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PubMed2006

Lysyl hydroxylase 3 (LH3) modifies proteins in the extracellular space, a novel mechanism for matrix remodeling.

Lysyl hydroxylase 3 (LH3), the multifunctional enzyme associated with collagen biosynthesis that possesses lysyl hydroxylase and collagen glycosyltransferase activities, has been characterized in the extracellular space in this study. Lysine modifications are known to occur in the endoplasmic reticulum (ER) prior to collagen triple-helix formation, but in this study we show that LH3 is also present and active in the extracellular space. Studies with in vitro cultured cells indicate that LH3, in addition to being an ER resident, is secreted from the cells and is found both in the medium and on the cell surface associated with collagens or other proteins with collagenous sequences. Furthermore, in vivo, LH3 is present in serum. LH3 protein levels correlate with the galactosylhydroxylysine glucosyltransferase (GGT) activity of mouse tissues. This, together with other data, indicates that LH3 is responsible for GGT activity in the tissues and that GGT activity assays can be used to quantify LH3 in tissues. LH3 in vivo is located in two compartments, in the ER and in the extracellular space, and the partitioning varies with tissue type. In mouse kidney the enzyme is located mainly intracellularly, whereas in mouse liver it is located solely in the extracellular space. The extracellular localization and the ability of LH3 to modify lysyl residues of extracellular proteins in their native, nondenaturated conformation reveals a new dynamic in extracellular matrix remodeling, suggesting a novel mechanism for adjusting the amount of hydroxylysine and hydroxylysine-linked carbohydrates in collagenous proteins.

Animals↗
PubMed1994

Minimum structural requirements for minoxidil inhibition of lysyl hydroxylase in cultured fibroblasts.

The structural features of minoxidil contributing to its inhibitory effect on lysyl hydroxylase in cultured fibroblasts were investigated. Minoxidil analogs in which the pyrimidine ring (two nitrogens) was replaced with a pyridine ring (one nitrogen) or a sym-triazine ring (three nitrogens) suppressed lysyl hydroxylase activity without affecting prolyl hydroxylase activity, as did an analog in which the piperidine ring was replaced with an N,N-diethyl group. By contrast, minoxidil analogs bearing an N-monoalkyl (ethyl or butyl) group in place of the piperidine substituent failed to suppress lysyl hydroxylase activity. The results indicate that nitroxides of pyridine and triazine, in addition to pyrimidine, having an ortho amino group can act as specific inhibitors of lysyl hydroxylase in the cell. The minimum structural requirement for inhibitory activity appears to be an organic moiety containing a tertiary nitrogen para to the nitroxide oxygen, a condition that is best fulfilled by the piperidine ring in minoxidil. Hydroxylation of minoxidil at the 3 or 4 position of the piperidine ring had no impact on its ability to inhibit the post-translational hydroxylation of lysine during collagen biosynthesis. Fibroblasts treated with minoxidil, 3'-hydroxyminoxidil, or 4'-hydroxyminoxidil synthesized a collagen specifically deficient in hydroxylysine by approximately 70%, which completely accounted for the diminished lysyl hydroxylase activity. The percentage of total proteins synthesized as collagen was reduced minimally by minoxidil but not by 3'- or 4'-hydroxyminoxidil. The studies offer a potential means for therapeutic intervention of excessive collagen deposition during fibrosis, using minoxidil or preferably its hydroxy derivatives to limit the supply of hydroxylysine for collagen crosslink formation.

Cell Line↗
PubMed1993

Characterization of acetyl-CoA: L-lysine N6-acetyltransferase, which catalyses the first step of carbon catabolism from lysine in Saccharomyces cerevisiae.

The carbon catabolism of L-lysine starts in Saccharomyces cerevisiae with acetylation by an acetyl-CoA:L-lysine N6-acetyltransferase. The enzyme is strongly induced in cells grown on L-lysine as sole carbon source and has been purified about 530-fold. Its activity was specific for acetyl-CoA and, in addition to L-lysine, 5-hydroxylysine and thialysine act as acetyl acceptor. The following apparent Michaelis constants were determined: acetyl-CoA 0.8 mM, L-lysine 5.8 mM, DL-5-hydroxylysine, 2.8 mM, L-thialysine 100 mM. The enzyme had a maximum activity at pH 8.5 and 37 degrees C. Its molecular mass, estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was 52 kDa. Since the native molecular mass, determined by gel filtration, was 48 kDa, the enzyme is a monomer.

