Epsilon-amino Groups Of Lysyl Residues Research Articles

Gas-Phase Fragmentation Characteristics of Benzyl-Aminated Lysyl-Containing Tryptic Peptides

The fragmentation characteristics of peptides derivatized at the side-chain epsilon-amino group of lysyl residues via reductive amination with benzaldehyde have been examined using collision-induced dissociation (CID) tandem mass spectrometry. The resulting MS/MS spectra exhibit peaks representing product ions formed from two independent fragmentation pathways. One pathway results in backbone fragmentation and commonly observed sequence ion peaks. The other pathway corresponds to the unsymmetrical, heterolytic cleavage of the C(zeta)-N(epsilon) bond that links the benzyl derivative to the side-chain lysyl residue. This results in the elimination of the derivative as a benzylic or tropylium carbocation and a (n - 1)(+)-charged peptide product (where n is the precursor ion charge state). The frequency of occurrence of the elimination pathway increases with increasing charge of the precursor ion. For the benzyl-modified tryptic peptides analyzed in this study, peaks representing products from both of these pathways are observed in the MS/MS spectra of doubly-charged precursor ions, but the carbocation elimination pathway occurs almost exclusively for triply-charged precursor ions. The experimental evidence presented herein, combined with molecular orbital calculations, suggests that the elimination pathway is a charge-directed reaction contingent upon protonation of the secondary epsilon-amino group of the benzyl-derivatized lysyl side chain. If the secondary epsilon-amine is protonated, the elimination of the carbocation is observed. If the precursor is not protonated at the secondary epsilon-amine, backbone fragmentation persists. The application of appropriately substituted benzyl analogs may allow for selective control over the relative abundance of product ions generated from the two pathways.

Effects of Heating and Glycation of β-Lactoglobulin on Its Recognition by IgE of Sera from Cow Milk Allergy Patients

beta-Lactoglobulin (beta-LG) is one of the cow's major milk proteins and the most abundant whey protein. This globular protein of about 18 kDa is folded, forming a beta-barrel (or calyx) structure. This structure is stabilized by two disulfide bonds and can be altered by heating above 65 degrees C. beta-LG is also one of the major allergens in milk. Heating is one of the most common technologic treatments applied during many milk transformations. During heating in the presence of reducing sugars, beta-LG is also submitted to the Maillard reaction, which at the first stage consists of the covalent fixation of sugars on the epsilon-amino groups of lysyl residues. The following steps are condensation and polymerization reactions leading to the formation of melanoidins (brown pigments). Despite the frequency of use of heating during milk transformation, the effects of heat-induced denaturation and of glycation of beta-LG on its recognition by IgE from cow's milk allergy (CMA) patients are not fully understood. The objectives of our work were to evaluate the effect of heat-induced denaturation of bovine beta-LG on binding of IgE from CMA patients and to determine the effect of moderate glycation on the degree of recognition by IgE. We showed that heat-induced denaturation (loss of tertiary and secondary structures) of beta-LG is associated with weaker binding of IgE from CMA patients. It was also shown that moderate glycation of beta-LG in early stages of Maillard reaction has only a small effect on its recognition by IgE, whereas a high degree of glycation has a clear "masking" effect on the recognition of epitopes. This demonstrates the importance of epsilon-amino groups of lysines in the definition of epitopes recognized by IgE.

Aging, Diabetes, and Renal Failure Catalyze the Oxidation of Lysyl Residues to 2‐Aminoadipic Acid in Human Skin Collagen

The epsilon-amino group of lysyl residues oxidatively deaminates in the presence of alpha-dicarbonyl sugars and redox-active metals forming alpha-aminoadipic acid-delta-semialdehyde (allysine; Suyama's hypothesis), which can further oxidize into 2-aminoadipic acid. Here we show that 2-aminoadipic acid is significantly (P < 0.05) correlated with 6-hydroxynorleucine, carboxyethyllysine (CEL), and carboxymethyllysine (CML) in human skin collagen. Since CEL and CML can originate from carbohydrate and lipid by oxidative decomposition and alpha-dicarbonyl formation, these results provide support for Suyama's hypothesis. Allysine, in turn, is readily converted by oxidation into 2-aminoadipic acid, which accumulates to high levels in skin (i.e., > 2 nmol/mg collagen).

