Structural analysis of human apolipoprotein A-I variants. Amino acid substitutions are nonrandomly distributed throughout the apolipoprotein A-I primary structure

In the course of an electrophoretic mutation screening program of 32,000 dried blood samples from newborns, 17 genetic variants of apolipoprotein A-I (apoA-I) were found and structurally analyzed. The following defects were identified by the combined use of high performance liquid chromatography, time-of-flight secondary ion mass spectrometry, and sequence analysis: Pro3----Arg (1 x), Pro4----Arg (1 x), Asp89----Glu (1 x), Lys107----0 (4 x), Lys107----Met (2 x), Glu139----Gly (2 x), Glu147----Val (1 x), Pro165----Arg (4 x), and Glu198----Lys (1 x). The distribution of point mutations in the apoA-I gene leading to these 9 and 11 other variants of apoA-I reported previously was statistically analyzed. Substitutions are overrepresented in the 10 amino-terminal amino acids (p less than 0.001, chi 2-test) and in residues 103-177 (p less than 0.025, chi 2-test) or residues 103-198 (p less than 0.05, chi 2-test), respectively. We further noted the following. (i) Prolines were substituted by arginine or histidine residues at a frequency much higher than expected on the basis of random nucleotide substitutions (5 out of 18 "electrically non-neutral" amino acid substitutions, p less than 0.001, chi 2-test). These substitutions are the result of transversions of cytosines contained within stretches of at least 5 consecutive cytosines in the apoA-I gene. The observed hypervariability of the apoA-I amino terminus, therefore, might be caused by a hot spot for mutation formed by the 7 subsequent cytosines in codons 3, 4, and 5. (ii) CpG dinucleotides were overrepresentatively affected by C----T transitions (5 out of 18 electrically nonneutral amino acid substitution, p less than 0.001, chi 2-test). The hypervariability of the apoA-I alpha-helical domain might therefore be caused by CpG dinucleotides predominantly occurring in codons 120-208 of apoA-I (82 out of 125). (iii) Comparison of mutation sites in the human apoA-I gene with sites of nonsynonymous substitutions revealed that amino acid substitutions found in human apoA-I were predominantly localized in areas that were little conserved during mammalian evolution. These regions may therefore represent areas of less structural constraint for the function of apoA-I.     

Structural and functional properties of reconstituted high density lipoprotein discs prepared with six apolipoprotein A-I variants

Six apolipoprotein A-I (apoA-I) variants containing the following amino acid changes: Pro3----Arg, Pro4----Arg, Lys107----0 (Lys deletion) Lys107----Met, Pro165----Arg, and Glu198----Lys, and the corresponding normal allele products, were isolated by preparative isoelectric focusing from heterozygous individuals. The apoA-I samples were reconstituted with palmitoyloleoyl phosphatidylcholine (POPC) or dipalmitoyl phosphatidylcholine (DPPC), and small amounts of cholesterol, into discoidal high density lipoprotein (HDL) complexes in order to examine their lipid binding and structural properties as well as their ability to activate lecithin:cholesterol acyltransferase (LCAT). Starting with initial molar ratios around 100:5:1 for phosphatidylcholine-cholesterol-apolipoprotein, all the normal and variant apoA-Is were completely incorporated into reconstituted HDL (rHDL). The rHDL particle sizes and their distributions were examined by nondenaturing gradient gel electrophoresis, before and after incubation with LDL, to assess the folding of apoA-I in the complexes. Intrinsic Trp fluorescence properties of the rHDL were measured, as a function of temperature and guanidine hydrochloride concentration, to detect conformational differences in the apoA-I variants. In addition, the LCAT reaction kinetics were measured with all the rHDL, and the apparent kinetic constants were compared. In terms of the structure of the rHDL particles, all the normal variant apoA-Is had similar sizes (94, 96 A) and size distributions, and indistinguishable fluorescence properties, with the exception of the Lys107----0 mutant. This variant formed slightly larger particles that were resistant to rearrangements in the presence of LDL, and had an altered apoA-I conformation in the vicinity of the Trp residues. The kinetic experiments with LCAT indicated that the apoA-I variants, Lys107----0 and Pro165----Arg, in rHDL particles had statistically different (30 to 90%) kinetic constants from the corresponding normal allele products; however, the variability in the kinetic constants among the normal apoA-I products was even greater (40 to 430%). Therefore, we conclude that the effects of these six mutations in apoA-I on the activation of LCAT are minor, and that the structural effects on rHDL, and possibly native HDL, are insignificant with the exception of the Lys107----0 mutation.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