Acetylation↗
PubMed1979

Biochemical characteristics of Ehlers-Danlos syndrome type VI in a family with one affected infant.

The parents of a child with the clinical symptoms of Ehlers-Danlos syndrome type VI were identified as third-degree cousins. Biochemical analysis of the dermis of the patient revealed a complete lack of hydroxylysine in the dermal collagen. The dermis of both parents contained only half the amount of hydroxylysine found in healthy individuals. Hydroxylation of prolyl residues was normal in the skin of the patient and his parents. Investigation of the collagen synthesized by fibroblasts derived from the skin of the patient showed a normal proportion of type I and type III collagen. However, while hydroxylation of prolyl residues was normal in type I and type III collagen, hydroxylation of lysyl residues was markedly lower than normal in both type I and type III collagen.

Collagen↗
PubMed1987

Lectin binding to collagen strands in histologic tissue sections.

Histologic sections from human skin and uterine ligaments were stained with the following FITC conjugated lectins: Con A, WGA, s-WGA, SBA, DBA, UEA I, PNA, RCA I, BPA, GSA I, GSA II, MPA and LPA. The staining of the connective tissue was similar in the dermis and the uterine ligaments and it was most intense in the extracellular matrix containing collagen strands whereas the fibrocytes remained unstained. The staining was clear with glucose or N-acetylglucosamine binding lectins like Con A, WGA, s-WGA and GSA II, which may be related to the presence of glucose residues in collagenous hydroxylysine. The staining with some of the galactose or N-acetylgalactosamine binding lectins like RCA I, DBA, and BPA was less intense. This may reflect the presence of terminal galactose sugars in the hydroxylysine of collagen. No staining was found with SBA, UEA I, PNA, GSAI, MPA or LPA. The results show that different particularly glucose specific lectins bind to the extracellular matrix and especially to collagenous strands in connective tissue. It is suggested that this might be used in histochemical studies of connective tissue and particularly concerning the changes that may occur in different disease states.

Collagen↗
PubMed1977

Collagen glycopeptides from the sea cucumber Stichopus japonicus.

Structures of several hydroxylysine-containing glycopeptides derived from the body wall of the sea cucumber have been determined. The glycosylated hydroxylysines were all the Y positions of Gly-X-Y triplets. They are thought to be distributed throughout the peptide chain, in contrast to vertebrate skin collagens. One glycopeptide demonstrated the existence of the sequence Gly-Gly-Hyp in invertebrates for the first time.

Amino Acid Sequence↗
PubMed1983

The nonenzymatic glycosylation of collagen.

Calf skin acid-soluble collagen, containing about 34 residues of lysine plus hydroxylysine per 100,000 dalton polypeptide chain, was treated with [14C]glucose in the presence or absence of NaCNBH3. In 144 h, under the conditions employed, the presence of NaCNBH3 increased the extent of glycosylation from 8 to 15% of the total residues of lysine plus hydroxylysine. The extent of glycosylation was estimated, using acid hydrolysates of the protein, by isolation and determination of reduced adducts (1-lysinohexitols) employing a system of paper chromatography followed by chromatography on an amino acid analyzer. By those means the difficulties of using specific color reactions such as that with thiobarbituric acid were obviated. Identification of the reduced adducts as forms of 1-lysinohexitol was made by comparison of that substance prepared by treatment of polylysine with [14C]glucose in the presence of NaCNBH3. Of interest is the fact that treatment of the polymer with glucose for 144 h under conditions similar to those used for the collagen, resulted in an increase of extent of glycosylation from 3 to about 50% of the total lysine residues when NaCNBH3 was present in the incubation medium. The greater degree of glycosylation of lysine residues in polylysine as compared with collagen (15 versus about 50%) may be ascribed to the different orders of macromolecular structure in the protein that could sequester certain of the residues from reaction with glucose. 1-Lysinohexitol was also identified in hydrolysates of neutral salt-soluble guinea pig skin collagen that had been reacted with glucose and then treated with NaB3H4. The glycosylated collagens were fragmented by treatment with CNBr, and modified lysine residues were found to occur along the entire length of the collagen chains. The use of NaCNBH3 in the manner described above permits measurement of both aldimine and ketoamine forms of the adducts made with glucose. The possible physiological significance of the reversibility of the ketoamine form of adduct is discussed briefly.

