Functional Contributions of Noncysteine Residues within the Cystine Knots of Human Chorionic Gonadotropin Subunits*

Ryan J. DarlingDagger §||||, Jason A. WilkenDagger , Amanda K. Miller-LindholmDagger ||, Teresa M. UrlacherDagger **, Raymond W. RuddonDagger §DaggerDagger, Simon A. ShermanDagger , and Elliott BedowsDagger §§§¶¶

From the Dagger  Eppley Institute for Research in Cancer and Allied Diseases, § Department of Pharmacology,  Department of Biochemistry and Molecular Biology, and the §§ Department of Obstetrics and Gynecology, University of Nebraska Medical Center, Omaha, Nebraska 68198

Received for publication, November 8, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human chorionic gonadotropin (hCG) is a heterodimeric member of a family of cystine knot-containing proteins that contain the consensus sequences Cys-X1-Gly-X2-Cys and Cys-X3-Cys. Previously, we characterized the contributions that cystine residues of the hCG subunit cystine knots make in folding, assembly, and bioactivity. Here, we determined the contributions that noncysteine residues make in hCG folding, secretion, and assembly. When the X1, X2, and X3 residues of hCG-alpha and -beta were substituted by swapping their respective cystine knot motifs, the resulting chimeras appeared to fold correctly and were efficiently secreted. However, assembly of the chimeras with their wild type partner was almost completely abrogated. No single amino acid substitution completely accounted for the assembly inhibition, although the X2 residue made the greatest individual contribution. Analysis by tryptic mapping, high performance liquid chromatography, and SDS-polyacrylamide gel electrophoresis revealed that substitution of the central Gly in the Cys-X1-Gly-X2-Cys sequence of either the alpha - or beta -subunit cystine knot resulted in non-native disulfide bond formation and subunit misfolding. This occurred even when the most conservative change possible (Gly right-arrow Ala) was made. From these studies we conclude that all three "X" residues within the hCG cystine knots are collectively, but not individually, required for the formation of assembly-competent hCG subunits and that the invariant Gly residue is required for efficient cystine knot formation and subunit folding.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The cystine knot motif defines a superfamily of dimeric proteins and appears to function as a structural scaffold that stabilizes the 3-loop structures of the individual subunits (1). As shown in Fig. 1, the cystine knot consists of three disulfide (S-S)1 bonds; two of these bonds bridge adjacent polypeptide strands, creating a ring that includes the intervening polypeptide backbone, and the third bond penetrates this ring (1, 2). The cystine knot is common to a biologically diverse set of dimeric proteins including transforming growth factor-beta , vascular endothelial growth factor, platelet-derived growth factor, and human chorionic gonadotropin (hCG) (1-4). Additionally, more than 30 other proteins are predicted to contain this motif (2). The functional importance of the cystine knots of hCG (5-9), transforming growth factor-beta 1 (10), and platelet-derived growth factor (11) is evident from studies where cysteine residues within the knot were mutated, thus preventing a particular S-S bond from forming. Disruption of the cystine knot disulfides results in the synthesis of nonfunctional proteins that are usually degraded intracellularly. Thus, a more detailed understanding of the functional components of cystine knots will help to understand how members of this emerging protein family fold and assemble into biologically active molecules.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1.   Schematic diagram of hCG subunits. GPH-alpha and hCG-beta each contain 3 loops (labeled L1, L2, and L3) and are arranged in a head-to-tail orientation in the hCG dimer (17, 18). Both subunits also contain a cystine knot formed by three S-S bonds (displayed as lines connecting the solid circles; numbers indicate the cysteine residue number). Noncystine knot S-S bonds are represented by the lines connecting open circles. The noncysteine residues of the cystine knots are labeled X1, X2, and X3. A particularly interesting feature of the hCG dimer is the C-terminal region of hCG-beta that wraps around the GPH-alpha L2, creating the so-called "seat-belt."

The subunits of hCG are prototypes for the cystine knot growth factor family (1). Heterodimeric hCG forms when hCG-beta assembles with the common glycoprotein hormone alpha -subunit (GPH-alpha ). GPH-alpha also assembles with luteinizing hormone beta -subunit, thyroid-stimulating hormone beta -subunit (TSH-beta ), and follicle-stimulating hormone beta -subunit to form luteinizing hormone, TSH, and follicle-stimulating hormone beta -subunit, respectively (12). Luteinizing hormone-beta , TSH-beta , and follicle-stimulating hormone beta  also contain the consensus residues required to form a cystine knot, but the actual presence of the knot has not yet been confirmed by structural studies.

Previously, we described the folding pathways of both hCG-beta (9, 13-15) and GPH-alpha (6, 16) using S-S bond formation as an index of folding. The methods established in our laboratory for the folding of these two subunits now allow us to determine the importance of noncysteine residues of the cystine knot in hCG subunit folding, secretion, and assembly. Fig. 1 illustrates the location of the cystine knot motifs for both subunits of hCG, as well as the noncystine knot disulfides (17, 18). The 8-residue ring of the cystine knot consists of four cysteines that form two S-S bridges, a Gly residue common to all 8-membered cystine knot rings, and three nonconserved residues (termed X1, X2, and X3). Thus, the consensus sequences for this motif can be defined as C-X1-G-X2-C and C-X3-C (3, 19).

The residues at the X1, X2, and X3 positions vary among cystine knot-containing proteins and their functional importance is largely unknown. The sequence containing X1 and X2 in all four glycoprotein beta -subunits is CAGYC. The equivalent sequence in GPH-alpha is CMGCC, where the Cys at the X2 position forms a noncystine knot S-S bond with Cys7 (Fig. 1). In this report, we investigated the contribution of hCG X1, X2, and X3 residues in folding, secretion, and assembly by employing a chimeric strategy where the residues within the hCG-beta and GPH-alpha cystine knots were interchanged individually or collectively. Furthermore, the role of the intervening Gly residue in both cystine knots was determined using various amino acid substitutions. We report that: (i) there is a subunit-specific complement of three "X" residues, all of which are needed for efficient assembly and (ii) the presence of the central invariant Gly is an absolute requirement for efficient folding, cystine knot formation, and hCG assembly.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- 293T cells (20) were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, and penicillin (100 units/ml)/streptomycin (100 µg/ml) (Life Technologies, Inc.).

Site-directed Mutagenesis-- Mutations were made using a "megaprimer" polymerase chain reaction methodology (21) with Pfu polymerase (Stratagene). The GPH-alpha polymerase chain reaction products were cloned into pcDNA3 (Invitrogen) and the hCG-beta polymerase chain reaction products were cloned into pGS (9). DNA sequencing confirmed incorporation of desired mutations. Plasmid DNA was purified using the Maxi Plasmid Kit (Qiagen) according to the manufacturer's protocol and used for transient transfection as described below.