A study of the Asp110-Glu112 region of EcoRII restriction endonuclease by site-directed mutagenesis

Site-directed mutagenesis of the ecoRII gene has been used to search for the active site of the EcoRII restriction endonuclease. Plasmids with point mutations in ecoRII gene resulting in substitutions of amino acid residues in the Asp110-Glu112 region of the EcoRII endonuclease (Asp110 --> Lys, Asn, Thr, Val, or Ile; Pro111 --> Arg, His, Ala, or Leu; Glu112 --> Lys, Gln, or Asp) have been constructed. When expressed in E. coli, all these plasmids displayed EcoRII endonuclease activity. We also constructed a plasmid containing a mutant ecoRII gene with deletion of the sequence coding the Gln109-Pro111 region of the protein. This mutant protein had no EcoRII endonuclease activity. The data suggest that Asp110, Pro111, and Glu112 residues do not participate in the formation of the EcoRII active site. However, this region seems to be relevant for the formation of the tertiary structure of the EcoRII endonuclease.     

Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis

Computer analysis of the crystallographic structure of the A subunit of Escherichia coil heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53 → Glu or Asp, Ser-63 → Lys, Val-97 → Lys, Tyr-104 → Lys or Asp, and Ser-14 → Lys or Glu. This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41 → Phe, Ala-45 → Tyr or Glu, Val-53 → Tyr, Val-60 → Gly, Ser-68 → Pro, His-70 → Pro, Val-97 → Tyr and Ser-114 → Tyr. The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54 → Lys or Ala, Tyr-59 → Met, Ser-68 → Lys, Ala-72 → Arg, His or Asp and Arg-192 → Asn. The results provide a further understanding of the structure–function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.     

Oxidation of methionine residues to methionine sulfoxides does not decrease potential antiatherogenic properties of apolipoprotein A-I

The initial stage of oxidation of high density lipoproteins (HDL) is accompanied by the lipid hydroperoxide-dependent, selective oxidation of two of the three Met residues of apolipoprotein A-I (apoA-I) to Met sulfoxides (Met(O)). Formation of such selectively oxidized apoA-I (i.e. apoA-I(+32)) may affect the antiatherogenic properties of HDL, because it has been suggested that Met(86) and Met(112) are important for cholesterol efflux and Met(148) is involved in the activation of lecithin:cholesterol acyl transferase (LCAT). We therefore determined which Met residues were oxidized in apoA-I(+32) and how such oxidation of apoA-I affects its secondary structure, the affinity for lipids, and its ability to remove lipids from human macrophages. We also assessed the capacity of discoidal reconstituted HDL containing apoA-I(+32) to act as substrate for LCAT, and the dissociation of apoA-I and apoA-I(+32) from reconstituted HDL. Met(86) and Met(112) were present as Met(O), as determined by amino acid sequencing and mass spectrometry of isolated peptides derived from apoA-I(+32). Selective oxidation did not alter the alpha-helicity of lipid-free and lipid-associated apoA-I as assessed by circular dichroism, and the affinity for LCAT was comparable for reconstituted HDL containing apoA-I or apoA-I(+32). Cholesteryl ester transfer protein mediated the dissociation of apoA-I more readily from reconstituted HDL containing apoA-I(+32) than unoxidized apoA-I. Also, compared with native apoA-I, apoA-I(+32) had a 2- to 3-fold greater affinity for lipid (as determined by the rate of clearance of multilamellar phospholipid vesicles) and its ability to cause efflux of [(3)H]cholesterol, [(3)H]phospholipid, and [(14)C]alpha-tocopherol from lipid-laden human monocyte-derived macrophages was significantly enhanced. By contrast, no difference was observed for cholesterol and alpha-tocopherol efflux to lipid-associated apolipoproteins. Together, these results suggest that selective oxidation of Met residues enhances rather than diminishes known antiatherogenic activities of apoA-I, consistent with the overall hypothesis that detoxification of lipid hydroperoxides by HDL is potentially antiatherogenic.     