Animals↗
PubMed1985

A paradoxical effect of hydralazine on prolyl and lysyl hydroxylase activities in cultured human skin fibroblasts.

The effect of hydralazine on several parameters of collagen biosynthesis has been studied in cultured human skin fibroblasts. Cells treated with hydralazine synthesized procollagen which was severely deficient in hydroxyproline and hydroxylysine, indicating inhibition of prolyl and lysyl hydroxylase reactions in the cell. Assays of prolyl and lysyl hydroxylase activities, however, revealed markedly increased levels in hydralazine-treated cells. The stimulatory effect of hydralazine could not be simulated in cell extracts, demonstrating its requirement for intact cells. The effect occurred slowly over a period of 96 h and was dependent on hydralazine concentration between 10 and 100 microM. This phenomenon was also observed in lysyl hydroxylase-deficient mutants. In both normal and mutant cells the relative magnitude of the hydralazine effect could be modified by ascorbic acid in the culture medium. Ascorbic acid increased the response of prolyl hydroxylase to hydralazine from 1.5- to 2-fold to 3- to 7-fold, whereas it decreased the response of lysyl hydroxylase to hydralazine from 4- to 8-fold to 2- to 3-fold. Total collagen synthesis was substantially reduced in hydralazine-treated cells; the time course and the dose-response relationship were similar to those observed for the hydroxylases. alpha, alpha'-Dipyridyl, an iron chelator, mimicked these effects of hydralazine. The studies suggest the existence in cultured cells of a compensatory mechanism for overproduction of these crucial enzymes in collagen biosynthesis, a mechanism which remains functional in cells derived from patients afflicted with hydroxylysine-deficient collagen disease.

Ascorbic Acid↗
PubMed1976

Pepsin-solubilized collagen of human nucleus pulposus and annulus fibrosus.

Human nucleus pulposus and annulus fibrosus, obtained at autopsy from patients 7-30 years of age, were extracted with 2 M guanidine-HCl (pH 5.82) to remove proteoglycans, then stirred with pepsin in 0.5 M acetic acid, followed by three 24-h extractions with 1 M NaCl (pH 7.5) and one 24-h extraction with 2 M KSCN (potassium thiocyanate) (pH 7.2). Pepsin and NaCl solubilized an average of about 30% of nucleus pulposus collagen and 18% of annulus fibrosus collagen. KSCN extracted a further 34% of nucleus pulposus collagen and only 4% of annulus fibrosus collagen. CM-cellulose chromatography of nucleus and annulus collagen purified from the pepsin, NaCl and KSCN supernatants consistently revealed only one peak, always appearing slightly ahead of the alpha1 position for rat tail tendon type I collagen. Polyacrylamide and SDS-gel electrophoresis consistently revealed only one band with the mobility of alpha1 chains. Amino acid composition of collagen from nucleus and annulus is comparable to those of mammalian and avian cartilage type II collagen, and distinctly different from those of rat tail tendonand guinea pig skin type I collagens. Periodate oxidation of nucleus and annulus collagens showed that 81% and 67%, respectively, of the hydroxylysine residues survive treatment, compared to 71% for bovine articular cartilage collagen and 17% for guinea pig skin collagen. Total hexose analysis revealed 1.8 muM and 2.0 muM hexose per muM periodate-stable hydroxylysine in nucleus and annulus collagens, respectively. Ion exchange chromatography showed the presence of glucose and galactose in a ratio of 0.92:1 in nucleas collagen and 1.07:1 in annulus collagen. Pepsin-solubilized, NaCl-extracted collagen from nucleus and annulus formed native-type fibrils in vitro. The banding patterns of ATP-induced segment-long-spacing precipitates of nucleus and annulus collagens were identical to each other and indistinguishable from those of cartilage (type II) collagen, but distinctly different from those of rat tail tendon (type I) collagen. These data suggest that the collagen which can be extracted after limited pepsin attack of human nucleus and annulus is of the form [alpha1 (II)]3.

