Reductive Methylation of the Lysyl Residues in the fd Gene 5 DNA-Binding Protein: CD and 13C NMR Results on the Modified Protein

Abstract

The ∈-amino groups of the six lysyl residues of the fd gene 5 DNA-binding protein have been modified by reductive methylation to form N^∈, N^∈-dimethyl lysyl derivatives containing ¹³C-labeled methyl groups. The α-amino terminus of the protein was not accessible to methylation. Circular dichroism studies show that the modified protein binds to fd DNA, but with a slightly reduced affinity compared with that of unmodified gene 5 protein. We also find that both the modified and unmodified proteins bind to an oligodeoxynucleotide, d(A)₇, but in neither case does binding cause a decrease in the 228 nm CD band of the protein as occurs when the protein binds to long DNA polymers. ¹³C NMR spectra at 50.1 MHz of [¹³C]methylated gene 5 protein show five distinct resonances between 43.30 and 42.76 ppm originating from the six N^∈, N^∈-dimethyl lysyl residues. We attribute one of the resonances to two solvated lysyl residues and the other four to individual lysyl residues in different microenvironments. All four of these latter resonances are affected by the binding of d(A)₇. However, since two of these resonances are similarly affected by the presence of salt in the absence of DNA, only two are uniquely affected by DNA binding.     

Metabolism of Phosphatidylglycerol and Lysyl Phosphatidylglycerol in Staphylococcus aureus

The metabolism of phosphatidylglycerol and lysyl phosphatidylglycerol was studied in Staphylococcus aureus under four conditions: growing at pH 7.0 and 5.2, and not growing (resting) at pH 7.0 and 5.2. Measurements of the amounts of phosphatidylglycerol and lysyl phosphatidylglycerol, as well as labeling and pulsechase experiments, revealed that the phosphate group of the former and the lysyl group of the latter were in a state of active turnover. A marked decline in the cellular level of phosphatidylglycerol observed when cells were resting at pH 5.2 was found to be caused by both a decrease in synthesis and an increase in catabolism. The level of lysyl phosphatidylglycerol was found to be relatively constant under the four incubation conditions, although the lysyl moiety was in a state of turnover. Experiments designed to test the possible role of lysyl phosphatidylglycerol as a lysyl group donor in biosynthetic processes or in lysine transport were negative; no evidence to support the hypothesis that lysyl phosphatidylglycerol serves as an intermediate was obtained.     

Induction of human monocyte motility by lysyl oxidase

Lysyl oxidase highly purified from calf aorta was found to be a potent chemotactic agent for unstimulated human peripheral blood mononuclear cells, determined in in vitro assays in Boyden chambers. A typical chemotactic bell-shaped curve was observed, with a maximal migratory response of 237% of control occurring at 10⁻¹⁰ M lysyl oxidase. The chemotactic response was prevented by prior heat inactivation of the enzyme, by treatment of the enzyme with β-aminopropionitrile or ethylenediamine, which are active site-directed inhibitors of lysyl oxidase, and by a competing, lysine-containing peptide substrate of lysyl oxidase. The chemoattractant reponse to lysyl oxidases was characterized by both chemokinetic and chemotactic components. These results raise the possibility that extracellular lysyl oxidase may have important roles to play in biology in addition to its established function in the crosslinking of elastin and collagen.     

Estimation of the circular dichroism exhibited by statistical coils of poly(L-alanine) and unionized poly(L-lysine) in water

