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PubMed2002

The third activity for lysyl hydroxylase 3: galactosylation of hydroxylysyl residues in collagens in vitro.

Lysyl hydroxylase (LH, EC 1.14.11.4), galactosyltransferase (EC 2.4.1.50) and glucosyltransferase (EC 2.4.1.66) are enzymes involved in posttranslational modifications of collagens. They sequentially modify lysyl residues in specific positions to hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosyl hydroxylysyl residues. These structures are unique to collagens and essential for their functional activity. Lysines and hydroxylysines form collagen cross-links. Hydroxylysine derived cross-links, usually as glycosylated forms, occur especially in weight-bearing and mineralized tissues. The detailed functions of the hydroxylysyl and hydroxylysyl linked carbohydrate structures are not known, however. Hydroxylysine linked carbohydrates are found mainly in collagens, but recent reports indicate that these structures are also present and probably have an important function in other proteins. Earlier we have shown that human LH3, but not isoforms LH1, LH2a and LH2b, possesses both LH and glucosyltransferase activity (J. Biol. Chem. 275 (2000) 36158). In this paper we demonstrate that galactosyltransferase activity is also associated with the same gene product, thus indicating that one gene product can catalyze all three consecutive steps in hydroxylysine linked carbohydrate formation. In vitro mutagenesis experiments indicate that Cys(144) and aspartates in positions 187-191 of LH3 are important for the galactosyltransferase activity. Our results suggest that manipulation of the gene for LH3 can be used to selectively alter the glycosylation and hydroxylation reactions, and provides a new tool to clarify the functions of the unique hydroxylysine linked carbohydrates in collagens and other proteins.

Amino Acid Sequence↗
PubMed1977

In vitro inhibition of collagen cross links by catechol analogs.

Catechol analogs inhibit the formation of hydroxylysine-derived intermolecular collagen cross links in tissue cultures of chick embryo calvaria. Formation of intermolecular collagen cross links was measured following incorporation of [14C]lysine, reduction with sodium borohydride, and elution from an ion exchange column with a pyridine-formate gradient. Cultures grown in the presence of 10(-3) M catechol, 10(-3) M dopamine, 10(-3) M L-dopa, or 10(-3) M D,L-serine-(2,3,4-trihydroxybenzyl)-hydrazide demonstrated between 43 and 84% inhibition of hydroxylysine formation. Collagen biosynthesis was not diminished in these cultures as compared to controls without additions or with beta-aminopropionitrile when measured by collagenase digestion. The formation of hydroxylysine-derived intermolecular cross links was inhibited 34 to 93% for 5,5'-dihydroxylysinonorleucine and 7 to 71% for 5-hydroxylysinonorleucine. The catechol analogs also inhibit the activity of lysyl hydroxylase as measured by specific tritium release as triated water from an L-[4,5-3H]lysine-labeled unhydroxylated collagen substrate prepared from chick calvaria. Since catechol analogs inhibit the formation of hydroxylysine in a cell-free assay, these compounds must pass into the cells of calvaria in this culture system to inhibit intracellular hydroxylysine formation and subsequently to diminish the reducible intermolecular cross links of the newly synthesized collagen.

Aminopropionitrile↗
PubMed1975

[Isolation and properties of a preparation of arterial collagen solubilized by pretreatment with alkali (author's transl)].

