hydroxylysine and glucosylgalactosylhydroxylysine

Hydroxylysine and its glycosylated forms, galactosylhydroxylysine and glucosylgalactosylhydroxylysine, are post-translational modifications unique to collagenous sequences. They are found in collagens and in many proteins having a collagenous domain in their structure. Since the last published reviews, significant new data have accumulated regarding these modifications. One of the lysyl hydroxylase isoforms, lysyl hydroxylase 3 (LH3), has been shown to possess three catalytic activities required sequentially to produce hydroxylysine and its glycosylated forms, that is, the lysyl hydroxylase (LH), galactosyltransferase (GT), and glucosyltransferase (GGT) activities. Studies on mouse models have revealed the importance of these different activities of LH3 in vivo. LH3 is the main molecule responsible for GGT activity in mouse embryos. A lack of this activity causes intracellular accumulation of type IV collagen, which disrupts the formation of basement membranes (BMs) during mouse embryogenesis and leads to embryonic lethality. The specific inactivation of the LH activity of LH3 causes minor alterations in the structure of the BM and collagen fibril organization, but does not affect the lifespan of mutated mice. Recent data from zebrafish demonstrate that growth cone migration depends critically on the LH3 glycosyltransferase domain. LH3 is located in the ER loosely associated with the membranes, but, unlike the other isoforms, LH3 is also found in the extracellular space in some tissues. LH3 is able to adjust the amount of hydroxylysine and hydroxylysine-linked carbohydrates of extracellular proteins in their native conformation, suggesting that it may have a role in matrix remodeling.

Topics: Amino Acid Sequence; Animals; Basement Membrane; Catalytic Domain; Collagen; Embryonic Development; Endoplasmic Reticulum; Extracellular Space; Galactosyltransferases; Glucosyltransferases; Glycosylation; Humans; Hydroxylysine; Mice; Molecular Sequence Data; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; Protein Processing, Post-Translational; Sequence Homology, Amino Acid; Substrate Specificity

Lysyl oxidase-generated intermolecular cross-links are essential for the tensile strength of collagen fibrils. Two cross-linking pathways can be defined, one based on telopeptide lysine aldehydes and another on telopeptide hydroxylysine aldehydes. Since the 1970s it has been accepted that the mature cross-linking structures on the lysine aldehyde pathway, which dominates in skin and cornea, incorporate histidine residues. Here, using a range of MS-based methods, we re-examined this conclusion and found that telopeptide aldol dimerization is the primary mechanism for stable cross-link formation. The C-telopeptide aldol dimers formed labile addition products with glucosylgalactosyl hydroxylysine at α1(I)K87 in adjacent collagen molecules that resisted borohydride reduction and after acid hydrolysis produced histidinohydroxylysinonorleucine (HHL), but only from species with a histidine in their α1(I) C-telopeptide sequence. Peptide MS analyses and the lack of HHL formation in rat and mouse skin, species that lack an α1(I) C-telopeptide histidine, revealed that HHL is a laboratory artifact rather than a natural cross-linking structure. Our experimental results also establish that histidinohydroxymerodesmosine is produced by borohydride reduction of N-telopeptide allysine aldol dimers in aldimine intermolecular linkage to nonglycosylated α1(I) K930. Borohydride reduction of the aldimine promotes an accompanying base-catalyzed Michael addition of α1(I) H932 imidazole to the α,β-unsaturated aldol. These aldehydes are intramolecular at the N terminus but at the C terminus they can be both intramolecular and intermolecular according to present and earlier findings.

