Molecular characterization of tissue-nonspecific alkaline phosphatase with an Ala to Thr substitution at position 116 associated with dominantly inherited hypophosphatasia

Mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene are responsible for hypophosphatasia, an inborn error of bone and teeth metabolism associated with reduced levels of serum alkaline phosphatase activity. A missense mutation (c.346G>A) of TNSALP gene, which converts Ala to Thr at position 116 (according to standardized nomenclature), was reported in dominantly transmitted hypophosphatasia patients (A.S. Lia-Baldini et al. Hum Genet. 109 (2001) 99-108). To investigate molecular phenotype of TNSALP (A116T), we expressed it in the COS-1 cells or Tet-On CHO K1 cells. TNSALP (A116T) displayed not only negligible alkaline phosphatase activity, but also a weak dominant negative effect when co-expressed with the wild-type enzyme. In contrast to TNSALP (W, wild-type), which was present mostly as a non-covalently assembled homodimeric form, TNSALP (A116T) was found to exist as a monomer and heterogeneously associated aggregates covalently linked via disulfide bonds. Interestingly, both the monomer and aggregate forms of TNSALP (A116T) gained access to the cell surface and were anchored to the cell membrane via glycosylphosphatidylinositol (GPI). Co-expression of secretory forms of TNSALP (W) and TNSALP (A116T), which are engineered to replace the C-terminal GPI anchor with a tag sequence (his-tag or flag-tag), resulted in the release of heteromeric complexes consisting of TNSALP (W)-his and TNSALP (A116T)-flag. Taken together, these findings strongly suggest that TNSALP (A116T) fails to fold properly and forms disulfide-bonded aggregates, though it is indeed capable of interacting with the wild-type and reaching the cell surface, therefore explaining its dominant transmission.     

Novel aggregate formation of a frame-shift mutant protein of tissue-nonspecific alkaline phosphatase is ascribed to three cysteine residues in the C-terminal extension. Retarded secretion and proteasomal degradation

In the majority of hypophosphatasia patients, reductions in the serum levels of alkaline phosphatase activity are caused by various missense mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. A unique frame-shift mutation due to a deletion of T at cDNA number 1559 [TNSALP (1559delT)] has been reported only in Japanese patients with high allele frequency. In this study, we examined the molecular phenotype of TNSALP (1559delT) using in vitro translation/translocation system and COS-1 cells transiently expressing this mutant protein. We showed that the mutant protein not only has a larger molecular size than the wild type enzyme by approximately 12 kDa, reflecting an 80 amino acid-long extension at its C-terminus, but that it also lacks a glycosylphosphatidylinositol anchor. In support of this, alkaline phosphatase activity of the cells expressing TNSALP (1559delT) was localized at the juxtanucleus position, but not on the cell surface. However, only a limited amount of the newly synthesized protein was released into the medium and the rest was polyubiquitinated, followed by degradation in the proteasome. SDS/PAGE and analysis by sucrose-density-gradient analysis indicated that TNSALP (1559delT) forms a disulfide-bonded high-molecular-mass aggregate. Interestingly, the aggregate form of TNSALP (1559delT) exhibited a significant enzyme activity. When all three cysteines at positions of 506, 521 and 577 of TNSALP (1559delT) were replaced with serines, the aggregation disappeared and instead this modified mutant protein formed a noncovalently associated dimer, strongly indicating that these cysteine residues in the C-terminal region are solely responsible for aggregate formation by cross-linking the catalytically active dimers. Thus, complete absence of TNSALP on cell surfaces provides a plausible explanation for a severe lethal phenotype of a homozygote hypophosphatasia patient carrying TNSALP (1559delT).     