The circular dichroism of Ac-(Ala)_x-OMe and H-Lys-(Lys)_x-OH with x = 1, 2, 3, and 4 has been measured in aqueous solutions. The oligomers with x = 4 show similar circular dichroism spectra in water when the lysyl amino groups are protonated, and they respond in similar fashion to heating and to sodium perchlorate. Both oligomers at 15°C exhibit a positive circular dichroism band at 217–218 nm, which is eliminated by the isothermal addition of 4 M sodium perchlorate or by heating. The positive circular dichroism of the lysine oligomer is also eliminated when the pH is elevated to deprotonate the amino groups. Positive circular dichroism is still observed for Ac-(Ala)₄-OMe at elevated pH. Circular dichroism spectra have been estimated for poly(L -alanine) and poly(L -lysine) as statistical coils under the above conditions, based on the trends established with the oligomers. Poly(L -lysine) and poly(L -alanine) are predicted to exhibit similar circular dichroism behavior in aqueous solution so long as the lysyl amino groups are protonated. The circular dichroism of the statistical coil of poly(L -lysine), but not poly(L -alanine), is predicted to change when the pH is elevated sufficiently to deprotonate the lysyl amino groups. These results suggest that the unionized lysyl side chains participate in interactions that are not available to poly(L -alanine). Hydrophobic interactions may occur between the unionized lysyl side chains. Protonation of the lysyl amino groups is proposed to disrupt these interactions, causing poly(L -alanine) and protonated poly(L -lysine) to have similar circular dichroism properties.     

Lysyl oxidase activity regulates oncogenic stress response and tumorigenesis

Cellular senescence, a stable proliferation arrest, is induced in response to various stresses. Oncogenic stress-induced senescence (OIS) results in blocked proliferation and constitutes a fail-safe program counteracting tumorigenesis. The events that enable a tumor in a benign senescent state to escape from OIS and become malignant are largely unknown. We show that lysyl oxidase activity contributes to the decision to maintain senescence. Indeed, in human epithelial cell the constitutive expression of the LOX or LOXL2 protein favored OIS escape, whereas inhibition of lysyl oxidase activity was found to stabilize OIS. The relevance of these in vitro observations is supported by in vivo findings: in a transgenic mouse model of aggressive pancreatic ductal adenocarcinoma (PDAC), increasing lysyl oxidase activity accelerates senescence escape, whereas inhibition of lysyl oxidase activity was found to stabilize senescence, delay tumorigenesis, and increase survival. Mechanistically, we show that lysyl oxidase activity favors the escape of senescence by regulating the focal-adhesion kinase. Altogether, our results demonstrate that lysyl oxidase activity participates in primary tumor growth by directly impacting the senescence stability.     

Modification of the tritium-release assays for prolyl and lysyl hydroxylases using Dowex-50 columns.

The tritium release assay of Hutton et al. (Anal. Biochem.16, 384, 1966) for prolyl hydroxylase and of Miller (Anal. Biochem.45, 202, 1972) for lysyl hydroxylase have been modified. The reaction is carried out on a microscale, and tritiated water is collected after passage of the trichloroacetic acid-soluble reaction products through a small Dowex-50 (H⁺) column instead of using a vacuum distillation apparatus as described in the original procedures. When measured by the modified procedures, both the prolyl and lysyl hydroxlyase reactions showed regions of linearity with respect to enzyme concentration, time and substrate concentration and were almost completely dependent on ascorbate and α-ketoglutarate. In addition, both reactions were completely inhibited by the iron chelator, α,α′-dipyridyl. The results indicate that these assay procedures are valid means of measuring prolyl and lysyl hydroxylase activities.     

The interaction of chemically modified human plasma alpha1-antitrypsin with porcine pancreatic elastase.

The elastase inhibitory capacity of human plasma α₁-antitrypsin was determined following chemical modification of lysyl and arginyl residues. Modification of the guanidino group had no effect upon the inhibitory activity, while acetylation, citraconylation, and trinitrophenylation of the lysyl ?-amino group brought about a loss of elastase inhibitory capacity.     