Functional consequences of alterations to amino acids located in the hinge domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis of the sarcoplasmic reticulum Ca(2+)-ATPase was used to investigate the functional roles of 18 amino acid residues located at or near the "hinge-domain," a highly conserved region of the cation-transporting ATPases. Mutation of Lys684 to arginine, alanine, histidine, and glutamine resulted in complete loss of calcium transport function and ATPase activity. For the Lys684----Ala, histidine, and glutamine mutants, this coincided with a loss of the ability to form a phosphorylated intermediate from ATP or Pi. The Lys684----Arg mutant retained the ability to phorphorylate from ATP with normal apparent affinity, demonstrating the importance of the positive charge. On the other hand, no phosphorylation was observed with Pi as substrate in this mutant. Examination of the partial reactions after phosphorylation from ATP in the Lys684----Arg mutant demonstrated a reduction of the rate of transformation of the ADP-sensitive phosphoenzyme intermediate (E1P) to the ADP-insensitive phosphoenzyme intermediate (E2P), which could account for the loss of transport function. Once accumulated, the E2P intermediate was able to decompose rapidly in the presence of K+ at neutral pH. These results may be interpreted in terms of a preferential destabilization of protein phosphate interactions in the E2P form of this mutant. The Asp703----Ala and Asn-Asp707----Ala-Ala mutants were completely inactive and unable to form phosphoenzyme intermediates from ATP or Pi. In these mutants as well as in the Lys684----Ala mutant, nucleotides were found to protect with normal affinity against intramolecular cross-linking induced with glutaraldehyde, indicating that the nucleotide binding site was intact. Mutation of Glu646, Glu647, Asp659, Asp660, Glu689, Asp695, Glu696, Glu715, and Glu732 to alanine did not affect the maximum rates of calcium transport and ATP hydrolysis or the apparent affinities for calcium and ATP. Mutation of the 2 highly conserved proline residues, Pro681 and Pro709, as well as Lys728, to alanine resulted in partially inhibited Ca(2+)-ATPase enzymes with retention of the ability to form a phosphoenzyme intermediate from ATP or Pi and with normal apparent affinities for ATP and calcium. The proline mutants retained the biphasic ATP concentration dependence of ATPase activity, characteristic of the wild-type, and therefore the partial inhibition of turnover could not be ascribed to a disruption of the low affinity modulatory ATP site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased methionine sulfoxide content of apoA-I in type 1 diabetes

Cardiovascular disease is a major cause of morbidity and premature mortality in diabetes. HDL plays an important role in limiting vascular damage by removing cholesterol and cholesteryl ester hydroperoxides from oxidized low density lipoprotein and foam cells. Methionine (Met) residues in apolipoprotein A-I (apoA-I), the major apolipoprotein of HDL, reduce peroxides in HDL lipids, forming methionine sulfoxide [Met(O)]. We examined the extent and sites of Met(O) formation in apoA-I of HDL isolated from plasma of healthy control and type 1 diabetic subjects to assess apoA-I exposure to lipid peroxides and the status of oxidative stress in the vascular compartment in diabetes. Three tryptic peptides of apoA-I contain Met residues: Q(84)-M(86)-K(88), W(108)-M(112)-R(116), and L(144)-M(148)-R(149). These peptides and their Met(O) analogs were identified and quantified by mass spectrometry. Relative to controls, Met(O) formation was significantly increased at all three locations (Met(86), Met(112), and Met(148)) in diabetic patients. The increase in Met(O) in the diabetic group did not correlate with other biomarkers of oxidative stress, such as N(epsilon)-malondialdehyde-lysine or N(epsilon)-(carboxymethyl)lysine, in plasma or lipoproteins. The higher Met(O) content in apoA-I from diabetic patients is consistent with increased levels of lipid peroxidation products in plasma in diabetes. Using the methods developed here, future studies can address the relationship between Met(O) in apoA-I and the risk, development, or progression of the vascular complications of diabetes.     