Transient Transfection-- 293T cells (1.9 × 106) were plated into 60-mm plastic dishes and grown overnight to 70-80% confluency. Plasmid DNA was precipitated as described previously (6). For coexpression of hCG-beta and GPH-alpha , both plasmids were included in the precipitation. To obtain comparable expression levels in coexpression studies, a 40:1 GPH-alpha to hCG-beta ratio of plasmid was used. The resulting precipitate was added dropwise to the dishes and agitated gently to mix. To ensure a uniform precipitate exposure, one large-scale precipitation was distributed equally among all dishes. Cells were incubated for 2 days at 37 °C and used for metabolic labeling.

Metabolic Labeling with [35S]Cysteine-- Transiently transfected 293T cells were pulse-labeled for the times indicated in the text with L-[35S]cysteine (~1100 Ci/mmol; PerkinElmer Life Sciences), at a concentration of 100-150 µCi/ml, in serum-free medium lacking cysteine (9). For experiments using dithiothreitol (DTT), the DTT was added with the [35S]cysteine at a final concentration of 2.0 mM. Pulse incubations were carried out as described previously (13) and cells were incubated for the chase times indicated; the chase medium was saved for secretion studies. Cells were harvested by rinsing with cold phosphate-buffered saline and immediately lysed in 2.5 ml of phosphate-buffered saline containing detergents (1.0% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS), protease inhibitors (20 mM EDTA and 2 mM phenylmethanesulfonyl fluoride), and 5 mM N-ethylmaleimide (NEM), pH 7.0, or 50 mM iodoacetate, pH 8.0, to trap free thiols in folding intermediates that contained unformed S-S bonds. NEM was used for GPH-alpha because it results in efficient alkylation of GPH-alpha thiols and better separation of folding intermediates by HPLC (6). Similarly, iodoacetate was used for hCG-beta because it efficiently alkylates beta -subunit thiols and facilitates mapping of hCG-beta tryptic peptides (13, 14). Cell lysates were incubated for 10-20 min at room temperature in the dark, followed by disruption through a 22-gauge needle (5 times) and centrifuged for 1 h at 100,000 × g. The collected chase medium was also clarified by centrifugation.

Immunoprecipitation of hCG Subunits from Cell Lysates and Chase Media-- Immunoreactive forms of GPH-alpha or hCG-beta were immunoprecipitated with polyclonal antibodies specific for each respective subunit (6, 22). Immunoprecipitations were carried out at 4 °C for 16-20 h with rotation in the dark. Immune complexes were precipitated with protein A-Sepharose (Sigma) and prepared for SDS-polyacrylamide gel electrophoresis (PAGE) or reversed-phase HPLC as described below.

SDS-PAGE and Quantitation of [35S]Cysteine-labeled Subunits-- Radiolabeled folding forms that adsorbed to protein A-Sepharose beads were prepared as described previously (13). Briefly, protein A-Sepharose beads containing immunopurified subunits were resuspended in twice concentrated SDS gel sample buffer (125 mM Tris-HCl, pH 6.8, containing 2% SDS, 20% glycerol, and 40 µg/ml bromphenol blue). For reducing conditions, beta -mercaptoethanol was included at a final concentration of 2%. Samples were boiled for 5 min, loaded on polyacrylamide gradient slab gels (5-20%), and run by the method of Laemmli (23). Gels were dried in vacuo on filter paper and exposed to a phosphorscreen (Molecular Dynamics). The phosphorscreen was scanned on a Molecular Dynamics Storm 860 and bands were quantitated using the Molecular Dynamics ImageQuant (version 5.0) volume report. To determine the percentage of GPH-alpha that had combined with hCG-beta in Figs. 3 and 7, the following formula was used: [10/12 (amount of hCG-beta that coimmunoprecipitated with GPH-alpha ) + (amount of GPH-alpha in anti-beta immunoprecipitation)]/[total GPH-alpha ]. For the alpha -C31Y mutant, 8/12 was used instead of 10/12 because this mutant has only 8 cysteine residues.

Reversed-phase HPLC Analysis-- Radiolabeled folding intermediates that adsorbed to protein A-Sepharose beads were prepared as described previously (13). Briefly, protein A-Sepharose beads/antibody-antigen immunocomplexes were washed three times with phosphate-buffered saline containing detergents (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) followed by four washes with phosphate-buffered saline lacking detergents. Immunocomplexes were pelleted between washes by centrifugation for 1 min at 2000 × g. To dissociate the Sepharose/antibody/antigen interactions, immunocomplexes were treated with 6 M guanidine HCl, pH 3.0 (sequenal grade; Pierce), for 16-20 h while rotating at room temperature. 100 µg of myoglobin was added as a carrier. The guanidine eluates were injected onto a Vydac 300-Å C4 reversed-phase column equilibrated with 0.1% trifluoroacetic acid and eluted using an acetonitrile gradient as described previously (14). Fractions were collected in 1-min intervals and quantitated by scintillation counting. Samples were stored at -20 °C until further characterization.

Tryptic Digestion and Reversed-phase HPLC Analysis of Tryptic Peptides-- HPLC fractions from C4 reversed-phase HPLC representing hCG-beta folding intermediates pbeta 1 or pbeta 2 were pooled, concentrated, and digested for 16-20 h in silanized polypropylene tubes containing 100-200 µg of myoglobin, 0.03% trypsin (Sigma), 5 mM CaCl2, and 50-150 mM Tris-HCl, pH 8. Digestions were continued for 2 h with two additional aliquots of 25 µg of trypsin (0.06% final concentration) (13, 14). hCG-beta tryptic peptides were separated on a Vydac C18 reversed-phase column as described previously (14). Amino acid sequencing was used previously to identity the peptide(s) in each peak (14).

