Cross-linking Amino Acids Research Articles

Methylglyoxal Inhibits the Binding Step of Collagen Phagocytosis

Bacterial infection-induced fibrosis affects a wide variety of tissues, including the periodontium, but the mechanisms that dysregulate matrix turnover and mediate fibrosis are not defined. Since collagen turnover by phagocytosis is an important pathway for matrix remodeling, we studied the effect of the bacterial and eukaryotic cell metabolite, methylglyoxal (MGO), on the binding step of phagocytosis by periodontal fibroblasts. Type 1 collagen was treated with various concentrations of methylglyoxal, an important glucose metabolite that modifies Arg and Lys residues. The extent of MGO-induced modifications was authenticated by amino acid analysis, solubility, and cross-linking. Cells were incubated with fluorescent beads coated with collagen, and the percentage of phagocytic cells was estimated by flow cytometry. MGO inhibited collagen binding (20% of control for 10 mm MGO) in a time- and concentration-dependent manner. MGO-induced inhibition of binding was prevented by aminoguanidine, which blocks the formation of collagen cross-links. MGO reduced collagen binding strength and blocked intracellular calcium signaling. MGO modified the Arg residue in the critical alpha2beta1 integrin-binding recognition sequence of triple helical collagen peptides, whereas MGO-induced cross-linking of Lys residues played only a small role in binding inhibition. Thus, MGO modifications of Arg residues in collagen could be a key factor in the impaired degradation of collagen that promotes fibrosis in chronic infections, such as periodontitis.

Progress in the methodological strategies for the detection in real samples of desmosine and isodesmosine, two biological markers of elastin degradation

Desmosines are crosslinking amino acids unique to mature elastin in humans. Owing to this unicity, they have been discussed as potentially attractive indicators of connective tissue disorders whose clinical manifestations are mostly the result of elastin degradation. This review covers advances in immunochemical, chromatographic, and electrophoretic procedures applied in the last 25 years to detect and quantitate these crosslinksin a variety of biological samples. Recent applications of CE with LIF detection (CE-LIF) for investigating the content of desmosines in different fluids will also be discussed.

Organoboron Reagents in the Preparation of Functionalized ?-Amino Acids

Over the past decade, major advances in the preparation and utilization of organoboron reagents have been applied to virtually all areas of organic synthesis. The present review collates recent examples of the use of organoboron reagents in the synthesis of α-amino acids and their derivatives. Aryl- and alkenylboronic acids have been used in the asymmetric synthesis of α-amino acids through conjugate addition to unsaturated amino acids and the Petasis three-component coupling reaction. Additionally, α-amino acid derivatives with organoboron functionality on the side-chain have been prepared and used in metal-catalyzed cross-coupling reactions to prepare cross-linked amino acids and complex cyclic peptide natural products.

Identification of Precise Electrostatic Recognition Sites between Cytochrome c6 and the Photosystem I Subunit PsaF Using Mass Spectrometry

The reduction of the photo-oxidized special chlorophyll pair P700 of photosystem I (PSI) in the photosynthetic electron transport chain of eukaryotic organisms is facilitated by the soluble copper-containing protein plastocyanin (pc). In the absence of copper, pc is functionally replaced by the heme-containing protein cytochrome c6 (cyt c6) in the green alga Chlamydomonas reinhardtii. Binding and electron transfer between both donors and PSI follows a two-step mechanism that depends on electrostatic and hydrophobic recognition between the partners. Although the electrostatic and hydrophobic recognition sites on pc and PSI are well known, the precise electrostatic recognition site on cyt c6 is unknown. To specify the interaction sites on a molecular level, we cross-linked cyt c6 and PSI using a zero-length cross-linker and obtained a cross-linked complex competent in fast and efficient electron transfer. As shown previously, cyt c6 cross-links specifically with the PsaF subunit of PSI. Mass spectrometric analysis of tryptic peptides from the cross-linked product revealed specific interaction sites between residues Lys27 of PsaF and Glu69 of cyt c6 and between Lys23 of PsaF and Glu69/Glu70 of cyt c6. Using these new data, we present a molecular model of the intermolecular electron transfer complex between eukaryotic cyt c6 and PSI.