Disulfide bonds are critical for tissue-nonspecific alkaline phosphatase function revealed by analysis of mutant proteins bearing a C(201)-Y or C(489)-S substitution associated with severe hypophosphatasia

Hypophosphatasia (HPP), a rare genetic disease characterized by reduced serum alkaline phosphatase (ALP) activity and failure in bone and tooth mineralization, is caused by mutations in tissue-nonspecific ALP (TNSALP) gene. Two missense mutations (C201Y and C489S, standardized nomenclature) of TNSALP, involved in intra-chain disulfide bonds, were reported in patients diagnosed with perinatal HPP (Taillandier A. et al. Hum. Mutat. 13 (1999) 171-172, Hum. Mutat. 15 (2000) 293). To investigate the role of the disulfide bond in TNSALP, we expressed TNSALP (C201Y) and TNSALP (C489S) in COS-1 cells transiently. Compared with the wild-type enzyme [TNSALP (W)], both the TNSALP mutants exhibited a diminished ALP activity in the cells, where a 66kDa immature form was predominant with a marginal amount of a 80kDa mature form of TNSALP. Detailed studies on Tet-On CHO established cell line expressing TNSALP (W) or TNSALP (C201Y) showed that the 66kDa form of TNSALP (C201Y) exists as a monomer in contrast to a dimer of TNSALP (W). Only a small fraction of the TNSALP (C201Y) reached cell surface as the 80kDa mature form, though most of the 66kDa form was found to be endo-β-N-acetylglucosaminidase H sensitive and rapidly degraded in proteasome following polyubiquitination. Collectively, these results indicate not only that the intra-subunit disulfide bonds are crucial for TNSALP to properly fold and assemble into the dimeric enzyme, but also that the development of HPP associated with TNSALP (C201Y) or TNSALP (C489S) is attributed to decreased cell surface appearance of the functional enzyme.     

Disulfide bonds are critical for tissue-nonspecific alkaline phosphatase function revealed by analysis of mutant proteins bearing a C-Y or C-S substitution associated with severe hypophosphatasia

Hypophosphatasia (HPP), a rare genetic disease characterized by reduced serum alkaline phosphatase (ALP) activity and failure in bone and tooth mineralization, is caused by mutations in tissue-nonspecific ALP (TNSALP) gene. Two missense mutations (C201Y and C489S, standardized nomenclature) of TNSALP, involved in intra-chain disulfide bonds, were reported in patients diagnosed with perinatal HPP (Taillandier A. et al. Hum. Mutat. 13 (1999) 171-172, Hum. Mutat. 15 (2000) 293). To investigate the role of the disulfide bond in TNSALP, we expressed TNSALP (C201Y) and TNSALP (C489S) in COS-1 cells transiently. Compared with the wild-type enzyme [TNSALP (W)], both the TNSALP mutants exhibited a diminished ALP activity in the cells, where a 66 kDa immature form was predominant with a marginal amount of a 80 kDa mature form of TNSALP. Detailed studies on Tet-On CHO established cell line expressing TNSALP (W) or TNSALP (C201Y) showed that the 66 kDa form of TNSALP (C201Y) exists as a monomer in contrast to a dimer of TNSALP (W). Only a small fraction of the TNSALP (C201Y) reached cell surface as the 80 kDa mature form, though most of the 66 kDa form was found to be endo-β-N-acetylglucosaminidase H sensitive and rapidly degraded in proteasome following polyubiquitination. Collectively, these results indicate not only that the intra-subunit disulfide bonds are crucial for TNSALP to properly fold and assemble into the dimeric enzyme, but also that the development of HPP associated with TNSALP (C201Y) or TNSALP (C489S) is attributed to decreased cell surface appearance of the functional enzyme.     

The active site cysteine of the proapoptotic protein glyceraldehyde-3-phosphate dehydrogenase is essential in oxidative stress-induced aggregation and cell death