Topics: Aldehydes; Animals; Artifacts; Cattle; Collagen Type I; Dipeptides; Histidine; Hydroxylysine; Peptides; Protein-Lysine 6-Oxidase; Skin

Collagens are the most abundant glycoproteins in the body. One characteristic of this protein family is that the amino acid sequence consists of repeats of three amino acids -(X-Y-Gly)n. Within this motif, the Y residue is often 4-hydroxyproline (HyP) or 5-hydroxylysine (HyK). Glycosylation in collagen occurs at the 5-OH group in HyK in the form of two glycosides, galactosylhydroxylysine (Gal-HyK) and glucosyl galactosylhydroxylysine (GlcGal-HyK). In collision induced dissociation (CID), collagen tryptic glycopeptides exhibit unexpected gas-phase dissociation behavior compared to typical N- and O-linked glycopeptides (i.e., in addition to glycosidic bond cleavages, extensive cleavages of the amide bonds are observed). The Gal- or GlcGal- glycan modifications are largely retained on the fragment ions. These features enable unambiguous determination of the amino acid sequence of collagen glycopeptides and the location of the glycosylation site. This dissociation pattern was consistent for all analyzed collagen glycopeptides, regardless of their length or amino acid composition, collagen type or tissue. The two fragmentation pathways-amide bond and glycosidic bond cleavage-are highly competitive in collagen tryptic glycopeptides. The number of ionizing protons relative to the number of basic sites (i.e., Arg, Lys, HyK, and N-terminus) is a major driving force of the fragmentation. We present here our experimental results and employ quantum mechanics calculations to understand the factors enhancing the labile character of the amide bonds and the stability of hydroxylysine glycosides in gas phase dissociation of collagen glycopeptides.

Topics: Amino Acid Sequence; Animals; Binding Sites; Cattle; Chromatography, High Pressure Liquid; Collagen; Glycopeptides; Glycosylation; Hydroxylysine; Molecular Sequence Data; Molecular Structure; Peptide Fragments; Quantum Theory; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Trypsin

In recent years, glycopeptide purification by hydrazide chemistry has become popular in structural studies of glycoconjugates; however, applications of this method have been almost completely restricted to analysis of the N-glycoproteome. Here we report a novel method for analyzing O-glycosylations unique to collagen, which are attached to hydroxylysine and include galactosyl-hydroxylysine and glucosyl-galactosyl-hydroxylysine. We established a hydrazide chemistry-based glycopeptide purification method using (1) galactose oxidase to introduce an aldehyde into glycopeptides and (2) formic acid with heating to elute the bound glycopeptides by cleaving the hydrazone bond. This method allows not only identification of O-glycosylation sites in collagen but also concurrent discrimination of two types of carbohydrate substitutions. In bovine type I and type II collagens, galactosyl-hydroxylysine /glucosyl-galactosyl-hydroxylysine -containing peptides were specifically detected on subsequent comprehensive liquid chromatography (LC)/MS analysis, and many O-glycosylation sites, including unreported ones, were identified. The position of glycosylated hydroxylysine, which is determined by our unambiguous and simple method, could provide insight into the physiological role of the modifications.

Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Cattle; Chromatography, Affinity; Collagen; Collagen Type I; Collagen Type II; Glycoproteins; Glycosylation; Hydrazines; Hydroxylysine; Molecular Sequence Data; Oxidation-Reduction; Peptide Fragments; Peptide Mapping; Protein Processing, Post-Translational; Tandem Mass Spectrometry

This paper reports the first chemical synthesis of alpha-D-glucopyranosyl-(1-->2)-beta-D-galactopyranosyl-O-hydroxylysine, a glycoside of hydroxylysine important as indicator of skin and bone collagen turnover, starting with commercial compounds.

Topics: Collagen; Hydroxylysine

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.

