A mutated transferrin receptor lacking asparagine-linked glycosylation sites shows reduced functionality and an association with binding immunoglobulin protein

The function of the transferrin receptor is to transport iron-bound transferrin into the cell. In order to function properly, this dimeric glycoprotein must be expressed on the cell surface and be able to bind transferrin. Site-directed mutagenesis was performed to abolish the three asparagine-linked glycosylation consensus sequences of the human transferrin receptor. The DNA encoding the mutated transferrin receptor was stably transfected into mouse fibroblasts. This form of the human transferrin receptor shows reduced transferrin binding, reduced intersubunit bond formation, and reduced cell surface expression, indicating that the transferrin receptor which lacks asparagine-linked glycosylation is not fully functional. In addition, the mutated form of the receptor is not processed as quickly. It shows an association with an endoplasmic reticular chaperone protein, binding immunoglobulin protein, leading to the hypothesis that the mutated transferrin receptor experiences increased retention in the endoplasmic reticulum.     

Characterization of the transferrin receptor in tunicamycin-treated A431 cells

The transferrin receptor undergoes extensive co- and post-translational modifications during its biosynthesis. In this study, the functional and structural properties of the transferrin receptor from tunicamycin-treated A431 cells were examined. Incubation of A431 cells with this inhibitor of asparagine-linked glycosylation results in a shift of the apparent molecular weight of the transferrin receptor from 94,000 to 79,000. The electrophoretic mobility of the receptor from treated cells is that of a monomer under nonreducing conditions, whereas the transferrin receptor in untreated cells has the mobility of a dimer under identical conditions. This result indicates a lack of disulfide bond formation between subunits of the receptor from tunicamycin-treated cells. In solution no dimers can be detected with cross-linking studies. This unglycosylated receptor does not appear to stably bind transferrin as demonstrated by a lack of isolation of this form of the receptor with transferrin-linked Sepharose. It is not transported to the surface of A431 cells.     

Structural characteristics of the mouse transferrin receptor

Rat monoclonal antibodies against mouse transferrin receptor have been used to isolate and characterize the mouse receptor molecule. The molecule is a dimeric glycoprotein of Mr 200 000 resembling its human homolog of Mr 190 000. Receptor molecules prepared from different lymphoid cell populations show structural differences which can be explained by variations in the carbohydrate moiety of the molecule. Both the antibody-binding site and the transferrin-binding site are located on tryptic fragments of Mr 80 000 on the extracellular part of the molecule. After trypsin treatment, these fragments are partially retained at the cell surface, probably non-covalently bound to one intact receptor subunit, but they are released at higher trypsin concentrations. The soluble fragments retain their ability to bind transferrin and appear to exist as dimers. In this fragment, there are no disulfide bonds present. Disulfide bonds are located near the plasma membrane. Studies using a cleavable cross-linker indicated the presence of cross-linking sites at the intramembranous or the cytoplasmic part of the molecule.     

Changes in glycosylation alter the affinity of the human transferrin receptor for its ligand

When transferrin receptors of human erythroleukemic cells were pulse-labeled with [35S]methionine and then chased in the absence of radioactive precursor, the first detectable immunoprecipitable form of the receptor had a molecular mass of 85 kDa. This form of the receptor was converted to the mature form of 93 kDa with a half-time of about 40-60 min. Both the immature (85 kDa) and mature (93 kDa) receptors associated as dimers, the native form of the receptor. The 85-kDa, as well as the 93-kDa, receptors bound to a monoclonal antibody raised against the transferrin receptor or to transferrin-Sepharose. In order to determine whether glycosylation was necessary for ligand binding, purified receptors were isolated from cells grown in the presence of tunicamycin. When K562 cells were grown in the presence of tunicamycin, an 80-kDa nonglycosylated form of the receptor was synthesized. This nonglycosylated receptor was also capable of dimer formation; however, much less of it reached the cell surface than the fully glycosylated form, although both untreated and tunicamycin-grown cells appeared to synthesize transferrin receptors at similar rates. Although the number of receptor molecules/cell was similar in control and tunicamycin-treated cells, the nonglycosylated receptors exhibited a much lower affinity for transferrin than those of untreated cells; in contrast, when receptors were purified by immunoprecipitation and digested with bacterial alkaline phosphatase, no difference was observed between the affinity of these receptors and undigested immunoprecipitated receptors. These results suggest that glycosylation is not necessary for specific binding of transferrin to its receptor, but the affinity of this binding can be influenced greatly by the presence or absence of carbohydrate residues.     