The acid-soluble, highly cross-linked aorta collagen, of which about 30% can be converted into a soluble form by alkali treatment, followed by extraction with aetic acid, was obtained predominantly in the form of monomeric, helical molecules, as indicated by the value for the intrinsic viscosity and its behaviour in sodium dodecylsulphate disc electrophoresis. Apart from decreased values for tyrosine (0.26%), arginine (4.4%) and aspartic acid (3.9%), the amino acid composition of the aorta collagen fraction was similar to that of the acid-soluble calf skin collagen. This finding, together with the cyanogen bromide peptide pattern, shows that the collagen extracted from the artery is predominantly type I. Treatment with alkali probably shortens the alpha1-CB6-peptide by about 45 amino acids. The collagen extracted from artery was compared with acid soluble skin collagen by sodium dodecylsulphate polyacrylamide electrophoresis. The arterial collagen showed a marked increase in the rations alpha1 to alpha2 (4:1), alpha to beta (3:1) and beta11 to beta12 (2.5:1). Compared with acid soluble skin collagen, the aorta collagen contained twice as much galactose and glucose (13.5 and 9.6 nmol/mg protein respectively), which are bound to hydroxylysine. 50% of the hydroxylysine residues are unsubstituted, 15% are present as galactosyl hydroxylysine, and 35% as glucosyl-galactosyl hydroxylysine. On the basis of its reported properties, arterial collagen obtained by the method of Fujii appears to be a suitable substrate for the study of the enzymic synthesis and enzymic degradation of hydroxylysine glycosides of native arterial collagen.

Amino Acids↗
PubMed1985

Urinary excretions of hydroxylysylglycosides in rats with experimentally induced collagen-like syndrome.

The excretion of the two hydroxylysine glycosides hydroxylysine-galactose-glucose and hydroxylysine-galactose was studied in the urine of normal healthy and experimental rats chronically treated with collagen-like syndrome inductors, hydralazine or binazine. The elevated urinary hydroxylysine-galactose-glucose and hydroxylysine-galactose strongly suggests an altered rate of collagen degradation during induced collagen-like syndrome.

Animals↗
PubMed1993

Tendon hypertrophy is associated with increased hydroxylation of nonhelical lysine residues at two specific cross-linking sites in type I collagen.

This study was designed to investigate whether the changes in lysine hydroxylation known to occur in hypertrophic tendon occur randomly or at specific lysine residues in the type I collagen molecule. Peptides corresponding to the two known major cross-linking sites of type I collagen (a lysine (or hydroxylysine) at position 9N cross-linked to a hydroxylysine at 930 and a lysine (or hydroxylysine) at position 16C cross-linked to a hydroxylysine at position 87) were prepared by collagenase digestion, size fractionation, and separation by high performance liquid chromatography from normal chicken tendon and from chicken tendon subjected to increased tensile load as a result of muscle hypertrophy. The ratio of the difunctional cross-links dihydroxylysinonorleucine to hydroxylysinonorleucine in normal tendon is 0.75:1; this ratio is increased to 6:1 in hypertrophic tendon. The dihydroxylysinonorleucine to hydroxylysinonorleucine ratio is increased to the same extent in samples of the purified cross-linked peptides derived from both the N-terminal and C-terminal lysine aldehyde residues. On the other hand, the relative hydroxylysine content of preparations of the pooled larger helical peptides obtained by cyanogen bromide digestion of normal and hypertrophic tendons was essentially identical. These results demonstrate that there is a specific increase in hydroxylation of only the N- and C-terminal non-helical lysine residues that participate in the formation of the reducible difunctional cross-links of type I collagen in hypertrophic tendon, while the extent of hydroxylation of lysine residues in the helical regions is not affected. The specific mechanism by which the enzyme lysyl hydroxylase acting on its substrate can distinguish between lysine residues destined to be in non-helical versus helical regions in a nascent collagenous peptide that has not yet attained its final secondary structure remains to be defined.

Amino Acid Sequence↗
PubMed1993

Rat cortical and trabecular bone collagen glycosylation are differently influenced by ovariectomy.

This study sought to evaluate whether estrogen depletion influences the hydroxylysine glycosydes content of rat bone collagen. For this reason thirty 100 day old female rats were divided in 6 groups of 5 rats each. Three groups were ovariectomized and 3 groups were sham-operated. The animals were sacrificed at 115, 130 and 145 days of age (i.e., 15, 30 and 45 days after surgery). Cortical and trabecular bone was prepared from tibiae and femurs. Hydroxylysine glycosydes content was measured by HPLC. Ovariectomy is followed, in the rat, by an increased hydroxylysine glycosylation in trabecular bone but by a constant or slightly decreased hydroxylysine glycosylation in the cortical bone. In view of the different effects of estrogens on the two bone compartments previously reported, a possible functional explanation of these findings is proposed.