Recent studies have revealed that the redox-sensitive glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is involved in neuronal cell death that is triggered by oxidative stress. GAPDH is locally deposited in disulfide-bonded aggregates at lesion sites in certain neurodegenerative diseases. In this study, we investigated the molecular mechanism that underlies oxidative stress-induced aggregation of GAPDH and the relationship between structural abnormalities in GAPDH and cell death. Under nonreducing in vitro conditions, oxidants induced oligomerization and insoluble aggregation of GAPDH via the formation of intermolecular disulfide bonds. Because GAPDH has four cysteine residues, including the active site Cys(149), we prepared the cysteine-substituted mutants C149S, C153S, C244A, C281S, and C149S/C281S to identify which is responsible for disulfide-bonded aggregation. Whereas the aggregation levels of C281S were reduced compared with the wild-type enzyme, neither C149S nor C149S/C281S aggregated, suggesting that the active site cysteine plays an essential role. Oxidants also caused conformational changes in GAPDH concomitant with an increase in beta-sheet content; these abnormal conformations specifically led to amyloid-like fibril formation via disulfide bonds, including Cys(149). Additionally, continuous exposure of GAPDH-overexpressing HeLa cells to oxidants produced disulfide bonds in GAPDH leading to both detergent-insoluble and thioflavin-S-positive aggregates, which were associated with oxidative stress-induced cell death. Thus, oxidative stresses induce amyloid-like aggregation of GAPDH via aberrant disulfide bonds of the active site cysteine, and the formation of such abnormal aggregates promotes cell death.     

An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of lambda repressor

Site-directed mutagenesis has been used to replace Tyr-88 at the dimer interface of the N-terminal domain of lambda repressor with cysteine. Computer model building had suggested that this substitution would allow formation of an intersubunit disulfide without disruption of the dimer structure [Pabo, C. O., & Suchanek, E. G. (1986) Biochemistry (preceding paper in this issue)]. We find that the Cys-88 protein forms a disulfide-bonded dimer that is very stable to reduction by dithiothreitol and has increased operator DNA binding activity. The covalent Cys88-Cys88' dimer is also considerably more stable than the wild-type protein to thermal denaturation or urea denaturation. As a control, Tyr-85 was replaced with cysteine. A Cys85-Cys85' disulfide cannot form without disrupting the wild-type structure, and we find that this disulfide bond reduces the DNA binding activity and stability of the N-terminal domain.     

Molecular basis of perinatal hypophosphatasia with tissue-nonspecific alkaline phosphatase bearing a conservative replacement of valine by alanine at position 406. Structural importance of the crown domain

Hypophosphatasia, a congenital metabolic disease related to the tissue-nonspecific alkaline phosphatase gene (TNSALP), is characterized by reduced serum alkaline phosphatase levels and defective mineralization of hard tissues. A replacement of valine with alanine at position 406, located in the crown domain of TNSALP, was reported in a perinatal form of hypophosphatasia. To understand the molecular defect of the TNSALP (V406A) molecule, we examined this missense mutant protein in transiently transfected COS-1 cells and in stable CHO-K1 Tet-On cells. Compared with the wild-type enzyme, the mutant protein showed a markedly reduced alkaline phosphatase activity. This was not the result of defective transport and resultant degradation of TNSALP (V406A) in the endoplasmic reticulum, as the majority of newly synthesized TNSALP (V406A) was conveyed to the Golgi apparatus and incorporated into a cold detergent insoluble fraction (raft) at a rate similar to that of the wild-type TNSALP. TNSALP (V406A) consisted of a dimer, as judged by sucrose gradient centrifugation, suggestive of its proper folding and correct assembly, although this mutant showed increased susceptibility to digestion by trypsin or proteinase K. When purified as a glycosylphosphatidylinositol-anchorless soluble form, the mutant protein exhibited a remarkably lower Kcat/Km value compared with that of the wild-type TNSALP. Interestingly, leucine and isoleucine, but not phenylalanine, were able to substitute for valine, pointing to the indispensable role of residues with a longer aliphatic side chain at position 406 of TNSALP. Taken together, this particular mutation highlights the structural importance of the crown domain with respect to the catalytic function of TNSALP.     