Amino Acid Analysis Procedure to Determine S-S Bond Content-- A modified protocol, similar to the one used in determining the S-S folding pathways for potato carboxypeptidase inhibitor and human epidermal growth factor (24, 25) was used. [35S]Cysteine-containing folding forms isolated from reversed-phase HPLC were dried in vacuo and hydrolyzed as described previously (6). Quantitation of [35S]cystine and succinyl-[35S]cysteine (hydrolysis product of NEM-Cys) was performed using a modification of the method described by Cohen and Michaud (26). Hydrolysates were resuspended in 10 µl of 10 mM HCl. To this, 70 µl of 0.2 N borate buffer, pH 8.8, was added. Derivatization of amino acids was performed by adding 30 µl of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (8 mg/ml in anhydrous acetonitrile). Samples were dried in vacuo. Before injection, samples were resuspended in 110 µl of buffer A (140 mM sodium acetate, 17 mM triethylamine, pH 4.9). Derivatized amino acids were separated by HPLC as described previously (6) using buffer A and buffer B (60% acetonitrile in water). The column was eluted at 1.0 ml/min at 30 °C and 1-ml fractions were collected and quantitated by scintillation counting. Recovery of [35S]cystine represented S-S bonded cysteine residues, whereas succinyl-[35S]cysteine represented cysteine residues of unformed S-S bonds. The percentage of [35S]cystine and succinyl-[35S]cysteine was calculated by dividing the counts/min recovered for each species by the total counts/min recovered. Fully folded [35S]cysteine-labeled hCG-beta was used as a positive control for [35S]cystine content.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Swapping of hCG-beta and GPH-alpha Cystine Knot Motifs-- The only conserved sequences in cystine knot-containing proteins that have an 8-membered ring are the C-X1-G-X2-C and C-X3-C sequences (3, 19). The contribution that the X residues make toward attaining a native conformation is unknown. Furthermore, it is not known whether cystine knot sequences are protein-specific, or whether the residues of the knot motif are functionally interchangeable. To address this, we used a chimeric strategy wherein cystine knot residues of GPH-alpha and hCG-beta were swapped singly or collectively.

Swapping of hCG cystine knot motifs was accomplished by site-directed mutagenesis at the X1, X2, and X3 positions to match the residue(s) of the other subunit. Three single GPH-alpha mutants (alpha -M29A, alpha -C31Y, and alpha -H83Q) and a GPH-alpha mutant containing all three mutations (termed alpha beta knot) were constructed (Table I). Thus, the amino acid sequence of the alpha beta knot cystine knot matched that of hCG-beta . Cys7 (which normally pairs with Cys31) was also changed to Ala in the alpha -C31Y mutant to prevent a free thiol at Cys7 from causing S-S rearrangements or retention of the subunit in the endoplasmic reticulum (27). Importantly, removal of S-S bond 7-31 does not affect GPH-alpha folding, secretion, or hCG biological activity (6, 7). In addition, the hCG-beta cystine knot residues were replaced with those of GPH-alpha to give beta -A35M, beta -Y37A, beta -Q89H single mutants and a beta alpha knot triple mutant (Table I). beta -Y37 was replaced with Ala instead of Cys to avoid introduction of a free thiol for the reasons alluded to above. Ala was selected because its side chain properties closely resemble those of Cys, and because Ala and Ser substitutions have similar effects on hCG-beta folding and assembly when they are substituted for Cys (8).


                              
View this table:
[in this window]
[in a new window]
 
Table I
GPH-alpha and hCG-beta cystine knot chimeras
Three single mutants and a triple mutant were constructed for GPH-alpha such that the residues at the X1, X2, and X3 positions matched that of hCG-beta . Additionally, three single mutants and a triple mutant were constructed for hCG-beta such that the residues at the X1, X2, and X3 positions matched that of GPH-alpha . The residues within the boxes represent those that were changed by site-directed mutagenesis.

The six single mutants (three GPH-alpha and three hCG-beta ), two triple mutants (alpha beta knot and beta alpha knot), and wild type (WT) subunits were analyzed for proper folding as described previously (6, 13, 14, 16). Briefly, GPH-alpha folding was monitored using S-S bond formation and HPLC elution times (6, 16), while hCG-beta folding was monitored by shifts in migration on nonreducing SDS-PAGE (14, 22). No significant differences in folding were observed for any of the mutants (data not shown), suggesting that these cystine knots may be interchangeable. To test this further, we determined the efficiency of subunit secretion. Secretion was assayed by pulse labeling transiently transfected 293T cells for 10 min, followed by a 10-min or 8-h chase. The immunopurified cell lysates and media were analyzed by SDS-PAGE (Fig. 2A), and the bands were quantitated as described under "Experimental Procedures." The percent secretion for the GPH-alpha and hCG-beta chimeras are shown in Fig. 2, B and C. Consistent with previous studies, about 80% of WT GPH-alpha and hCG-beta were secreted by 8 h. Furthermore, swapping of single residues or the entire cystine knot did not significantly affect subunit secretion of GPH-alpha or hCG-beta (Fig. 2). This efficient secretion is another indicator that these subunits folded to a native or native-like conformation, since misfolded or incompletely folded hCG subunits are generally retained intracellularly and degraded (5, 6).



View larger version (59K):
[in this window]
[in a new window]
 
Fig. 2.   Secretion of hCG-beta and GPH-alpha cystine knot chimeras. 293T cells expressing WT or cystine knot chimeric hCG subunits were pulse-labeled with [35S]cysteine and chased for 10 min or 8 h. Immunopurified subunits were analyzed by reducing SDS-PAGE and quantitated. A, secretion of beta -WT, beta alpha knot, alpha -WT, and alpha beta  knot. The lysate (L) from the 10-min chase represents the total intracellular [35S]cysteine-labeled protein. The amount of radioactivity in this band was used to calculate the percentage of protein that was secreted into the medium (M) after 8 h. By 8 h, <5% of total protein remaining intracellular (data not shown), indicating that secretion of the radiolabeled protein was essentially complete. SDS-PAGE analysis of the six single mutants was comparable to WT and the triple mutants (gels not shown). The slower mobility of the secreted subunits is due to the addition of O-linked oligosaccharides (38) and/or differential processing of N-linked oligosaccharides (39). B and C, quantitation of the percentage secreted for the respective hCG-beta and GPH-alpha WT subunits (solid bars), composite chimeras (open bars), and single mutants (shaded bars). The percentage secreted is defined as the amount of 35S-labeled subunit recovered in the medium after 8 h relative to that present at 10 min intracellular. Each bar represents the mean ± S.D. of at least three experiments.