Animals↗
PubMed2000

Posttranslational modifications of bone collagen type I are related to the function of rat femoral regions.

This study analyzes the relationship between the function of femoral regions in the rat and the extent of collagen type I posttranslational modifications, to assess whether the different functional roles, i.e., mechanical or metabolic, of the bone tissues are related to the molecular structure of the matrix. For this purpose, 18 female, 100-day-old Sprague-Dawley rats were sacrificed, under anesthesia, and their femurs were removed and dissected free of adhering tissue. The spongy bone of the proximal metaphysis and the diaphysis were then selected as regions exerting prevalently a mechanical function, and the spongy bone of the distal metaphysis was selected as mainly related to metabolic function. Bone prepared from these regions was used to extract and purify the major component of the matrix, type I collagen. The content of hydroxyproline, hydroxylysine, glycosylated hydroxylysine, and pyridinium crosslinks was evaluated and the amount of each compound was expressed as a molar ratio to hydroxyproline. The amount of glycosylated hydroxylysine and pyridinium crosslinks in the distal metaphysis are significantly different from the amounts measured both in the diaphysis and the proximal metaphysis. On the contrary, the amounts of the same compounds in the diaphysis and the proximal metaphysis are statistically the same. The amount of free hydroxylysine, however, appears to be different in the proximal metaphysis and in the diaphysis. The conclusion is that matrix composition differs among different skeletal regions according to the main function they exert.

Analysis of Variance↗
PubMed1975

Collagen synthesis and secretion by isolated rat renal glomeruli.

Glomeruli were isolated from rat renal cortex and incubated with radioactive lysine to study in vitro collagen synthesis in these preparations. Glomerular basement membrane was obtained by sonication, and the appearance of [-14C]lysine and hydroxylysine in medium, membrane and intracellular proteins was determined. Total glomerular incorporation of [-14C]lysine into protein linearly increased for up to 2-h period, and membrane hydroxylysine content gradually rose during this time. Hydroxy[-14C]lysine was recovered in the 105 000 times g pellet, reaching a hydroxylysine content of 22 percent in this intracellular fraction after 90 min of incubation. 60 percent of the protein secreted into the medium, and about 75 percent of newly synthesized sonicated basement membrane was acetic acid soluble. Hydroxylysine content was 33 percent in the acetic acid-insoluble fraction of sonicated membrane, suggesting that basement-membrane collagen was a significant component of total collagen synthesized by these preparation, The ability of isolated glomeruli to synthesize and secrete basement-membrane protein will be useful for studies concerning control of glomerular collagen and basement-membrane synthesis.

Animals↗
PubMed1976

Effect of L-azetidine-2-carboxylic acid on glycosylations of collagen in chick-embryo tendon cells.

The glycosylations of hydroxylysine during collagen biosynthesis in isolated chick-embryo tendon cells were studied by using pulse-chase labelling experiments with [14C]-lysine. The hydroxylation of lysine and the glycosylations of hydroxylysine continued after a 5 min pulse label for up to about 10 min during the chase period. These data differ from those obtained previously in isolated chick-embryo cartilage cells, in which, after a similar 5 min pulse label, these reactions continued during the chase period for up to about 20 min. The collagen synthesized by the isolated chick-embryo tendon cells differed markedly from the type I collagen of adult tissues in its degree of hydroxylation of lysine residues and glycosylations of hydroxylysine residues. When the isolated tendon cells were incubated in the presence of L-azetidine-2-carboxylic acid, the degree of glycosylations of hydroxylysine during the first 10 min of the chase period was identical with that in cells incubated without thcarboxylic acid for at least 60 min, whereas no additional glycosylations took place in the control cells after the 10 min time-point. As a consequence, the collagen synthesized in the presence of this compound contained more carbohydrate than did the collagen synthesized by the control cells. Additional experiments indicated that azetidine-2-carboxylic acid did not increase the collagen glycosyltransferase activities in the tendon cells or the rate of glycosylation reactions when added directly to the enzyme incubation mixture. Control experiments with colchicine indicated that the delay in the rate of collagen secretion, which was observed in the presence of azetidine-2-carboxylic acid, did not in itself affect the degree of glycosylations of collagen. The results thus suggest that the increased glycosylations were due to inhibition of the collagen triple-helix formation, which is known to occur in the presence of azetidine-2-carboxylic acid.

