Intersubunit Disulfide Linkage Research Articles

Gastrointestinal Tract Stabilized Protein Delivery Using Disulfide Thermostable Exoshell System.

Thermostable exoshells (tES) are engineered proteinaceous nanoparticles used for the rapid encapsulation of therapeutic proteins/enzymes, whereby the nanoplatform protects the payload from proteases and other denaturants. Given the significance of oral delivery as the preferred model for drug administration, we structurally improved the stability of tES through multiple inter-subunit disulfide linkages that were initially absent in the parent molecule. The disulfide-linked tES, as compared to tES, significantly stabilized the activity of encapsulated horseradish peroxidase (HRP) at acidic pH and against the primary human digestive enzymes, pepsin, and trypsin. Furthermore, the disulfide-linked tES (DS-tES) exhibited significant intestinal permeability as evaluated using Caco2 cells. In vivo bioluminescence assay showed that encapsulated Renilla luciferase (rluc) was ~3 times more stable in mice compared to the free enzyme. DS-tES collected mice feces had ~100 times more active enzyme in comparison to the control (free enzyme) after 24 h of oral administration, demonstrating strong intestinal stability. Taken together, the in vitro and in vivo results demonstrate the potential of DS-tES for intraluminal and systemic oral drug delivery applications.

DPP6A Confers Redox Sensitivity to Kv4 Channel Inactivation

Reactive oxygen species (ROS) are important modulators of excitability and may play a critical role in the etiology of many neurodegenerative disorders. Previous studies have shown that the fast N-type inactivation of Kv1.4 channels is suppressed by ROS-mediated cysteine oxidation whereas Kv4 channel N-type inactivation is not. However, in native neurons Kv4 channel subunits form large macromolecular complexes with KChIP and DPL proteins, and fast N-type inactivation is conferred to Kv4 channel complexes by a specific isoform of DPL proteins, either DPP10a or DPP6a. To investigate whether the DPP6a-mediated fast inactivation is regulated by ROS, tert- butyl hydroperoxide (tBHP) was applied to oocytes expressing Kv4.2+KChIP3a+DPP6a channels. tBHP (1 mM) application dramatically increases the peak current amplitude by ∼ 44% and slowed inactivation kinetics. The effects of tBHP are reversed by the reducing agent dithiothreitol (DTT, 10 mM). In contrast, ternary complex channels containing another DPP6 isoform (DPP6K) are not affected by tBHP, indicating the importance of the DPP6a variable N-terminal domain for the tBHP effect. Alignment of N-terminal sequences from DPP6a and DPP10a with Kv1.4 reveals a common cysteine residue in position 13 (Cys-13), which in Kv1.4 is critical for the redox-regulation of N-type inactivation. Substituting Cys-13 of DPP6a with serine (DPP6a/C13S) results in a loss of regulation by tBHP, consistent with a similar critical role for DPP6a Cys-13. To test if other cysteines in the channel are also required for this regulation, we switched to mutant Kv4.1 (C11xA) and KChIP3a (KChIP3a/del2-59) constructs that remove most intracellular cysteine residues. Channels composed of C11xA, KChIP3a/del2-59, and normal DPP6a show disrupted regulation by tBHP, suggesting that ROS likely induces an intersubunit disulfide linkage to regulate DPP6a-mediated N-type inactivation. This work was supported by a grant from the National Institute of Health (R01 GM090029).