Finally, we assayed for the ability of the GPH-alpha cystine knot chimeras to assemble with beta -WT and, conversely, for the ability of hCG-beta cystine knot chimeras to assemble with alpha -WT. To do this, 293T cells coexpressing both subunits were pulse-labeled and chased for 8 h. Unassembled GPH-alpha and intact hCG alpha /beta dimer were first precipitated from the collected medium with a polyclonal alpha -antiserum. This step was followed by a second precipitation with a polyclonal beta -antiserum to recover any excess, unassembled hCG-beta . There was a dramatic decrease in the ability of alpha beta knot and beta alpha knot to combine with beta -WT and alpha -WT, respectively (Fig. 3, A and B). Furthermore, this decreased combination was not due to altering any one residue, as all of the single mutations were significantly less deleterious than the triple mutations. However, the single mutations at the X2 position (alpha -C31Y and beta -Y37A) did decrease assembly to a level intermediate to that of WT and the triple mutants. Co-transfection of alpha beta knot and beta alpha knot together resulted in <5% assembly (data not shown). Taken together, these data indicate that the noncysteine residues within the GPH-alpha and hCG-beta cystine knots contribute to an assembly-competent conformation in a subunit-specific manner. Furthermore, this contribution appears to be a function of the set of all noncysteine residues, as opposed to arising from the contribution of a single residue.



View larger version (54K):
[in this window]
[in a new window]
 
Fig. 3.   Assembly of hCG-beta and GPH-alpha cystine knot chimeras. 293T cells expressing WT GPH-alpha and hCG-beta (WT or cystine knot chimeras) (panel A) or WT hCG-beta and GPH-alpha (WT or cystine knot chimeras) (panel B) were pulse-labeled with [35S]cysteine and chased for 8 h. The medium was collected and immunoprecipitated with polyclonal antibody to GPH-alpha . This immunoprecipitation pulls down unassembled and assembled GPH-alpha and co-precipitates hCG-beta combined with GPH-alpha . Shown above each bar is a representative SDS-PAGE gel. The remaining unassembled hCG-beta was immunoprecipitated with anti-beta to ensure that there was excess hCG-beta such that it was not a limiting factor in assembly (data not shown). In some cases, a portion of the total GPH-alpha was present in the anti-beta immunoprecipitation and was included in calculating the percentage of combined GPH-alpha . Quantitation was performed as described under "Experimental Procedures" and is shown in the bar graphs for WT subunits (solid bars), composite chimeras (open bars), and single mutants (shaded bars). In both A and B, the results are displayed as the percentage of total secreted GPH-alpha that had dimerized with hCG-beta . Each bar represents the mean ± S.D. of at least three experiments.

The Central Gly Residue in the hCG-beta Cystine Knot Is Critical for Folding, Secretion, and Assembly-- As alluded to above, the central Gly residue in hCG-beta is conserved among all known cystine knots that contain an 8-membered ring structure (1, 2). A mutation resulting in the conversion of the central Gly residue to Arg in TSH-beta causes congenital isolated TSH deficiency, wherein the mutant TSH-beta fails to be secreted and assemble with GPH-alpha (28). This demonstrates that the central Gly of the CAGYC region is critical for TSH activity and suggests that it may also be essential to the function of the other glycoprotein hormones.

Knowledge of the hCG-beta folding pathway (13, 14) provides a novel system to determine the contribution that residues of the cystine knot make in attaining an assembly-competent conformation. To determine whether substitution of the Gly of the CAGYC region of hCG-beta alters folding, secretion, and/or assembly, we created and analyzed three Gly mutants: beta -G36A, beta -G36N, and beta -G36R. Mutation to Arg was chosen because this mutation is observed in the naturally occurring TSH-beta mutant (28), G36N was chosen because Asn has a smaller neutral side chain in comparison with the positively charged Arg, and G36A was chosen because it is the most conservative change possible; however, Ala in most cases can adopt the required positive phi  torsion angle only under conditions of unfavorable steric hindrance (29). 293T cells expressing beta -WT, beta -G36N, beta -G36R, or beta -G36A were pulse-labeled with [35S]cysteine and chased for 0, 5, 15, 30, 60, 120, or 480 min. Fig. 4A shows the progression of beta -WT from pbeta 1 (the earliest detectable folding intermediate) to pbeta 2 to mature beta . At chase times >= 60 min, beta -WT was detectable in the media as mature, secreted beta . Fig. 4B shows that most of beta -G36A did not progress beyond pbeta 1 and was not secreted. However, a pbeta 2-like species of beta -G36A was isolated by reversed-phase HPLC (Fig. 4C). This species was termed "pbeta 2-like" since it eluted from HPLC at a similar time to that of WT pbeta 2 (13, 14). Folding and secretion data for beta -G36N and beta -G36R subunits yielded similar results to those shown in Fig. 4 (not shown).



View larger version (66K):
[in this window]
[in a new window]
 
Fig. 4.   SDS-PAGE analysis of WT hCG-beta and beta -G36A folding and secretion. 293T cells transiently expressing WT hCG-beta or beta -G36A were pulse-labeled for 5 min and chased for 0-480 min. Folding intermediates were immunoprecipitated with a polyclonal hCG-beta antibody from the cell lysate (intracellular) and the medium (secreted) and analyzed by nonreducing SDS-PAGE. A, SDS-PAGE analysis of WT hCG-beta . Note the pbeta 1 right-arrow pbeta 2 right-arrow beta  progression as a function of time. Mature hCG-beta (beta ) was primarily secreted and was present in the medium beginning at the 60-min chase. B, SDS-PAGE analysis of beta -G36A. Only a small portion of beta -G36A converted from pbeta 1 to pbeta 2. Furthermore, beta -G36A was not secreted, as evidenced by the lack of bands present in the 30-480 min secreted material. The lack of any significant radiolabeled intracellular or secreted beta -G36A at 480 min indicates that beta -G36A was almost entirely degraded by this time. C, C4 reversed-phase HPLC of beta -G36A showing the presence of a pbeta 2-like material that eluted at 30-40 min. This peak was designated as pbeta 2-like because it elutes at a similar position to that of WT pbeta 2 (14). The left-most lane in A and B contain carbonic anhydrase (Mr = 29,000).