Animals↗
PubMed1976

Urinary excretion of hydroxylysyl glycosides during acute inflammation in the rat.

1. Subcutaneous inflammatory granuloma were induced in young rats and the urinary excretion of hydroxyproline and hydroxylysyl glycosides was observed during the period of acute inflammation. 2. All collagen metabolites were increased in the urine and excretion of glucosyl-galactosyl-hydroxylysine was much greater than excretion of galactosyl-hydroxylysine in the first days. 3. It is argued that urinary glucosyl-galactosyl-hydroxylysine is probably derived from hydroxylysyl residues of soluble collagen. 4. This study affords new arguments in favour of the dermal origin of urinary glucosyl-galactosyl-hydroxylysine, at least in skin inflammation.

Acute Disease↗
PubMed1981

Biochemical properties of glomerular basement membrane in daunomycin nephrosis and nephrotoxic serum nephritis.

Glomerular basement membrane (GBM) were isolated from the kidneys of rats suffering from daunomycin nephrosis or nephrotoxic serum nephritis. The GBM from daunomycin nephrotic rats contained significantly less hydroxyproline, hydroxylysine and glycine than that of control rats. There was an increase in glucosamine content in the membrane. No significant change was found in the neutral sugar content. In nephrotoxic serum nephritis, the relative amounts of hydroxyproline, glycine and half-cystine were decreased, whereas the relative amounts of aspartic acid, alanine, lysine and hydroxylysine were increased. The ratio of hydroxyproline to proline and the ratio of hydroxylysine to lysine were decreased. An increase in sialic acid content and a decrease in fucose and hexosamine content and glucosyl-galactosyl-hydroxylysine content were noted in nephrotoxic nephritic GBM. These chemical structural alterations could accounted for the functional disorders of diseased GBM.

Animals↗
PubMed1976

Glomerular basement membrane: biosynthesis and chemical composition in the streptozotocin diabetic rat.

To study the effect of streptozotocin induced diabetes on glomerular basement membrane (GBM) synthesis, an isolated rat glomerular preparation has been developed, and its metabolic properties have been defined. The chemical composition of normal rat GBM isolated from this preparation closely resembles human GBM. Incubation with [U-14C] lysine leads to prompt incorporation of label into GBM and the subsequent appearance of labeled hydroxylysine. A 1-h lag before detection of labeled hydroxylysine in GBM suggests a delay in the release of GBM precursors. Significantly lower counts appeared in the nondialyzable fraction of the medium than in insoluble GBM during pulse-chase experiments, and labeled hydroxylysine accounted for a lower portion of the total counts in the medium (0.85%) than in the GBM (1.98%). Isolated glomeruli were prepared from streptozotocin diabetic rats of 4-6 wks duration. After incubation with [ U-14C] lysine recovery of label in diabetic GBM (88.98+/-8.26 nmol/g GBM) did not differ from age matched controls (82.52 +/- 8.26 nmol/g GBM). In pulse-chase experiments recovery of label in hydroxylysine of diabetic GBM (o.473 +/- 0.082 nmol/g GBM) did not differ from age matched controls (0567+/-0.065 nmol/g GBM). These findings indicate normal rates of GBM synthesis and hydroxylation of lysine residues in animals with streptozotocin diabetes.

Amino Acids↗
PubMed1988

Nonenzymatic glycation of basement membranes from human glomeruli and bovine sources. Effect of diabetes and age.

