Elastase-like enzymes in human neutrophils localized by ultrastructural cytochemistry.

The thiol ester N-t-Boc-L-alanine-p-nitrothiophenyl ester (Boc-Ala-SNp) was synthesized and applied as an ultrastructural cytochemical substrate for intracellular elastase-like enzymes. Mature human neutrophils incubated with Boc-Ala-SNp and gold ions generate an electron-dense reaction product, gold p-nitrothiophenolate, which is found in the nuclear membrane, Golgi complex, endoplasmic reticulum, mitochondria, and granules of these cells. Enzyme activity against Boc-Ala-SNp is also observed in developing monkey bone marrow neutrophils and in other blood cells. The intracellular neutrophil enzyme activity is elastase-like because it is characterized by a slightly alkaline pH optimum and is inactivated by exposure of the cells to general and specific active site inhibitors of neutrophil elastase. This substrate appears to have important potential for use in ultrastructural studies of intracellular elastase-like enzymes.

Ultrastructural immunolocalization of lysyl oxidase in vascular connective tissue.

The localization of lysyl oxidase was examined in calf and rat aortic connective tissue at the ultrastructural level using polyclonal chicken anti-lysyl oxidase and gold conjugated rabbit anti-chicken immunoglobulin G to identify immunoreactive sites. Electron microscopy of calf aortic specimens revealed discrete gold deposits at the interface between extracellular bundles of amorphous elastin and the microfibrils circumferentially surrounding these bundles. The antibody did not react with microfibrils which were distant from the interface with elastin. There was negligible deposition of gold within the bundles of amorphous elastin and those few deposits seen at these sites appeared to be associated with strands of microfibrils. Lysyl oxidase was similarly localized in newborn rat aorta at the interface between microfibrils and nascent elastin fibers. Gold deposits were not seen in association with extracellular collagen fibers even after collagen-associated proteoglycans had been degraded by chondroitinase ABC. However, the antibody did recognize collagen-bound lysyl oxidase in collagen fibers prepared from purified collagen to which the enzyme had been added in vitro. No reaction product was seen if the anti-lysyl oxidase was preadsorbed with purified lysyl oxidase illustrating the specificity of the antibody probe. The present results are consistent with a model of elastogenesis predicting the radial growth of the elastin fiber by the deposition and crosslinking of tropoelastin units at the fiber-microfibril interface.

A crosslinked cofactor in lysyl oxidase: redox function for amino acid side chains.

A previously unknown redox cofactor has been identified in the active site of lysyl oxidase from the bovine aorta. Edman sequencing, mass spectrometry, ultraviolet-visible spectra, and resonance Raman studies showed that this cofactor is a quinone. Its structure is derived from the crosslinking of the epsilon-amino group of a peptidyl lysine with the modified side chain of a tyrosyl residue, and it has been designated lysine tyrosylquinone. This quinone appears to be the only example of a mammalian cofactor formed from the crosslinking of two amino acid side chains. This discovery expands the range of known quino-cofactor structures and has implications for the mechanism of their biogenesis.

PQQ, the elusive coenzyme.

The recently discovered redox coenzyme, PQQ (methoxatin), is widely distributed. Quantitation of protein-bound PQQ has been difficult, but unique redox cycling reactions, which reflect its striking biological properties, reveal trace amounts. PQQ is a potential target for drugs.

Amidated carboxyl groups in elastin.

Dicarboxylic amino acids constitute the most numerous residues of insoluble elastin in which are potentially ionizable in the physiological range of pH. These residues are essential in facilitating productive electrostatic interaction between elastase and elastin. The present study has investigated the possibility that the glutamic and aspartic acid residues of elastin are amidated. Acid-labile amide-bound ammonia of elastin was quantitated after hydrolysis of the insoluble protein with 2 M HC1 by incubating aliquots of microdistilled hydrolysates with glutamate dehydrogenase, excess alpha-ketoglutarate, and reduced nicotinamide adenine dinucleotide and measuring the resultant decrease in A340 due to oxidation of the dinucleotide cofactor. It was found that ligament elastin purified by repeated autoclaving contains approximately 2.29 mumol of acid-labile amide nitrogen per 10 mg of protein, a value equivalent to approximately 70% of the total number of dicarboxylic amino acid residues. Independent analysis of the amide content was obtained by amino acid analysis of an esterified and reduced elastin sample in which the free dicarboxylic amino acid residues had been converted to the corresponding alcohol derivatives. This analysis indicated that autoclaved ligament elastin contains approximately 18 glutamine, 3 asparagine, 4 glutamic acid and 5 aspartic acid residues per 1000 residues, in good agreement with the analysis of total acid-labile ammonia. The esterified and reduced elastin derivative was nearly inert as an elastase substrate, consistent with a lack of free dicarboxylic amino acid residues. However, addition of sodium dodecyl sulfate to this elastin derivative restores enzyme-substrate charge complementarity, and the elastin-ligand complex was readily hydrolyzed by elastase at the fully stimulated rate, emphasizing the control such ligands can exert in elastolysis. The amide bonds of elastin were found to be significantly more resistant to hydrolysis by 0.1 M NaOH at 98 degrees C than were those of lysozyme or free amidated amino acids. The finding that most of dicarboxylic amino acid residues of elastin exist at neutral amides further emphasizes the apolar character of elastin and has bearing upon the metabolic susceptibility, ligand-binding ability and structural aspects of this connective tissue protein.