Characterization of Novel Hexadecameric Thioredoxin Peroxidase from Aeropyrum pernix K1

A gene (APE2278) encoding the peroxiredoxin (Prx) homologous protein of yeast and human was identified in the genome data base of the aerobic hyperthermophilic archaeon Aeropyrum pernix. We cloned the gene and produced the encoded protein in Escherichia coli cells. The isolated recombinant protein showed peroxidase activity in vitro and used the thioredoxin system of A. pernix as an electron donor. These results indicate that the recombinant protein is in fact thioredoxin peroxidase (ApTPx) of A. pernix. Immunoblot analysis revealed that the expression of ApTPx was induced as a cellular adaptation in response to the addition of exogenous H2O2 and may exert an antioxidant activity in vivo. An analysis of the ApTPx oligomers by high pressure liquid chromatography and electron microscopic studies showed that ApTPx exhibited the hexadecameric protein forming 2-fold toroid-shaped structure with outer and inner diameters of 14 and 6 nm, respectively. These results indicated that ApTPx is a novel hexadecameric protein composed of two identical octamers. Although oligomerization of individual subunits does not take place through an intersubunit-disulfide linkage involving Cys50 and Cys213, Cys50 is essential for the formation of the hexadecamer. Mutagenesis studies suggest that the sulfhydryl group of Cys50 is the site of oxidation by peroxide and that oxidized Cys50 reacts with the sulfhydryl group of Cys213 of another subunit to form an intermolecular disulfide bond. The resulting disulfide can then be reduced by thioredoxin. In support of this hypothesis, ApTPx mutants lacking either Cys50 or Cys213 showed no TPx activity, whereas the mutant lacking Cys207 had a TPx activity. This is the first report on the biochemical and structural features of a novel hexadecameric thioredoxin peroxidase from the archaea.

Moloney murine leukemia virus envelope protein subunits, gp70 and Pr15E, form a stable disulfide-linked complex.

The nature and stability of the interactions between the gp70 and Pr15E/p15E molecules of murine leukemia virus (MLV) have been disputed extensively. To resolve this controversy, we have performed quantitative biochemical analyses on gp70-Pr15E complexes formed after independent expression of the amphotropic and ecotropic Moloney MLV env genes in BHK-21 cells. We found that all cell-associated gp70 molecules are disulfide linked to Pr15E whereas only a small amount of free gp70 is released by the cells. The complexes were resistant to treatment with reducing agents in vivo, indicating that the presence and stability of the disulfide interaction between gp70 and Pr15E are not dependent on the cellular redox state. However, disulfide-bonded Env complexes were disrupted in lysates of nonalkylated cells in a time-, temperature-, and pH-dependent fashion. Disruption seemed not to be caused by a cellular factor but is probably due to a thiol-disulfide exchange reaction occurring within the Env complex after solubilization. The possibility that alkylating agents induce the formation of the intersubunit disulfide linkage was excluded by showing that disulfide-linked gp70-Pr15E complexes exist in freshly made lysates of nonalkylated cells and that disruption of the complexes can be prevented by lowering the pH. Together, these data establish that gp70 and Pr15E form a stable disulfide-linked complex in vivo.

Localization of the labile disulfide bond between SU and TM of the murine leukemia virus envelope protein complex to a highly conserved CWLC motif in SU that resembles the active-site sequence of thiol-disulfide exchange enzymes.

Previous studies have indicated that the surface (SU) and transmembrane (TM) subunits of the envelope protein (Env) of murine leukemia viruses (MuLVs) are joined by a labile disulfide bond that can be stabilized by treatment of virions with thiol-specific reagents. In the present study this observation was extended to the Envs of additional classes of MuLV, and the cysteines of SU involved in this linkage were mapped by proteolytic fragmentation analyses to the CWLC sequence present at the beginning of the C-terminal domain of SU. This sequence is highly conserved across a broad range of distantly related retroviruses and resembles the CXXC motif present at the active site of thiol-disulfide exchange enzymes. A model is proposed in which rearrangements of the SU-TM intersubunit disulfide linkage, mediated by the CWLC sequence, play roles in the assembly and function of the Env complex.