Effect of diabetes andinsulin on rat renal glomerular protocollagen hydroxylase activities

The effect of diabetes and insulin on the activities of both prolyl hydroxylase (trivial name; proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) and lysyl hydroxylase (trivial name; lysine,2-oxoglutarate dioxygenase, EC 1.14.11.4) in isolated rat renal glomeruli was determined. Three groups of experimental animals were used: age-matched controls, streptozotocin-diabetic, and insulin-treated streptozotocin-diabetic. Using ¹⁴C-labeled lysine or proline hydroxylase substrate prepared from chick embryo tibiae, glomerular 17 000 × g supernatant enzyme was incubated in a complete hydroxylating system for 60 and 120 min. Lysyl hydroxylase activity was significantly increased in diabetic preparations, but prolyl hydroxylase activity did not differ from control. Administration of insulin to streptozotocin-injected animals completely restored glomerular lysyl hydroxylase to normal levels. The results suggest that the specific elevation of lysyl hydroxylase relates to the biochemical changes contributory to diabetic nephropathy, and that insulin may reverse this process.     

Proteolytic activity in human skin fibroblasts harvested with trypsin and its effect on measurements of collagen hydroxylase activities.

Lysates of human skin fibroblasts harvested without the use of trypsin do not contain detectable proteolytic activity, but when trypsin is used, lysates may contain activity equal to 10 ng of trypsin/10⁷ cells. The amount of cell lysate ordinarily examined for collagen prolyl and lysyl hydroxylase activity is sufficiently small that such amounts of trypsin have no observable effect on the unhydroxylated collagen substrate. Larger amounts of trypsin cause proteolysis of the unhydroxylated collagen substrate and a reduction of both prolyl and lysyl hydroxylation with lysyl hydroxylation more affected at low trypsin concentration than prolyl hydroxylation.     

Reaction of aspirin with cysteinyl residues of lens γ-crystallins: a mechanism for the proposed anti-cataract effect of aspirin

Incubation of lens crystallins with aspirin inhibits the development of opacities caused by cyanate. Cyanate-induced opacities are thought to be due to carbamylation of the lysyl residues which causes a decrease in the protein charge and subsequent conformational changes that permit disulfide bonding. Because aspirin can also react with lysyl residues, it has been proposed that the aspiring inhibition of cataractogonesis is due to acetylation of the lysyl which would block their reaction with cyanata. However, acetylation oflysyl residues also lowers the protein charge and would be expected to effect changes in protein conformation similar to those caused by carbamylation. Therefore, acetylation of the lysyl residues is not a satisfactory explanation for the inhibitory effect of aspirin on lens opacification. Our investigations of the reactions of cyanate and aspirin with bovine γ_II-crystallins show that the cysteinyl residues are also carbamylated and acetylated at pH 7.4. At this pH, the carbamylation at the cysteinyl residues is reversible, leading to regeneration of the thiol group and disulfide bonding. In contrast, the acetylation at cysteinyl residues is stable at pH 7.4 and can prevent disulfide bonding. This difference in stability explains how cyanate promotes, and aspirin inhibits, cataractogenesis.     

Regulation of the glycosylations of collagen hydroxylysine in chick embryo tendon and cartilage cells

The regulation of the glycosylations of hydroxylysine was studied in isolated chick-embryo cells by labelling with a [¹⁴C]lysine pulse. The course of the procollagen lysyl modifications was compared in tendon and cartilage cells, and the effect on the glycosylations of the degree of lysyl hydroxylation and the concentration of Mn²⁺ and Fe²⁺ were also studied, in tendon cells. Procollagen triple helix formation was inhibited in most experiments in order to eliminate the effect of this process on the continuation of the reactions.Both in the tendon and cartilage cells the intracellular lysyl modifications proceeded in a biphasic fashion. After an initial sharp linear increase, the reactions did not cease but were protracted at a slower but constant rate. Lysyl hydroxylation was followed by rapid galactosylation in both cell types and this was followed almost immediately by rapid glucosylation, suggesting a close association of the corresponding enzymes. The data further suggest that other factors must also exist, in addition to the differences in the timing of triple helix formation and the actual hydroxylysine content, which are responsible for the different amounts of galactose in the collagens synthesized by these cell types. The amount of glycosylgalactosylhydroxylysine nevertheless seemed to be determined by the available acceptor sites, i.e., the amount of galactosylhydroxylysine.In further experiments wiht tendon cells the oxygen participating in lysyl hydroxylation was displaced by nitrogen at various points in time. When the degree of lysyl hydroxylation was reduced to less than one-third of the original, the total amounts of glycosylated residues decreased correspondingly, but their proportion relative to total hydroxylysine remained unchanged.Extra Mn²⁺ increased the proportion of galactosylated hydroxylysine, suggesting that the activity of hydroxylysyl galactosyltransferase is not saturating in respect of the catalyzed reaction. Experiments on the addition of Fe²⁺ or its chelation by α, α′-dipyridyl gave indications that the presence of this co-factor is not required for either glycosylation reaction in isolated tendon cells.     