Extracellular cysteines of CCR5 are required for chemokine binding, but dispensable for HIV-1 coreceptor activity.

CCR5 is the major coreceptor for macrophage-tropic human immunodeficiency virus type I (HIV-1). For most G-protein-coupled receptors that have been tested so far, the disulfide bonds linking together the extracellular loops (ECL) are required for maintaining the structural integrity necessary for ligand binding and receptor activation. A natural mutation affecting Cys20, which is thought to form a disulfide bond with Cys269, has been described in various human populations, although the consequences of this mutation for CCR5 function are not known. Using site-directed mutagenesis, we mutated the four extracellular cysteines of CCR5 singly or in combination to investigate their role in maintaining the structural conformation of the receptor, its ligand binding and signal transduction properties, and its ability to function as a viral coreceptor. Alanine substitution of any single Cys residue reduced surface expression levels by 40-70%. However, mutation of Cys101 or Cys178, predicted to link ECL1 and ECL2 of the receptor, abolished recognition of CCR5 by a panel of conformation sensitive anti-CCR5 antibodies. The effects of the mutations on receptor expression and conformation were partially temperature-sensitive, with partial restoration of receptor expression and conformation achieved by incubating cells at 32 degrees C. All cysteine mutants were unable to bind detectable levels of MIP-1beta, and did not respond functionally to CCR5 agonists. Surprisingly, all cysteine mutants did support infection by R5 strains of HIV, though at reduced levels. These results indicate that both disulfide bonds of CCR5 are necessary for maintaining the structural integrity of the receptor necessary for ligand binding and signaling. Env binding and the mechanisms of HIV entry appear much less sensitive to alterations of CCR5 conformation.     

Characterization of the human transferrin receptor produced in a baculovirus expression system

Recombinant human transferrin receptor has been produced in a baculovirus expression system. Magnetic particles coated with an anti-transferrin receptor monoclonal antibody were used to immunoselect virus-infected Sf9 insect cells expressing the human transferrin receptor on their cell surface. Recombinant virus containing the human transferrin receptor cDNA was then plaque-purified from these cells. Biosynthetic labeling studies of infected cells showed that the human transferrin receptor is one of the major proteins made 2-3 days postinfection. The recombinant receptor made in insect cells is glycosylated and is also posttranslationally modified by the addition of a fatty acid moiety. However, studies with tunicamycin and endoglycosidases H and F showed that the oligosaccharides displayed on the recombinant receptor differ from those found on the naturally occurring receptor in human cells. As a consequence, the human receptor produced in the baculovirus system has an Mr of 82,000 and is smaller in size than the authentic receptor. About 30% of human transferrin receptors made in insect cells do not form intermolecular disulfide bonds, but are recognized by the anti-transferrin receptor antibody, B3/25, and bind specifically to a human transferrin-Sepharose column. Binding studies using 125I-labeled human transferrin showed that insect cells infected with the recombinant virus expressed an average of 5.8 +/- 0.9 X 10(5) transferrin receptors (Kd = 63 +/- 9 nM) on their cell surface. Thus, the human transferrin receptor produced in insect cells is biologically active and appears suitable for structural and functional studies.     

Cell-free synthesis and assembly of prolyl 4-hydroxylase: the role of the beta-subunit (PDI) in preventing misfolding and aggregation of the alpha-subunit.