Structure-function relationships in reconstituted HDL: Focus on antioxidative activity and cholesterol efflux capacity

AimsHigh-density lipoprotein (HDL) contains multiple components that endow it with biological activities. Apolipoprotein A-I (apoA-I) and surface phospholipids contribute to these activities; however, structure-function relationships in HDL particles remain incompletely characterised.MethodsReconstituted HDLs (rHDLs) were prepared from apoA-I and soy phosphatidylcholine (PC) at molar ratios of 1:50, 1:100 and 1:150. Oxidative status of apoA-I was varied using controlled oxidation of Met112 residue. HDL-mediated inactivation of PC hydroperoxides (PCOOH) derived from mildly pre-oxidized low-density lipoprotein (LDL) was evaluated by HPLC with chemiluminescent detection in HDL + LDL mixtures and re-isolated LDL. Cellular cholesterol efflux was characterised in RAW264.7 macrophages.ResultsrHDL inactivated LDL-derived PCOOH in a dose- and time-dependent manner. The capacity of rHDL to both inactivate PCOOH and efflux cholesterol via ATP-binding cassette transporter A1 (ABCA1) increased with increasing apoA-I/PC ratio proportionally to the apoA-I content in rHDL. Controlled oxidation of apoA-I Met112 gradually decreased PCOOH-inactivating capacity of rHDL but increased ABCA1-mediated cellular cholesterol efflux.ConclusionsIncreasing apoA-I content in rHDL enhanced its antioxidative activity towards oxidized LDL and cholesterol efflux capacity via ABCA1, whereas oxidation of apoA-I Met112 decreased the antioxidative activity but increased the cholesterol efflux. These findings provide important considerations in the design of future HDL therapeutics.Non-standard abbreviations and acronyms: AAPH, 2,2′-azobis(-amidinopropane) dihydrochloride; ABCA1, ATP-binding cassette transporter A1; apoA-I, apolipoprotein A-I; BHT, butylated hydroxytoluene; CV, cardiovascular; EDTA, ethylenediaminetetraacetic acid; HDL-C, high-density lipoprotein cholesterol; LOOH, lipid hydroperoxides; Met(O), methionine sulfoxide; Met112, methionine 112 residue; Met86, methionine 86 residue; oxLDL, oxidized low-density lipoprotein; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PL, phospholipid; PCOOH, phosphatidylcholine hydroperoxide; PLOOH, phospholipid hydroperoxide.     

Studies on D-tetrose metabolism. VI. Crystallization and some properties of D-erythrulose reducatase from beef liver.

D-Erythrulose reductase of beef liver was crystallized from ammonium sulfate solution at pH 8.17. The crystals are needle-shaped. The enzyme protein contains 851 amino acid residues per mole of the enzyme: Lys28, His11, Arg52, Asp79, Thr58, Ser56, Glu68, Pro20, Gly80, Ala107, Val112, Met24, Ile31, Leu88, Tyr7, Phe22, Trp4, and Cys4. The enzyme is inactivated by exposure to temperatures below 12degrees. The inactivation is accelerated by increasing the salt concentration and decreasing the enzyme concentration. The pH of the medium also has a pronounced effect, the maximum stability of the enzyme is obtained at pH 8.5. NADP+ protected the enzyme from cold inactivation at all stages of the process and also afforded protection against inactivation by heat and pH. The cold inactivation of the enzyme is accompanied by dissociation of the enzyme protein to subunits.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

Selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein D: implications for lipid antioxidant activity and Alzheimer's disease