Tryptic digestion of fully folded native hCG-beta (i.e. all native S-S bonds formed) produces [35S]cysteine-labeled peptides, all of which are linked by S-S bonds (13, 14). If a particular S-S bond has not yet formed in a given intermediate, then digestion with trypsin results in the release of a [35S]cysteine-containing peptide from the S-S-linked core material (13, 14). Thus, HPLC separation of released peptides from the S-S-linked peptides identifies the S-S bonds of a given hCG-beta folding intermediate that are unformed. Fig. 5, A and C, show the respective reversed-phase HPLC profiles of trypsin-treated WT hCG-beta that eluted from HPLC at the positions of pbeta 1 and pbeta 2. As defined in previous studies (13, 14), the release of peptides 9-20, 69-74, 87-94, 96-104, and 105-114 from G36A pbeta 1 (Fig. 5B) indicates that Cys10, Cys72, Cys90, Cys93, Cys100, and Cys110, respectively, were not part of a S-S bond. Additionally, unidentified peaks were present (labeled with an asterisk in Fig. 5, B and D), which suggest the presence of non-native S-S bonds. Peptides 9-20 and 87-94 were not present in the WT hCG-beta pbeta 2 tryptic profile (Fig. 5C), consistent with our previous report that the cysteines in these peptides are involved in S-S bonds in the pbeta 2 intermediate (14). The tryptic profile of beta -WT pbeta 2 clearly differed from that of beta -G36A pbeta 2-like (Fig. 5, compare C and D), suggesting that non-native S-S bonds had formed in beta -G36A. Furthermore, unlike WT-beta , the 87-94 and 9-20 peptides (Fig. 5D, peaks 3 and 4, respectively) were recovered in G36A pbeta 2-like digested material, indicating that S-S bonds 38-90 and 9-57 had not formed completely. The tryptic map of G36A pbeta 2-like material was similar to G36A pbeta 1 (Fig. 5, compare B and D), demonstrating that the material that eluted from HPLC at the pbeta 2 locus (i.e. pbeta 2-like) more closely resembled pbeta 1 than the more folded pbeta 2. Tryptic maps of beta -G36N and beta -G36R revealed that non-native S-S bonds had similarly formed (data not shown).



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 5.   Tryptic analysis of WT and beta -G36A pbeta 1 and pbeta 2 folding intermediates. WT and beta -G36A pbeta 1 and pbeta 2 species were isolated from reversed-phase HPLC and fractions representing each species were pooled and concentrated in vacuo. The isolated species were digested with trypsin to release peptides not linked by S-S bonds from the otherwise S-S linked core. The resulting mixture of peptides and S-S linked peptides were separated by C18 reversed-phase HPLC. A, tryptic map of WT pbeta 1. B, tryptic map of beta -G36A pbeta 1. C, tryptic map of WT pbeta 2. D, tryptic map of beta -G36A pbeta 2-like. The identities of the peaks (13, 14) are: peak 1a and 1b, peptide 96-104; peak 2, peptides 69-74 and 105-114; peak 3, peptide 87-94; peak 4, peptide 9-20; peak 5, S-S-linked peptides. Peaks marked with an asterisk are peaks that appear to represent S-S-linked peptides connected by non-native S-S bonds (13, 14).

Taken together, these data indicate that the central Gly of the hCG-beta CAGYC region is critical for the proper formation of S-S bonds and thus, is important for its folding and secretion. This also implies that the Gly right-arrow Arg mutation observed in congenital isolated TSH deficiency (28) results from improper folding of TSH-beta , which prevents its assembly with GPH-alpha .

Mutation of the Invariant GPH-alpha Cystine Knot Gly Residue-- The previous section demonstrated that the Gly residue in the CAGYC sequence of hCG-beta is critical for proper folding. This implies that this Gly may be critical for the folding of other cystine knot-containing proteins as well. To test this, we made the equivalent G30A mutation in the CMGCC sequence of GPH-alpha .

Unlike hCG-beta folding intermediates, GPH-alpha intermediates do not migrate differently on SDS-PAGE (6). However, GPH-alpha folding can be monitored by changes in reversed-phase HPLC elution times; unfolded GPH-alpha containing no S-S bonds and being more hydrophobic, elutes later than the native conformation and folding intermediates (6, 16). Moreover, HPLC elution position correlates with the formation of S-S bonds as GPH-alpha folds to its less hydrophobic conformation (i.e. earlier eluting species contain more S-S bonds than the later eluting, less folded species) (6, 16). Shown in Fig. 6 are the HPLC profiles generated for alpha -G30A after a 10-min pulse in the presence of DTT, followed by 0-, 5-, and 30-min chases after DTT removal. DTT was used in the pulse to delay the formation of S-S bonds until its removal during the chase (6). alpha -G30A folding did not generate a species that eluted at the position of native alpha -WT (Fig. 6, vertical dotted line). Additionally, a late eluting peak (Fig. 6, peak *) was observed at all chase times. This peak migrated at a relative molecular weight of about twice that of alpha -WT, G30A-alpha 1, and G30A-alpha 2 when analyzed by nonreducing SDS-PAGE (data not shown). This suggests that some of alpha -G30A had formed a homodimer. In contrast, reduction of S-S bonds before SDS-PAGE resulted in migration at the molecular weight of monomeric GPH-alpha . These data suggest that a significant proportion of alpha -G30A forms dimers that arise from non-native, intermolecular S-S bonds.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 6.   HPLC analysis of alpha -G30A folding intermediates. 293T cells expressing alpha -G30A were pulse-labeled with [35S]cysteine in the presence of 2 mM DTT and chased for 0, 5, or 30 min after DTT removal. The immunoprecipitated folding forms were separated by reversed-phase HPLC and the amount of [35S]cysteine-labeled alpha -G30A in each 1-min fraction was quantitated. Reducing and nonreducing SDS-PAGE were used to demonstrate that the peak marked with an asterisk (*) represents alpha -G30A aggregates that were mostly S-S linked homodimers with some multimeric species (data not shown). The unfolded monomeric alpha -G30A eluted at 57-58 min (0 min chase, top panel) and converted to two folding forms termed alpha 1 and alpha 2 that contained 3.8 and 4.8 S-S bonds, respectively. The vertical dotted line represents the elution position of native WT GPH-alpha (6).

Amino acid analysis of alpha -G30A alpha 1 and alpha 2 was used to quantitate the number of S-S bonds that had formed in each species. Recovery of 97% of the 35S label as cystine and 3% as succinyl-cysteine (hydrolysis product of NEM-alkylated cysteine) indicates that alpha 2 contained five intact S-S bonds (Table II). As expected, the later eluting species, alpha 1, contained less than five S-S bonds (3.7 out of 5). Although it is not obvious whether the bonds present in these two species are native, the differences in HPLC elution times compared with native WT GPH-alpha (Fig. 6) nonetheless implies that these folding forms have non-native conformations.


                              
View this table:
[in this window]
[in a new window]
 
Table II
Quantitation of S-S content in alpha -G30A folding intermediates
Amino acid analysis was used to determine the relative amount of S-S bond formation as described under "Experimental Procedures." The folding forms listed below correspond to the intermediates referred to on the HPLC chromatograms in Fig. 6. Shown are the calculated percentages of S-S bond formation (S-S), unformed S-S bonds (NEM-Cys), and the calculated number of S-S bonds formed. Native alpha -WT represents the fully folded, assembly-competent conformation (6).