Prolyl 4-hydroxylase (P4-H) catalyses a vital post-translational modification in the biosynthesis of collagen. The enzyme consists of two distinct polypeptides forming an alpha 2 beta 2 tetramer (alpha = 64 kDa, beta = 60 kDa), the beta-subunit being identical to the multifunctional enzyme protein disulfide isomerase (PDI). By studying the cell-free synthesis of the rat alpha-subunit of P4-H we have shown that the alpha-subunit can be translocated, glycosylated and the signal peptide cleaved by dog pancreatic microsomal membranes to yield both singly and doubly glycosylated forms. When translations were carried out under conditions which prevent disulfide bond formation, the product synthesized formed aggregates which were associated with the immunoglobulin heavy chain binding protein (BiP). Translations carried out under conditions that promote disulfide bond formation yielded a product that was not associated with BiP but formed a complex with the endogenous beta-subunit (PDI). Complex formation was detected by co-precipitation of the newly synthesized alpha-subunit with antibodies raised against PDI, by sucrose gradient centrifugation and by chemical cross-linking. When microsomal vesicles were depleted of PDI, BiP and other soluble endoplasmic reticulum proteins, no complex formation was observed and the alpha-subunit aggregated even under conditions that promote disulfide bond formation. We have therefore demonstrated that the enzyme P4-H can be assembled at synthesis in a cell-free system and that the solubility of the alpha-subunit is dependent upon its association with PDI.     

Redox manipulation of DNA binding activity and BuGR epitope reactivity of the glucocorticoid receptor.

Treatment of the transformed glucocorticoid receptor with hydrogen peroxide promotes the formation of disulfide bonds and inhibits the ability of the receptor to bind to DNA (Tienrungroj, W., Meshinchi, S., Sanchez, E. R., Pratt, S. E., Grippo, J. F., Holmgren, A., and Pratt, W. B. (1987) J. Biol. Chem. 262, 6992-7000). It has not been determined whether the inhibition of DNA binding activity is due to disulfide bonds formed within the DNA binding domain or between the DNA binding domain and another region of the receptor. In this paper, we examined the ability of hydrogen peroxide to inactivate the DNA binding activity of the mouse glucocorticoid receptor. We show that inhibition of DNA binding activity caused by hydrogen peroxide can be accounted for entirely by the formation of disulfide bonds between cysteine residues lying within the 15-kDa tryptic fragment containing the DNA binding domain of the receptor. Reversal of the peroxide-induced inactivation of DNA binding activity requires both zinc and a thiol-disulfide exchange reagent, such as dithiothreitol. Peroxide also eliminates recognition of the intact receptor and the 15-kDa tryptic fragment by the BuGR monoclonal antibody, and the reactivity of the BuGR epitope is restored by reduction without a requirement for zinc. Pretreatment of the receptor with methyl methanethiosulfonate inhibits much of the peroxide-mediated inactivation of the BuGR epitope but pretreatment with N-ethylmaleimide does not. Similarly, DNA binding activity of the receptor is inhibited by methyl methanethiosulfonate but not by N-ethylmaleimide. These results are consistent with the proposal that peroxide promotes the formation of disulfide bonds between thiols that lie spatially close to one another in the 15-kDa tryptic fragment, resulting in rapid elimination of zinc. Restoration of the zinc finger structure restores DNA-binding activity but restoration of the BuGR epitope requires only reduction without restoration of the zinc fingers.     

Disulfide Bonds Play a Critical Role in the Structure and Function of the Receptor-binding Domain of the SARS-CoV-2 Spike Antigen

The current coronavirus pandemic is exerting a tremendously detrimental impact on global health. The Spike proteins of coronaviruses, responsible for cell receptor binding and viral internalization, possess multiple and frequently conserved disulfide bonds raising the question about their role in these proteins. Here, we present a detailed structural and functional investigation of the disulfide bonds of the SARS-CoV-2 Spike receptor-binding domain (RBD). Molecular dynamics simulations of the RBD predict increased flexibility of the surface loops when the four disulfide bonds of the domain are reduced. This flexibility is particularly prominent for the disulfide bond-containing surface loop (residues 456–490) that participates in the formation of the interaction surface with the Spike cell receptor ACE2. In vitro, disulfide bond reducing agents affect the RBD secondary structure, lower its melting temperature from 52 °C to 36–39 °C and decrease its binding affinity to ACE2 by two orders of magnitude at 37 °C. Consistent with these in vitro findings, the reducing agents tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT) were able to inhibit viral replication at low millimolar levels in cell-based assays. Our research demonstrates the mechanism by which the disulfide bonds contribute to the molecular structure of the RBD of the Spike protein, allowing the RBD to execute its viral function.     