Dose‐dependent utilisation of casein‐linked lysinoalanine, N(epsilon)‐fructoselysine and N(epsilon)‐carboxymethyllysine in rats

During the heat treatment of protein-containing foods, the amino acid lysine is most prone to undergo chemical reactions in the course of amino acid cross-linking or Maillard reactions. Among the reaction products formed, lysinoalanine (LAL), N(epsilon)-fructoselysine (FL) and N(epsilon)-carboxymethyllysine (CML) are those which serve as sensitive markers for the heat treatment applied. From a nutritional perspective, these compounds are ingested with the diet in considerable amounts but information about their metabolic transit and putative in vivo effects is scarce. In the present study, casein-linked LAL, FL and CML were administered to rats in two different doses for 10 days. Quantitation of LAL, FL and CML in plasma, tissue and faeces samples revealed that the kidneys are the predominant sites of accumulation and excretion. The maximum percent of dietary LAL, FL and CML excreted in the urine was 5.6, 5.2 and 29%, whereas the respective recoveries in the kidneys were 0.02, 26 and 1.4%. The plasma and tissue analyses revealed that the endogenous load of either compound is increased by its dietary intake. But the dose-dependent utilisation of dietary protein-linked LAL, FL and CML in rats has been demonstrated for the first time to vary substantially from each other.

Measurement of Skin Desmosine as an Indicator of Altered Cutaneous Elastin in Draft Horses With Chronic Progressive Lymphedema

Chronic progressive lymphedema in Clydesdale and Shire draft horses causes severe disability of the limbs which leads to premature death of these horses. Since appropriate function of lymph vessels is dependent on the presence of viable elastin fibers, the goal of this study was to document differences in skin elastin fibers in affected horse breeds, compared to a nonaffected draft horse breed. Biochemical analysis of cutaneous desmosine, a cross-linking amino acid found only in elastin, was used to measure elastin in the skin from 110 draft horses. This included 7 normal, 38 mildly affected, 30 moderately, and 15 severely affected horses, and 20 horses of a nonaffected draft breed. Desmosine concentrations in neck, considered a nonaffected skin region, and left forelimb, an affected skin region, were compared between the groups. A significantly lower desmosine concentration was found in the skin of the neck and limb of clinically normal animals of affected draft breeds compared to a nonaffected draft horse breed. During the progression of the disease in the affected breeds, cutaneous desmosine concentrations most prominently increased in the skin of the distal limbs. Chronic progressive lymphedema in draft horses was associated with an initially systemic lower cutaneous elastin level and a deposition of elastin during the progression of the disease. A failure of elastic fibers to appropriately support the skin and its lymphatics is proposed as a possible contributing factor for chronic progressive lymphedema in Shires and Clydesdales.

The characteristics of elastic fiber assembled with recombinant tropoelastin isoform

Objective: It is known that elastin mRNA is transcribed from a single gene. The variety of tropoelastin isoforms results from multiple alternative splicing of the primary transcript. The purpose of this study was to investigate the characteristics of elastic fiber assembled with tropoelastin isoform, which is full-length human tropoelastin (HTE), exon 26A missing tropoelastin (Δ26A), and exon 32 missing tropoelastin (Δ32). Design and methods: We demonstrated the process of elastic fiber assembly and the existence of elastic fiber resistant to pancreatic elastase with HTE, Δ26A, or Δ32 fiber using an in vitro model of elastic fiber assembly. These elastic fibers were evaluated by immunofluorescent staining, the quantitative analysis of cross-linked amino acids, and semi-quantitative analysis of matrix-associated tropoelastin. Results: There were no big differences getting into the matrix among these tropoelastins in immunofluorescence microscopy and semi-quantitative analysis. In the comparison with the HTE, the Δ26A and the Δ32 significantly increased and decreased, respectively, the formation of cross-linking amino acids and the binding to scaffold proteins. Furthermore, it was found that it is difficult to degrade the Δ26A assembly with pancreatic elastase as compared with HTE or Δ32 assembly. Conclusion: The elastic fiber assembled with the tropoelastin isoforms was characterized using an in vitro model. The present study provides important information regarding the pathology of human diseases including emphysema and atherosclerosis.