Cysteine residues required for the attachment of the light chain in human IgA2

In humans, there are two subclasses of IgA, IgA1 and IgA2, with IgA2 existing as three allotypes, IgA2m(1), IgA2m(2) and IgA2(n). In IgA1, Cys(133) in C(H)1 forms the disulfide bond to the L chain. Our previous studies indicated that in IgA2 lacking Cys(133), a disulfide bond forms between the alpha-chain and the L chain when Cys(220) is followed by Arg(221), but not when Cys(220) is followed by Pro(221), suggesting that the Cys in C(H)1 might be involved in disulfide bonding to the L chain. However, here we show that covalent assembly of the H and L chains in IgA2(n) requires hinge-proximal Cys(241) and Cys(242) in C(H)2 and not Cys(196) or Cys(220) in C(H)1. Using pulse-chase experiments, we have demonstrated that wild-type IgA2(n) with Arg(221) and Cys(241) and Cys(242) assembles through a disulfide-bonded HL intermediate. In contrast, the major intermediate for IgA2 m(1) with Pro(221) assembly was H(2) even though both Cys(241) and Cys(242) were present. Only a small fraction of IgA2 m(1) assembles through disulfide-bonded HL. Overall, our studies indicate that for IgA2 covalent assembly of the H and L chains requires the hinge-proximal cysteines in C(H)2 and that the structure of C(H)1 influences the efficiency with which this covalent bond forms.     

Highly conserved cysteines of mouse core 2 beta1,6-N-acetylglucosaminyltransferase I form a network of disulfide bonds and include a thiol that affects enzyme activity

Core 2 beta1,6-N-acetylglucosaminyltransferase I (C2GnT-I) plays a pivotal role in the biosynthesis of mucin-type O-glycans that serve as ligands in cell adhesion. To elucidate the three-dimensional structure of the enzyme for use in computer-aided design of therapeutically relevant enzyme inhibitors, we investigated the participation of cysteine residues in disulfide linkages in a purified murine recombinant enzyme. The pattern of free and disulfide-bonded Cys residues was determined by liquid chromatography/electrospray ionization tandem mass spectrometry in the absence and presence of dithiothreitol. Of nine highly conserved Cys residues, under both conditions, one (Cys217) is a free thiol, and eight are engaged in disulfide bonds, with pairs formed between Cys59-Cys413, Cys100-Cys172, Cys151-Cys199, and Cys372-Cys381. The only non-conserved residue within the beta1,6-N-acetylglucosaminyltransferase family, Cys235, is also a free thiol in the presence of dithiothreitol; however, in the absence of reductant, Cys235 forms an intermolecular disulfide linkage. Biochemical studies performed with thiolreactive agents demonstrated that at least one free cysteine affects enzyme activity and is proximal to the UDP-GlcNAc binding site. A Cys217 --> Ser mutant enzyme was insensitive to thiol reactants and displayed kinetic properties virtually identical to those of the wild-type enzyme, thereby showing that Cys217, although not required for activity per se, represents the only thiol that causes enzyme inactivation when modified. Based on the pattern of free and disulfide-linked Cys residues, and a method of fold recognition/threading and homology modeling, we have computed a three-dimensional model for this enzyme that was refined using the T4 bacteriophage beta-glucosyltransferase fold.     

Role of an intrachain disulfide bond in the conformation and stability of ovalbumin

Ovalbumin, which contains one intrachain disulfide bond and four cysteine sulfhydryls, was reduced with dithiothreitol under non-denaturing conditions, and its conformation and stability were compared with those of the disulfide-bonded form. The CD spectrum in the far-UV region revealed that the overall conformation of the reduced form is similar to that of the disulfide-bonded one. Likewise, the inaccessibility to trypsin and the non-reactivity of the four cysteine sulfhydryls, exhibited by the native disulfide-bonded ovalbumin, were still retained in the disulfide-reduced form. Thus, the reduced ovalbumin appeared to substantially take the native-like conformation. However, the near-UV CD spectrum slightly differed between the native and disulfide-reduced forms. Protein alkylation with a fluorescent dye and subsequent sequence analysis showed that the two sulfhydryls (Cys73 and Cys120) originating from the disulfide bond are highly reactive in the reduced form. Furthermore, upon proteolysis with subtilisin, the N-terminal side of Cys73 was cleaved in the reduced form, but not in the disulfide-bonded one. Upon heat denaturation, the transition temperature of the reduced form was lower, by 6.8 degrees C, than that of the disulfide-bonded one. Thus, we concluded that ovalbumin has a native-like conformation in its disulfide-reduced form, but that the local conformation of the reduced form fluctuates more than that of the disulfide-bonded one. Such local destabilization may be related to the decreased stability against heat denaturation.     