Iso[7]LGD2-protein adducts are abundant in vivo and free radical-induced oxidation of an arachidonyl phospholipid generates this D series isolevuglandin in vitro.

Isolevuglandins (isoLGs) are a family of gamma-ketoaldehydes, aka isoketals or neuroketals, that are generated by free radical-induced oxidation of polyunsaturated fatty acid-containing lipids. Because of their high reactivity toward epsilon-amino groups of lysyl residues, isoLGs are found as protein adducts in vivo. Plasma levels of isoLG-derived protein modifications are orders of magnitude higher than levels of the corresponding isoprostane. This suggests that while isoprostanes are rapidly cleared from the circulation, isoLG-protein adducts accumulate over the lifetime of the protein, which can be weeks, and this may provide a dosimeter for oxidant stress. We now confirm the postulated formation of the first D series isoLG, iso[7]LGD(2), by free radical-induced oxidation of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine in vitro. We also show that iso[7]LGD(2)-protein adduct levels in blood are the highest known for an isoLG-derived epitope. They average 30-fold higher than isoLGE(2)-protein and 3-fold higher than iso[4]LGE(2)-protein levels. Similarly, iso[7]LGD(2)-derived epitope levels in oxidized low density lipoprotein are 20 times higher than isoLGE(2)-protein and five times higher than iso[4]LGE(2)-protein levels. Previous studies showed that plasma levels of protein-bound E series isoLGs, i.e., isoLGE(2) and iso[4]LGE(2), are elevated in individuals with atherosclerosis as compared with age-matched controls. Plasma iso[7]LGD(2)-protein immunoreactivity in individuals with atherosclerosis averages 8.5 +/- 3.1 nmol/mL, significantly higher (P = 0.01) than the 3.5 +/- 0.1 nmol/mL in healthy controls. Plasma levels of iso[7]LGD(2)-protein adducts are strongly correlated with iso[4]LGE(2)- (r = 0.933) and isoLGE(2)-protein adducts (r = 0.877). This supports the hypothesis that isoLGs are generated in vivo by parallel competing radical-induced pathways.

Mutational hotspot in the human biotinidase gene causes profound biotinidase deficiency.

Biotinidase deficiency is an autosomal recessive inherited disorder that is characterized by neurological and cutaneous symptoms. Biotinidase-deficient children cannot recycle endogenous biotin, an essential water-soluble B vitamin. Biotin is covalently attached to epsilon-amino groups of lysyl residues of four carboxylases. These carboxylases are subsequently degraded to biocytin (biotin-epsilon-lysine). Biotinidase cleaves biocytin to biotin and lysine, thereby completing the biotin cycle. The symptoms of biotinidase deficiency can be resolved or prevented by treatment with biotin. Therefore, it is important that biotinidase deficiency is diagnosed early so that permanent neurological damage can be prevented. Many states and countries currently perform newborn screening for biotinidase deficiency. We have recently isolated and characterized the cDNA for normal human biotinidase and localized the gene to chromosome 3p25 (ref. 9). We have now identified the first mutation that causes profound biotinidase deficiency. It occurs in a distinct region of the gene that encodes the putative signal peptide. Fifty percent of symptomatic children studied have a 7-bp deletion coupled with a 3-bp insertion in at least one of their alleles of the biotinidase gene. This mutation appears to be a common cause of biotinidase deficiency in symptomatic children.

The role of pyrrole formation in the alteration of neurofilament transport induced during exposure to 2,5-hexanedione.