To determine the efficiency of secretion for alpha -G30A, 293T cells expressing alpha -G30A or alpha -WT were pulse-labeled for 10 min with [35S]cysteine and chased for 10 min or 8 h. The immunopurified cell lysates and media containing GPH-alpha were analyzed by reducing SDS-PAGE and bands quantitated as described under "Experimental Procedures." About 80% of alpha -WT present at 10 min was secreted into the medium by 8 h (Fig. 7A). In contrast, only about 40% of alpha -G30A was secreted (Fig. 7A). Furthermore, <10% of alpha -G30A remained in the cell after 8 h (data not shown), indicating that about 50% had been degraded. This is consistent with our previous reports (6, 16) demonstrating that mutant forms of GPH-alpha that do not migrate at the position of alpha -WT on HPLC (i.e. they contain a non-native conformation) are readily degraded.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 7.   Secretion and assembly of alpha -WT and alpha -G30A. A, 293T cells expressing alpha -WT or alpha -G30A were pulse-labeled 10 min and chased for 10 min or 8 h. [35S]Cysteine-labeled GPH-alpha was immunoprecipitated from the 10-min cell lysate (representing intracellular GPH-alpha ) and 8 h medium (representing secreted GPH-alpha ). The immunopurified subunits were analyzed by reducing SDS-PAGE and quantitated. The percentage secreted is defined as the amount of 35S-labeled subunit recovered in the medium after 8 h relative to that present at 10 min intracellular. Following an 8-h chase, <5% of the total GPH-alpha remained in the cell lysate (data not shown), indicating that by 8 h the majority of the radiolabeled subunit had either been secreted or degraded intracellularly. B, 293T cells expressing alpha -WT or alpha -G30A and hCG-beta were pulse-labeled with [35S]cysteine and chased for 8 h. The percentage of GPH-alpha that had combined with WT hCG-beta was determined as described in the legend to Fig. 3. Each bar represents the mean ± S.D. of at least three experiments.

To determine whether alpha -G30A could assemble with hCG-beta , both subunits were coexpressed in 293T cells, pulse-labeled with [35S]cysteine for 20 min, and chased for 8 h. Immunopurified subunits were analyzed by SDS-PAGE, the bands were quantitated, and the percentage of GPH-alpha that had combined with hCG-beta was calculated (see "Experimental Procedures"). Results of this analysis are shown in Fig. 7B. Only 30% of the secreted alpha -G30A combined with WT hCG-beta , compared with 80% for alpha -WT. These results further demonstrate the importance of the central cystine knot Gly residue in protein folding and heterodimer formation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The growth factor cystine knot superfamily of dimeric proteins contain similar structural topologies but lack significant sequence similarity other than the spacing of the six cysteine residues that form the three cystine residues of the knot (1). The importance of the cystine residues in producing functional proteins has been well documented (6, 7, 9-11, 16), but the role of the noncysteine residues located within these cystine knots is largely unknown.

All known growth factors containing a cystine knot motif have an 8-amino acid ring structure, with the exception of nerve growth factor, which contains a 14-membered ring. For the 8-residue rings, such as those found in GPH-alpha and hCG-beta , the two sides of the ring contain 5- and 3-residues and are linked by two S-S bridges. Other than the cysteine residues, the only conserved residue of the ring is a Gly located at the central position of the 5-residue stretch, such that this sequence is termed C-X1-G-X2-C. Studies showing that this Gly residue is essential for producing functional TSH (28, 30) have been repeated in hCG-beta using identical (Gly right-arrow Arg) and similar (Gly right-arrow Asn) mutations (31, 32). However, a lack of structural data and knowledge of the folding mechanisms for these subunits at the time of these observations failed to define why this Gly is critical. In light of more recent findings, including the hCG crystal structure (17, 18) and knowledge of GPH-alpha and hCG-beta folding (6, 9, 14, 16), we can now address the mechanism by which specific residues within the hCG cystine knots contribute to hormone function. In particular, results from mutational analyses can be more precisely interpreted because we can distinguish between two general consequences of these mutations: (i) the mutation removes a key residue important for a direct subunit interaction; or (ii) the mutation causes global misfolding such that the protein cannot attain native structure.

The central Gly residue located between X1 and X2 is thought to be necessary because, in contrast with other amino acids, Gly can readily adopt a positive phi  torsion angle, which allows it to avoid steric hindrance with the penetrating S-S bond of the cystine knot (1). The biological importance of this Gly can be inferred from several observations. First, a naturally occurring Gly right-arrow Arg mutation in TSH-beta causes congenital isolated TSH deficiency (28). Second, mutation of the equivalent Gly in GPH-alpha prevents the production of functional hCG (31) and TSH (30). Third, mutation of this Gly to Arg or Asp in hCG-beta results in undetectable levels of heterodimeric hCG being secreted from Xenopus laevis oocytes (32).

In this report, we investigated the role of the invariant cystine knot Gly residue in the folding, secretion, and assembly of GPH-alpha and hCG-beta , two prototypes of the growth factor cystine knot superfamily (1). Even the most conservative substitution possible, Gly right-arrow Ala, resulted in nearly 100% of hCG-beta being misfolded and degraded intracellularly. The misfolding was evident from the non-native S-S bonds that had formed (Fig. 5), as well as the failure to efficiently convert to the pbeta 2 folding intermediate (Fig. 4B). The resulting non-native S-S bond formation and misfolding provides an explanation for why the central Gly residue is essential for hCG function.

Mutation of the equivalent Gly in GPH-alpha (alpha -G30A) gave similar results, although, the deleterious effects were less pronounced; 40% of alpha -G30A was secreted, 30% of the which assembled with WT hCG-beta . This result is consistent with a study that detected immunoreactive hCG when alpha -G30A and WT hCG-beta were coexpressed in X. laevis oocytes (31). However, the mutant hCG heterodimer displayed no bioactivity in a murine testosterone production-based Leydig cells bioassay, suggesting that native hCG conformation was not attained (31).

A notable effect of the G30A mutation on GPH-alpha folding was that it slowed folding significantly. Following a 30-min chase, less than half of the alpha -G30A synthesized converted to the most folded form that contained five S-S bonds. This compares with a t1/2 of about 90 s for WT GPH-alpha folding. Previously, we reported that disruption of the GPH-alpha cystine knot S-S bonds results in inefficient folding and secretion (6). The observation that alpha -G30A was inefficiently folded and secreted implies that the alpha -G30A mutation also interfered with formation of the GPH-alpha cystine knot.

