The Disulfide Linkage and the Free Sulfhydryl Accessibility of Acyl-Coenzyme A:Cholesterol Acyltransferase 1 As Studied by Using mPEG 5000 -Maleimide † (original) (raw)
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Journal of The American Society for Mass Spectrometry, 2013
Cystine knots or nested disulfides are structurally difficult to characterize, despite current technological advances in peptide mapping with high-resolution liquid chromatography coupled with mass spectrometry (LC-MS). In the case of recombinant human arylsulfatase A (rhASA), there is one cystine knot at the Cterminal, a pair of nested disulfides at the middle, and two out of three unpaired cysteines in the N-terminal region. The statuses of these cysteines are critical structure attributes for rhASA function and stability that requires precise examination. We used a unique approach to determine the status and linkage of each cysteine in rhASA, which was comprised of multi-enzyme digestion strategies (from Lys-C, trypsin, Asp-N, pepsin, and PNGase F) and multi-fragmentation methods in mass spectrometry using electron transfer dissociation (ETD), collision induced dissociation (CID), and CID with MS 3 (after ETD). In addition to generating desired lengths of enzymatic peptides for effective fragmentation, the digestion pH was optimized to minimize the disulfide scrambling. The disulfide linkages, including the cystine knot and a pair of nested cysteines, unpaired cysteines, and the post-translational modification of a cysteine to formylglycine, were all determined. In the assignment, the disulfide linkages were Cys138-Cys154, Cys143-Cys150, Cys282-Cys396, Cys470-Cys482, Cys471-Cys484, and Cys475-Cys481. For the unpaired cysteines, Cys20 and Cys276 were free cysteines, and Cys51 was largely converted to formylglycine (970 %). A successful methodology has been developed, which can be routinely used to determine these difficult-to-resolve disulfide linkages, ensuring drug function and stability.
Journal of Mass Spectrometry, 2000
Cysteine residues and disulfide bonds are important for protein structure and function. We have developed a simple and sensitive method for determining the presence of free cysteine (Cys) residues and disulfide bonded Cys residues in proteins (<100 pmol) by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with protein database searching using the program Sequest. Free Cys residues in a protein were labeled with PEO-maleimide biotin immediately followed by denaturation with 8 M urea. Subsequently, the protein was digested with trypsin or chymotrypsin and the resulting products were analyzed by capillary LC/ESI-MS/MS for peptides containing modified Cys and/or disulfide bonded Cys residues. Although the MS method for identifying disulfide bonds has been routinely employed, methods to prevent thiol-disulfide exchange have not been well documented. Our protocol was found to minimize the occurrence of the thiol-disulfide exchange reaction. The method was validated using well-characterized proteins such as aldolase, ovalbumin, and b-lactoglobulin A. We also applied this method to characterize Cys residues and disulfide bonds of b 1,4-galactosyltransferase (five Cys), and human blood group A and B glycosyltransferases (four Cys). Our results demonstrate that b 1,4-galactosyltransferase contains one free Cys residue and two disulfide bonds, which is in contrast to work previously reported using chemical methods for the characterization of free Cys residues, but is consistent with recently published results from x-ray crystallography. In contrast to the results obtained for b 1,4-galactosyltransferase, none of the Cys residues in A and B glycosyltransferases were found to be involved in disulfide bonds.
Biochemistry, 2007
Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a resident enzyme in the endoplasmic reticulum. ACAT1 is a homotetrameric protein and contains nine transmembrane domains (TMDs). His460 is a key active residue and is located within TMD7. Human ACAT1 has seven free Cys, but the recombinant ACAT1 devoid of free Cys retains full enzyme activity. To further probe the functionality of TMD7 (amino acids 446-460) and TMD8 (amino acids 466-481), we used a parental ACAT1 devoid of free Cys as the template to perform Cys-scanning mutagenesis within these regions. Each of the single Cys mutants was expressed in Chinese hamster ovary (CHO) cell line AC29 lacking endogenous ACAT1. We measured the effect of single Cys substitution on enzyme activity and used the Cu(1,10-phenanthroline) 2 SO 4-mediated disulfide cross-linking method to probe possible interactions of engineered Cys between the two identical subunits. The results show that several residues in one subunit closely interact with the same residues in the other subunit; mutating these residues to Cys does not lead to large loss in enzyme activity. Helical wheel analysis suggests that these residues are located at one side of the coil. In contrast, mutating residues F453, A457, or H460 to Cys causes large loss in enzyme activity; the latter residues are located at the opposite side of the coil. A similar arrangement is found for residues in TMD8. Thus, helical coils in TMD7 and TMD8 have two distinct functional sides: one side is involved in substrate-binding/catalysis, while the other side is involved in subunit interaction.