Molecular mechanism of oxidative stress perception by the Orp1 protein

In this study we investigated the molecular mechanism by which the Orp1 (Gpx3) protein in Saccharomyces cerevisiae senses and reacts with hydrogen peroxide. Upon exposure to H(2)O(2) Orp1(Cys36) forms a disulfide-bonded complex with the C-terminal domain of the Yap1 protein (Yap1-cCRD). We used 4-nitrobenzo-2-oxa-1,3-diazole to identify a cysteine sulfenic acid (Cys-SOH) modification that forms on Cys(36) of Orp1(Cys36) upon exposure to H(2)O(2). Under similar conditions, neither Cys(82) of Orp1(Cys82) nor Cys(598) of Yap1 forms Cys-SOH. A homology-based molecular model of Orp1 suggests that the structure of the active site of Orp1 is similar to that found in mammalian selenocysteine glutathione peroxidases. Proposed active site residues Gln(70) and Trp(125) form a catalytic triad with Cys(36) in the Orp1 molecular model. The remainder of the active site pocket is formed by Phe(38), Asn(126), and Phe(127), which are evolutionarily conserved residues. We made Q70A and W125A mutants and tested the ability of these mutants to form Cys-SOH in response to H(2)O(2). Both mutants were unable to form Cys-SOH and did not form a H(2)O(2)-inducible disulfide-bonded complex with Yap1-cCRD. The pK(a) of Cys(36) was determined to be 5.1, which is 3.2 pH units lower than that of a free cysteine (8.3). In contrast, Orp1 Cys(82) (the resolving cysteine) has a pK(a) value of 8.3. The pK(a) of Cys(36) in the Q70A and W125A mutants is also 8.3, demonstrating the importance of these residues in modulating the nucleophilic character of Cys(36). Finally, we show that S. cerevisiae strains with ORP1 Q70A and W125A mutations are less tolerant to H(2)O(2) than those containing wild-type ORP1. The results of our study suggest that attempts to identify novel redox-regulated proteins and signal transduction pathways should focus on characterization of low pK(a) cysteines.     

Use of a site-directed triple mutant to trap intermediates: demonstration that the flavin C(4a)-thiol adduct and reduced flavin are kinetically competent intermediates in mercuric ion reductase

A mutant form of mercuric reductase, which has three of its four catalytically essential cysteine residues replaced by alanines (ACAA: Ala135Cys140Ala558Ala559), has been constructed and used for mechanistic investigations. With disruption of the Hg(II) binding site, the mutant enzyme is devoid of Hg(II) reductase activity. However, it appears to fold properly since it binds FAD normally and exhibits very tight binding of pyridine nucleotides as is seen with the wild-type enzyme. This mutant enzyme allows quantitative accumulation of two species thought to function as intermediates in the catalytic sequence of the flavoprotein disulfide reductase family of enzymes. NADPH reduces the flavin in this mutant, and a stabilized E-FADH- form accumulates. The second intermediate is a flavin C(4a)-Cys140 thiol adduct, which is quantitatively accumulated by reaction of oxidized ACAA enzyme with NADP+. The conversion of the Cys135-Cys140 disulfide in wild-type enzyme to the monothiol Cys140 in ACAA and the elevated pKa of Cys140 (6.7 vs 5.0 in wild type) have permitted detection of these intermediates at low pH (5.0). The rates of formation of E-FADH- and the breakdown of the flavin C(4a)-thiol adduct have been measured and indicate that both intermediates are kinetically competent for both the reductive half-reaction and turnover by wild-type enzyme. These results validate the general proposal that electrons flow from NADPH to FADH- to C(4a)-thiol adduct to the FAD/dithiol form that accumulates as the EH2 form in the reductive half-reaction for this class of enzymes.     