Multifunctional Tryptophan-synthesizing Enzyme: THE MOLECULAR WEIGHT OF THE EUGLENA GRACILIS PROTEIN IS UNEXPECTEDLY LOW

After developing a suitable procedure to produce large amounts of Euglena gracilis as well as a reliable protocol to purify the multifunctional tryptophan-synthesizing enzyme derived from it (Schwarz, T., Bartholmes, P., and Kaufmann, M. (1995) Biotechnol. Appl. Biochem. 22, 179-190), we here describe structural and catalytic properties of the multifunctional tryptophan-synthesizing enzyme. The kinetic parameters kcat of all five activities and Km for the main substrates were determined. The relative molecular weight under denaturing conditions as judged by SDS-polyacrylamide gel electrophoresis is 136,000. Cross-linking as well as gel filtration experiments revealed that the enzyme exists as a homodimer. Neither intersubunit disulfide linkages nor glycosylations were detected. On the other hand, the polypeptide chains are blocked N-terminally. Complete tryptic digestion of the protomer, high pressure liquid chromatography separation of the resulting peptides, and N-terminal sequence analysis of homogenous peaks as judged by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry was performed. Depending on the sequenced peptides, alignments to all entries of the SwissProt data base resulted in both strong sequence homologies to known Trp sequences and no similarities at all. Proteolytic digestion under native conditions using endoproteinase Glu-C uncovered one major cleavage site yielding a semistable, N-terminally blocked fragment with a molecular weight of 119,000. In addition, an increase in beta-elimination accompanied by a decrease in beta-replacement activity of the beta-reaction during proteolysis was observed.

Design of the helix-turn-helix motif: nonlocal effects of quaternary structure in DNA recognition investigated by laser raman spectroscopy

The operator-binding domain of phage lambda repressor provides a model for DNA recognition by the helix-turn-helix (HTH) motif. In the wild-type protein, dimerization is mediated by hydrophobic packing (of the dyad-related helix 5), which serves as an indirect determinant of operator affinity. The mutant repressor, Tyr88----Cys, forms an intersubunit disulfide linkage and exhibits enhancement of both structural stability and operator affinity. Yet the dimer-specific operator affinity of the mutant is 10-fold weaker than that of the wild-type (noncovalent) dimer, suggesting nonlocal effects of the intersubunit disulfide bond on HTH recognition (Sauer et al., 1986). To explore such nonlocal effects, we describe laser Raman studies of the Cys88 mutant repressor and its interaction with operator sites OL1 and OR3. The following results have been obtained: (i) Wild-type and mutant dimers exhibit similar secondary structures, indicated by quantitative comparison of Raman amide I and amide III bands. (ii) The engineered disulfide of the mutant lacks rigorous symmetry; we observe mainly the gauche/gauche/trans CC-S-S-CC rotamer. (iii) Remarkably, distinctive local and nonlocal differences are observed in the mechanisms of DNA recognition by wild-type and mutant repressors. These differences involve specific hydrogen-bonding interactions between the protein and DNA, including guanine N7 sites in the major groove of DNA, and alterations in DNA phosphodiester conformation induced by protein binding. We analyze these differences in relation to crystal structures of the wild-type dimer with and without bound DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Bovine brain acetylcholinesterase primary sequence involved in intersubunit disulfide linkages.

Three distinct classes of membrane-bound acetylcholinesterases (AChEs) have been identified. A12 AChE is composed of 12 catalytic subunits that are linked to noncatalytic collagen-like subunits through intersubunit disulfide bonds. G2 AChE is localized in membranes by a glycoinositol phospholipid covalently linked to the C-terminal amino acid. Brain G4 AChE involves two catalytic subunits linked by a direct intersubunit disulfide bond while the other two are disulfide-linked to a membrane-binding 20-kDa noncatalytic subunit. Molecular cloning studies have so far failed to find evidence of more than one AChE gene in any organism although alternative splicing of torpedo AChE mRNA results in different C-terminal sequences for the A12 and G2 AChE forms. Support for a single bovine AChE gene is provided in this report by amino acid sequencing of the N-terminal domains from the G2 erythrocyte, G4 fetal serum, and G4 brain AChE. Comparison of the 38-amino acid sequences reveals virtually complete identity among the three AChE forms. Additional extensive identity between the fetal serum and brain AChEs was demonstrated by sequencing several brain AChE peptides isolated by high performance liquid chromatography after trypsin digestion of nitrocellulose blots of brain AChE catalytic subunits. Cysteines involved in intersubunit disulfide linkages in brain AChE were reduced selectively with dithiothreitol in the absence of denaturants and radioalkylated with iodoacetamide. The observed sequence of the major radiolabeled tryptic peptide was C*SDL, where C* was the radioalkylated cysteine residue. This sequence is precisely the same as that observed at the C terminus of fetal bovine serum AChE and shows close homology to the C-terminal sequence of torpedo A12 AChE. We conclude that the mammalian brain G4 AChEs utilize the same exon splicing pattern as the A12 AChEs and that factors other than the primary sequence of the AChE catalytic subunits dictate assembly with either the collagen-like or the 20-kDa noncatalytic subunits.