Changes in lung lysyl oxidase activity in streptozotocin-diabetes and in starvation.

The effects of streptozotocin-induced diabetes and of starvation on the lysyl oxidase activity of rat lung were investigated. Enzyme activity was elevated 2--3 fold in the lungs of streptozotocin-diabetic rats. In contrast, starvation of rats produced a rapid loss of lung lysyl oxidase activity, with levels approximating 25% of control values after 48--72 h of starvation. Enzyme activity was essentially fully restored to control values upon refeeding the 48-h starved animals for 3 h. These studies demonstrate the responsiveness of lysyl oxidase to these physiological states and suggest a component, enzymatic basis of change in lung function known to occur in the diabetic state.

Oxidation of peptidyl lysine by copper complexes of pyrroloquinoline quinone and other quinones. A model for oxidative pathochemistry.

Various o- and p-quinones were assessed as oxidants of peptidyl lysine in elastin and collagen substrates in the presence and absence of divalent copper as paradigms of protein-lysine 6-oxidase (lysyl oxidase) which contains both quinone and copper cofactors. Pyrroloquinoline quinone was among the most active in the absence and the most active of the o- and p-quinones tested in the presence of copper. The optimal rate of elastin oxidation occurred at a 2:1 PQQ/Cu(II) ratio while Cu(II) itself oxidized elastin relatively slightly. Elastin oxidation by 2:1 PQQ/Cu(II) required aerobic conditions consistent with oxygen-dependent turnover of this catalytic pair. Dimethylsulfoxide and catalase individually or in combination inhibited elastin oxidation by PQQ/Cu(II) by approx. 50%, suggesting that oxygen free radical species participate in the reaction. Amino-acid analysis of elastin and collagen substrates oxidized by 2:1 PQQ/Cu and then reduced with borohydride revealed that alpha-aminoadipic-delta-semialdehyde and lesser amounts of covalent cross-linkages were generated by this oxidant. In contrast, lysine oxidase produced aldehydes and significantly greater quantities of cross-linkage products, consistent with the known specificity of the enzyme. These data, thus, indicate the potential for free quinones, such as PQQ, particularly when stimulated by appropriate metal ions, to act as adventitious oxidants of lysine side-chains in proteins.

The interaction of collagen with alpha1-acid glycoprotein.

The influence of alpha1-acid glycoprotein on the formation of fibrous long spacing fibers of collagen has been investigated. It was observed that addition of the glycoprotein to dialyzed collagen solutions caused a significant decrease in the intensity of the circular dichroic spectrum of collagen. This phenomenon, which displays an optimum with respect to glycoprotein, is consistent with previous observations of fibrous long spacing fiber formation. Changes in viscosity of collagen initially dissolved in acetic acid were monitored during dialysis. It was found that a significant increase in viscosity must occur during dialysis of collagen before fibrous long spacing formation could take place. This increase in viscosity can be related directly to removal of acetic acid from the collagen solution. Removal of all sialyl residues from the alpha1-acid glycoprotein with neuraminidase prevents fibrous long spacing formation while removal of up to 35% of the sialyl residues has no effect on the interaction of glycoprotein with collagen. Amino acid composition and radioactivity studies suggest that 45-55% of the insoluble fibrous long spacing fibers is glycoprotein. In contrast to native collagen fibers, reduced fibrous long spacing fibers do not contain histidinohydroxymerodesmosine or hydroxylysinonorleucine. Instead, they contain significant quantities of allysine aldol and epsilon-hydroxynorleucine.

Lysyl oxidase: properties, regulation and multiple functions in biology.