Allochromatium vinosum DsrC: solution-state NMR structure, redox properties, and interaction with DsrEFH, a protein essential for purple sulfur bacterial sulfur oxidation

Sequenced genomes of dissimilatory sulfur-oxidizing and sulfate-reducing bacteria containing genes coding for DsrAB, the enzyme dissimilatory sulfite reductase, inevitably also contain the gene coding for the 12-kDa DsrC protein. DsrC is thought to have a yet unidentified role associated with the activity of DsrAB. Here we report the solution structure of DsrC from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum determined with NMR spectroscopy in reducing conditions, and we describe the redox behavior of two conserved cysteine residues upon transfer to an oxidizing environment. In reducing conditions, the DsrC structure is disordered in the highly conserved carboxy-terminus. We present multiple lines of evidence that, in oxidizing conditions, a strictly conserved cysteine (Cys111) at the penultimate position in the sequence forms an intramolecular disulfide bond with Cys100, which is conserved in DsrC in all organisms with DsrAB. While an intermolecular Cys111-Cys111 disulfide-bonded dimer is rapidly formed under oxidizing conditions, the intramolecularly disulfide-bonded species (Cys100-Cys111) is the thermodynamically stable form of the protein under these conditions. Treatment of the disulfidic forms with reducing agent regenerates the monomeric species that was structurally characterized. Using a band-shift technique under nondenaturing conditions, we obtained evidence for the interaction of DsrC with heterohexameric DsrEFH, a protein encoded in the same operon. Mutation of Cys100 to serine prevented formation of the DsrC species assigned as an intramolecular disulfide in oxidizing conditions, while still allowing formation of the intermolecular Cys111-Cys111 dimer. In the reduced form, this mutant protein still interacted with DsrEFH. This was not the case for the Cys111Ser and Cys100Ser/Cys111Ser mutants, both of which also did not form protein dimers. Our observations highlight the central importance of the carboxy-terminal DsrC cysteine residues and are consistent with a role as a sulfur-substrate binding/transferring protein, as well as with an electron-transfer function via thiol-disulfide interchanges.     

Solution structure and backbone dynamics of the reduced form and an oxidized form of E. coli methionine sulfoxide reductase A (MsrA): structural insight of the MsrA catalytic cycle

Methionine sulfoxide reductases (Msr) reduce methionine sulfoxide (MetSO)-containing proteins, back to methionine (Met). MsrAs are stereospecific for the S epimer whereas MsrBs reduce the R epimer of MetSO. Although structurally unrelated, the Msrs characterized so far display a similar catalytic mechanism with formation of a sulfenic intermediate on the catalytic cysteine and a concomitant release of Met, followed by formation of at least one intramolecular disulfide bond (between the catalytic and a recycling cysteine), which is then reduced by thioredoxin. In the case of the MsrA from Escherichia coli, two disulfide bonds are formed, i.e. first between the catalytic Cys51 and the recycling Cys198 and then between Cys198 and the second recycling Cys206. Three crystal structures including E. coli and Mycobacterium tuberculosis MsrAs, which, for the latter, possesses only the unique recycling Cys198, have been solved so far. In these structures, the distances between the cysteine residues involved in the catalytic mechanism are too large to allow formation of the intramolecular disulfide bonds. Here structural and dynamical NMR studies of the reduced wild-type and the oxidized (Cys51-Cys198) forms of C86S/C206S MsrA from E. coli have been carried out. The mapping of MetSO substrate-bound C51A MsrA has also been performed. The data support (1) a conformational switch occurring subsequently to sulfenic acid formation and/or Met release that would be a prerequisite to form the Cys51-Cys198 bond and, (2) a high mobility of the C-terminal part of the Cys51-Cys198 oxidized form that would favor formation of the second Cys198-Cys206 disulfide bond.     

Juxtaposition of the two distal CX3C motifs via intrachain disulfide bonding is essential for the folding of Tim10