Human Liver Growth Hormone Receptor and Plasma Binding Protein: Characterization and Partial Purification*

The human liver GH receptor has been further characterized using several biochemical approaches. Crosslinking of [125 I]human GH (hGH) to microsomal receptors and to particulate and solubilized plasma membrane receptors, followed by gel electrophoresis and autoradiography, revealed two predominant receptor-hormone complexes with a mol wt of 124,000 and 75,000, respectively. As previously shown, the 70-80 k band appears to be generated from the 124 k band in the presence of beta-mercaptoethanol, suggesting intersubunit disulfide linkages. A minor complex of mol wt 150,000 was sometimes found. By immunoblot, using a polyclonal antibody raised against a synthetic peptide (residues 391-405 of the mature human GH receptor), a single band of 100 k was detected. Human liver GH receptor and plasma GH-binding protein (BP) were purified 1,000- and 4,000-fold, respectively. The partially purified membrane receptor, analyzed by ligand-blot, showed two major bands of 55 and 32 k and minor bands of 68 and 47 k. Crosslinking of the purified GH-BP or purified receptor with [125I]hGH revealed a 75 k receptor-hormone complex. Polyclonal antibodies raised against the hepatic GH receptor inhibited the binding of hGH to the human receptor and were able to immunoprecipitate the GH receptor and also the GH-BP complex. Our findings demonstrate the existence of multiple forms of the GH receptor in human liver (major components of 100 and 50-55 k, minor component of 130 k); they lend more support to the possible subunit structure of the GH receptor; and finally, they also suggest a close relationship, with common antigenic properties, of the membrane receptor and the plasma GH-BP.

Acetylcholinesterase from bovine caudate nucleus is attached to membranes by a novel subunit distinct from those of acetylcholinesterases in other tissues.

Acetylcholinesterase extracted with Triton X-100 from bovine brain caudate nuclei was purified by affinity chromatography to apparent homogeneity. The purified enzyme was labeled with [3H]diisopropyl fluorophosphate at the active sites and with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, a compound which has been shown to be selective for the hydrophobic membrane-binding domains of several other proteins. The subunit structure was analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate before and after disulfide reduction. After reduction, a single 3H-labeled band at 70 kDa was stained by silver, but most of the 125I label corresponded to a 20-kDa species. Prior to reduction, five 3H-labeled and silver-stained bands were apparent at 70, 140, 160, 260, and greater than 360 kDa. These species were presumed to represent monomer and disulfide-linked oligomers of 70-kDa catalytic subunits. 125I label was selectively associated with the 160-, 260-, greater than 360-, and a 90-kDa species. Quantitative gel slicing of 3H- and 125I-labeled nonreduced enzyme supported a structural model in which the tetrameric enzyme is a dimer of nonidentical catalytic subunit dimers, one of which involves a direct intersubunit disulfide linkage between two 70-kDa catalytic subunit monomers and the second of which contains two disulfide linkages through an intervening 125I-labeled 20-kDa noncatalytic subunit. This 20-kDa subunit is proposed to contain the membrane attachment site. The brain enzyme did not contain components characteristic of the glycolipid anchors of erythrocyte acetylcholinesterases. However, part of the 125I label was associated with fatty acids, indicating that at least a portion of the brain enzyme membrane anchor is composed of nonamino acid components.