Topics: Amino Acid Sequence; Animals; Aspartic Acid; Cell Line; Cysteine; Drug Residues; Enzymes; Galactose; Galactosyltransferases; Humans; Hydroxylysine; Insecta; Mutation; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase

Collagen abnormalities of the spinal cord and the skin have been reported in patients with amyotrophic lateral sclerosis (ALS). The urinary concentrations of the hydroxylysine glycosides, i.e., glucosylgalactosyl hydroxylysine (glu-gal Hyl) and galactosyl hydroxylysine (gal Hyl), indicate the tissue origin of the collagen metabolites and the rate of the degradation of collagen. We measured the urinary levels of glu-gal Hyl and gal Hyl in 12 ALS patients, 10 diseased control subjects with other neurologic or muscular diseases (Control Group A), and 10 healthy control subjects (Control Group B). The urinary level of glu-gal Hyl in ALS patients was significantly lower than in the two control groups. In addition, a significant negative relationship between glu-gal Hyl urinary level and duration of illness was found in ALS patients. There was no marked difference in the urinary level of gal Hyl between ALS patients and the control groups. Our data suggest that the decreased urinary level of glu-gal Hyl may be useful in assessing the alteration in collagen metabolism in ALS and may have a relationship with the progression of ALS.

Topics: Aged; Biomarkers; Collagen; Female; Humans; Hydroxylysine; Male; Middle Aged; Motor Neuron Disease; Reference Values; Regression Analysis; Time Factors

Some glycosides of hydroxylysine, viz., alpha-1, 2-glucosylgalactosyl-O-hydroxylysine and beta-1-galactosyl-O-hydroxylysine, appear to be good indicators of collagen turnover. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method is proposed for measuring these analytes in urine, with no sample preparation except for a dilution step. Quantitation is performed using external calibration with no internal standard. A preliminary survey indicates good intra- and inter-day reproducibility (better than 5 and 8%, respectively). With the present method, the estimated limits of detection (S/N > 3) in urine are 0.8 and 0.5 microM/L for beta-1-galactosyl-O-hydroxylysine and alpha-1,2-glucosylgalactosyl-O-hydroxylysine, respectively. The method is proposed as a robust tool for a large-scale research investigation on collagen turnover.

Topics: Chromatography, Liquid; Collagen; Humans; Hydroxylysine; Mass Spectrometry

The ovariectomized rat is the most commonly used animal model of human postmenopausal osteoporosis, exhibiting a high rate of bone turnover with resorption exceeding formation. At present, bone turnover is quantified directly by dynamic histomorphometry. The aim of the present study was to determine whether the measurement of the urinary output of some specific bone collagen catabolites--pyridinolines and hydroxylysine glycosides--could be used to indirectly monitor the initial phase of bone turnover increase in ovariectomized 90-day-old rats. Ninety-day-old female rats were randomly divided into three groups (n = 6): ovariectomized, sham-operated and non-treated controls. Urine samples (24 h) were collected 6 days before surgery and twice weekly for the 4 weeks following ovariectomy. Urinary excretion of pyridinoline (PYD), deoxypyridinoline (DPD), glucosyl-galactosyl-hydroxylysine (GGHYL) and galactosyl-hydroxylysine (GHYL) were measured. As expected, ovariectomy was associated with a significant decrease in bone mineral density in both the proximal tibial and distal femoral metaphysis. Compared with both sham-operated and control animals, ovariectomized rats showed significant increases in PYD, GGHYL, and GHYL urinary output 8 days after surgery and in DPD output after 15 days. These changes were maintained throughout the study. The results confirm that measurement of the urinary excretion of pyridinolines and hydroxylysine glycosides represents a powerful tool for detecting the onset of bone turnover in ovariectomized 90-day-old rats.

Topics: Amino Acids; Animals; Bone and Bones; Female; Glycosides; Hydroxylysine; Ovariectomy; Rats; Rats, Sprague-Dawley; Time Factors