Lysyl oxidase (LO) is a copper-dependent amine oxidase that plays a critical role in the biogenesis of connective tissue matrices by crosslinking the extracellular matrix proteins, collagen and elastin. Levels of LO increase in many fibrotic diseases, while expression of the enzyme is decreased in certain diseases involving impaired copper metabolism. While the three-dimensional structure of the enzyme is not yet available, many of its physical-chemical properties, its novel carbonyl cofactor, and its catalytic mechanism have been described. Lysyl oxidase is synthesized as a preproprotein, secreted as a 50 kDa, N-glycosylated proenzyme and then proteolytically cleaved to the 32 kDa, catalytically active, mature enzyme. Within the past decade, the gene encoding LO has been cloned, facilitating investigations of the regulation of expression of the enzyme in response to diverse stimuli and in numerous disease states. Transforming growth factor-beta, platelet-derived growth factor, angiotensin II, retinoic acid, fibroblast growth factor, altered serum conditions, and shear stress are among the effectors or conditions that regulate LO expression. New, LO-like genes have also been identified and cloned, suggesting the existence of a multigene family. It has also become increasingly evident that LO may have other important biological functions in addition to its role in the crosslinking of elastin and collagen in the extracellular matrix.

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 beta-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 response 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.

Salivary histatin 5 is a potent competitive inhibitor of the cysteine proteinase clostripain.

Histatin 5 is a low molecular weight salivary protein which is known to exhibit inhibitory activity against several proteinases, including the cysteine proteinases gingipains. The purpose of this study was to characterize the effect of salivary histatin on the proteolytic activity of the cysteine proteinase clostripain derived from the pathogen Clostridium histolyticum. Using a synthetic nitroanilide substrate, we studied in detail the inhibition of clostripain by histatin 5 and compared the effect of this peptide to that of leupeptin, a known competitive inhibitor of clostripain. It was found that the concentration of histatin 5 required to inhibit 50% of clostripain activity was 23.6+/-1.6 nM. Kinetic analysis revealed that histatin 5 is a competitive inhibitor of clostripain with an inhibition constant (K(i)) of 10 nM. The K(i) for the inhibition of clostripain activity against nitroanilide substrate by leupeptin was found to be 60 nM, significantly higher than that of histatin 5. Thus, histatin 5 inhibits clostripain more effectively than leupeptin and other cysteine protease inhibitors studied here. No significant proteolysis of histatin 5 was observed when histatin 5 was incubated at physiologic concentrations with clostripain. The potent inhibition of clostripain by histatin 5 points towards the possibility that this protein may prevent establishment of clostridial infections and therefore may have significant potential for the treatment of diseases associated with this enzyme.

Aminoalkylaziridines as substrates and inhibitors of lysyl oxidase: specific inactivation of the enzyme by N-(5-aminopentyl)aziridine.

The reaction of lysyl oxidase was assessed with members of a series of aminoalkylaziridines in which the primary amino group and the aziridinyl nitrogen were separated by 3-7 methylene carbons. Among these, N-(5-aminopentyl)aziridine proved to be the poorest substrate by far and to inhibit the enzyme activity. Aminoalkylaziridines with chain lengths shorter or longer than five carbons did not inhibit the enzyme. The resulting inhibition was competitive with productive substrates and became irreversible with time, following pseudo first order kinetics with a KI of 0.22 mM. N-(5-aminopentyl)aziridine appears to act as a bifunctional affinity label covalently interacting with the active site of this enzyme.

Ultrastructural localization of elastase-like enzymes in human neutrophils.

The thiol ester, N-t-butyloxycarbonyl-L-alanine-p-nitrothiophenyl ester (Boc-Ala-SNp) has been synthesized and applied as an ultrastructural cytochemical substrate for leukocytic elastase-like enzymes. Incubation of fixed human neutrophils with Boc-Ala-SNp in the presence of gold ions generates electron-dense deposits of gold p-nitrothiophenolate in the nuclear membrane, endoplasmic reticulum, Golgi complex, mitochondria, and granules. Deposition of product is inhibited by pretreatment of cells with general and specific chemical inactivators of neutrophil elastase. This substrate appears to have significant potential as a probe for the ultrastructural localization of elastase-like activity.

Detection of elastin in the human fetal membranes: proposed molecular basis for elasticity.

The human fetal membranes provide a sterile biomechanical container which adjust by growth to mid-pregnancy to the increase in fetal size, and by elasticity to the forceful movements of the fetus. The molecular basis for this elasticity is not known, yet reduced elasticity may lead to their premature rupture and preterm birth, a major problem in perinatal medicine. Classically, elastin confers the property of elastic recoil to elastic fibres which are assembled from a family of tropoelastin precursors. These are covalently cross-linked to form insoluble elastin by formation of desmosine and isodesmosine, catalysed by the enzyme lysyl oxidase. The amnion, chorion and decidua were shown by Northern analysis and RT-PCR to contain detectable levels of tropoelastin mRNA and the mRNA encoding lysyl oxidase. The proteins encoded by these mRNAs were also identified by Western blotting and immunolocalization. Further, insoluble elastin was extracted from the human fetal membranes and shown by comparison to elastin preparations from other elastic tissues to have a reasonable desmosine content. Finally, scanning electron microscopy confirmed the presence of multiple layers of an apparently very thin elastic system in this tissue. This biochemical and histopathologic study has demonstrated therefore that the human fetal membranes synthesize and deposit a novel elastic fibre. The presence of such an elastic system in these tissues provides, for the first time, a probable molecular basis for the elastic properties of this tissue.