Exposure to the gamma-diketone, 2,5-hexanedione (HD), results in the accumulation of neurofilaments within the distal axon and is associated with acceleration of neurofilament transport within the proximal axon. The epsilon-amino groups of lysyl residues react with HD forming pyrrole adducts, followed by pyrrole-mediated protein crosslinking. Both reaction steps have been proposed as mechanisms causing neurofilament accumulation and acceleration of transport. In order to assess the importance of these steps on neurofilament transport, we compared transport in the optic system of rats exposed to HD and 3-acetyl-2,5-hexanedione (AcHD), a non-toxic analog of HD which forms pyrroles but does not crosslink proteins. Control, HD-treated, and AcHD-treated rats received intraoptic injections of [35S]-methionine and were exposed to saline, HD, or AcHD by intraperitoneal injections before and during the period of neurofilament transport. Neurofilament triplet proteins in the optic nerve and tract were identified by polyacrylamide gel electrophoresis followed by fluorography. The rate of neurofilament transport was accelerated in HD-treated animals over that of controls. However, despite higher levels of protein-bound pyrroles in AcHD-treated animals, the rate of transport was indistinguishable from that of controls. These findings indicate that pyrrole formation alone is not sufficient to cause acceleration of neurofilament transport.

Chemical Modification of Pseudomonas ochraceae 4-Hydroxy-4-Methyl-2-0xoglutarate Aldolase by Diethyl Pyrocarbonate

Diethyl pyrocarbonate inactivates Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17] by a simple bimolecular reaction. The inactivation is not reversed by hydroxylamine. The pH curve of inactivation indicates the involvement of a residue with a pK of 8.8. Several lines of evidence show that the inactivation is due to the modification of epsilon-amino groups of lysyl residues. Although histidyl residue is also modified, this is not directly correlated to the inactivation. No cysteinyl, tyrosyl, or tryptophyl residue or alpha-amino group is significantly modified. The modification of three lysyl residues per enzyme subunit results in the complete loss of aldolase activity toward various 4-hydroxy-2-oxo acid substrates, whereas oxaloacetate beta-decarboxylase activity associated with the enzyme is not inhibited by this modification. Statistical analysis suggests that only one of the three lysyl residues is essential for activity. l-4-Carboxy-4-hydroxy-2-oxoadipate, a physiological substrate for the enzyme, strongly protects the enzyme against inactivation. Pi as an activator of the enzyme shows no specific protection. The molecular weight of the enzyme, Km for substrate or Mg2+, and activation constant for Pi are virtually unaltered after modification. These results suggest that the modification occurs at or near the active site and that the essential lysyl residue is involved in interaction with the hydroxyl group but not with the oxal group of the substrate.

Targeting Sod by Gene and Protein Engineering and Inhibition of Free Radical Injury

Although oxygen toxicity of tissues can be decreased by a variety of antioxidants and some enzymes, such as SOD and catalase, their protective effect on tissue injury in various diseases are fairly small predominantly because of their unfavorable in vivo behavior. To minimize oxidative stress in various diseases, such as ischemic myocardial injury, circulatory disturbance and corneal inflammation, we synthesized three types of SOD derivatives by gene and protein engineering technique. One type of SOD (SM-SOD covalently linked with hydrophobic anions) circulates bound to albumin with a half life of 6 h and accumulates in tissues whose local pH is decreased. The other type of SOD (AC-SOD covalently linked with long chain fatty acids via the epsilon-amino group of lysyl residues) anchors onto membrane/lipid bilayers of various cells. The last type of SOD (HB-SOD synthesized by constructing a fusion gene coding human CuZn-type SOD and a C-terminal heparin-binding domain) binds to heparin-like proteoglycans on vascular endothelial cell surface. Intravenous administration of either SM-SOD or HB-SOD markedly inhibited postischemic reflow arrhythmias in the rat. When the left anterior descending artery was occluded permanently, about 65% of animals died within 30 min predominantly due to irreversible ventricular fibrillation; the motality of animals decreased to 15% by administering SM-SOD either before or after occlusion. Topically administered AC-SOD bound to the corneal epithelial cell surface and polymorphonuclear leukocytes and efficiently dismutated superoxide radicals at their cell surface. Thus, endotoxin-induced keratitis was inhibited markedly by topical instillation of AC-SOD.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of fructose in glycation and cross-linking of proteins.

