Characterization of a membrane-associated 3,3',5-triiodo-L-thyronine binding protein by use of monoclonal antibodies

Four mouse hybridoma cell lines have been isolated which secrete antibodies to the membrane-associated thyroid hormone binding protein (Mr 55,000) from human epidermoid carcinoma A431 cells. J6 is rat specific; J2 is human and monkey specific; J8 and J9 have a wider specificity and react with similar thyroid hormone binding proteins (p55) from human, monkey, rat, and hamster. None of these antibodies reacts with mouse cells. J2, J6, and J9 are of the IgG1k class, and J8 is an IgAk antibody. p55 was characterized by using these monoclonal antibodies. It is not posttranslationally processed by glycosylation, phosphorylation, or sulfation. It has a cellular degradation rate t1/2 approximately equal to 3.2 h. Using immunofluorescence and electron microscopic immunocytochemistry, p55 was found to be associated with the lumenal face of the endoplasmic reticulum and nuclear envelope. When cell homogenates were prepared, significant amounts of p55 were released into the 110000g supernatant, indicating that p55 is loosely associated with the endoplasmic reticulum and nuclear envelope.     

Molecular cloning of cDNA encoding a 55-kDa multifunctional thyroid hormone binding protein of skeletal muscle sarcoplasmic reticulum.

A cDNA clone encoding 55-kDa multifunctional, thyroid hormone binding protein of rabbit skeletal muscle sarcoplasmic reticulum was isolated and sequenced. The cDNA encoded a protein of 509 amino acids, and a comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of the purified protein indicates that an 18-residue NH2-terminal signal sequence was removed during synthesis. The deduced amino acid sequence of the rabbit muscle clone suggested that this protein is related to human liver thyroid hormone binding protein, rat liver protein disulfide isomerase, human hepatoma beta-subunit of prolyl 4-hydroxylase and hen oviduct glycosylation site binding protein. The protein contains two repeated sequences Trp-Cys-Gly-His-Cys-Lys proposed to be in the active sites of protein disulfide isomerase. Northern blot analysis showed that the mRNA encoding rabbit skeletal muscle form of the protein is present in liver, kidney, brain, fast- and slow-twitch skeletal muscle, and in the myocardium. In all tissues the cDNA reacts with mRNA of 2.7 kilobases in length. The 55-kDa multifunctional thyroid hormone binding protein was identified in isolated sarcoplasmic reticulum vesicles using a monoclonal antibody specific to the 55-kDa thyroid hormone binding protein from rat liver endoplasmic reticulum. The mature protein of Mr 56,681 contains 95 acidic and 61 basic amino acids. The COOH-terminal amino acid sequence of the protein is highly enriched in acidic residues with 17 of the last 29 amino acids being negatively charged. Analysis of hydropathy of the mature protein suggests that there are no potential transmembrane segments. The COOH-terminal sequence of the protein, Arg-Asp-Glu-Leu (RDEL), is similar to but different from that proposed to be an endoplasmic reticulum retention signal; Lys-Asp-Glu-Leu (KDEL) (Munro, S., and Pelham, H.R.B. (1987) Cell 48, 899-907). This variant of the retention signal may function in a similar manner to the KDEL sequence, to localize the protein to the sarcoplasmic or endoplasmic reticulum. The positively charged amino acids Lys and Arg may thus interchange in this retention signal.     

Glycosylation site binding protein, a component of oligosaccharyl transferase, is highly similar to three other 57 kd luminal proteins of the ER

A 57 kd component of oligosaccharyl transferase, termed glycosylation site binding protein, specifically recognizes a photoaffinity probe containing the N-glycosylation site sequence Asn-Lys-Thr. It is present in the lumen of the ER (endoplasmic reticulum) and its release from this compartment results in a loss of N-glycosylation. Antibodies against this protein were used to identify cDNA clones from a lambda gt11 expression library. Analysis of its cDNA sequence reveals high sequence similarity to three other 57 kd luminal endoplasmic reticulum proteins: protein disulfide isomerase, the beta-subunit of prolyl hydroxylase, and thyroid hormone binding protein. This finding suggests that the capacity to recognize multiple polypeptide domains may reside in a single luminal protein that participates in co- and/or posttranslational modifications of newly synthesized proteins.     