Archives of Biochemistry and Biophysics, 1996
measurements provided further evidence that the disulfide-bonded enzyme retains structure in the pres-Chinese hamster ovary cells were stably transence of denaturants. Taken together, these results fected with the cDNA for human carbonic anhydrase show that the disulfide bonds contribute significantly IV that was engineered to encode a secretory form of both to the retention of structure and of catalytic acthe normally glycosylphosphatidylinositol-anchored tivity in the presence of denaturants, and to the ability membrane protein. Overexpression was achieved by to renature following removal of denaturants. ᭧ 1996 amplification of the cDNA and its dihydrofolate re-Academic Press, Inc. ductase-containing expression vector by growth in the presence of methotrexate. The 33-kDa secretory form of the enzyme was purified to homogeneity from cellular secretions by inhibitor affinity chromatography. Occasional CA IV preparations contained proteo-Carbonic anhydrase IV (CA IV) 2 is expressed on the lytic fragments of 18 and 15 kDa held together by diplasma membrane of epithelial and endothelial cells sulfide bonds. N-terminal sequencing identified the of microcapillaries of several organs and anchored to 18-kDa fragment as the N-terminus and the 15-kDa membranes by a glycosylphosphatidylinositol (GPI) anfragment as the C-terminal portion. The specific acchor (1-13). It plays a key role in reabsorption of tivity of the purified enzyme preparations (2587 { 149 HCO 0 3 in kidney (6). It is also implicated in homeostasis U/mg protein) was comparable to that of enzyme puriof CO 2 and HCO 0 3 in brain (10), in catalysis of CO 2 fied from human tissues. exchange and local pH regulation in lung (9), in acidifi-In order to identify the cysteines involved in the two cation of epididymal fluid in reproductive organs (8), disulfide bonds, enzyme purified following metabolic and in catalysis of the hydration of metabolically prolabeling with [ 35 S]cysteine was subjected to proteolytic duced CO 2 in muscle during exercise (12). cleavage and the N-terminal amino acid sequence de-The remarkable resistance of CA IV to SDS concentermined on the labeled peptides isolated by HPLC. trations that inactivate other CAs, first noted by Whit-Results indicated that the disulfide bonds in the native ney and Briggle (1), greatly facilitates its purification phatidylinositol; SDS, sodium dodecyl sulfate; DTT, dithiothreitol;
Journal of Biological Chemistry, 1994
The complexity of protein folding is often aggravated reticulum (Marquardt, T., and Helenius, A. (1992) J. by the low solubility of the denatured state. The ineffi-Cell. Biol. 117, 506-513). ciency of the oxidative refolding of reduced, denatured lysozyme results from a kinetic partitioning of the unfolded Protein between Pathways leading to aggrega-The uncatalyzed in vitro refolding of denatured proteins fretion and Pathways leading to the native structure. Proquently yields low levels of native protein and often is kinetitein disulfide isomerase (PDI), a resident foldase of the cally incompetent when compared with the in vivo reaction. endoplasmic reticulum, catalyzes the in vitro oxidative one of the major obstacles to the folding of many 'luding lysozyme' Depending On the results in hydrophobic aggregation (1). The efficacy of folding trations of foldase and denatured substrate and the ordepends on the partitioning of the unfolded protein between der in which they are added to initiate folding, PDI can exhibit either a chaperone activity or an anti-chaper-productive pathways leading to the native structure and non-Chem 269, 7764-7771). PDI,s chaperone activity leads to vivo folding and assembly of newly synthesized proteins often quantitative recovery of native lysozyme. Its anti-chapinvolves a variety of cellular folding enzymes and molecular erOne activity diverts substrate away from productive chaperones (2). Cellular foldases increase the rate and yield of folding and facilitates disulfide cross-linking of lyproductive folding by catalyzing slow chemical steps, such as sozyme into large, inactive aggregates that specifically disulfide formation and proline isomerization, that accompany incorporate PDI. A mutant PDI (N,C,.pDI), in which the correct folding and maturation of proteins (3, 4). On the both the Nand C-terminal active site cysteines have other hand, molecular chaperones bind to unfolded proteins been changed to serines, loses all chaperone activity and prevent nonproductive folding and aggregation (2, 5). In and behaves as an anti-chaperone at all substrate and uivo, the proper interplay between unfolded substrates, fol-PDI concentrations tested. The dithiolldisulfide sites of dases, and molecular chaperones normally ensures a proper PDI are essential for the chaperone activity observed balance between productive and nonproductive pathways. at high