Lysyl oxidase, the extracellular matrix-forming enzyme, in rat brain injury sites.

Lysyl oxidase is an extracellular enzyme that catalyzes cross-linkages of extracellular matrix proteins. We hypothesized that this enzyme is secreted by cells attracted to central nervous system injury sites and is involved in extracellular matrix modulation and in scar formation. Specific antibodies for immunohistochemistry and enzyme activity measurements were used to detect the presence of lysyl oxidase after longitudinal knife cuts in adult rat forebrain. Immunoreactivity was observed within the core of injury sites from 1 and up to 30 days postoperative, with less staining at 2 and 5 days, and was not associated with glial fibrillary acidic protein-positive astrocytes. Enzyme activity increased transiently in injury site regions with a peak (200% of control) at 10 days postoperative. These results are the first to provide evidence for a time-dependent appearance of active extracellular lysyl oxidase in brain injury sites. They imply that enzyme molecules are synthesized and secreted by cells attracted to brain injury sites and participate in extracellular matrix modulation.

Intra- and extracellular enzymes of collagen biosynthesis as biological and chemical targets in the control of fibrosis.

The several steps in the pathway for the biosynthesis of fibrillar collagen are reviewed to illustrate potential sites for the chemotherapeutic control of fibrosis. Particular emphasis is placed upon the properties and inhibition of lysyl oxidase, the enzyme which initiates the covalent crosslinking of extracellular collagen molecules converting these to insoluble fibers, and upon the properties and inhibition of prolyl hydroxylase, the intracellular enzyme which hyroxylates proline residues within collagen.

Lysyl oxidase: mechanism, regulation and relationship to liver fibrosis.

Lysyl oxidase oxidizes peptidyl lysine in collagen and elastin substrates to residues of alpha-aminoadipic-delta-semialdehyde. The peptidyl aldehydes can then undergo spontaneous condensations with unreacted epsilon-amino groups and with neighboring aldehyde functions, thus forming the covalent crosslinkages which convert elastin and collagen into insoluble fibers. The unique role of lysyl oxidase in the post-translational modification of these proteins qualifies this enzymatic reaction as a potentially pivotal site of biological and/or chemotherapeutic control of collagen fiber deposition. Recent advances in the study of the catalytic mechanism, in the development of active site inhibitors, and in the biosynthesis and regulation of this unusual catalyst are reviewed as are studies on the response of lysyl oxidase in fibrotic liver.

The fate of intratracheal 14C-guanidinated pancreatic elastase in hamster lung.

Native pancreatic elastase and guanidinated elastase have similar in vitro and in vivo properties and produce emphysema of similar severity in hamsters. 14C-guanidinated pancreatic elastase (16,000 cpm/0.2 mg) was instilled into the trachea of anesthetized hamsters. Within 24 hours the radiolabel found in the lungs rapidly decreases to 12% of the original 16,000 cpm and to 1% after 96 hours. Most of the radiolabel and elastase activity found in the lungs can be removed by bronchopulmonary lavage up to 48 hours after installation. Although seemingly very small, there is a significant amount of radiolabel (1-2%) which cannot be removed from the lungs by extensive bronchopulmonary lavage.

Studies on lysyl oxidase of bovine ligamentum nuchae and bovine aorta.

Lysyl oxidase had been purified to near homogeneity from bovine aorta and bovine ligamentum nuchae employing a modification of methods described by Harris et al., and Stassen and his colleagues. The aortic enzyme gives rise to at least three peaks and the ligament enzyme resolves into at least four peaks upon chromatography on DEAE cellulose. The molecular weight of each peak of both enzymes is approximately 30,000 daltons in sodium dodecyl sulfate. The aortic enzyme aggregates to species with molecular weights varying from approximately 60,000 to 1,000,000 daltons upon dialysis out of urea into phosphate-buffered saline. Temperature studies reveal that lysyl oxidase is stable to temperatures as high as 80 degrees C, although the assay optimum is 52 degrees C. Studies in progress suggest the temperature dependency of assay may reflect conformational changes in the elastin substrate.