Glucosylgalactosylhydroxylysine (GGHYL), galactosylhydroxylysine (GHYL), pyridinoline (PYD) and deoxypyridinoline (DPD) were measured in the urine (6 h serial specimens over 96 and 24 h urine specimens for 4 days) collected from four adult Sprague Dawley rats and in the femoral and tibia] bone as well as in the dorsal skin of the same rats. No significant daily variations were found in the urine excretion of GGHYL, GHYL, PYD and DPD but significant diurnal variations. The GGHYL/GHYL ratio in rat urine (0.46 +/- 0.1) reflected neither the bone collagen ratio (1.9 to 2.4) nor the skin collagen ratio (1.22 +/- 1.07), a finding that may reflect GGHYL conversion into GHYL. The content of both pyridinolines was very low in the skin and high in the bone collagen and the urinary PYD/DPD ratio (1.46 +/- 0.15) reflected essentially the bone collagen ratio (0.8-3.0). These results suggest the usefulness of measuring GGHYL, GHYL, PYD and DPD in 24 h urine specimen and, based on the inter-animal variations, the necessity to consider each animal as its own control when bone turnover needs to be monitored.

Topics: Amino Acids; Animals; Biomarkers; Bone and Bones; Collagen; Cross-Linking Reagents; Glycosides; Humans; Hydroxylysine; Male; Pyridinium Compounds; Rats; Rats, Sprague-Dawley; Skin; Time Factors

This study was undertaken to assess the sensitivity of hydroxylysylpyridinoline (HP), lysylpyridinoline (LP), galactosylhydroxylysine (GHyl) and glucosylgalactosylhydroxylysine (GGHyl) to monitor bone response to estrogen deficiency and replacement by comparing their excretory patterns in ovariectomized aged (11-14 months old) rats. The ovariectomized (OVX) rats were randomized into two groups: (1) OVX plus vehicle; (2) OVX plus 17 beta-estradiol (17-beta E, 10 micrograms/kg, s.c., 4 days/week). Treatment with 17-beta E started immediately after OVX and continued for 60 days. The collagen catabolites were measured in urine for 1 month before OVX and thereafter for 60 days. In temporal coincidence with urine collection, bone area and bone mineral density (BMD) of lumbar vertebrae, femoral diaphysis and distal metaphysis were measured by dual-energy X-ray absorptiometry. In the untreated rats, BMD of the femoral metaphysis and lumbar vertebrae decreased significantly and the urinary excretion of LP, HP, GHyl and GGHyl increased with different patterns. In the treated rats, 17-beta E replacement prevented the increment in LP excretion, partially prevented the increase in HP excretion, but had no effect on the excretion of GHyl and GGHyl. In conclusion pyridinolines and glycosides have different sensitivities to the bone response to OVX. Glycoside excretion after OVX also reflects metabolic processes not strictly related to bone loss and, in contrast with LP, is not sensitive to estrogen replacement.

Topics: Absorptiometry, Photon; Amino Acids; Animals; Biomarkers; Bone and Bones; Estradiol; Estrogen Replacement Therapy; Female; Hydroxylysine; Ovariectomy; Pyridinium Compounds; Random Allocation; Rats; Rats, Sprague-Dawley

The composition of collagen was analyzed and the degree of lysyl hydroxylation of individual collagen chains was determined in four osteosarcomas and two osteofibrous dysplasias. In addition, the tumor proliferation (number of mitoses, proliferating-nuclear-antigen-positive cells, MIB) as well as the response to chemotherapy (morphological regression grade) were checked. All tumors contained a high proportion of collagen III and, in all but one osteosarcoma, pepsin-extracted collagens I and III were overmodified. Furthermore, the proportion of diglycosides in collagen I was about four times higher than in controls. The collagen composition and modification resembled those of bones at early stages of human development. One osteosarcoma and both osteofibrous dysplasias were in the normal range of lysyl hydroxylation. There was no correlation between the collagen properties and the histopathological marker of tumor proliferation.