Adolescent↗
PubMed1983

Selective solubilization of two populations of polypeptides from bovine retinal basement membranes.

Basement membranes isolated from the entire neural retinae of normal cows consist of a heterogeneous mixture of polypeptides resolvable as two major populations, one collagenous and the other non-collagenous. The non-collagenous population consists of disulfide-bonded aggregates of which the major fraction is soluble in lithium dodecyl sulfate (LDS) at 4 degrees C without reducing agent, with the remainder requiring reduction for solubilization at 4 degrees C. Only the second 4 degrees C extract is contaminated with small amounts of hydroxylated amino acids. The non-collagenous population consists of up to 25 polypeptides detectable in sodium dodecyl sulfate (SDS) gels, all of which stain blue with Coomassie Blue R-250, are insensitive to bacterial collagenase and migrate on or slightly below the central diagonal in urea-SDS/SDS electrophoresis. The collagenous population is soluble in LDS at 100 degrees C under reducing conditions and has a collagenous amino acid composition, although consisting of only 272 glycine residues/1000. This fraction consists of four major and four minor polypeptides detectable in SDS gels, all of which stain red (metachromatically) with Coomassie Blue R-250, are degraded by bacterial collagenase and migrate below the central diagonal in urea-SDS/SDS electrophoresis. The four major collagenous polypeptides are larger than the alpha-chains of type I collagen. In total, the solubilized proteins account for 60% of the general protein, but only 30% of the sum of hydroxyproline and hydroxylysine. The insoluble residue has a collagenous amino acid composition similar to that of the 100 degrees C extract, but with lower 3-hydroxyproline and hydroxylysine contents.

Amino Acids↗
PubMed1985

Connective tissue metabolism in muscular dystrophy. Amino acid composition of native types I, III, IV and V collagen isolated from the gastrocnemius muscle of embryonic chickens with genetic muscular dystrophy.

The amino acid composition data on types I, III, IV and V collagen isolated from embryonic dystrophic skeletal muscle strongly indicate that alterations in collagen synthesis occur in intramuscular connective tissue of developing muscles in embryonic dystrophic chickens. The changes observed in the amino acid composition of dystrophic collagen were: (a) a selective removal of polar amino acids and substitution with non-polar amino acids; (b) significant decreases in basic (lysine, hydroxylysine and arginine) and hydroxylated (4-hydroxyproline and hydroxylysine) amino acids; and (c) significant increases in the amounts of glycine, proline and alanine. The amino acid substitutions suggest a genetic alteration in the collagen synthesizing process and a change in its structure. The variations in amino acid composition of collagen from dystrophic chickens could give rise to a decrease in both inter- and intramolecular cross-linking, thus decreasing the stability and functionality of newly formed collagen fibrils. The differences associated with the dystrophic collagen reported in this study are probably due to the differences in primary structure in terms of amino acid sequence rather than post-translational modifications. The structural differences noted would also lead to an alteration of the role collagen plays in regulating the differentiation of developing muscles. The changes in amino acid structure strongly suggest that the 'collagen' formed by dystrophic chickens should be considered a collagen-like protein or 'collagenoid'.

Amino Acids↗
PubMed1997

Denatured thiolated collagen. I. Synthesis and characterization.