Affinity purification and structural characterization of a specific binding protein for human growth hormone in human serum

A highly specific binding protein for human growth hormone (hGH) has been isolated from human serum by hGH-affinity chromatography. A purification of approximately 1500-fold with a 30-40% recovery was obtained with essentially no alteration in binding characteristics. Covalent cross-linking of 125I-hGH to the binding protein, followed by analysis by SDS-polyacrylamide gel electrophoresis and autoradiography, revealed two specifically labeled complexes. Allowing for a 1:1 binding stoichiometry the binding proteins themselves had mean mol wts of 57,000 and 69,300. These increased slightly to mol wt 60,300 and 72,000 respectively in the presence of 100 mM dithiothreitol, suggesting the presence of intramolecular but not inter-subunit disulfide linkages. These data confirm the presence of the hGH binding protein(s) in human serum and define their gross structural nature.

Characterization of pepsin-resistant collagen-like tail subunit fragments of 18S and 14S acetylcholinesterase from Electrophorus electricus.

Digestion of 18S and 14S acetylcholinesterase from eel electric organ with pepsin at 15 degrees C for 6 h results in extensive degradation of the catalytic subunits, but a major portion of the collagen-like tail structure associated with these enzyme forms resists degradation. The pepsin-resistant structures partially aggregate and can be isolated by gel exclusion chromatography on Sepharose CL-6B in buffered 1 M sodium chloride. The largest structure, denoted F3, has a molecular weight of 72 000 according to gel electrophoresis in sodium dodecyl sulfate and is composed of three 24 000 molecular weight polypeptides linked by intersubunit disulfide bonds. This structure is largely, but not completely, a collagen-like triple helix as indicated by a circular dichroism spectrum typical of triple-helical collagen and an amino acid composition characterized by 27% glycine, 5% hydroxyproline, and 5% hydroxylysine. Continued pepsin action results in degradation of the disulfide linkage region such that disulfide-linked dimers F2 and finally F1 monomers become the predominant forms in sodium dodecyl sulfate. Digested samples in which either F3 or F2 predominate have virtually identical circular dichroic spectra and amino acid compositions and generate similar diffuse 24 000 molecular weight polypeptides following disulfide reduction. Thus the intersubunit disulfide linkages in F3 must occur close to the end(s) of the fragment polypeptide chains. Pepsin conversion of F3 to F2 is particularly accelerated between 25 and 30 degrees C, suggesting that the triple-helical structure in the disulfide linkage region undergoes thermal destabilization in this temperature range. Digestion at 40 degrees C yields presumably triple-helical F1 structures devoid of disulfide linkages, although their degradation to small fragments can be detected at this temperature. The question of whether the three tail subunits that give rise to F1 polypeptides are identical remains open.

The Evolution of the Immune Response

Abstract 18.5S and 7S immunoglobulins were purified from sera of horned sharks immunized with Brucella abortus. Both preparations had antibody activity, which was abolished more readily by reduction with dithiothreitol than with cysteine. Chromatography on Sephadex G-100 columns, in 6 M guanidine-HCl, disclosed that partial reduction with dithiothreitol cleaved all interchain bonds, whereas cysteine mainly affected the inter-subunit disulfide linkages of the 18.5S antibody molecules. Although high reactivity of sulfhydryl groups toward reducing agents was found in these immunoglobulins, the non-covalent bonds linking their chains appeared more resistant to dissociation than those of mammalian IgM. These studies suggest that in the immunoglobulins of elasmobranchs, the interchain disulfide bridges are of greater importance for stabilization of the antibody-combining sites than they are in the immunoglobulins of more recent evolutionary stages.