There are several possible explanations for why mutation of the invariant Gly was more detrimental to hCG-beta folding than that of GPH-alpha . First, the inherent flexibility of the GPH-alpha loop 2 (residues 33-58) (33) may allow for greater perturbation of the GPH-alpha cystine knot and permit closure of the cystine knot ring in a portion of the molecules by adopting the positive phi  torsion angle needed at residue 30. Second, WT hCG-beta folds at a much slower rate compared with GPH-alpha (t1/2 = 5 min versus t1/2 = 90 s, respectively) and, therefore, when the rate of hCG-beta folding is slowed even further (as noted above for alpha -G30A), beta -G36A may be more readily degraded before the subunit has time to fold. The latter possibility is based upon a recently proposed model (34, 35) that suggests that glycoproteins only have a limited amount of time to fold and exit the endoplasmic reticulum before being degraded.

The other three noncysteine residues of the hCG cystine knots are X1, X2, and X3 of the C-X1-G-X2-C and C-X3-C sequences. To understand the role(s) of these residues, we constructed chimeras in which the X residues of GPH-alpha and hCG-beta were swapped individually or in combination, while leaving the central Gly unchanged. Folding and subunit secretion was not significantly affected in either the single or the triple mutants (alpha beta knot and beta alpha knot) (Fig. 2). However, assembly of alpha beta knot and beta alpha knot with their WT partners was decreased by about 90% (Fig. 3). The decrease in assembly was not due to any one particular substitution at the X1, X2, or X3 positions but, rather, was due to the combination of all three substitutions, suggesting that the set of all three X residues act in a subunit-specific manner.

The observation that alpha beta knot and beta alpha knot are efficiently secreted but do not assemble adds to a growing body of evidence that suggests that determinants necessary for assembly and secretion of hCG subunits are different. Recently, we reported two examples of modified GPH-alpha subunits that are efficiently secreted but do not assemble with hCG-beta (6); one modification changed residues in loop 2 necessary for combination while the other modification simultaneously removed both 7-31 and 59-87 S-S bonds. In addition, hCG-beta mutants lacking the 93-100 or 26-110 S-S bonds are also efficiently secreted but do not assemble with GPH-alpha (5, 9). Thus, structural determinants needed for hCG assembly are not necessarily required for subunit secretion.

Our data suggest that the noncysteine residues within the hCG-beta and GPH-alpha cystine knots are critical for intersubunit interactions that are necessary to form a stable dimer. This finding is further supported by an important feature of the hCG structure (17, 18). At the core of the dimer interface is a series of interchain beta -sheets. Intimately involved in this beta -sheet are the regions encompassing the C-X1-G-X2-C sequences of both subunits (residues 25-39 of GPH-alpha and 27-40 of hCG-beta ), which form a significant number of intersubunit hydrogen bonds. Thus, the simultaneous alteration of multiple X residues, as was done in alpha beta knot and beta alpha knot, could interfere with formation of this intersubunit beta -sheet, whereas single changes might be less disruptive because most other intersubunit interactions remain intact.

The set of all three X residues within both hCG cystine knots are required for biological activity since they are necessary for dimer formation (Fig. 3) and only the hCG heterodimer is functional (12). Whether or not this region is important for receptor binding and signal transduction is unknown. A single-chain model that tethers assembly-incompetent subunits to their WT partner has been used successfully to address similar questions. GPH-alpha subunits containing cysteine mutations that disrupt the cystine knot, such that the free subunits alone cannot assemble with hCG-beta , maintain in vitro biological activity when tethered to hCG-beta (36, 37). These data suggest that the cystine knot region is necessary for heterodimer formation, but not for receptor binding and signal transduction. Thus, it seems likely that the intervening X residues may also not be directly involved in receptor binding and signal transduction.

A complete understanding of the common cystine knot motif must take into account all residues of the knot. Previous studies have focused primarily on the S-S bonds and some aspects of the central Gly residue (6, 8, 9, 28, 30-32). The data presented in this report provides evidence that the intervening X residues located between the cystine knot also make important contributions to hCG biosynthesis. Specifically, these residues appear to play a critical role in dimer formation rather than directly influencing individual subunit folding or secretion. Thus, most noncysteine residues within cystine knots may not be interchangeable because they appear to have subunit-specific functions. Future studies aimed at elucidating the role of analogous residues in other cystine knot proteins may determine how universal a role these residues play in the function of other members of the cystine knot superfamily.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant CA32949 (to E. B.), NCI, National Institutes of Health Cancer Center Support Grant P 30 CA36727 to the Eppley Institute, a National Science Foundation Graduate Fellowship (to R. J. D.), and an Emley Fellowship (to A. K. M.-L.).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.

|| Current address: Transgenomic Inc., 12325 Emmet St., Omaha, NE 68164.

** Current address: LI-COR Inc., 4308 Progressive Avenue, Lincoln, NE 68504.

Dagger Dagger Current address: Corporate Office of Science and Technology, Johnson & Johnson, 410 George St., New Brunswick, NJ 08901.

|||| Current address: Eli Lilly and Co., Lilly Corporate Center, Indianapolis, IN 46295.

¶¶ To whom correspondence should be addressed: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805. Tel.: 402-559-6074; Fax: 402-559-4651; E-mail: ebedows@unmc.edu.