PDI concentrations, but they are not required Protein disulfide isomerase (PDI)' is a 55-kDa protein that for the anti-chaperone activity found at low PDI conresides in the endoplasmic reticulum (ER). PDI catalyzes the in centrations. Inactivation of PDI's peptide/protein bindvitro formation and rearrangement of disulfide bonds (6) and ing site by a specific Photoaffinity label (Noiva, R.9 has two functionally independent active sites, each of which Freedman, R. and Lennarz, J* (lgg3) J. Bioz. contains a dithiolldisulfide center. Both sites are present in Chem. 268, 19210-19217) inhibits the disulfide isomerthioredoxin-like domains, one near the N terminus and the * Supported by National Institutes of Health Grant GM40379 (to H. ~ F. G.) and by a grant from the National Science Foundation EPSCoR The abbreviations used are: PDI, protein disulfide isomerase; GSH, program and the South Dakota Futures Fund (to R. N.). The costs of glutathione; GSSG, glutathione disulfide; wt, wild-type; N,C,-PDI, publication of this article were defrayed in part by the payment of page mutant of PDI in which the N-and C-terminal active site cysteines have in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tide binding site; N U , monoiodo-N"-3-(4-hydroxyphenylpropionyl)-charges. This article must therefore be hereby marked "aduertisement" been changed to serines; pal-PDI, PDI photoaffinity labeled at the pep-1 To whom correspondence should be addressed: Dept. of Biochemis-Asn-Lys-(N-€-p-azidobenzoy1)-Ala-NH,; ER, endoplasmic reticulum; try,
Journal of Biological Chemistry, 2004
Bovine core 2 1,6-N-acetylglucosaminyltransferase-M (bC2GnT-M) catalyzes the formation of all mucin 1,6-Nacetylglucosaminides, including core 2, core 4, and blood group I structures. These structures expand the complexity of mucin carbohydrate structure and thus the functional potential of mucins. The four known mucin 1,6-N-acetylglucosaminyltransferases contain nine conserved cysteines. We determined the disulfide bond assignments of these cysteines in [ 35 S]cysteine-labeled bC2GnT-M isolated from the serum-free conditioned medium of Chinese hamster ovary cells stably transfected with a pSecTag plasmid. This plasmid contains bC2GnT-M cDNA devoid of the 5sequence coding the cytoplasmic tail and transmembrane domain. The C18 reversed phase high performance liquid chromatographic profile of the tryptic peptides of reducedalkylated 35 S-labeled C2GnT-M was established using microsequencing. Each cystine pair was identified by rechromatography of the C8 high performance liquid chromatographic radiolabeled tryptic peptides of alkylated bC2GnT-M on C18 column. Among the conserved cysteines in bC2GnT-M, the second (Cys 113) was a free thiol, whereas the other eight cysteines formed four disulfide bridges, which included the first (Cys 73) and sixth (Cys 230), third (Cys 164) and seventh (Cys 384), fourth (Cys 185) and fifth (Cys 212), and eighth (Cys 393) and ninth (Cys 425) cysteine residues. This pattern of disulfide bond formation differs from that of mouse C2GnT-L, which may contribute to the difference in substrate specificity between these two enzymes. Molecular modeling using disulfide bond assignments and the fold recognition/threading method to search the Protein Data Bank found a match with aspartate aminotransferase structure. This structure is different from the two major protein folds proposed for glycosyltransferases.
Journal of Biological Chemistry, 2001
The transport and intraluminal reduction of dehydroascorbate was investigated in microsomal vesicles from various tissues. The highest rates of transport and intraluminal isotope accumulation (using radiolabeled compound and a rapid filtration technique) were found in hepatic microsomes. These microsomes contain the highest amount of protein-disulfide isomerase, which is known to have a dehydroascorbate reductase activity. The steady-state level of intraluminal isotope accumulation was more than 2-fold higher in hepatic microsomes prepared from spontaneously diabetic BioBreeding/Worcester rats and was very low in fetal hepatic microsomes although the initial rate of transport was not changed. In these microsomes, the amount of protein-disulfide isomerase was similar, but the availability of protein thiols was different and correlated with dehydroascorbate uptake. The increased isotope accumulation was accompanied by a higher rate of dehydroascorbate reduction and increased protein thiol oxidation in microsomes from diabetic animals. The results suggest that both the activity of protein-disulfide isomerase and the availability of protein thiols as reducing equivalents can play a crucial role in the accumulation of ascorbate in the lumen of the endoplasmic reticulum. These findings also support the fact that dehydroascorbate can act as an oxidant in the proteindisulfide isomerase-catalyzed protein disulfide formation.