Topics: Adolescent; Adult; Bone Neoplasms; Cell Division; Child; Collagen; Electrophoresis, Polyacrylamide Gel; Fibrous Dysplasia of Bone; Glycosylation; Humans; Hydroxylation; Hydroxylysine; Lysine; Osteosarcoma; Proline; Protein Processing, Post-Translational; Sodium Dodecyl Sulfate

Topics: Animals; Bone Remodeling; Chromatography, High Pressure Liquid; Female; Humans; Hydroxylysine; Ovariectomy; Rats

Sixty men with spinal cord injury who had developed pressure ulcers in the past but whose skin was intact when they joined the study were followed for 2 years, or until a pressure ulcer developed. Forty of the men were contacted every 4-6 weeks to answer questions about their skin care practices and to provide a 24 hour urine sample. The others were only contacted at the beginning and the end of the study to answer a questionnaire and to provide a urine sample. Changes in skin collagen metabolism were monitored by measuring urinary excretion of a metabolite, glucosyl-galactosyl hydroxylysine (glu-gal Hyl), corrected for creatinine excretion. Sustained increases in levels of glu-gal Hyl excretion were detected at least 2 months and as much as 5 months in advance of overt clinical signs of ulcer development. Increased excretion of glu-gal Hyl was significantly associated (p < 0.05) with the development of a pressure ulcer. An increase in the urinary excretion of glu-gal Hyl is an indication of increased degradation of skin collagen. Body mass index (weight/height2) of 33% of subjects with pressure ulcers, and 12% of those without, was at least one standard deviation below the mean of all subjects. Thirty-six percent of those who smoked developed ulcers, while only 26% of the nonsmokers did.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Collagen; Humans; Hydroxylysine; Male; Models, Psychological; Patient Compliance; Pressure Ulcer; Prospective Studies; Risk Factors; Spinal Cord Injuries

Glucosyl-galactosyl-hydroxylysine (GGHYL) and galactosyl-hydroxylysine (GHYL) are constituents of collagen protein. The ratio of the two hydroxylysine glycosides varies with the collagen type and, moreover, for a given collagen type, it also varies according to the connective tissue. For example, in type I collagen (the most abundant in the body), the GGHYL/GHYL ratio tends to be greater in soft connective tissues and lower in bone. The hydroxylysine glycosides are not recycled during collagen turnover and are excreted in the urine. Therefore, the urinary GGHYL/GHYL ratio, which reflects the proportion of the two metabolites in the various collagens, may indicate the type of connective tissue affected by pathological turnover, and may thus be a promising marker of bone metabolism. In this paper a method is described for the measurement of urinary hydroxylysine glycosides by reversed-phase liquid chromatography after purification of the sample by solid-phase extraction. The method presented is analytically reliable and suitable for routine use in a clinical laboratory.

Topics: Adult; Biomarkers; Bone and Bones; Chromatography, High Pressure Liquid; Collagen; Female; Humans; Hydroxylysine; Indicators and Reagents; Osteoporosis; Reference Standards; Spectrophotometry, Ultraviolet

Glucosyl-galactosyl-hydroxylysine (GGHYL) and galactosyl-hydroxylysine (GHYL) are metabolites derived from collagen degradation. They are useful biochemical markers provided they are not further processed. Experiments were run to test the activity of alpha- and beta-glycosidases in human kidney cortex preparations. Results allow to exclude the presence of the specific enzymes, in contrast with what is reported for the rat kidney tissue.

Topics: Animals; Collagen; Glycoside Hydrolases; Humans; Hydroxylysine; In Vitro Techniques; Kidney Cortex; Male; Middle Aged; Rats; Rats, Sprague-Dawley

The effect of an organophosphorus pesticide (dimethoate) on the urinary excretion of hydroxyproline (total, nondialysable, dialysable and free fractions) and hydroxylysylglycosides, glucosylgalactosyl hydroxylysine and galactosehydroxylysine was investigated in two groups of female albino rats fed with normal and high protein diets. In comparison to controls, dimethoate treated animals were found to excrete significantly decreased amounts of urinary hydroxyproline fractions from 7th day onwards. The excretion of total hydroxylysylglycoside in urine parallels the excretion of hydroxyproline. The urinary output of both glu-gal-hyl and gal-hyl was also appreciably lower from dimethoate treated animals. The normal ratio of glu-gal-hyl and gal-hyl found in the urine of dimethoate treated animals was discussed in light of decreased turn over of collagen in both bone and skin. The effect of dimethoate in rats fed with high protein diet was comparatively less than those fed with normal diet.