Evidence for the presence of disulfide bridges in opioid receptors essential for ligand binding. Possible role in receptor activation

The mobility of purified mu opioid binding protein in SDS-polyacrylamide gek electrophoresis is sensitive to the presence of reducing agents. In the presence of increasing concentrations of DTT the apparent molecular weight increases in a stepwise fashion from 53 kDa to 65 kDa. This reduction in mobility is attributed to the successive breakage of disulfide bridges, resulting in an increasingly asymmetric molecule. Treatment of cell membranes from various brain areas with reducing agents, such as DTT, produced a concentration-dependent inhibition of opioid binding. Sensitivity to DTT inhibition varied between receptor types, mu greater than delta much greater than kappa. For mu receptors, agonist binding was considerably more sensitive to DTT than antagonist binding. Inhibition by DTT is readily reversible and is unaffected by Na+ and/or Mg2+ ions. Reversibility may be partially prevented by the inclusion of a low concentration of a reducing reagent such as glutathione which does not inhibit binding but blocks reformation of disulfide bonds. Scatchard analysis of saturation data shows that DTT causes a pronounced decrease in binding affinity with little effect on receptor number. It is suggested that disulfide bonds are essential for ligand binding and that cleavage of one or more of these bonds may play a role in opioid receptor activation by agonists.     

Chemical engineering of a three-fingered toxin with anti-alpha7 neuronal acetylcholine receptor activity

Though it possesses four disulfide bonds the three-fingered fold is amenable to chemical synthesis, using a Fmoc-based method. Thus, we synthesized a three-fingered curaremimetic toxin from snake with high yield and showed that the synthetic and native toxins have the same structural and biological properties. Both were characterized by the same 2D NMR spectra, identical high binding affinity (K(d) = 22 +/- 5 pM) for the muscular acetylcholine receptor (AChR) and identical low affinity (K(d) = 2.0 +/- 0.4 microM) for alpha7 neuronal AchR. Then, we engineered an additional loop cyclized by a fifth disulfide bond at the tip of the central finger. This loop is normally present in longer snake toxins that bind with high affinity (K(d) = 1-5 nM) to alpha7 neuronal AchR. Not only did the chimera toxin still bind with the same high affinity to the muscular AchR but also it displayed a 20-fold higher affinity (K(d) = 100 nM) for the neuronal alpha7 AchR, as compared with the parental short-chain toxin. This result demonstrates that the engineered loop contributes, at least in part, to the high affinity of long-chain toxins for alpha7 neuronal receptors. That three-fingered proteins with four or five disulfide bonds are amenable to chemical synthesis opens new perspectives for engineering new activities on this fold.     

Post-translational changes in tertiary and quaternary structure of the insulin proreceptor. Correlation with acquisition of function

Tertiary and quaternary structural changes that occur during post-translational processing of the insulin proreceptor were examined in 3T3-L1 adipocytes. In pulse-chase experiments with [35S]methionine, labeled insulin receptor species, isolated by immuno- and insulin-affinity adsorption, were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under conditions where intra- and intermolecular disulfide bonds remained intact or were cleaved by reduction. Reducing SDS-polyacrylamide gel electrophoresis confirmed that the insulin receptor is synthesized as a long-lived (t1/2 = 3 h) proreceptor precursor of 210 kDa which undergoes proteolytic cleavage and carbohydrate maturation to form the alpha- and beta-subunits of the mature receptor. The proreceptor acquires insulin binding activity through a subtle structural change (t1/2 = 45 min) detected only by an autoimmune antibody specific for an epitope of the active insulin binding site. Analysis of insulin receptor species by nonreducing SDS-polyacrylamide gel electrophoresis revealed that the proreceptor undergoes two additional structural changes not detected by reducing SDS-polyacrylamide gel electrophoresis. The proreceptor is synthesized as a monomer (M1) with an apparent molecular mass of 170 kDa that is converted by disulfide rearrangement to another monomeric form of 190-kDa apparent molecular mass (M2). N-Linked glycosylation is required for this transition, since aglycoproreceptor, synthesized in the presence of tunicamycin, does not undergo any detectable tertiary or quaternary structural changes. M2 self-associates to form a disulfide-linked proreceptor dimer (D) which is subsequently proteolytically processed, forming the mature, disulfide-linked alpha 2 beta 2 receptor tetramer. The mature receptor was distinguished from the three proreceptor species (M1, M2, and D) by its cell surface location and its ability to bind tightly to wheat germ agglutinin-agarose, indicating the presence of complex oligosaccharide chains. Subcellular fractionation indicated that both the M1 to M2 and M2 to D conversions occur in the endoplasmic reticulum. Separation of the nonreduced proreceptor species into "active" and "inactive" forms by affinity chromatography on insulin-agarose revealed that neither the transition of M1 to M2, nor of M2 to D, is correlated with the acquisition of insulin binding function. Rather, during its life-time, the M2 species acquires insulin binding activity and an epitope recognized by a binding site specific autoimmune antibody through a subtle structural change not detected by reducing or nonreducing SDS-polyacrylamide gel electrophoresis.     