Conserved Role of the Linker α-Helix of the Bacterial Disulfide Isomerase DsbC in the Avoidance of Misoxidation by DsbB

In the bacterial periplasm the co-existence of a catalyst of disulfide bond formation (DsbA) that is maintained in an oxidized state and of a reduced enzyme that catalyzes the rearrangement of mispaired cysteine residues (DsbC) is important for the folding of proteins containing multiple disulfide bonds. The kinetic partitioning of the DsbA/DsbB and DsbC/DsbD pathways partly depends on the ability of DsbB to oxidize DsbA at rates >1000 times greater than DsbC. We show that the resistance of DsbC to oxidation by DsbB is abolished by deletions of one or more amino acids within the alpha-helix that connects the N-terminal dimerization domain with the C-terminal thioredoxin domain. As a result, mutant DsbC carrying alpha-helix deletions could catalyze disulfide bond formation and complemented the phenotypes of dsbA cells. Examination of DsbC homologues from Haemophilus influenzae, Pseudomonas aeruginosa, Erwinia chrysanthemi, Yersinia pseudotuberculosis, Vibrio cholerae (30-70% sequence identity with the Escherichia coli enzyme) revealed that the mechanism responsible for avoiding oxidation by DsbB is a general property of DsbC family enzymes. In addition we found that deletions in the linker region reduced, but did not abolish, the ability of DsbC to assist the formation of active vtPA and phytase in vivo, in a DsbD-dependent manner, revealing that interactions between DsbD and DsbC are also conserved.

Mass Spectrometric Analysis of the Ubiquinol-binding Site in Cytochrome bd from Escherichia coli

Cytochrome bd is a heterodimeric terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. For understanding the unique catalytic mechanism of the quinol oxidation, mass spectrometry was used to identify amino acid residue(s) that can be labeled with a reduced form of 2-azido-3-methoxy-5-methyl-6-geranyl-1,4-benzoquinone or 2-methoxy-3-azido-5-methyl-6-geranyl-1,4-benzoquinone. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrated that the photo inactivation of ubiquinol-1 oxidase activity was accompanied by the labeling of subunit I with both azidoquinols. The cross-linked domain was identified by reverse-phase high performance liquid chromatography of subunit I peptides produced by in-gel double digestion with lysyl endopeptidase and endoproteinase Asp-N. Electrospray ionization quadrupole time-of-flight mass spectrometry determined the amino acid sequence of the peptide (m/z 1047.5) to be Glu(278)-Lys(283), where a photoproduct of azido-Q(2) was linked to the carboxylic side chain of I-Glu(280). This study demonstrated directly that the N-terminal region of periplasmic loop VI/VII (Q-loop) is a part of the quinol oxidation site and indicates that the 2- and 3-methoxy groups of the quinone ring are in the close vicinity of I-Glu(280).

Urinary desmosine as a biomarker in acute lung injury

Acute lung injury (ALI) is a complex disorder associated with an acute inflammatory response thought to contribute to tissue injury. Desmosine, a cross-linking amino acid present in elastin, is released during matrix degradation and cleared by the kidney. Results from animal models and human disease studies have suggested that ALI is associated with the release of desmosine, resulting in increased urinary desmosine. A radioimmunoassay was used to monitor urinary desmosine levels over 10 days in ten patients with ALI. The concentration of desmosine was measured with and without acid hydrolysis. Baseline urinary desmosine was increased in two of ten patients. The concentration of desmosine at baseline did not appear to be related to age, gender, neutrophil elastase (NE)/α1-antiprotease complex concentration or PaO2/FiO2 ratio. No meaningful changes in desmosine levels were noted after removal from mechanical ventilation. Baseline desmosine concentrations did not appear to correlate with the risk of death. The limited sensitivity, predictive correlations and dynamic modulation would suggest that urine desmosine has a limited role as a biomarker for ALI. Hydrolysis of urine samples appears necessary for optimal measurement of urine desmosine.