Topics: Animals; Bone and Bones; Carbohydrate Sequence; Collagen; Dietary Proteins; Dimethoate; Female; Hydroxylysine; Hydroxyproline; Molecular Sequence Data; Rats; Skin

Immediately after the trauma, spinal cord injury patients have an increased rate of collagen synthesis and an even greater increase of collagen degradation. Collagen lost from bone is implicated in the etiology of osteoporosis and heterotopic ossification, and collagen lost from skin might lead to a propensity to develop pressure ulcers. The urinary excretion of two collagen metabolites has been monitored and their fluctuation related to the onset of skin or bone complications in these patients. One metabolite, glucosyl-galactosyl hydroxylysine, is more abundant in skin collagen; the other, galactosyl hydroxylysine, is more abundant in bone collagen. The excretion of both metabolites increases after injury, reaching a peak between three and six months after injury, and declines gradually, reaching control values about a year after injury. If a skin pressure ulcer develops, the urinary excretion of the diglycoside remains elevated instead of gradually decreasing. Similarly, if osteoporosis or heterotopic ossification is diagnosed, the monoglycoside excretion does not return to control values until the bone turnover stabilizes. Monitoring of the urinary excretion of both glycosides might prove helpful in prompting early examination to establish the presence of emerging skin and bone complications. Thus, aggressive preventive therapy could be given sooner.

Topics: Adolescent; Adult; Bone Diseases, Metabolic; Collagen; Humans; Hydroxylysine; Male; Middle Aged; Pressure Ulcer; Spinal Cord Injuries

1. Assay of the activity of alpha-1,2-glucosidase was completed within 10 min using reversed-phase high performance liquid chromatography and purified dansylated glucosyl galactosyl hydroxylysine as the substrate. 2. A comparative study was made on the enzyme activity of liver homogenate from eight animal species, mouse, frog, chicken, rabbit, pig, rat, human, bovine and that of a spinach leaf homogenate. alpha-1,2-glucosidase activity in human and bovine liver was very low, and that of alpha-1,2-glucosidase could not be detected in the spinach homogenate as expected. 3. 1-deoxynojirimycin, a well known potent inhibitor of alpha-1,2-glucosidases which act on the N-glycosidic type carbohydrate chain, also inhibited alpha-1,2-glucosidase acting specificity on glucosyl galactosyl hydroxylysine derived from the collagen molecule.

Topics: alpha-Glucosidases; Animals; Bufo bufo; Cattle; Chickens; Chromatography, High Pressure Liquid; Humans; Hydroxylysine; Kinetics; Liver; Mice; Rabbits; Rats; Species Specificity; Substrate Specificity; Swine

The dansyl derivative of glucosyl galactosyl hydroxylysine (GGH) was separated into two components, as GP-I (monodansyl GGH) and GP-II (didansyl GGH) by paper chromatography. GP-I was further fractionated into four peaks (a, b, c and d) by reversed-phase liquid chromatography. These peaks corresponded to the dansyl derivatives at the alpha-amino (a and b) and epsilon-amino (c and d) groups of their hydroxylysine residues. There is the possibility that the fractions for b and d are diastereoisomers of a and c, respectively, since the monodansyl derivative from human urine consists of a and c. GP-II was fractionated into two peaks, e and f, which may possibly be diastereoisomers of each other. Treatment of the a, b, c and d fractions with crude chicken liver enzyme resulted in the preferential cleavage of a and b and the production of monodansyl galactosyl hydroxylysine. Components c and d were also cleaved slowly, resulting in the production of monodansyl hydroxylysine by the successive action of beta-galactosidase on dansyl galactosyl hydroxylysine. The detected alpha-glucosidase activity was strongly inhibited by free mannosamine. The method developed using the monodansyl GGH fraction a (or b) and high-performance liquid chromatography facilitated the detection of alpha-1,2-glucosidase, which acts specifically toward GGH even in a crude enzyme preparation.