The tethering arm of the EGF receptor is required for negative cooperativity and signal transduction

The EGF receptor is a classical receptor-tyrosine kinase. In the absence of ligand, the receptor adopts a closed conformation in which the dimerization arm of subdomain II interacts with the tethering arm in subdomain IV. Following the binding of EGF, the receptor opens to form a symmetric, back-to-back dimer. Although it is clear that the dimerization arm of subdomain II is central to the formation of receptor dimers, the role of the tethering arm of subdomain IV (residues 561-585) in this configuration is not known. Here we use (125)I-EGF binding studies to assess the functional role of the tethering arm in the EGF receptor dimer. Mutation of the three major residues that contribute to tethering (D563A,H566A,K585A-EGF receptor) did not significantly alter either the ligand binding properties or the signaling properties of the EGF receptor. By contrast, breaking the Cys(558)-Cys(567) disulfide bond through double alanine replacements or deleting the loop entirely led to a decrease in the negative cooperativity in EGF binding and was associated with small changes in downstream signaling. Deletion of the Cys(571)-Cys(593) disulfide bond abrogated cooperativity, resulting in a high affinity receptor and increased sensitivity of downstream signaling pathways to EGF. Releasing the Cys(571)-Cys(593) disulfide bond resulted in extreme negative cooperativity, ligand-independent kinase activity, and impaired downstream signaling. These data demonstrate that the tethering arm plays an important role in supporting cooperativity in ligand binding. Because cooperativity implies subunit-subunit interactions, these results also suggest that the tethering arm contributes to intersubunit interactions within the EGF receptor dimer.     

Reversible inactivation of AT(2) angiotensin II receptor from cysteine-disulfide bond exchange

Dithiothreitol (DTT) treatment of angiotensin II (Ang II) type 2 (AT(2)) receptor potentiates ligand binding, but the underlying mechanism is not known. Two disulfide bonds proposed in the extracellular domain were examined in this report. Based on the analysis of ligand affinity of cysteine (Cys, C) to alanine (Ala, A) substitution mutants, we provide evidence that Cys(35)-Cys(290) and Cys(117)-Cys(195) disulfide bonds are formed in the wild-type AT(2) receptor. Disruption of the highly conserved Cys(117)-Cys(195) disulfide bond linking the second and third extracellular segments leads to inactivation of the receptor. The Cys(35)-Cys(290) bond is highly sensitive to DTT. Its breakage results in an increased binding affinity for both Ang II and the AT(2) receptor-specific antagonist PD123319. Surprisingly, in the single Cys mutants, C35A and C290A, a labile population of receptors is produced which can be re-folded to high-affinity state by DTT treatment. These results suggest that the free -SH group of Cys(35) or Cys(290) competes with the disulfide bond formation between Cys(117) and Cys(195). This Cys-disulfide bond exchange results in production of the inactive population of the mutant receptors through formation of a non-native disulfide bond.     

The covalent tagging of the cell surface insulin receptor in intact cells with the generation of an insulin-free, functional receptor. A new approach to the study of receptor dynamics