Biochemical Analysis of Elastic Fiber Formation with a Frameshift-Mutated Tropoelastin (fmTE) at the C-Terminus of Tropoelastin

Elastic fibers play an important role in characteristic elastic properties of tissues such as skin, lungs, ligaments, and blood vessels. Many of the disorders related to elastic fibers are due to errors in assembly, yet the basic mechanisms of this process remain unclear. Cutis laxa, which is caused by an elastin gene mutation, includes a rare and heterogeneous group of the disorders characterized by lax, inelastic skin. To investigate whether elastic fiber assembly insufficiency is a possible factor in cutis laxa, we compared normal tropoelastin (nTE) and frameshift-mutated tropoelastin (fmTE) from a patient with autosomal dominant cutis laxa using an in vitro elastic fiber assembly model, in which purified recombinant tropoelastin was added to the culture medium. Assembly was evaluated by immunofluorescence staining, the quantitative analysis of cross-linked amino acids, and semi-quantitative analysis of matrix-associated tropoelastin. Immunofluorescence microscopy indicated an approximate 50% decrease in the deposition of fmTE in the extracelluar matrix, in contrast to that of nTE. The amount of cross-linked amino acids unique to mature insoluble elastin in the fmTE assembly was also decreased by approximately 20%. In addition, we clarified that the molecular interaction between fmTE and the amino-terminal domain of fibrillin-1 or full-length fibulin-5 was significantly decreased. Our results suggest that the defect of fmTE fiber assembly is due, at least in part, to decreased molecular interactions between tropoelastin and the microfibrillar components fibulin-5 and fibrillin.

Probing conformational changes in T7 RNA polymerase during initiation and termination by using engineered disulfide linkages

During the transition from an initiation complex to an elongation complex (EC), the single-subunit bacteriophage T7 RNA polymerase (RNAP) undergoes dramatic conformational changes. To explore the significance of these changes, we constructed mutant RNAPs that are able to form disulfide bonds that limit the mobility of elements that are involved in the transition (or its reversal) and examined the effects of the crosslinks on initiation and termination. A crosslink that is specific to the initiation complex conformation blocks transcription at 5-6 nt, presumably by preventing isomerization to an EC. A crosslink that is specific to the EC conformation has relatively little effect on elongation or on termination at a class I terminator (T), which involves the formation of a stable stem-loop structure in the RNA. Crosslinked ECs also pause and resume transcription normally at a class II pause site (concatamer junction) but are deficient in termination at a class II terminator (PTH, which is found in human preparathyroid hormone gene), both of which involve a specific recognition sequence. The crosslinked amino acids in the EC lie close to the upstream end of the RNA-DNA hybrid and may prevent a movement of the polymerase that would assist in displacing or releasing RNA from a relatively unstable DNA-RNA hybrid in the paused PTH complex.

Development of a new in vitro model of elastic fiber assembly in human pigmented epithelial cells

Objectives: We developed an in vitro model of elastic fiber assembly that provides a comparison of the efficiency of different tropoelastin molecules to organize into fibers. Design and methods: Recombinant tropoelastin was added to ARPE-19 cell culture medium. The elastic fiber assembly was evaluated by immunofluorescence staining, the quantitative analysis of cross-linking amino acids, and semi-quantitative analysis of matrix-associated tropoelastin. Results: We confirmed that ARPE-19 cells express fibrillin-containing microfibrils and lysyl oxidase, but they do not express tropoelastin. Immunofluorescence staining showed a dose- and time-dependent increase in the extracellular matrix. The quantity of cross-linking amino acids and matrix-associated tropoelastin also increased together with the matrix-associated elastin. Moreover, the analysis of a radioimmunoprecipitation assay (RIPA) buffer-soluble fraction indicated that tropoelastin interacted with microfibrils and cross-linked elastin was detected as a super molecular complex. Conclusion: These observations indicate that this in vitro model is especially useful for the analysis of mechanisms of elastic fiber formation.