Topics: alpha-Glucosidases; Animals; Chickens; Chromatography, High Pressure Liquid; Dansyl Compounds; Humans; Hydroxylysine; Liver

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aging; Amino Acids; Basement Membrane; Carbohydrates; Humans; Hydroxylysine; Kidney Glomerulus; Middle Aged

The core-specific lectin (CSL) synthesized and secreted by rat hepatocytes and the rat hepatoma H-4-II-E shows affinity for mannose and N-acetylglucosamine residues in the "core" region of asparagine-linked oligosaccharides. The CSL undergoes two stages of post-translational modification which result in an increase in its Mr from 24,000 to 26,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have determined that the lectin undergoes hydroxylation of proline and lysine and that the hydroxylysine is glycosylated to form glucosylgalactosylhydroxylysine (GlcGalHyLys). CSL metabolically labeled with [3H]lysine and [3H]proline contains hydroxylated forms of proline and lysine. The mature form of the lectin can also be metabolically labeled with [3H]galactose. alpha,alpha'-Dipyridyl, an inhibitor of collagen prolyl and lysyl hydroxylases, prevents the metabolic incorporation of [3H]galactose and the post-translational increases in the Mr of the CSL, indicating that both events are dependent upon hydroxylation of proline and lysine. Virtually all of the hydroxylysine present in the CSL is recovered as glucosylgalactosylhydroxylysine after alkaline hydrolysis. The post-translational modifications of the CSL place it in a select family of secreted proteins which contain collagen-like sequences, including the pulmonary surfactant proteins, complement component C1q, and the 18 S asymmetric form of acetylcholinesterase.

Topics: 2,2'-Dipyridyl; Animals; Hydroxylysine; Hydroxyproline; Lectins; Liver; Liver Neoplasms, Experimental; Mannose-Binding Lectin; Protein Processing, Post-Translational; Rats

As Alport's syndrome is probably a molecular disorder of basement membrane composition, investigation of the urine on basement membrane components might be a diagnostic aid. It was shown previously that measurement of the excretion of free hydroxylysine and its glycosides, glucosylgalactosylhydroxylysine and galactosylhydroxylysine, was not useful for this purpose. Because the urinary peptide-bound fraction may be more basement membrane specific, a subsequent study was performed with respect to the total (= free and peptide-bound) concentrations and fractions. To establish reference values, urinary specimens of 38 normal subjects of different ages were investigated. These values were used for comparison with data on 30 Alport patients, and 10 Alport siblings. 10 patients with benign recurrent hematuria were also investigated. No marked differences were observed in the excretory rates between normals and patients.

Topics: Adolescent; Adult; Age Factors; Child; Humans; Hydroxylysine; Nephritis, Hereditary; Protein Binding; Sex Factors

Alport's syndrome probably is a molecular disorder of basement membrane composition. Investigation of urine on basement membrane components such as hydroxylysine and its glycosides, glucosylgalactosylhydroxylysine and galactosylhydroxylysine, may be helpful for diagnosis of the disease. Urinary specimens of 33 patients and 12 siblings were investigated, and the results were compared with those of 14 healthy adults and of 29 healthy children. The urine of patients with glomerulopathies, occurring during childhood (IgA nephropathy, benign recurrent hematuria, poststreptococcal glomerulonephritis, Henoch-Schönlein nephropathy, membranoproliferative glomerulonephritis, and nephrotic syndrome due to minimal lesions), was also investigated. No marked differences between normal and diseased subjects could be demonstrated, with respect to excretion of hydroxylysine and its glycosides, in contrast to data reported in the literature.