Incubation of carbohydrate-free human serum albumin (HSA) with fructose in an aqueous buffer at pH 7.4 resulted in glycation of epsilon-amino groups of lysyl residues. A recently developed procedure, involving analysis of hexitol amino acids by high-performance liquid chromatography of phenylthiocarbamyl derivatives, was used to show that 85% of the bound hexose was attached to protein via carbon 2 (C-2). The remainder was attached to protein via carbon 1 (C-1). When incubations were conducted with glucose under identical conditions, all the hexose was attached via C-1. Examination of human ocular lens proteins showed that the majority of the covalently bound hexose was connected to epsilon-amino groups of lysyl residues via C-1; this was attributed mainly to nonenzymatic glucosylation in vivo, which has already been documented. A significant proportion (10-20%) of the bound hexose was connected via C-2. In view of the HSA-hexose incubation results (above), this indicated that the lens proteins had reacted with endogenous fructose; i.e., they had undergone nonenzymatic fructosylation in vivo. The model protein bovine pancreatic ribonuclease A reacted with fructose and glucose at similar rates under physiological conditions. However, covalent, non-disulfide cross-linking, which could be inhibited by D-penicillamine, was induced 10 times more rapidly by fructose than by glucose. It is postulated that some of the protein cross-linking that occurs in vivo is fructose-induced. The possible significance of these processes in diabetic subjects is discussed.

Phosphorylation site of eukaryotic initiation factor 4E.

Eukaryotic protein synthesis initiation factor 4E (eIF-4E) was labeled in situ with [32P]orthophosphate in cultured HeLa cells and rabbit reticulocytes and purified by affinity chromatography. Tryptic digestion yielded one labeled peptide which contained predominantly serine and lysine. After treatment of the protein with citraconic anhydride to block epsilon-amino groups of lysyl residues, tryptic digestion yielded a labeled peptide whose composition was consistent with the structure Trp-Ala-Leu-Trp-Phe-Phe-Lys-Asn-Asp-Lys-Ser(P)-Lys-Thr-Trp-Gln-Ala-Asn-L eu-Arg, one of the arginyl peptides predicted from the human eIF-4E cDNA sequence. The only serine in this peptide is located at position 53 of eIF-4E. Thus, it is concluded that eIF-4E contains a single site of phosphorylation for an endogenous protein kinase, which is Ser-53 in the human eIF-4E sequence.

Reaction of electron-transfer flavoprotein with electron-transfer flavoprotein-ubiquinone oxidoreductase.

The oxidative half-reaction of electron-transfer flavoprotein (ETF), electron transfer from ETF to electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), is dependent on complementary surface charges on the two proteins. ETF is the positively charged member of the redox pair. The evidence is based on the pH and ionic strength dependencies of the comproportionation of oxidized ETF and ETF hydroquinone catalyzed by ETF-QO and on the effects of chemical modification of ETF on the comproportionation reaction. Acetylation of one and five epsilon-amino groups of lysyl residues results in 3- and 13-fold increases, respectively, in the Km of ETF-QO for ETF but no change in Vmax. Amidination, which maintains positive charge at modified loci, has no effect on steady-state kinetic constants. These chemical modifications have no effect on the equilibrium constant for equilibration of ETF redox states. The Km of ETF-QO for ETF is pH dependent above pH 8.5, suggesting titration of lysyl residues as previously observed in studies of the reductive half-reaction of ETF [Beckmann, J. D., & Frerman, F. E. (1983) J. Biol. Chem. 258, 7563-7569]. The ionic strength dependence of TN/KmETF for the reaction follows the limiting Brønsted equation ln (TN/Km) = ln k0 + 2 alpha Z1Z2I1/2, and Z1Z2, the product of charges on the reacting proteins, is similar to the value of Z1Z2 for the reductive half-reaction of ETF by the general acyl-CoA dehydrogenase. The ETF-QO-catalyzed comproportionation reaction exhibits a primary deuterium isotope effect in D2O, perhaps indicating the participation of solvent water in the electron-transfer reaction.

Increased solubility of newly synthesized collagen in retinal capillary pericyte cultures by nonenzymatic glycosylation.