ApoD (apolipoprotein D) is up-regulated in AD (Alzheimer's disease) and upon oxidative stress. ApoD inhibits brain lipid peroxidation in vivo, but the mechanism is unknown. Specific methionine residues may inhibit lipid peroxidation by reducing radical-propagating L-OOHs (lipid hydroperoxides) to non-reactive hydroxides via a reaction that generates MetSO (methionine sulfoxide). Since apoD has three conserved methionine residues (Met(49), Met(93) and Met(157)), we generated recombinant proteins with either one or all methionine residues replaced by alanine and assessed their capacity to reduce HpETEs (hydroperoxyeicosatetraenoic acids) to their HETE (hydroxyeicosatetraenoic acid) derivatives. ApoD, apoD(M49-A) and apoD(M157-A) all catalysed the reduction of HpETEs to their corresponding HETEs. Amino acid analysis of HpETE-treated apoD revealed a loss of one third of the methionine residues accompanied by the formation of MetSO. Additional studies using apoD(M93-A) indicated that Met(93) was required for HpETE reduction. We also assessed the impact that apoD MetSO formation has on protein aggregation by Western blotting of HpETE-treated apoD and human brain samples. ApoD methionine oxidation was associated with formation of apoD aggregates that were also detected in the hippocampus of AD patients. In conclusion, conversion of HpETE into HETE is mediated by apoD Met(93), a process that may contribute to apoD antioxidant function.     

Formation of methionine sulfoxide during glycoxidation and lipoxidation of ribonuclease A

Chemical modification of proteins by reactive oxygen species affects protein structure, function and turnover during aging and chronic disease. Some of this damage is direct, for example by oxidation of amino acids in protein by peroxide or other reactive oxygen species, but autoxidation of ambient carbohydrates and lipids amplifies both the oxidative and chemical damage to protein and leads to formation of advanced glycoxidation and lipoxidation end-products (AGE/ALEs). In previous work, we have observed the oxidation of methionine during glycoxidation and lipoxidation reactions, and in the present work we set out to determine if methionine sulfoxide (MetSO) in protein was a more sensitive indicator of glycoxidative and lipoxidative damage than AGE/ALEs. We also investigated the sites of methionine oxidation in a model protein, ribonuclease A (RNase), in order to determine whether analysis of the site specificity of methionine oxidation in proteins could be used to indicate the source of the oxidative damage, i.e. carbohydrate or lipid. We describe here the development of an LC/MS/MS for quantification of methionine oxidation at specific sites in RNase during glycoxidation or lipoxidation by glucose or arachidonate, respectively. Glycoxidized and lipoxidized RNase were analyzed by tryptic digestion, followed by reversed phase HPLC and mass spectrometric analysis to quantify methionine and methionine sulfoxide containing peptides. We observed that: (1) compared to AGE/ALEs, methionine sulfoxide was a more sensitive biomarker of glycoxidative or lipoxidative damage to proteins; (2) regardless of oxidizable substrate, the relative rate of oxidation of methionine residues in RNase was Met29>Met30>Met13, with Met79 being resistant to oxidation; and (3) arachidonate produced a significantly greater yield of MetSO, compared to glucose. The methods developed here should be useful for assessing a protein's overall exposure to oxidative stress from a variety of sources in vivo.     

Plasminogen activator inhibitor-1 deficient mice are protected from angiotensin II-induced fibrosis

Fungal methionine synthase, Met6p, transfers a methyl group from 5-methyl-tetrahydrofolate to homocysteine to generate methionine. The enzyme is essential to fungal growth and is a potential anti-fungal drug design target. We have characterized the enzyme from the pathogen Candida albicans but were unable to crystallize it in native form. We converted Lys103, Lys104, and Glu107 all to Tyr (Met6pY), Thr (Met6pT) and Ala (Met6pA). All variants showed wild-type kinetic activity and formed useful crystals, each with unique crystal packing. In each case the mutated residues participated in beneficial crystal contacts. We have solved the three structures at 2.0–2.8 Å resolution and analyzed crystal packing, active-site residues, and similarity to other known methionine synthase structures. C. albicans Met6p has a two domain structure with each of the domains having a (βα)₈-barrel fold. The barrels are arranged face-to-face and the active site is located in a cleft between the two domains. Met6p utilizes a zinc ion for catalysis that is bound in the C-terminal domain and ligated by four conserved residues: His657, Cys659, Glu679 and Cys739.     