The nonenzymatic glycation of glomerular basement membranes (GBMs) from 14 diabetic and 19 nondiabetic human subjects was determined after boronic acid affinity and high-performance cation-exchange chromatography of their NaB[3H]4-reduced ketoamine adducts. The glucitol-lysine (Glc-Lys) and the glucitol-hydroxylysine (Glc-Hyl) content of diabetic GBM was found to be about twofold higher than that of nondiabetic samples (P less than .001). The content of these glycated amino acids did not correlate with age over the range examined (20-91 yr) or with the length of disease in diabetic subjects (2-16 yr). However, analyses of Glc-Lys and Glc-Hyl in calf and adult bovine GBM and lens capsules indicated that the levels of these glycated amino acids were several times greater in basement membranes from older animals. We also observed that guanidine-insoluble collagen of bovine GBM is more extensively glycated (approximately 4-fold) than primarily noncollagenous proteins that are extracted by this reagent. In all of the basement membranes examined, the percentage of glycation of lysine was greater than of hydroxylysine. Characterization of the components released by alkaline hydrolysis indicated that O-glycosylated hydroxylysine residues are nonenzymatically N-glycated to the same extent as those without an enzymatically attached carbohydrate unit. Our study indicates that more than a hundred times as many hydroxylysine residues are enzymatically glycosylated in human and bovine GBM as those containing the nonenzymatically formed ketoamine adduct.

Adult↗
PubMed1975

Contribution of kidney basement membranes and collagen to the urinary excretion of hydroxylysyl glycosides.

Glucosyl-galactosyl-hydroxylysine and galactosyl-hydroxylysine are two substances present only in collagen and in basement membranes. The purpose of this study is to estimate to what extent the kidney contributes to urinary hydroxylysyl glycoside excretion. "In vitro" perfusion of isolated kidneys allows the measurement of hydroxylysyl glycosides independently of the important amount of interstitial collagen present in the whole body. It is shown that the kidney parenchyma contributes no more than 10 percent of the glycosylated hydroxylysine excreted in urine. GBM is not the only renal source of glucosyl-galactosyl-hydroxylysine in urine. Tubular basement membrane and intersitial collagen of kidney may contribute to the excretion by the isolated organ.

Animals↗
PubMed1983

Differences in the extent and heterogeneity of lysyl hydroxylation in embryonic chick cranial and long bone collagens.

The hydroxylation of lysine in embryonic chick long bone and mandibular collagen was found to be approximately 3-fold greater than that of the collagens of adult animals. In contrast, no significant difference was found in extent of lysine hydroxylation of the collagens of frontal bones of embryos and postnatal animals. Both histochemical and biochemical evidence established that full thickness diaphyseal bone samples contained cartilage and, consequently, type II collagen which undoubtedly contributed to the higher hydroxylysine contents of young postnatal animals reported previously. DEAE ion exchange chromatography of the alpha 1(I) chains of lathyritic long bone and mandibular collagens isolated by carboxymethyl-cellulose ion exchange chromatography showed considerable heterogeneity, whereas the alpha 1(I) chains obtained from lathyritic frontal bone collagen did not. Three fractions of alpha 1(I) chains of long bones and mandibular collagen were isolated which differed significantly in their hydroxylysine contents. The relative proportion of the three peaks changed as a function of embryonic age and maturation: more of the alpha 1(I) chains with the highest hydroxylysine content was present in the collagen synthesized earliest during embryonic development. This is consistent with results which demonstrated that the collagens synthesized earliest during embryonic and postnatal development had the highest hydroxylysine contents.

Aging↗
PubMed1986

Effects of silica on lung collagen.