A new thiolating reagent is used to introduce sulphur groups into denatured atelocollagen. The procedure is easy to control and applicable on a large scale. The reagent is a reactive dicarboxylic acid compound containing sulphur in the form of a disulphide functionality. It is prepared by reacting N,N'-disuccinoylcystamine with 1,1'-carbonyldiimidazole. When this reagent is added to a solution of denatured atelocollagen in dimethylsulphoxide, amide bonds are formed between the carbonyl functions of the reagent and epsilon-NH2 of lysine and hydroxylysine residues from the protein. The disulphide groups introduced can then be reduced by reaction with 1,4-dithiothreitol to give the-SH form of the modified protein. Control of the stoichiometry between the reagent and the protein can lead to varying modification levels. A maximum level of 0.33 mmol SH per gram of protein can be attained, which corresponds to complete thiolation of the lysine and hydroxylysine residues. Thiolated denatured atelocollagen exhibits gelatin-like behaviour, by being highly soluble in water at all pH values and by forming heat-reversible gels.

Collagen↗
PubMed1982

Studies on the alpha-glucosidase specific for collagen disaccharide units: variations associated with capillary basement membrane thickening in kidney and brain of diabetic and aged rats.

The alpha-glucosidase specific for the hydroxylysine-linked disaccharide units of collagens (or 2-0-alpha-D-glucopyranosyl-5-0-beta-D-galactopyranosylhydroxy-L-lysine glucohydrolase) has been measured in kidney cortex and brain cortical tissue of streptozotocin diabetic rats after 19, 23 or 28 weeks of diabetes and of aged rats 22 months old. Increased specific activities of the enzyme have been found repeatedly in the dialyzed homogenates and the 7.2 X 10(6) g.min supernatants of kidney and brain at the various stages of diabetes when compared with age-matched controls; the specific activities returned to a normal level after insulin treatment. Similar increased specific activities were observed in kidney and brain of the aged normoglycemic rats when compared with young adult rats. In diabetic kidney cortex, beta-galactosidase and p-nitrophenyl-alpha-D-glucoside glucosidase specific activities were decreased in contrast to the increase of glucosyl-galactosyl-hydroxy-lysine glucohydrolase. In kidney cortex of the aged rats, beta-galactosidase activity was also decreased, but p-nitrophenyl-alpha-D-glucoside glucosidase was increased. In both diabetic and aged rats, thickening of the kidney glomerular basement membranes was confirmed; thickening of the brain cortical capillary basement membranes was also observed. Thus in the diabetic and aged animals, the increased glucosyl-galactosyl-hydroxylysine glucohydrolase specific activity was associated with basement membrane thickening in the kidney and the brain.

Aging↗
PubMed1986

Collagen crosslinking in lungs of rats with experimental silicosis.

Rats were intratracheally instilled with 50 mg of size-fractionated crystalline quartz to induce silicosis. Lungs were analyzed 1, 4, 6, and 9 months after instillation for their content of the reduced difunctional collagen crosslinks dihydroxylysinonorleucine (DHLNL) and hydroxylysinonorleucine (HLNL), of the nonreducible trifunctional (mature) crosslink, hydroxypyridinium (OHP), and of hydroxylysine. Ratios of DHLNL: HLNL were elevated in silicotic lung collagen at all times sampled, due both to increased levels of DHLNL and decreased amounts of HLNL. Hydroxylysine content of collagen in the silicotic lungs was also increased as compared with age-matched control rats. Hydroxypyridinium content of silicotic lung collagen was less than control values at 1 month, but was significantly increased to about 120%, 150%, and 175% of the age-matched control values at 4, 6, and 9 months after silica instillation, respectively. The increased levels of OHP in lung collagen were temporally correlated with the appearance of mature silicotic nodules in these lungs. We conclude that the large amounts of excess collagen deposited in silicotic lungs differs biochemically from normal lung collagen despite maintenance of the normal ratio of major collagen types in silicotic lungs.

Amino Acids↗
PubMed1992

Enzymatic production of alpha-aminoadipate-delta-semialdehyde and related compounds by lysine epsilon-dehydrogenase from Candida albicans.