Active Site Aspartate Residues Are Critical for Tryptophan Tryptophylquinone Biogenesis in Methylamine Dehydrogenase

The biosynthesis of methylamine dehydrogenase (MADH) requires formation of six intrasubunit disulfide bonds, incorporation of two oxygens into residue betaTrp57 and covalent cross-linking of betaTrp57 to betaTrp108 to form the protein-derived cofactor tryptophan tryptophylquinone (TTQ). Residues betaAsp76 and betaAsp32 are located in close proximity to the quinone oxygens of TTQ in the enzyme active site. These residues are structurally conserved in quinohemoprotein amine dehydrogenase, which possesses a cysteine tryptophylquinone cofactor. Relatively conservative betaD76N and betaD32N mutations resulted in very low levels of MADH expression. Analysis of the isolated proteins by mass spectrometry revealed that each mutation affected TTQ biogenesis. betaD76N MADH possessed the six disulfides but had no oxygen incorporated into betaTrp57 and was completely inactive. The betaD32N MADH preparation contained a major species with six disulfides but no oxygen incorporated into betaTrp57 and a minor species with both oxygens incorporated, which was active. The steady-state kinetic parameters for the betaD32N mutant were significantly altered by the mutation and exhibited a 1000-fold increase in the Km value for methylamine. These results have allowed us to more clearly define the sequence of events that lead to TTQ biogenesis and to define novel roles for aspartate residues in the biogenesis of a protein-derived cofactor.

Geometric preferences of crosslinked protein‐derived cofactors reveal a high propensity for near‐sequence pairs

Protein-derived cofactors that are composed of covalently crosslinked amino acid side chains are of increasing importance in protein science. These crosslinked protein-derived cofactors (CPDC) are formed either through direct oxidation by metal/O(2)-derived intermediates or through outer sphere oxidation by highly oxidizing cofactors. CPDCs that are formed by outer sphere oxidation do not require side-chain precursors to be coordinated by a metal center, and therefore are more difficult to identify than those formed by direct oxidation. To better understand the propensity for CPDC formation by outer sphere oxidation, the geometrical preferences of CPDCs were examined. The Dezymer algorithm has been used to identify all putative CPDC-forming mutations in 500 proteins. Geometrically, although chemically unrelated, these CPDCs were found to be similar to disulfide-bonded cysteine pairs. Additionally, the percentage of near-sequence pairs (i and i +1 to i and i + 5) increased as the average C(alpha)-C(alpha) distance between the amino acid pairs increased. This survey also examined the protein databank for proteins with pre-attack conformations for CPDCs, using non-bonded contacts reported by Procheck. A total of 323 unique proteins was identified, with 55 being near-sequence amino acid pairs. The high geometric propensity of near-sequence amino acid pairs for forming CPDCs is significant due to difficulties associated with detection by structural or mass spectrometric methods.

Interaction Between Proteoglycans and α-Elastin in Construction of Extracellular Matrix of Human Yellow Ligament

One type of large proteoglycan and three types of small proteoglycans (decorin, decorin-subtype, and biglycan) were purified by chromatography, and α-elastin was isolated by alkali treatment from human yellow ligaments taken at the time of operation. The interaction of the proteoglycans with immobilized α-elastin on a sensor was analyzed by surface plasmon resonance, and we confirmed that each of the small proteoglycans exhibited a specific binding to α-elastin. The binding sites of small proteoglycans were contained in the protein cores. In addition, the differences in the interaction of the small proteoglycans with α-elastin of normal and ossified ligaments were compared. The interactions of the small proteoglycans with α-elastin of the ossified ligaments were lower than those with α-elastin of the normal ligaments. In the ossified ligaments, neodesmosine, one of the cross-linking amino acids, was significantly less than in the normal ligaments (p < .05).