A new method is described in which the cell surface insulin receptor can be radioactively tagged in a specific manner with a small insulin-free probe. After protecting the amino groups of insulin essential for binding and bio-activity, insulin is coupled to the heterobifunctional, cleavable cross-linking reagent SASD (sulfosuccinimidyl 2-(p-azidosalicylamido)-1,3'-dithiopropionate), via displacement of the N-hydroxysuccinimide moiety of SASD. Removal of the protecting groups results in the formation of 2-(p-azidosalicylamido)-1,3'-dithiopropionate (ASD)-insulin with insulin receptor binding activity equivalent to unmodified insulin. Iodination of ASD-insulin results in the incorporation of 125I into both the azidohydroxybenzoyl moiety of SASD and a tyrosine residue of insulin. Following binding of 125I-ASD-insulin to intact monolayers of 3T3-C2 cells, radiolabel is incorporated exclusively into a 135-kDa protein in a manner dependent upon the length of exposure of the cells to short wavelength ultraviolet light. This protein corresponds in molecular weight to the alpha subunit of the insulin receptor. Labeling of this protein can be inhibited by excess unlabeled insulin. Reduction of the disulfide bond of ASD with 10 mM glutathione causes the release of the 125I-insulin portion of the reagent from the receptor complex, with the iodinated photoactivated end of ASD covalently attached to the receptor. Insulin receptor labeled in this manner retains its ability to bind insulin. General metabolic processes of the intact cells do not appear to be perturbed by this labeling procedure, and the cellular processing of the insulin receptor does not appear to be modified by the covalent labeling of the receptor protein. This procedure therefore provides a way to specifically label the cell surface insulin receptor in a manner which does not perturb the normal functioning of the labeled cell and equally importantly, does not perturb the normal cellular processing of the insulin receptor itself.     

Influence of a sulfhydryl cross-link across the allosteric-site interface of E. coli phosphofructokinase

To assess the role of quaternary stability on the properties of Escherichia coli phosphofructokinase (PFK), a disulfide bond has been introduced across the subunit interface containing the allosteric binding sites in E. coli phosphofructokinase by changing N288 to cysteine. N288 is located in close proximity to the equivalent residue on an adjacent subunit. Although SDS-PAGE of oxidized N288C indicates monomeric protein, blocking the six native cysteine residues with N-ethyl maleimide (NEM) reveals dimers of N288C on non-native gels. Subsequent addition of dithiothreitol (DTT) to NEM-labeled N288C regenerates the monomer on SDS-PAGE, reflecting the reversibility of intersubunit disulfide bond formation. KSCN-induced hybrid formation between N288C and the charged-tagged mutant E195,199K exhibits full monomer-monomer exchange only upon DTT addition, providing a novel assessment of disulfide bond formation without NEM treatment. N288C also exhibits a diminished tendency toward nonspecific aggregation under denaturing conditions, a phenomenon associated with monomer formation in PFK. Pressure-induced dissociation and urea denaturation studies further indicate that oxidized N288C exhibits increased quaternary stability along both interfaces of the tetramer, suggesting a synergistic relationship between active site and allosteric site formation. Although the apparent binding affinities of substrates and effectors change somewhat upon disulfide formation in N288C, little difference is evident between the maximally inhibited and activated forms of the enzyme in oxidizing versus reducing conditions. Allosteric influence, therefore, is not correlated to subunit-subunit affinity, and does not involve substantial interfacial rearrangement.     

Kinetic analyses of the surface-transmembrane disulfide bond isomerization-controlled fusion activation pathway in Moloney murine leukemia virus

The surface (SU) and transmembrane (TM) subunits of Moloney murine leukemia virus (Mo-MLV) Env are disulfide linked. The linking cysteine in SU is part of a conserved CXXC motif in which the other cysteine carries a free thiol. Recently, we showed that receptor binding activates its free thiol to isomerize the intersubunit disulfide bond into a disulfide within the motif instead (M. Wallin, M. Ekstr?m and H. Garoff, EMBO J. 23:54-65, 2004). This facilitated SU dissociation and activation of TM for membrane fusion. The evidence was mainly based on the finding that alkylation of the CXXC-thiol prevented isomerization. This arrested membrane fusion, but the activity could be rescued by cleaving the intersubunit disulfide bond with dithiothreitol (DTT). Here, we demonstrate directly that receptor binding causes SU-TM disulfide bond isomerization in a subfraction of the viral Envs. The kinetics of the isomerization followed that of virus-cell membrane fusion. Arresting the fusion with lysophosphatidylcholine did not arrest isomerization, suggesting that isomerization precedes the hemifusion stage of fusion. Our earlier finding that native Env was not possible to alkylate but required isomerization induction by receptor binding intimated that alkylation trapped an intermediate form of Env. To further clarify this possibility, we analyzed the kinetics by which the alkylation-sensitive Env was generated during fusion. We found that it followed the fusion kinetics. In contrast, the release of fusion from alkylated, isomerization-blocked virus by DTT reduction of the SU-TM disulfide bond was much faster. These results suggest that the alkylation-sensitive form of Env is a true intermediate in the fusion activation pathway of Env.     