A single intratracheal injection of 50 mg crystalline silica (quartz) into rats produces silicosis. This animal model may be used to study collagen metabolism during the early, middle, and late phases of lung injury, corresponding respectively to the stages of lung injury, development of discrete granulomas, and development of mature silicotic nodules. The early phase is characterized by a rapid increase in the rate of synthesis of lung collagen (within one week of instillation) and increased deposition of excess lung collagen (significant increases within two weeks of instillation). Later phases are characterized by a continuing increase in deposition of excess lung collagen for at least one year after instillation. Silica-induced fibrosis is unique among all the animal models (and in most human fibrotic diseases) thus far examined, in that the excess collagen deposited in the lung contains normal ratios of the two major collagen types of the lung: types I and III. This collagen is nonetheless biochemically different from normal lung collagen. There are reproducible and characteristic differences in the intermolecular cross-links of the collagen in lungs from rats injected with silica. Within one month of silica instillation (the earliest time point examined thus far), an increased hydroxylysine content of collagen can be appreciated. The reducible dysfunctional cross-links are also more likely to be derived from hydroxylysine (i.e. the ratio of dihydroxylated to monohydroxylated cross-links increases). Within four months of silica instillation (and increasingly thereafter), increased amounts of the mature trifunctional cross-link hydroxypyridinium (derived from three residues of hydroxylysine) can also be appreciated, seemingly paralleling the evolution of mature silicotic nodules in these lungs. These changes in cross-linking of lung collagen seem to be common to all the animal models of pulmonary fibrosis examined, and are also consistent with changes occurring in human fibrotic lungs. Preliminary observations suggest that the locus of cross-linking remains the same: hydroxylysine replaces lysine in the primary structure of a specific collagen alpha chain to form the altered cross-links. Thus, there may be molecular markers for the collagen of fibrosis in diseased lungs.

Animals↗
PubMed1981

The influence of 1-hydroxyethane-1,1-diphosphonate and dichloromethanediphosphonate on lysine hydroxylation and cross-link formation in rat bone, cartilage and skin collagen.

The effects in vivo of dichloromethanediphosphonate and 1-hydroxyethane 1,1-diphosphonate on collagen solubility, hydroxylation of lysine and proline and on the formation of collagen intermolecular cross-links were studied by using rat bone, cartilage and skin tissues. Dichloromethanediphosphonate decreased bone collagen solubility both in acetic acid and after pepsin treatment. Although none of the diphosphonates had any effect on the hydroxylation of proline, dichloromethane-diphosphonate, but not 1-hydroxyethane-1,1-diphosphonate, increased the number of hydroxylysine residues in the alpha-chains of bone, skin and cartilage collagen. The stimulatory effect was dose-dependent. The dichloromethanediphosphonate-mediated increase in hydroxylysine residues in bone and cartilage was manifested in an increase of dihydroxylysinonorleucine, the cross-link that is formed by the condensation of two hydroxylysine residues. The cross-link hydroxylysinonorleucine, a condensation product of hydroxylysine and lysine, on the other hand, was decreased. The total number of intermolecular cross-links was not changed by the diphosphonate.

Animals↗
PubMed1977

In vitro inhibition of chick embryo lysyl hydroxylase by homogentisic acid. A proposed connective tissue defect in alkaptonuria.

Homogentisic acid inhibits the in vitro activity of chick embryo lysyl hydroxylase, a microsomal enzyme which catalyzes the transformation of certain lysyl residues in collagen to hydroxylysine. Chick embryo lysyl hydroxylase activity was measured as specific tritium release as tritium water from a [4,5-(3)H]lysine-labeled unhydroxylated collagen substrate prepared from chick calvaria. Kinetic studies revealed a linear, noncompetitive type of inhibition with respect to collagen substrate with a Ki of 120-180 muM. The inhibition by homogentisic acid was reversible in that enzyme activity could be restored after dialysis of preincubated mixtures of homogentisic acid with enzyme or substrate. The inhibition by homogentisic acid was competitive with respect to ascorbic acid, and the addition of reducing agents, such as ascorbic acid or 1,4-dithiothreitol, protected lysyl hydroxylase activity from homogentisic acid inhibition.In organ cultures of embryonic chick calvaria, biosynthesis of hydroxylysine-derived intermolecular collagen cross-links was inhibited in a dose-dependent manner by 0.5-5 mM homogentisic acid. Because homogentisic acid inhibits the formation of hydroxylysine in a cell-free assay and in organ cultures, this compound must pass into the cells of calvaria to inhibit intracellular hydroxylysine formation and subsequently to diminish the reducible intermolecular cross-links of the newly synthesized collagen. We propose that the inhibition of lysyl hydroxylase and the resulting hydroxylsine-deficient, structurally modified collagen may be clinically significant in the defective connective tissue found in alkaptonuric patients.

Alkaptonuria↗