The effect of increasing glucose concentration on the solubility of newly synthesized collagen was studied in bovine retinal capillary pericyte (BRCP) cultures. Synchronized pericytes were labeled with [3H]-proline in media containing either 5, 10, 20 or 40 mM glucose, or 5 mM glucose and 100 micrograms/ml beta-aminopropionitrile fumarate (BAPN). The relative rate of collagen synthesis in the medium (soluble collagen) was significantly increased, and the rate in the cell layer (insoluble collagen) was decreased, by an elevated glucose concentration. The increase in collagen solubility with 20 and 40 mM glucose (over normal, 5 mM glucose) was 14 and 42%, respectively, of that obtained with BAPN. BAPN and 4-deoxypyridoxine inhibited BRCP lysyl oxidase activity, but elevated glucose concentrations did not. Increased nonenzymatic glycosylation of newly synthesized collagen was demonstrated in BRCP cultures co-labeled with L-[14C]-glucose and L-[3H]-proline. The increase in collagen solubility with elevated glucose concentrations therefore appears to be the result of the inhibition of lysyl-derived cross-link formation because of an increased nonenzymatic glycosylation of the epsilon-amino groups of lysyl residues of newly synthesized collagen in culture.

Effect of lysine modification on the activity of the sigma subunit of Escherichia coli RNA polymerase.

The function of lysyl residues of the sigma subunit of the RNA polymerase from Escherichia coli was investigated by chemical modification with trinitrobenzenesulfonic acid (TNBS). Following reaction with TNBS, analysis of the modified sigma indicated that trinitrophenylation was limited to the epsilon-amino groups of lysyl residues. Progressive loss in the activity of sigma followed increasing trinitrophenylation as assayed by the ability to stimulate RNA polymerase core enzyme in a reaction directed by T7 DNA. Modification of five lysyl groups resulted in the complete loss of sigma activity. Kinetic analysis indicated that one lysyl group is critical for the function of sigma. TNP-sigma was able to form a holoenzyme complex with a binding affinity comparable to that of sigma. Promoter recognition studies were done by using HindIII fragments from T5 DNA. The TNP-sigma core complex was unable to form a tight binary complex with the T5 promoters. Studies on RNA chain initiation were carried out by using d(A-T)n and T7 DNA templates. TNP-sigma was unable to stimulate RNA chain initiation by core polymerase. Limited proteolytic digests of TNP-sigma or sigma using Staphylococcus aureus V8 protease were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results suggested a change in the conformation of sigma following trinitrophenylation.

Tritium labeling of proteins to high specific radioactivity by reduction methylation.

The third component of human complement, hemoglobin A2, and IgG have been radiolabeled with tritium to specific activities of 179, 103, and 89 Ci/mmol, respectively, by reductive methylation. The labeling procedure is mild, requiring only brief exposure to formaldehyde (10 to 12 mM) and tritiated sodium borohydride (3 to 5 mM), and specific for the alpha amino groups of NH2-terminal residues and epsilon-amino groups of lysyl residues. The extent of modification for each protein ranged between 5 and 16% at available amino groups based on a stoichiometric reactivity of 2 mol of formaldehyde/mol of primary amine. Although the degree of substitution required to obtain these specific activities was high, no significant loss of antigenic or biological activities was apparent. Each labeled protein was 90 to 95% precipitable with antiserum elicited to the unreacted protein. The hemolytic activity of C3 was reduced by only 16%; the oxygen affinity of HbA2 was minimally decreased, and the phosphorylcholine binding affinity of an IgA myeloma protein (TEPC-15) was not significantly affected. Precise double antibody radioimmunoassays were developed with each tritiated protein capable of detecting less than 10(-8) g of antigen. Labeled protein samples have been stored for time periods up to 1 year without appreciable deterioration.

Oxidative deamination of epsilon-aminolysine residues and formation of Schiff base cross-linkages in cell envelopes of Escherichia coli.