Diastereoselective protein methionine oxidation by reactive oxygen species and diastereoselective repair by methionine sulfoxide reductase

Recent studies have shown that the "calcium-sensor" protein calmodulin (CaM) suffers an age-dependent oxidation of methionine (Met) to methionine sulfoxide (MetSO) in vivo. However, MetSO did not accumulate on the Met residues that show the highest solvent-exposure. Hence, the pattern of Met oxidation in vivo may give hints as to which reactive oxygen species and oxidation mechanisms participate in the oxidation of this important protein. Here, we have exposed CaM under a series of different reaction conditions (pH, [Ca(2+)], [KCl]) to various biologically relevant reactive oxygen species and oxidizing systems (peroxides, HOCl, peroxynitrite, singlet oxygen, metal-catalyzed oxidation, and peroxidase-catalyzed oxidation) to investigate whether one of these systems would lead to an oxidation pattern of CaM similar to that observed in vivo. However, generally, these oxidizing conditions led to a preferred or exclusive oxidation of the C-terminal Met residues, in contrast to the oxidation pattern of CaM observed in vivo. Hence, none of the employed oxidizing conditions was able to mimic the age-dependent oxidation of CaM in vivo, indicating that other, yet unidentified oxidation mechanisms may be important in vivo. Some oxidizing species showed a quite-remarkable diastereoselectivity for the formation of either L-Met-D-SO or L-Met-L-SO. Diastereoselectivity was dependent on the nature of the oxidizing species but was less a function of the location of the target Met residue in the protein. In contrast, diastereoselective reduction of L-Met-D-SO by protein methionine sulfoxide reductase (pMSR) was efficient regardless of the position of the L-Met-D-SO residue in the protein and the presence or absence of calcium. With only the L-Met-D-SO diastereomer being a substrate for pMSR, any preferred formation of L-Met-L-SO in vivo may cause the accumulation of MetSO unless the oxidized protein is substrate for (accelerated) protein turnover.     

Effects of orally administrated amino acids on myofibrillar proteolysis in chicks

We examined the effects of orally administrated amino acids on myfibrillar proteolysis in food-deprived chicks. Plasma N(tau)-methylhistidine concentration, as an index of myofibrillar proteolysis, was decreased by the administration of Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg but not by Asp, Val, Phe, Tyr or His to chicks. Orally administrated Cys was fatal to chicks. These results indicate that oral Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg administration suppressed myofibrillar proteolysis in chicks.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH₂-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH₂-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH₂-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala²Val³ residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH₂-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH₂-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH₂-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.     

Homologues of the human C and A apolipoproteins in the Macaca fascicularis (cynomolgus) monkey

We used antisera to human A and C apolipoproteins to identify homologues of these proteins among the high-density lipoprotein apoproteins of Macaca fascicularis (cynomolgus) monkeys, and NH2-terminal analysis was used to verify the homology. The NH2-terminal sequence of the M. fascicularis apoA-I is identical with that of another Old World species, Erythrocebus patas, and differs from human apoA-I at only 4 of the first 24 residues. M. fascicularis apoA-II contains a serine for cysteine replacement at position 6 and is therefore monomeric like the apoA-II from all species below apes. Human and monkey apoA-II are not otherwise different through their first 25 residues. About 20% of M. fascicularis apoC-I aligns with human apoC-I through residue 22, and 80% lacks an NH2-terminal dipeptide. Otherwise, the monkey apoC-I differs from the human protein at only 2 of 25 positions. Two forms of M. fascicularis apoC-II were identified. ApoC-II1 is highly homologous with human apoC-II, whereas an NH2-terminal hexapeptide is absent from apoC-II2. ApoC-II2 was the predominant species, and apoC-II1 appears to represent a propeptide from which a hexapeptide prosegment is cleaved at a Gln-Asp bond. Both forms of monkey apoC-II are potent activators of lipoprotein lipase. There are two polymorphic forms of M. fascicularis apoC-III, and their electrophoretic mobilities become identical after treatment with neuraminidase. Except for a glycine for serine substitution at position 10, the first 15 NH2-terminal residues of M. fascicularis and human apoC-III are the same.