Nepsilon-formylation of lysine is a widespread post-translational modification of nuclear proteins occurring at residues involved in regulation of chromatin function

Post-translational modification of histones and other chromosomal proteins regulates chromatin conformation and gene activity. Methylation and acetylation of lysyl residues are among the most frequently described modifications in these proteins. Whereas these modifications have been studied in detail, very little is known about a recently discovered chemical modification, the N^ε-lysine formylation, in histones and other nuclear proteins. Here we mapped, for the first time, the sites of lysine formylation in histones and several other nuclear proteins. We found that core and linker histones are formylated at multiple lysyl residues located both in the tails and globular domains of histones. In core histones, formylation was found at lysyl residues known to be involved in organization of nucleosomal particles that are frequently acetylated and methylated. In linker histones and high mobility group proteins, multiple formylation sites were mapped to residues with important role in DNA binding. N^ε-lysine formylation in chromosomal proteins is relatively abundant, suggesting that it may interfere with epigenetic mechanisms governing chromatin function, which could lead to deregulation of the cell and disease.     

A [3H]lysine-containing synthetic peptide substrate for human protocollagen lysyl hydroxylase

A tridecapeptide containing tritium-labelled lysine and corresponding closely to residues 98 to 110 of the α chain of type I collagen was synthesized by the solid-phase method. Gly-Leu-Hyp-Gly-Nle-[4,5-³H]Lys-Gly-His-Arg-Gly-Phe-Ser-Gly was used as a substrate of human protocollagen lysyl hydroxylase (peptidyllysine, 2-oxoglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4) obtained from dermal fibroblasts. L-[4,5-³H]Lysine was converted to $\text{N}{}^{\mathrm{\alpha }}\text{-t-}\text{butyloxycarbonyl}\text{-N}{}^{\mathrm{?}}\text{-o-}\text{chlorobenzyloxycarbonyl}\text{[}{}^3\text{H}\text{]}\text{lysine}$ which was incorporated during stepwise synthesis of the peptide. The chemical and radiochemical purities and specific activity of the completed peptide were characterized. A non-radiolabelled analogue of the peptide inhibited the hydroxylation of [³H]lysine-containing protocollagen by human lysyl hydroxylase, indicating that the synthetic peptide interacted with the enzyme. The peptide containing [³H]lysine was a substrate for lysyl hydroxylase and permitted direct measurement of enzyme activity in relatively crude cell extracts by a tritium-release assay. Extracts of cultured fibroblasts from a patient with an autosomal recessive pattern of inheritance for Ehlers-Danlos syndrome type VI had activities for tritium release from either the radiolabelled synthetic peptide or from [³H]lysine-containing protocollagen that were only 30% of those from control cells. These data indicate that a stable, well-defined synthetic peptide containing [³H]lysine is a useful substrate for studies of genetically variant lysyl hydroxylase from cultured human cells.     