Peptide binding by protein disulfide isomerase, a resident protein of the endoplasmic reticulum lumen

Previously we had demonstrated by photoaffinity labeling that a 57-kDa protein of the endoplasmic reticulum can bind and become covalently linked to glycosylatable photoreactive peptides containing the sequence-Asn-Xaa-Ser/Thr-. Subsequently, it was found that this protein, called glycosylation site-binding protein, was a multifunctional protein, i.e. it was identical to protein disulfide isomerase (PDI), the beta-subunit of prolyl hydroxylase and thyroid hormone-binding protein. In this study, the peptide specificity for binding to this 57-kDa protein, hereafter called PDI, has been investigated in more detail using photoaffinity probes. The results reveal that although N-glycosylation by oligosaccharyl transferase in the endoplasmic reticulum has an absolute requirement for an hydroxyamino acid in the third amino acid residue of the glycosylation site sequence, no such specificity is observed in the binding of such peptides to PDI. In addition to the lack of specificity for an hydroxyamino acid in the third residue position, no specificity was observed for the asparagine residue in the first position. Thus, binding is not restricted to peptides containing N-glycosylation sites. We have investigated the discrepancy between this apparent lack of sequence specificity and earlier results indicating that binding of peptides to PDI was specific for N-glycosylation site sequences. We now demonstrate that PDI in the lumen of microsomes is more efficiently labeled by peptides containing photoreactive-Asn-Xaa-Ser/Thr- sequences than by nonacceptor site sequences because the former become glycosylated. This increased labeling does not occur because the glycosylated form of the probes are preferentially recognized by PDI. Rather, it appears that increased polarity of the affinity probe after attachment of the oligosaccharide chain prevents its exit from the sealed microsomes, in effect concentrating it within the lumen of the microsome. These results, coupled with other studies on the multifunctional nature of PDI, suggest that the observed peptide binding may be a manifestation of the ability of PDI to recognize the backbone of polypeptides in the lumen of the endoplasmic reticulum.     

Hormone binding by protein disulfide isomerase, a high capacity hormone reservoir of the endoplasmic reticulum

Protein disulfide isomerase (PDI) is a folding assistant of the eukaryotic endoplasmic reticulum, but it also binds the hormones, estradiol, and 3,3',5-triiodo-l-thyronine (T(3)). Hormone binding could be at discrete hormone binding sites, or it could be a nonphysiological consequence of binding site(s) that are involved in the interaction PDI with its peptide and protein substrates. Equilibrium dialysis, fluorescent hydrophobic probe binding (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS)), competition binding, and enzyme activity assays reveal that the hormone binding sites are distinct from the peptide/protein binding sites. PDI has one estradiol binding site with modest affinity (2.1 +/- 0.5 microm). There are two binding sites with comparable affinity for T(3) (4.3 +/- 1.4 microm). One of these overlaps the estradiol site, whereas the other binds the hydrophobic probe, bis-ANS. Neither estradiol nor T(3) inhibit the catalytic or chaperone activity of PDI. Although the affinity of PDI for the hormones estradiol and T(3) is modest, the high local concentration of PDI in the endoplasmic reticulum (>200 microm) would drive hormone binding and result in the association of a substantial fraction (>90%) of the hormones in the cell with PDI. High capacity, low affinity hormone sites may function to buffer hormone concentration in the cell and allow tight, specific binding to the true receptor while preserving a reasonable number of hormone molecules in the very small volume of the cellular environment.     

A platform for compartmentalized protein synthesis: protein translation and translocation in the ER

Recent advances in the study of protein translocation across the membrane of the endoplasmic reticulum include insights into the mechanism of signal-sequence function. Biochemical and genetic studies have provided further evidence that lumenal proteins perform direct roles in secretory protein translocation and in the regulation of protein-conducting-channel permeability during membrane protein integration. A hypothesis identifying the endoplasmic reticulum as a site of mRNA localization and compartmentalized protein synthesis has been suggested.     

A modeling framework for the study of protein glycosylation

The key step in the asparagine-linked glycosylation of secretory proteins is the transfer of oligosaccharide from a dolichol precursor to the polypeptide at an Asp-X-Ser/Thr (NXS/T) consensus sequence. It is often the case, both in cultured cells and in vivo, that this reaction does not occur for every molecule of a given protein. Thus, the cell may create two protein populations, one bearing and one lacking oligosaccharide, for each potential glycosylation site. We present a structured kinetic modeling framework of the initial glycosylation event based on a balance of available glycosylation sites through the region of endoplasmic reticulum lumen proximal to the membrane. Oligosaccharyltransferase, a multimeric protein complex, catalyzes the sugar transfer. This enzyme is integral to the endoplasmic reticulum membrane, and it is thought to act cotranslationally. The nascent polypeptide may also fold in such a way as to prevent glycosylation from occurring. The net result is a potentially complex spatial and temporal relationship among translation, glycosylation, and other cotranslational events. Model results predict how fractional glycosylation site occupancy may depend on protein synthesis rate, oligosaccharyldolichol availability, and mRNA elongation rate. Although we are currently unable to quantitatively compare predicted to experimentally obtained fractional site occupancy, we are able to determine qualitative trends which may be confirmed experimentally. (c) 1996 John Wiley & Sons, Inc.     