Topics: Adolescent; Adult; Child; Child, Preschool; Female; Glomerulonephritis; Glomerulonephritis, IGA; Hematuria; Humans; Hydroxylysine; Kidney Diseases; Male; Nephritis, Hereditary; Nephrotic 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.

Topics: Animals; Collagen; Collagen Diseases; Glycosides; Hydralazine; Hydroxylysine; Male; Neutrophils; Rats; Rats, Inbred Strains; Syndrome; Todralazine

Topics: Chromatography, High Pressure Liquid; Collagen; Humans; Hydroxylysine

A new technique to evaluate the degradation of skin or bone collagen by measuring glucosylgalactosyl hydroxylysine and galactosyl hydroxylysine is presented. The method utilizes an automated amino acid analyzer. Eluents used are lithium buffers, and the color reagent is ninhydrin. Both glycosides elute in 3.5 h. Samples require minimum preparation. Urinary concentrations of both glycosides in ten patients with cervical spinal cord injuries of less than six months duration were higher than in five healthy controls. Proportional increases were different for each of the two glycosides. Variations in the proportional increase of each glycoside indicate different rates of degradation of skin and bone collagen. Repeated evaluations of the two urinary glycosides may help to predict whether patients are likely to develop skin- or bone-related clinical complications.

Topics: Adult; Amino Acids; Chromatography; Hexoses; Humans; Hydroxylysine; Male; Spinal Cord Injuries

Topics: Adult; Bone and Bones; Child; Collagen; Humans; Hydroxylysine; Hydroxyproline

Topics: Animals; Cadmium; Collagen; Female; Hydroxylysine; Hydroxyproline; Kidney; Rats; Rats, Inbred Strains

Members of a family with dominant epidermolysis bullosa simplex were found to have a deficiency of galactosylhydroxylysyl glucosyltransferase (GGT), an enzyme catalyzing the glucosylation of galactosylhydroxylysyl residues in the biosynthesis of collagen. The enzyme's activity was low in serum, skin tissue, and cultured skin fibroblasts, although no abnormality was found in three other intracellular enzymes of collagen biosynthesis. Mixtures of serum samples from patients and healthy controls gave the expected GGT activity, indicating that the low values were not due to inhibitors. GGT deficiency was accompanied by decreased product formation in vivo, as shown by a markedly decreased urinary excretion of glucosylgalactosylhydroxylysine. Six of 12 affected members had definite GGT deficiency, and five had some evidence suggestive of this abnormality; 13 of 15 unaffected members had no such manifestations. No similar GGT deficiency was found in three other families with the same disease. We conclude that GGT deficiency may be etiologically related to this disease in some families, but that different defects must be the cause in other cases.

Topics: Adolescent; Adult; Child; Collagen; Epidermolysis Bullosa; Female; Fibroblasts; Galactosides; Glucosyltransferases; Glycosides; Humans; Hydroxylysine; Male; Middle Aged; Skin

Topics: Adolescent; Basement Membrane; Child; Child, Preschool; Connective Tissue; Female; Glycosaminoglycans; Humans; Hydroxylysine; Hydroxyproline; Infant; Kidney Glomerulus; Male; Nephritis, Hereditary

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.

Topics: Animals; Basement Membrane; Carbohydrates; Cysteine; Daunorubicin; Glomerulonephritis; Glycine; Hydroxylysine; Hydroxyproline; Kidney Glomerulus; Nephrosis; Rats

Topics: Alcohol Oxidoreductases; Amino Acids; Animals; Cattle; Chromatography, Gas; Chromatography, Ion Exchange; Cnidaria; Collagen; Cornea; Disaccharides; Echinodermata; Galactose; Glycopeptides; Glycosides; Hydroxylysine; Kinetics; Lysine; Magnetic Resonance Spectroscopy; Mass Spectrometry; Methods; Optical Rotation; Oxidation-Reduction; Porifera; Species Specificity; Stereoisomerism