The TIM10 complex, composed of the homologous proteins Tim10 and Tim9, chaperones hydrophobic proteins inserted at the mitochondrial inner membrane. A salient feature of the TIM10 complex subunits is their conserved "twin CX3C" motif. Systematic mutational analysis of all cysteines of Tim10 showed that their underlying molecular defect is impaired folding (demonstrated by circular dichroism, aberrant homo-oligomer formation, and thiol trapping assays). As a result of defective folding, clear functional consequences were manifested in (i) complex formation with Tim9, (ii) chaperone activity, and (iii) import into tim9ts mitochondria lacking both endogenous Tim9 and Tim10. The organization of the four cysteines in intrachain disulfides was determined by trypsin digestion and mass spectrometry. The two distal CX3C motifs are juxtaposed in the folded structure and disulfide-bonded to each other rather than within each other, with an inner cysteine pair connecting Cys44 with Cys61 and an outer pair between Cys40 and Cys65. These cysteine pairs are not equally important for folding and assembly; mutations of the inner Cys are severely affected and form wrong, non-native disulfides, in contrast to mutations of the outer Cys that can still maintain the native inner disulfide pair and display weaker functional defects. Taken together these data reveal this specific intramolecular disulfide bonding as the crucial mechanism for Tim10 folding and show that the inner cysteine pair has a more prominent role in this process.     

The structure of the murine Fc receptor for IgG. Assignment of intrachain disulfide bonds, identification of N-linked glycosylation sites, and evidence for a fourth form of Fc receptor

The Fc receptor (Fc gamma R) of the murine macrophage cell line, J774, was purified by immunoaffinity chromatography then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequencing. FcR material judged to be pure by these criteria was digested with a number of enzymes to identify the cysteine residues engaged in disulfide bonds within the native structure. The results clearly establish that the mouse macrophage Fc gamma R contains two intrachain disulfide bonds, each of which connects adjacent cysteine residues within the two putative extracellular domains of the molecule. In addition, each disulfide-bonded domain was shown to contain two authentic sites of N-linked glycosylation. Extensive peptide sequencing resulted in the unexpected identification of peptide fragments from a fourth Fc gamma R whose sequences were highly homologous to sequences surrounding the two Cys residues in the amino-terminal domain of both alpha and beta 1 Fc gamma R. The fourth Fc gamma R contains a disulfide-bonded amino-terminal domain similar to beta 1 Fc gamma R.     

Importance of cysteine residues for the stability and catalytic activity of human pancreatic beta cell glucokinase

The low-affinity glucose phosphorylating enzyme glucokinase has the function of a physiological glucose sensor in pancreatic beta cells and in liver. In contrast to the high-affinity hexokinase types I-III glucokinase shows extraordinary sensitivity toward SH group oxidizing compounds. To characterize the function of sulfhydryl groups cysteine residues in the vicinity of the sugar binding site (Cys 213, Cys 220, Cys 230, Cys 233, and Cys 252) as well as cysteine residues a distance from the active site (Cys 364, Cys 371, and Cys 382), they were replaced in human beta cell glucokinase by serine through site-directed mutagenesis. Controlled proteolysis of wild-type glucokinase by proteinase K revealed that the SH group oxidizing agent alloxan can induce the formation of multiple intramolecular disulfide bridges corresponding to a double-band pattern of glucokinase protein in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The formation of intramolecular disulfide bridges altered the mobility of the protein. None of the cysteine mutations could prevent the formation of the 49-kDa glucokinase conformation after alloxan treatment. The cysteine mutants Cys 233, Cys 252, and Cys 382 showed nearly complete loss of catalytic activity, whereas the V(max) values of the Cys 213, Cys 220, Cys 364, and Cys 371 mutants were decreased by 30-60%. Only the Cys 230 mutant showed kinetic characteristics comparable to those of wild-type glucokinase. The sensitivity of the Cys 213, Cys 230, Cys 364, and Cys 371 mutants toward alloxan-induced inhibition of enzyme activity was up to 10-fold lower compared with wild-type glucokinase. d-Glucose and dithiotreitol provided protection against alloxan-induced inhibition of wild-type glucokinase and all catalytically active cysteine mutants. Conclusively our data demonstrate the functional significance of the cysteine residues of beta cell glucokinase for both structural instability of the enzyme and catalytic function. Knowledge of sensitive cysteine targets may help to develop strategies that improve glucokinase enzyme function under conditions of oxidative stress.     