Published, JBC Papers in Press, December 29, 2000, DOI 10.1074/jbc.M010168200


    ABBREVIATIONS

The abbreviations used are: S-S, disulfide; hCG, human chorionic gonadotropin; GPH-alpha , glycoprotein hormone alpha -subunit; TSH, thyroid-stimulating hormone; DTT, dithiothreitol; NEM, N-ethylmaleimide; PAGE, polyacrylamide gel electrophoresis; WT, wild type; HPLC, high performance liquid chromatography.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Sun, P. D., and Davies, D. R. (1995) Annu. Rev. Biophys. Biomol. Struct. 24, 269-291[CrossRef][Medline] [Order article via Infotrieve]
2. Isaacs, N. W. (1995) Curr. Opin. Struct. Biol. 5, 391-395[CrossRef][Medline] [Order article via Infotrieve]
3. Murray-Rust, J., McDonald, N. Q., Blundell, T. L., Hosang, M., Oefner, C., Winkler, F., and Bradshaw, R. A. (1993) Structure 1, 153-159[Medline] [Order article via Infotrieve]
4. Muller, Y. A., Christinger, H. W., Keyt, B. A., and de Vos, A. M. (1997) Structure 5, 1325-1338[Medline] [Order article via Infotrieve]
5. Bedows, E., Norton, S. E., Huth, J. R., Suganuma, N., Boime, I., and Ruddon, R. W. (1994) J. Biol. Chem. 269, 10574-10580[Abstract/Free Full Text]
6. Darling, R. J., Ruddon, R. W., Perini, F., and Bedows, E. (2000) J. Biol. Chem. 275, 15413-15421[Abstract/Free Full Text]
7. Furuhashi, M., Ando, H., Bielinska, M., Pixley, M. R., Shikone, T., Hsueh, A. J., and Boime, I. (1994) J. Biol. Chem. 269, 25543-25548[Abstract/Free Full Text]
8. Suganuma, N., Matzuk, M. M., and Boime, I. (1989) J. Biol. Chem. 264, 19302-19307[Abstract/Free Full Text]
9. Bedows, E., Huth, J. R., Suganuma, N., Bartels, C. F., Boime, I., and Ruddon, R. W. (1993) J. Biol. Chem. 268, 11655-11662[Abstract/Free Full Text]
10. Brunner, A. M., Lioubin, M. N., Marquardt, H., Malacko, A. R., Wang, W. C., Shapiro, R. A., Neubauer, M., Cook, J., Madisen, L., and Purchio, A. F. (1992) Mol. Endocrinol. 6, 1691-1700[Abstract]
11. Giese, N. A., Robbins, K. C., and Aaronson, S. A. (1987) Science 236, 1315-1318[Medline] [Order article via Infotrieve]
12. Pierce, J. G., and Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465-495[CrossRef][Medline] [Order article via Infotrieve]
13. Bedows, E., Huth, J. R., and Ruddon, R. W. (1992) J. Biol. Chem. 267, 8880-8886[Abstract/Free Full Text]
14. Huth, J. R., Mountjoy, K., Perini, F., and Ruddon, R. W. (1992) J. Biol. Chem. 267, 8870-8879[Abstract/Free Full Text]
15. Huth, J. R., Perini, F., Lockridge, O., Bedows, E., and Ruddon, R. W. (1993) J. Biol. Chem. 268, 16472-16482[Abstract/Free Full Text]
16. Darling, R. J., Wilken, J. A., Ruddon, R. W., and Bedows, E. (2001) Biochemistry 40, 577-585[CrossRef][Medline] [Order article via Infotrieve]
17. Lapthorn, A. J., Harris, D. C., Littlejohn, A., Lustbader, J. W., Canfield, R. E., Machin, K. J., Morgan, F. J., and Isaacs, N. W. (1994) Nature 369, 455-461[CrossRef][Medline] [Order article via Infotrieve]
18. Wu, H., Lustbader, J. W., Liu, Y., Canfield, R. E., and Hendrickson, W. A. (1994) Structure 2, 545-558[Medline] [Order article via Infotrieve]
19. McDonald, N. Q., and Hendrickson, W. A. (1993) Cell 73, 421-424[Medline] [Order article via Infotrieve]
20. Pear, W. S., Nolan, G. P., Scott, M. L., and Baltimore, D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8392-8396[Abstract/Free Full Text]
21. Sarkar, G., and Sommer, S. S. (1990) BioTechniques 8, 404-407[Medline] [Order article via Infotrieve]
22. Beebe, J. S., Mountjoy, K., Krzesicki, R. F., Perini, F., and Ruddon, R. W. (1990) J. Biol. Chem. 265, 312-317[Abstract/Free Full Text]
23. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
24. Chang, J. Y., Canals, F., Schindler, P., Querol, E., and Aviles, F. X. (1994) J. Biol. Chem. 269, 22087-22094[Abstract/Free Full Text]
25. Chang, J. Y., Schindler, P., Ramseier, U., and Lai, P. H. (1995) J. Biol. Chem. 270, 9207-9216[Abstract/Free Full Text]
26. Cohen, S. A., and Michaud, D. P. (1993) Anal. Biochem. 211, 279-287[CrossRef][Medline] [Order article via Infotrieve]
27. Brewer, J. W., and Corley, R. B. (1996) J. Cell Sci. 109, 2383-2392[Abstract/Free Full Text]
28. Hayashizaki, Y., Hiraoka, Y., Endo, Y., Miyai, K., and Matsubara, K. (1989) EMBO J. 8, 2291-2296[Abstract]
29. Ramachandran, G. N., and Sasisekharan, V. (1968) Adv. Protein Chem. 23, 283-438[Medline] [Order article via Infotrieve]
30. Miyai, K., Kumazawa, I., Saji, F., Azuma, C., Koyama, M., Kimura, T., Narizuka, Y., Kusunoki, M., and Murata, Y. (1998) Endocr. J. 45, 467-473[Medline] [Order article via Infotrieve]
31. Kikuchi, T., Koyama, M., Miyai, K., Kimura, T., Nishikiori, N., Kimura, T., Azuma, C., Kusunoki, M., Saji, F., and Tanizawa, O. (1994) Mol. Cell. Endocrinol. 102, 1-7[Medline] [Order article via Infotrieve]
32. Azuma, C., Miyai, K., Saji, F., Kamiura, S., Tokugawa, Y., Kimura, T., Ohashi, K., Koyama, M., Iijima, Y., Kashiwai, T., Hayashizaki, Y., and Tanizawa, O. (1990) J. Mol. Endocrinol. 5, 97-102[Abstract]
33. Erbel, P. J., Karimi-Nejad, Y., Beer, T. D., Boelens, R., Kamerling, J. P., and Vliegenthart, J. F. (1999) Eur. J. Biochem. 260, 490-498[Abstract/Free Full Text]
34. Ellgaard, L., Molinari, M., and Helenius, A. (1999) Science 286, 1882-1888[Abstract/Free Full Text]
35. Liu, Y., Choudhury, P., Cabral, C. M., and Sifers, R. N. (1999) J. Biol. Chem. 274, 5861-5867[Abstract/Free Full Text]
36. Sato, A., Perlas, E., Ben-Menahem, D., Kudo, M., Pixley, M. R., Furuhashi, M., Hsueh, A. J., and Boime, I. (1997) J. Biol. Chem. 272, 18098-18103[Abstract/Free Full Text]
37. Jackson, A. M., Berger, P., Pixley, M., Klein, C., Hsueh, A. J., and Boime, I. (1999) Mol. Endocrinol. 13, 2175-2188[Abstract/Free Full Text]
38. Peters, B. P., Krzesicki, R. F., Perini, F., and Ruddon, R. W. (1989) Endocrinology 124, 1602-1612[Abstract]
39. Corless, C. L., Bielinska, M., Ramabhadran, T. V., Daniels-McQueen, S., Otani, T., Reitz, B. A., Tiemeier, D. C., and Boime, I. (1987) J. Biol. Chem. 262, 14197-14203[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.