Recombinant expression of hydroxylated human collagen in Escherichia coli

Collagen is the most abundant protein in the human body and thereby a structural protein of considerable biotechnological interest. The complex maturation process of collagen, including essential post-translational modifications such as prolyl and lysyl hydroxylation, has precluded large-scale production of recombinant collagen featuring the biophysical properties of endogenous collagen. The characterization of new prolyl and lysyl hydroxylase genes encoded by the giant virus mimivirus reveals a method for production of hydroxylated collagen. The coexpression of a human collagen type III construct together with mimivirus prolyl and lysyl hydroxylases in Escherichia coli yielded up to 90 mg of hydroxylated collagen per liter culture. The respective levels of prolyl and lysyl hydroxylation reaching 25 % and 26 % were similar to the hydroxylation levels of native human collagen type III. The distribution of hydroxyproline and hydroxylysine along recombinant collagen was also similar to that of native collagen as determined by mass spectrometric analysis of tryptic peptides. The triple helix signature of recombinant hydroxylated collagen was confirmed by circular dichroism, which also showed that hydroxylation increased the thermal stability of the recombinant collagen construct. Recombinant hydroxylated collagen produced in E. coli supported the growth of human umbilical endothelial cells, underlining the biocompatibility of the recombinant protein as extracellular matrix. The high yield of recombinant protein expression and the extensive level of prolyl and lysyl hydroxylation achieved indicate that recombinant hydroxylated collagen can be produced at large scale for biomaterials engineering in the context of biomedical applications.     

Structure and Expression of the Human Lysyl Hydroxylase Gene (PLOD): Introns 9 and 16 Contain Alu Sequences at the Sites of Recombination in Ehlers-Danlos Syndrome Type VI Patients

Lysyl hydroxylase (EC 1.14.11.4) catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in peptide linkages. This enzyme activity is known to be reduced in patients with the type VI variant of the Ehlers-Danlos syndrome, and the first mutations in the lysyl hydroxylase gene (PLOD) have recently been identified. We have now isolated genomic clones for human lysyl hydroxylase and determined the complete structure of the gene, which contains 19 exons and a 5′ flanking region with characteristics shared by housekeeping genes. The constitutive expression of the gene in different tissues further suggests that lysyl hydroxylase has an essential function. We have sequenced the introns of the gene in the region where many mutations and rearrangements analyzed to date are concentrated. Intron 9 and intron 16 show extensive homology resulting from the many Alu sequences found in these introns. Intron 9 contains five and intron 16 eight Alu sequences. The high homology and many short identical or complementary sequences in these introns generate many potential recombination sites with the gene. The delineation of the structure of the lysyl hydroxylase gene contributes significantly to our understanding of the rearrangements in the genome of Ehlers-Danlos type VI patients.     

Reaction of lysyl oxidase with soluble protein substrates: effect of neutral salts.

Soluble proteins released into the medium of aortic tissues in culture behave as substrates for the enzyme lysyl oxidase. The reaction shows an unusual dependence on the concentration of neutral salts in the assay medium. Practically no enzyme activity was observed in Tris-HCl, 0.005 m, pH 7.6 buffer. However, supplementing the buffer with high concentrations of KCl, KBr, NaCl, and (NH₄)₂SO₄ (in decreasing order of effectiveness) accelerated velocities as much as 10-fold. CaCl₂, KSCN, and KI at increasing concentrations became strongly inhibitory. β-Aminopropionitrile, a specific inhibitor of lysyl oxidase, effectively blocked the catalysis in low and high KCl. The salt-stimulated effects on lysyl oxidase activity were not as noticeable when insoluble proteins were used as substrates. Kinetic studies employing double reciprocal plots revealed that high KCl concentrations (2.0 m) raised the maximum velocity of the reaction but did not alter the apparent K_m. Thus high salt concentrations did not affect the binding of the soluble substrate to the enzyme. In high salts, however, more radioactive substrate proteins appeared to bind to the enzyme, suggesting that the high salt environment increases the fraction of the total enzyme potentially capable of binding to and catalyzing a reaction with the substrate.