High level expression of p55, a thyroid hormone binding protein which is homologous to protein disulfide isomerase in a retroviral vector

To develop an efficient system for a high level expression of a human cellular thyroid hormone binding protein (p55) in eukaryotic cells, a full-length p55 cDNA was inserted into a Harvey murine sarcoma virus-derived vector (pHTBr) and transfected into mouse NIH 3T3 cells. The expressed p55 has a molecular weight of 55,000 and is recognized by the human specific anti-p55 monoclonal antibody. Similar to the endogenous p55, the expressed p55 is localized on endoplasmic reticulum and nuclear envelope. Moreover, p55 was specifically labeled by N-bromoacetyl-3,3',5-triiodo-L-thyronine. Thus, the expressed p55 is structurally indistinguishable from the endogeneous p55. pHTBr was packaged into a virus with the aide of an amphotropic virus. Infection by pHTBr-containing virus yielded a 2-11 fold higher expression than the endogeneous p55 in NIH3T3, rat GH3, human HepG2 cells and a mouse monoclonal antibody secreting hybridoma.     

Estradiol dependent anchoring of the goat uterine estrogen receptor activation factor (E-RAF) at the endoplasmic reticulum by a 55 kDa anchor protein (ap55)

The primary intracellular site of localization of the estrogen receptor activation factor (E-RAF) is shown here to be the endoplasmic reticulum where the protein remains anchored through an estrogen dependent mechanism. The retention of E-RAF by the endoplasmic reticulum is facilitated by two proteins: (1) a 55 kDa anchor protein (ap55) which is an integral membrane protein of the endoplasmic reticulum. ap55 is a high affinity estrogen binding protein. A conformational change induced by estrogen binding is thought to favor the anchoring process. (2) The anchoring of E-RAF by ap55 is mediated by yet another protein. This is the 66 kDa transport protein (tp66) which recognizes ap55 on the one hand and E-RAF on the other. The presence of estradiol that saturates the hormone binding sites on ap55 appears to favor the anchoring of tp66-E-RAF complex to ap55. This interaction appears to be weakened by levels of estradiol below 7 nM concentration leading to the dissociation of the tp66-E-RAF complex from ap55. The tp66-E-RAF complex moves towards the nucleus.     

Identification of ERp29, an endoplasmic reticulum lumenal protein, as a new member of the thyroglobulin folding complex

Folding and post-translational modification of the thyroid hormone precursor, thyroglobulin (Tg), in the endoplasmic reticulum (ER) of the thyroid epithelial cells is facilitated by several molecular chaperones and folding enzymes, such as BiP, GRP94, calnexin, protein disulfide isomerase, ERp72, and others. They have been shown to associate simultaneously and/or sequentially with Tg in the course of its maturation, thus forming large heterocomplexes in the ER of thyrocytes. Here we present evidence that such complexes include a novel member, an ER-resident lumenal protein, ERp29, which is present in all mammalian tissues with exceptionally high levels of expression in the secretory cells. ERp29 was induced upon treatment of FRTL-5 rat thyrocytes with the thyroid-stimulating hormone, which is essential for the maintenance of thyroid cells and Tg biosynthesis. Chemical cross-linking followed by the cell lysis and immunoprecipitation of ERp29 or Tg revealed association of these proteins and additionally, immunocomplexes that also included major ER chaperones, BiP and GRP94. Sucrose density gradient analysis indicated co-localization of ERp29 with Tg and BiP in the fractions containing large macromolecular complexes. This was supported by immunofluorescent microscopy showing co-localization of ERp29 with Tg in the putative transport vesicular structures. Affinity chromatography using Tg as an affinity ligand demonstrated that ERp29 might be selectively isolated from the FRTL-5 cell lysate or purified lumenal fraction of rat liver microsomes along with the other ER chaperones. Preferential association with the urea-denatured Tg-Sepharose was indicative of either direct or circuitous ERp29/Tg interactions in a chaperone-like manner. Despite the presence of the C-terminal ER-retrieval signal, significant amounts of ERp29 were also recovered from the culture medium of stimulated thyrocytes, indicating ERp29 secretion. Based on these data, we suggest that the function of ERp29 in thyroid cells is connected with folding and/or secretion of Tg.     