L-Lysine epsilon-dehydrogenase [L-lysine:NADP+ oxidoreductase (epsilon-deaminating), EC 1.4.1.15] of Candida albicans was studied emphasizing its application for the production of alpha-aminoadipate-delta-semialdehyde and related compounds. A high enzyme level (240 pkat/mg of protein in the crude extract) could be attained during growth in the presence of L-lysine as sole nitrogen source. After optimization of the reaction conditions a partial purified enzyme (1.5 nkat/mg of protein) was used to produce alpha-aminoadipate-delta-semialdehyde, S-(beta-acetaldehyde)-cysteine, alpha-amino-delta-hydroxyadipate-semialdehyde and alpha-amino-gamma-hydroxyadipate-semialdehyde from the corresponding substrates lysine, S-(beta-aminoethyl)-cysteine, 5-hydroxylysine and 4-hydroxylysine, respectively. After purification of the compounds using Dowex 50 x 4 chromatography a yield of the products between 4.6 and 6.8% was achieved.

2-Aminoadipic Acid↗
PubMed1970

Chemistry of the collagen cross-links. Isolation and characterization of two intermediate intermolecular cross-links in collagen.

This paper describes the isolation from reduced collagen of two new amino acids believed to be involved, in their non-reduced form, as intermolecular cross-links stabilizing the collagen fibre. The reduction of intact collagen fibrils with tritiated sodium borohydride was found to stabilize the aldehyde-mediated cross-links to acid hydrolysis and thus allowed their location and isolation from acid hydrolysates on an automatic amino acid analyser. Comparison of the radioactive elution patterns from the autoanalyser of collagen treated in various ways before reduction permitted a preliminary classification of the peaks into cross-link precursors, intramolecular and intermolecular cross-links. The techniques employed to isolate the purified components on a large scale and to identify them structurally are described in detail. Two labile intermolecular cross-links were isolated in their reduced forms, one of which was identified by high-resolution mass spectrometry as N(in)-(5-amino-5-carboxypentyl)hydroxylysine. The structure of this compound was confirmed by chemical synthesis. The cross-link precursor alpha-aminoadipic delta-semialdehyde was isolated in its reduced form, in-hydroxynorleucine, together with its acid degradation product in-chloronorleucine. A relatively stable intermolecular cross-link was isolated and partially characterized by mass spectrometry as an aldol resulting from the reaction of the delta-semialdehyde derived from lysine and hydroxylysine.

Achilles Tendon↗
PubMed1978

Further characterization of collagen galactosyltransferase from chick embryos.

Optimum extraction of collagen galactosyltransferase activity from chick embryos required relatively high concentrations of detergent and salt. The activity was inhibited by concanavalin A, and the enzyme had a high affinity for columns of this lectin coupled to agarose; these results suggest the presence of carbohydrate units in the enzyme molecule. Collagen galactosyltransferase was highly labile, and only 1% of the originally bound enzyme activity could be eluted from the concanavalin A-agarose column with a buffer containing methyl glucoside and ethylene glycol. The purification of the activity over the original supernatant of chick embryo homogenate was 250-300-fold, with the optimum reaction conditions for the purified transferase differing somewhat from those for crude enzyme preparations. The reaction was inhibited by glucose-free basement-membrane collagen, UDP and galactosylhydroxylsine, and also by Co2+ and a number of compounds resembling UDP-galactose. Hydroxylysine was also a weak inhibitor. Immobilized hydroxylysine and UDP-glucuronic acid did not bind the collagen galactosyltransferase, but the enzyme was retarded in a column of UDP-galacturonic acid linked to agarose.

Animals↗
PubMed1978

Amino acid sequence of the N-terminal 108 amino acid residues of the B chain of subcomponent C1q of the first component of human complement.

The amino acid sequence of the N-terminal 108 residues of the B chain of subcomponent C1q of the first component of human complement was determined. The B chain has a blocked N-terminal amino acid, which was judged to be 5-oxopyrrolidine-2-carboxylic acid. A collagen-like region of 84 residues was found, which started at position B-6, and all of the six hydroxylysine residues and 12 hydroxyproline residues present in the chain were found in this region. Four of the six hydroxylysine residues may be glycosylated. The repeating nature of the collagen-like region is broken at position B-9, where alanine is found in a position where glycine would be expected. The exact position of the interchain disulphide bond joining the A and B chains of human subcomponent C1q was shown to be between residues A4 and B4.

Amino Acid Sequence↗