Di-isodityrosine Is the Intermolecular Cross-link of Isodityrosine-rich Extensin Analogs Cross-linked in Vitro

Extensins are cell wall hydroxyproline-rich glycoproteins that form covalent networks putatively involving tyrosyl and lysyl residues in cross-links catalyzed by one or more extensin peroxidases. The precise cross-links remain to be chemically identified both as network components in muro and as enzymic products generated in vitro with native extensin monomers as substrates. However, some extensin monomers contain variations within their putative cross-linking motifs that complicate cross-link identification. Other simpler extensins are recalcitrant to isolation including the ubiquitous P3-type extensin whose major repetitive motif, Hyp)(4)-Ser-Hyp-Ser-(Hyp)(4)-Tyr-Tyr-Tyr-Lys, is of particular interest, not least because its Tyr-Tyr-Tyr intramolecular isodityrosine cross-link motifs are also putative candidates for further intermolecular cross-linking to form di-isodityrosine. Therefore, we designed a set of extensin analogs encoding tandem repeats of the P3 motif, including Tyr --> Phe and Lys --> Leu variations. Expression of these P3 analogs in Nicotiana tabacum cells yielded glycoproteins with virtually all Pro residues hydroxylated and subsequently arabinosylated and with likely galactosylated Ser residues. This was consistent with earlier analyses of P3 glycopeptides isolated from cell wall digests and the predictions of the Hyp contiguity hypothesis. The tyrosine-rich P3 analogs also contained isodityrosine, formed in vivo. Significantly, these isodityrosine-containing analogs were further cross-linked in vitro by an extensin peroxidase to form the tetra-tyrosine intermolecular cross-link amino acid di-isodityrosine. This is the first identification of an inter-molecular cross-link amino acid in an extensin module and corroborates earlier suggestions that di-isodityrosine represents one mechanism for cross-linking extensins in muro.

Regional differences in degree of resilin cross-linking in the desert locust, Schistocerca gregaria

Various cuticular regions from the desert locust, Schistocerca gregaria, were quantitatively analyzed for two cross-linking amino acids, dityrosine and trityrosine, characteristic constituents of the rubberlike cuticular protein, resilin. These amino acids were found in all regions of cuticle investigated, but in widely varying amounts. In fully mature adult locusts the largest amounts of di- and trityrosine were obtained from the prealar arms and wing-hinges, structures possessing long-range elasticity and being involved in energy storage in the flight system. In structures where deformations tend to occur more slowly, such as the clypeo-labral springs and tracheae, di- and trityrosine are less abundant. In sclerotized cuticle from femur and tibia, as well as in cornea and in the highly stretchable intersegmental membranes of mature females, they are only found in trace amounts and are probably unrelated to elasticity. The trityrosine-to-dityrosine ratio in the variuos cuticular regions vary from nearly equal amounts of the two amino acids to about ten times more dityrosine than trityrosine, indicating that the regions differ in degree of cross-linking; the tracheal wall is the material with the highest trityrosine-to-dityrosine ratio. In some cuticular regions the ratio increases during maturation from newly moulted (teneral) adults to reproductively active locusts; the most pronounced increase was observed for the wing-hinges, and only a small increase was observed for the abdominal tergal plates. In most cuticular regions in fifth instar locust nymphs the contents of di- and trityrosine corresponded to the contents measured for the adult cuticular regions, but only trace amounts of the two amino acids were obtained from the region of the nymphal wing base which corresponds to the wing-hinge containing cuticular region in adult locusts.

Synthesis of Non‐Natural Amino Acids from N‐(p‐Tolylsulfonyl)‐α,β‐didehydroamino Acid Derivatives.

AbstractFor Abstract see ChemInform Abstract in Full Text.

LC/ESI-MS analysis of two elastin cross-links, desmosine and isodesmosine, and their radiation-induced degradation products.

In this work, the effect of Fenton reaction on two elastin cross-linked amino acids, desmosine (DES) and isodesmosine (IDE), in the absence or presence of different wavelength radiations generated from artificial sources has been evaluated using LC/ESI-MS. Irradiation as well as incubation of DES or IDE solutions in the presence of Fe(2+) and H(2)O(2) resulted in products with m/z 497.1 and 481.1 for [M+H](+). A strongly dose-dependent degradation of both amino acids was observed upon exposure to UVB at doses ranging from 0 to 3 J/cm(2) and a moderate dose-dependent degradation upon exposure to UVA at doses 10 times higher than that of UVB. A significant time-dependent degradation of DES and IDE was also observed upon exposure of these amino acids to a lamp emitting visible light similar to sunlight. Exposure of both amino acids to IR radiation (520 W) for 8 h did not cause significant degradation.