Monoglucosylated glycans in the secreted human complement component C3: implications for protein biosynthesis and structure

The monoglucosylated oligomannose N-linked oligosaccharide (Glc(1)Man(9)GlcNAc(2)) is a retention signal for the calnexin-calreticulin quality control pathway in the endoplasmic reticulum. We report here the presence of such monoglucosylated N-glycans on the human complement serum glycoprotein C3. This finding represents the first report of monoglucosylated glycans on a human serum glycoprotein from non-diseased individuals. The presence of the glucose moiety in 5% of the human C3 glycoprotein suggests that this glycosylation site is sequestered within the protein and is consistent with previous studies identifying a cryptic conglutinin binding site on C3 that becomes exposed upon its conversion to iC3b.     

Physicochemical properties of 57 kDa thyroid hormone binding protein from rat liver nuclei

The nuclear thyroid hormone binding protein (NTHB) with the molecular weight of 57 kDa was obtained from rat liver nuclear extracts by using HPLC and DEAE-Sephadex A-25 ion exchange chromatography methods. Fluorescein isothiocyanate-labeled 3,5,3'-triiodo-L-thyronine (F-T3) was used as a fluorescent probe to identify the hormone binding protein. Purified NTHB has a single binding site for T3 with the apparent binding constant of (3.3 +/- 0.7) X 10(8) M-1. NTHB is an acidic protein with a pI of 5.0. The secondary structure of NTHB is characterized by about 42% helical and 18% beta-structure from CD measurements.     

Post-translational modification of the S-layer glycoprotein occurs following translocation across the plasma membrane of the haloarchaeon Haloferax volcanii.

The halophilic archaeon Haloferax volcanii is surrounded by a protein shell solely comprised of the S-layer glycoprotein. While the gene sequence and glycosylation pattern of the protein and indeed the three-dimensional structure of the surface layer formed by the protein have been described, little is known of the biosynthesis of the S-layer glycoprotein. In the following, pulse-chase radiolabeling and cell-fractionation studies were employed to reveal that newly synthesized S-layer glycoprotein undergoes a maturation step following translocation of the protein across the plasma membrane. The processing step, detected as an increase in the apparent molecular mass of the S-layer glycoprotein, is unaffected by inhibition of protein synthesis and is apparently unrelated to glycosylation of the protein. Maturation requires the presence of magnesium ions, involved in membrane association of the S-layer glycoprotein, and results in increased hydrophobicity of the protein as revealed by enhanced detergent binding. Thus, along with protein glycosylation, additional post-translational modifications apparently occur on the external face of the haloarchaeal plasma membrane, the proposed topological homologue of the lumenal face of the eukaryal endoplasmic reticulum membrane.     

The endoplasmic reticulum retention signal of the E3/19K protein of adenovirus-2 is microtubule binding.

The signal for retention in the endoplasmic reticulum of the E3/19K protein of adenovirus type 2 is located within the carboxyl-terminal cytoplasmic extension. A synthetic peptide corresponding to this sequence showed affinity for beta-tubulin, could promote tubulin polymerization in vitro, and bound to taxol-polymerized microtubules. When compared with the microtubule binding sequences from two microtubule-associated proteins (MAPs; MAP2 and tau), we found similarities suggesting that the cytoplasmic tail might bind to tubulin/microtubules in a MAPs-like fashion. A synthetic peptide corresponding to the cytoplasmic tail of an E3/19K deletion mutant not retained in the endoplasmic reticulum was also tested. It had the same net charge but did not promote tubulin polymerization in vitro nor did it show measurable affinity for tubulin or microtubules. This indicates that binding to microtubules is important for retention of the E3/19K protein in the endoplasmic reticulum.     