Oxidative deamination of the epsilon-amino group of lysyl residues to form allysine is the initial reaction in the cross-linking of collagen and elastin in vertebrates. The allysyl residues, generated by lysyl oxidase in this reaction, condense with either other allysyl residues or epsilon-amino groups of lysyl or hydroxylysyl to form aldol or Schiff base cross-links. This paper presents evidence that similar allysyl residues and Schiff base cross-links are synthesized in cell envelopes of Escherichia coli. Acid hydrolysis followed by amino acid analysis of envelopes either reduced with NaB[3H]4 or labeled with [14C]lysine and reduced with NaBH4 yielded allysine and two labeled fragments with elution profiles and molecular weights (250 and 330) consistent with Schiff base products derived at least in part from allysine. When [6-3H]lysine-labeled cell envelopes were incubated at 37 degrees C, gradual release of tritiated water occurred. This suggests that an enzymatic reaction catalyzes the deamination of lysine in E. coli membranes and that the higher molecular weight proteins detected in stationary phase or in log phase cell envelopes after NaBH4 reduction occur as a result of formation of Schiff base cross-links.

Active site studies of ribulose-1,5-bisphosphate carboxylase/oxygenase with pyridoxal 5'-phosphate

There are 16 epsilon-amino groups of lysyl residues which are essential for the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. These lysyl residues formed a Schiff base with pyridoxal 5'-phosphate which was stabilized by NaBH4 reduction. The stoichiometry of covalently bound pyridoxal 5'-phosphate after NaBH4 reduction was determined spectrophotometrically with a derived molar extinction coefficient of 4800 M-1 cm-1. The incorporation of pyridoxal 5'-phosphate into the protein was accompanied by loss of the carboxylase and oxygenase activities, but the ratio of their activities remained constant. Vmax, but not Km, values were changed by this modification of the amino acid groups. Half of the epsilon-amino groups of lysine appeared to be at the 8 catalytic sites and half at the 8 activator sites for CO2, as indicated by kinetics of reactivation of the enzyme activity during dissociation of the Schiff base between pyridoxal 5'-phosphate and the protein. Reduction with NaB3H4 revealed that all 16 of the lysyl residues were on the large subunit. Ribulose-1,5-bis-phosphate alone protected 16 amino groups from Schiff base formation with pyridoxal 5'-phosphate, and the enzyme activity was fully conserved. NaHCO3 increased and MgCl2 lowered slightly the protective effect of ribulose-1,5-bisphosphate. Modification of sulfhydryl groups by p-chloromercuribenzoic acid inhibited the enzyme and excluded binding of 8 equivalents of pyridoxal 5'-phosphate. Upon removal of the mercuribenzoate groups with excess dithiothreitol, the loss of enzyme activity was exponentially correlated with the binding of 8 mol of pyridoxal 5'-phosphate/mol of enzyme. In contrast to p-chloromercuribenzoic acid, iodoacetamide, which inhibited the catalysis, had no influence on the binding of the 16 pyridoxal 5'-phosphate/mol of enzyme. It is suggested that the CO2 activator site with one epsilon-amino group for binding of CO2 and the catalytic site with one epsilon-amino group of a yet unknown function are located closely together on the large subunits of the enzyme. The results are consistent with a sulfhydryl group as the proton acceptor opposite carbon 3 of ribulose-1,5-bisphosphate, and when this sulfhydryl group is blocked by the bulky mercuribenzoate group, but not by the small carboxyamidomethyl group, pyridoxal 5'-phosphate binding is modified.

(Lysyl-Glycyl-Glycyl)5 and (Lysyl-Glycyl-Glycyl)10 as models of histone. Interaction with DNA and acetylation by calf thymus enzyme.

Sequential polypeptides (Lys-Gly-Gly)5 and (Lys-Gly-Gly)10 were synthesized as models for the N-terminal region of H4 histone and their interactions with DNA were studied. The ability of (Lys-Gly-Gly)10 to precipitate DNA was found to be much higher than that of (Lys-Gly-Gly)5. For example, at the physiological salt concentration, (Lys-Gly-Gly)10 precipitates DNA but (Lys-Gly-Gly)5 does not. Both peptides could be acetylated by a partially purified histone acetyltransferase preparation derived from calf thymus. epsilon-Amino groups of lysyl residues were the sites of the acetylation. DNA-cellulose chromatography of the products indicated that acetylation weakened the interaction of the peptides with DNA.