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2002
A rapid in vitro assay was developed for monitoring protein-mediated cholesterol monomerization from bile acid aggregates. This assay uses a fluorescent cholesterol analog, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3h-ol (NBD-cholesterol), which was shown to be absorbed by hamster in a fashion similar to cholesterol. The fluorescence of aggregates of NBD-cholesterol was strongly quenched in 2.5 mM of taurocholic acid. Addition of proteins from enterocytes of hamster small intestine led to a time-and dosedependent dequenching of NBD-cholesterol fluorescence. Comparable dequenching can be detected with SDS and appears to involve monomerization of the NBD-cholesterol. Purification of enterocyte extract by sequential chromatography revealed a f 140-kDa protein complex (p140) able to mediate the monomerization of NBD-cholesterol. Each p140 complex mediated monomerization of 2.7 NBDcholesterol molecules. The p140 complex appeared to be formed by dimerization of two f 58-kDa molecules since SDS-PAGE revealed a single dominant band at 58 kDa (p58). Protein sequence analysis suggested that p58 is protein-disulfide isomerase (PDI), and this conclusion was confirmed by cloning of hamster PDI, and detection of PDI enzyme activity in the purified fraction. Additional studies with either pure PDI or lysates of cells transfected with hamster PDI showed that PDI by itself was not sufficient for monomerizing cholesterol. Further, despite a similar mobility on SDS-PAGE ( f 58 kDa), the p140 complex appeared f 45-kDa larger than pure PDI ( f 95 kDa) when analyzed by a gel-filtration chromatography. The p140 complex may thus contain an unidentified molecule(s) in addition to PDI that may contribute importantly to cholesterol monomerization. D
Journal of Biological Chemistry, 2011
The ATP-binding cassette (ABC) transporter ABCB6 is a mitochondrial porphyrin transporter that activates porphyrin biosynthesis. ABCB6 lacks a canonical mitochondrial targeting sequence but reportedly traffics to other cellular compartments such as the plasma membrane. How ABCB6 reaches these destinations is unknown. In this study, we show that endogenous ABCB6 is glycosylated in multiple cell types, indicating trafficking through the endoplasmic reticulum (ER), and has only one atypical site for glycosylation (NXC) in its amino terminus. ABCB6 remained glycosylated when the highly conserved cysteine (Cys-8) was substituted with serine to make a consensus site, NXS. However, this substitution blocked ER exit and produced ABCB6 degradation, which was mostly reversed by the proteasomal inhibitor MG132. The amino terminus of ABCB6 has an additional highly conserved ER luminal cysteine (Cys-26). When Cys-26 was mutated alone or in combination with Cys-8, it also resulted in instability and ER retention. Further analysis revealed that these two cysteines form a disulfide bond. We discovered that other ABC transporters with an amino terminus in the ER had similarly configured conserved cysteines. This analysis led to the discovery of a disease-causing mutation in the sulfonylurea receptor 1 (SUR1)/ABCC8 from a patient with hyperinsulinemic hypoglycemia. The mutant allele only contains a mutation in a conserved amino-terminal cysteine, producing SUR1 that fails to reach the cell surface. These results suggest that for ABC transporters the propensity to form a disulfide bond in the ER defines a unique checkpoint that determines whether a protein is ER-retained. ATP-binding cassette (ABC) 2 transporters utilize ATP to facilitate the transmembrane movement of a variety of biologically important molecules (1). ABC transporters are required for many essential biological processes such as heme biosynthesis, [Fe-S] cluster formation, antigen presentation, and insulin secretion. Some point mutations in ABC genes produce only alterations in substrate specificity (e.g. P-glycoprotein (ABCB1) and ABCG2), whereas others cause profound conformational changes producing defects in trafficking (e.g. ⌬508-CFTR) (2, 3). The endoplasmic reticulum (ER) has a protein quality control system that monitors conformational changes in proteins. Membrane proteins have multiple domains (cytoplasmic, ER lumen, and membrane-spanning) that are recognized by the ER and are currently being elucidated. Some of these domains may determine the fate of a proteins within the ER (e.g. retention or degradation), and it is likely that ABC transporters contain characteristic domains determining their fate. Recent studies suggested that the ER contains multiple protein "quality control" checkpoints, each with defined criteria for recognizing protein folding (4-6). In membrane proteins, an initial ER checkpoint appears to require interrogation of the cytoplasmic domains to scan for lesions in folding that could activate protein degradation processes. A second checkpoint monitors domains located in the ER lumen. Some post-translational modifications occurring in the ER (e.g. disulfide bond formation) may be required by a domain to ensure proper folding, avoid activation of a checkpoint, and escape ER-associated protein degradation. Typically, disulfide bonds are maintained by the oxidizing environment of the ER and protein-disulfide isomerases. If the necessary disulfide bonds are not formed, the proteins might be retained in the ER and then degraded (7). Among the ABC transporters, it was discovered that ABCG2 contains an intramolecular disulfide bond, which is critical for protein stability (8). In the absence of one of the cysteines in the