Role of disulfide bonds in biologic activity of human interleukin-6.

We have examined the functional importance of the two disulfide bonds formed by the four conserved cysteines of human interleukin (IL-6). Using a bacterial expression system, we have synthesized a series of recombinant IL-6 mutants in which the constituent cysteines of the first (Cys45-Cys51), second (Cys74-Cys84), or both disulfide bonds of recombinant human interleukin-6 were replaced by other amino acids. Each mutant was partially purified and tested in four representative bioassays. While mutants lacking Cys45 and Cys51 retained activity similar to nonmutated recombinant IL-6, the activity of mutants lacking Cys74 and Cys84 was significantly reduced, especially in assays involving human cell lines. These results indicate that the first disulfide bond of human interleukin-6 is not required for maintenance of normal biologic activity. However, the fact that mutants lacking Cys45 and Cys51 were more active than corresponding cysteine-free mutants indicates that the disulfide bond formed by these residues contributes to biologic activity in the absence of the second disulfide bond. Competition binding studies with representative mutants indicate that their affinity for the human IL-6 receptor parallels their biologic activities on human cells.     

Post-translational acquisition of insulin binding activity by the insulin proreceptor. Correlation to recognition by autoimmune antibody

The post-translational acquisition of ligand binding activity by the insulin receptor was examined in 3T3-L1 adipocytes. In pulse-chase experiments with [35S] methionine, labeled receptor species were separated into "active" and "inactive" forms by affinity chromatography on insulin-agarose and then were characterized and quantitated. It was found that the newly translated high molecular weight proreceptor lacks the capacity to bind insulin. The acquisition of binding activity is relatively slow (t1/2 = 45 min) and occurs prior to conversion of the proreceptor to the mature alpha- and beta-subunits by proteolytic cleavage and maturation of its N-linked oligosaccharide chains (t1/2 = 3 h). Glycosylation appears to be required for this activation since the aglycoproreceptor, synthesized in the presence of tunicamycin, does not acquire insulin binding activity. However, once the proreceptor has acquired ligand binding activity, removal of its N-linked oligosaccharide chains with endoglycosidase H has no effect on the ability of the proreceptor to bind insulin. The modification of the proreceptor to bind insulin. The modification of the proreceptor that gives rise to insulin binding activity most likely involves a conformational change in the binding domain. A human autoimmune antibody that recognizes only the active insulin binding site does not interact with the inactive proreceptor, whereas a rabbit polyclonal antireceptor antibody recognizes all forms. Thus, the autoimmune antibody must recognize a new epitope created during conversion of the inactive proreceptor to the active form.     

Co-expression of molecular chaperones does not improve the heterologous expression of mammalian G-protein coupled receptor expression in yeast

The limitations to high-level expression of integral membrane proteins are not well understood. The human A(2)a adenosine receptor (A(2)a) and mouse Substance P receptor (SPR) were individually expressed in S. cerevisiae to identify potential cellular bottlenecks for G-protein coupled receptors. In the yeast system, A(2)a was not N-linked glycosylated but was functional and plasma membrane-localized. A(2)a also contained an intramolecular disulfide bond. Substance P receptor was also not N-linked glycosylated in yeast, but, unlike A(2)a, SPR was intracellularly retained, nonfunctional, and did not appear to contain an intramolecular disulfide bond. Since both receptors contain N-linked glycosylation and disulfide bonds in mammalian systems, machinery responsible for interacting with these modifications was investigated-specifically, the potential interactions between the nascent receptor and ER-resident proteins were explored. The chaperones calnexin and protein disulfide isomerase were co-overexpressed with the GPCRs to determine the effect on total and active yields of A(2)a and SPR, as well as on receptor trafficking. The effect of co-expressing the chaperone BiP on the total yields of A(2)a as well as intracellular fates of both receptors were determined. The co-expression of ER resident proteins did not improve A(2)a yields nor did they restore SPR activity or improve SPR cell surface expression. Taken together, these results indicate that an ER-folding bottleneck does not limit the expression of the mammalian receptors in yeast.