receptor function. In contrast, studies on the influenza hemagglutinin have shown that all the disulfide bonds are required for proper folding and secretion of the protein(20) . However, when certain cysteine residues are mutated in the beta subunit of human chorionic gonadotropin (40) and human lysozyme (41) , the rate of secretion is enhanced. Our study also demonstrates that loss of a disulfide linkage can lead to enhanced folding and formation of a fully functional protein.

The presence of the C1 cysteines in the wild-type ASGP receptor H2 subunit may hinder protein folding and processing. That they have been selected in evolution suggests that higher efficiency in folding and processing may not always be advantageous. Perhaps the presence of the C1 cysteines serves to reduce the turnover rate of a receptor whose expression need not be highly regulated. Nevertheless, it would be interesting to study the role of the C1 cysteines during the folding of the protein by determining and comparing the folding pathways of the wild-type and C1 mutant proteins.


FOOTNOTES

*
This work was supported by Grant CDR 88-03014 from the National Science Foundation to the Massachusetts Institute of Technology Biotechnology Process Engineering Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142. Tel.: 617-258-5216; Fax: 617-258-9872.

(^1)
The abbreviations used are: ASGP, asialoglycoprotein; ER, endoplasmic reticulum; DTT, dithiothreitol; SHPP, N-succinimidyl-3-(4-hydroxyphenyl)propionate; Cy3, cyanine dye Cy3.18; Endo H, endoglycosidase H; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; TLCK, N-tosyl-L-lysine chloromethyl ketone; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone.

(^2)
M. H. Yuk and H. F. Lodish, unpublished observations.


ACKNOWLEDGEMENTS

We thank R. Lin and D. Hirsch for critical reading of the manuscript and all of the members of the Lodish laboratory for their support and encouragement.


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