Identification and reactivity of the catalytic site of pig liver thioltransferase

The active site cysteine of pig liver thioltransferase was identified as Cys22. The kinetics of the reaction between Cys22 of the reduced enzyme and iodoacetic acid as a function of pH revealed that the active site sulfhydryl group had a pKa of 2.5. Incubation of reduced enzyme with [1-14C]cysteine prevented the inactivation of the enzyme by iodoacetic acid at pH 6.5, and no stable protein-cysteine disulfide was found when the enzyme was separated from excess [1-14C]cysteine, suggesting an intramolecular disulfide formation. The results suggested a reaction mechanism for thioltransferase. The thiolated Cys22 first initiates a nucleophilic attack on a disulfide substrate, resulting in the formation of an unstable mixed disulfide between Cys22 and the substrate. Subsequently, the sulfhydryl group at Cys25 is deprotonated as a result of micro-environmental changes within the active site domain, releasing the mixed disulfide and forming an intramolecular disulfide bond. Reduced glutathione, the second substrate, reduces the intramolecular disulfide forming a transient mixed disulfide which is then further reduced by glutathione to regenerate the reduced enzyme and form oxidized glutathione. The rate-limiting step for a typical reaction between a disulfide and reduced glutathione is proposed to be the reduction of the intramolecular disulfide form of the enzyme by reduced glutathione.     

Essential cysteine residues for human RNase κ catalytic activity

Human RNase κ is an endoribonuclease expressed in almost all tissues and organs and belongs to a highly conserved protein family bearing representatives in all metazoans. To gain insight into the role of cysteine residues in the enzyme activity or structure, a recombinant active form of human RNase κ expressed in Pichia pastoris was treated with alkylating agents and dithiothreitol (DTT). Our results showed that the human enzyme is inactivated by DDT, while it remains fully active in the presence of alkylating agents. The unreduced recombinant protein migrates on SDS/PAGE faster than the reduced form. This observation in combination with the above findings indicated that human RNase κ does not form homodimers through disulfide bridges, and cysteine residues are not implicated in RNA catalysis but participate in the formation of intramolecular disulfide bond(s) essential for its ribonucleolytic activity. The role of the cysteine residues was further investigated by expression and study of Cys variants. Ribonucleolytic activity experiments and SDS/PAGE analysis of the wild-type and mutant proteins under reducing and non-reducing conditions demonstrated that Cys7, Cys14 and Cys85 are not essential for RNase activity. On the other hand, replacement of Cys6 or Cys69 with serine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of human RNase κ by forming a disulfide bond. Due to the absolute conservation of these cysteine residues, the Cys6-Cys69 disulfide bond is likely to exist in all RNase κ family members.     

Human RNase H1 activity is regulated by a unique redox switch formed between adjacent cysteines

Human RNase H1 is active only under reduced conditions. Oxidation as well as N-ethylmaleimide (NEM) treatment of human RNase H1 ablates the cleavage activity. The oxidized and NEM alkylated forms of human RNase H1 exhibited binding affinities for the heteroduplex substrate comparable with the reduced form of the enzyme. Mutants of human RNase H1 in which the cysteines were either deleted or substituted with alanine exhibited cleavage rates comparable with the reduced form of the enzyme, suggesting that the cysteine residues were not required for catalysis. The cysteine residues responsible for the observed redox-dependent activity of human RNase H1 were determined by site-directed mutagenesis to involve Cys(147) and Cys(148). The redox states of the Cys(147) and Cys(148) residues were determined by digesting the reduced, oxidized, and NEM-treated forms of human RNase H1 with trypsin and analyzing the cysteine containing tryptic fragments by micro high performance liquid chromatography-electrospray ionization-Fourier transform ion cyclotron mass spectrometry. The tryptic fragment Asp(131)-Arg(153) containing Cys(147) and Cys(148) was identified. The mass spectra for the Asp(131)-Arg(153) peptides from the oxidized and reduced forms of human RNase H1 in the presence and absence of NEM showed peptide masses consistent with the formation of a disulfide bond between Cys(147) and Cys(148). These data show that the formation of a disulfide bond between adjacent Cys(147) and Cys(148) residues results in an inactive enzyme conformation and provides further insights into the interaction between human RNase H1 and the heteroduplex substrate.