N-linked glycosylation of G. mellonella juvenile hormone binding protein - comparison of recombinant mutants expressed in P. pastoris cells with native protein

Juvenile hormone (JH) regulates insect growth and development. JH present in the hemolymph is bound to juvenile hormone binding protein (hJHBP) which protects JH from degradation. In G. mellonella, this protein is glycosylated only at one (Asn(94)) of the two potential N-linked glycosylation sites (Asn(4) and Asn(94)). To investigate the function of glycosylation, each of the two potential glycosylation sites in the rJHBP molecule was examined by site-directed mutagenesis. MS analysis revealed that rJHBP overexpressed in the P. pastoris system may appear in a non-glycosylated as well as in a glycosylated form at both sites. We found that mutation at position Asn(94) reduces the level of protein secretion whereas mutation at the Asn(4) site has no effect on protein secretion. Purified rJHBP and its mutated forms (N4W and N94A) have the same JH binding activities similar to that of hJHBP. However, both mutants devoid of the carbohydrate chain are more susceptible to thermal inactivation. It is concluded that glycosylation of JHBP molecule is important for its thermal stability and secretion although it is not required for JH binding activity.     

Heavy chain binding protein recognizes incompletely disulfide-bonded forms of vesicular stomatitis virus G protein

To investigate the function of heavy chain binding protein (BiP, GRP 78) in the endoplasmic reticulum, we have characterized its interaction with a model plasma membrane glycoprotein, the G protein of vesicular stomatitis virus. We used a panel of well characterized mutant G proteins and immunoprecipitation with anti-BiP antibodies to determine if BiP interacted with newly synthesized G protein and/or mutant G proteins retained in the endoplasmic reticulum. We made three major observations: 1) BiP bound transiently to folding intermediates of wild-type G protein which were incompletely disulfide-bonded; 2) BiP did not bind stably to all mutant G proteins which remain in the endoplasmic reticulum; and 3) BiP bound stably only to mutant G proteins which do not form correct intrachain disulfide bonds.     

Slow translocon gating causes cytosolic exposure of transmembrane and lumenal domains during membrane protein integration

Integral membrane proteins are cotranslationally inserted into the endoplasmic reticulum via the protein translocation channel, or translocon, which mediates the transport of lumenal domains, retention of cytosolic domains and integration of transmembrane spans into the phospholipid bilayer. Upon translocon binding, transmembrane spans interact with a lateral gate, which regulates access to membrane phospholipids, and a lumenal gate, which controls the translocation of soluble domains. We analyzed the in vivo kinetics of integration of model membrane proteins in Saccharomyces cerevisiae using ubiquitin translocation assay reporters. Our findings indicate that the conformational changes in the translocon that permit opening of the lumenal and lateral channel gates occur less rapidly than elongation of the nascent polypeptide. Transmembrane spans and lumenal domains are therefore exposed to the cytosol during integration of a polytopic membrane protein, which may pose a challenge to the fidelity of membrane protein integration.     

ER protein quality control and proteasome-mediated protein degradation

A variety of mutant polypeptides that are associated with human disease are targeted for degradation by an endoplasmic reticulum (ER) quality control system. In addition, physiological signals and viral gene products can target the degradation of several ER resident proteins and secreted proteins passing through the ER. Although the mechanism of protein quality control and the site of degradation were obscure, recent data indicate that degradation requires the cytosolic proteasome. Biochemical and genetic analyses have indicated that both lumenal and integral membrane proteins are selected for proteolysis and exported to the cytosol by a process that in several cases requires ER associated molecular chaperones.     

Linkage between the hormone binding site and the reactive center loop of thyroxine binding globulin

Thyroxine binding globulin (TBG) is the major carrier of the thyroid hormones triiodothyronine (T3) and thyroxine (T4) in plasma. TBG is member of the serpin family of proteins although it has no proteinase inhibitory activity. In this study we show that TBG has properties typical of a metastable serpin and provide evidence that occupancy of the hormone binding site alters the conformation of the reactive center loop. After reactive center loop cleavage by endoproteinase Asp-N or neutrophil elastase the protein became more stable to guanidine hydrochloride denaturation compared to the native protein, as a result of loop insertion. In addition, incubation of the native protein with a reactive center loop peptide, caused a change in mobility on a native gel. This is consistent with the idea that thyroxine binding globulin is able to form a binary complex with the peptide as a result of beta-sheet A expansion. To assess the effect of cleavage and loop insertion on the hormone binding site we used the specific binding of a fluorophore, 1,8-anilinonaphthalene sulfonic acid (ANS). Loop insertion itself had no effect on ANS affinity, but cleavage with elastase at the P4'-P5' bond caused a reduction in affinity, presumably because this cleavage site is located within the hormone binding site. These data support the concept that cleavage of TBG by proteinases released in inflammation is a mechanism to deliver thyroid hormones to target tissues. A linkage between the occupancy state of the hormone binding site and the conformation of the reactive center loop was indicated by the observation that binding of T3 to native TBG reduced proteolytic susceptibility by both endoproteinase Asp-N and elastase.