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PubMed1977

Estimation of hydroxylysine in urine and serum of patients with chronic uremia.

Free hydroxylysine and hydroxylysing glycosides were separated from urine and serum extracts on cation exchange resin and assayed spectrophotometrically. The method in conjunction with gel filtration in Bio-Gel P2 allowed to separate from urine also polypeptide hydroxylysine and hydroxylysine bound in small molecules of neutral or acidic character. Glycosylgalactosylhydroxylysineand galactosylhydroxylysine were separated by partition and/or ion exchange chromatography. Patients with chronic renal insufficiency had elevated serum levels and urinary excretion of hydroxylysine glycosides with increased excretion of hydroxylysine bound in polypeptides and in small molecules of neutral or acidic character. The excretion of free hydroxylysine was often within normal limits. When compared to values found in normal growing subjects and in adult patients with increased bone turnover and normal renin function the urinary excretion of hydroxylysine glycosides in chronic uremia was more markedly increased than excretion of hydroxyproline polypeptides and total hydroxyproline.

Bone Diseases↗
PubMed1989

Urinary hydroxyproline and hydroxylysine excretions in relation to hepatic hydroxyproline content in chronic liver disease.

In patients with or without various chronic liver diseases, the total urinary excretion of hydroxyproline and hydroxylysine and the hepatic content of hydroxyproline were examined. In 7 patients without liver disease, the urinary excretion of hydroxyproline and hydroxylysine were 10.3 +/- 1.5 and 1.31 +/- 0.21 mmol/mol creatinine, respectively, and the hepatic content of hydroxyproline was 4.9 +/- 0.6 mumol/g of wet liver. In 33 patients with liver disease, the urinary excretion of hydroxyproline and hydroxylysine and the hepatic content of hydroxyproline were increased in proportion to the severity of liver disease. The hepatic content of hydroxyproline showed a significant correlation with the urinary excretion of hydroxyproline and hydroxylysine (r = +0.406 and r = +0.531, respectively). These results suggest that the study of urinary hydroxyproline and hydroxylysine excretion may yield useful information on the metabolism of hepatic collagen in chronic liver disease. Moreover, urinary hydroxylysine excretion seemed to be a better index of hepatic collagen metabolism than urinary hydroxyproline excretion; perhaps urinary hydroxylysine excretion is not much affected by dietary collagen intake.

Adult↗
PubMed1965

INCORPORATION OF HYDROXYLYSINE INTO THE CELL WALL AND A CELL-WALL PRECURSOR IN STAPHYLOCOCCUS AUREUS.

Smith, W. Grady (University of Minnesota, St. Paul), Daniel P. Gilboe, and L. M. Henderson. Incorporation of hydroxylysine into the cell wall and a cell-wall precursor in Staphylococcus aureus. J. Bacteriol. 89:136-140. 1965.-Recent work has shown that hydroxylysine can substitute for lysine in cell-wall synthesis of Streptococcus faecalis, apparently becoming incorporated into cell-wall mucopeptide. This paper extends these observations to investigate the metabolism of hydroxylysine in Staphylococcus aureus, an organism from which sufficiently large quantities of cell-wall precursors. uridine diphosphate-N-acetylmuramyl peptides, could be obtained. Hydroxylysine has been shown to be incorporated into the cell-wall precursor uridine diphosphate-N-acetylmuramyl l-ala.d-glu. l-lys.d-ala.d-ala from S. aureus (Copenhagen) apparently in lieu of lysine. Hydroxylysine was also incorporated into the cell-wall mucopeptides of S. aureus in resting cultures. This incorporation was inhibited by penicillin or lysine, but not by chloramphenicol. Hydroxylysine had little effect on the incorporation of lysine into S. aureus. Hydroxylysine acted as a growth inhibitor in this organism; the inhibition was reversed by lysine.

Alanine↗
PubMed1979

Hydroxylysine-linked glycosides of human complement subcomponent C1q and various collagens.

1. Human C1q, a subcomponent of the first component of complement, contains 67 disaccharides (glucosylgalactose) and 2.4 monosaccharides (galactose) linked to hydroxylysine in one molecule. It was found that 82.6% of the hydroxylsine residues were glycosylated. The suggestion of the possible existence of glucosylgalactosylhydroxylysine reported previously [Yonemasu, Stroud, Niedermeir & Butler (1971) Biochem. Biophys. Res. Commun. 43, 1388--1394] was confirmed. 2. The hydroxylysine-glycosides are not detected in the C-terminal, non-collagen-like, globular regions, but only in the collagen-like regions in the subcomponent C1q molecule. 3. Alpha 1(I) and alpha 2 in pig skin, alpha 1(II) in bovine cartilage and alpha 1(III) in bovine skin collagens contain 2.0, 2.2, 13.2 and 2.0 residues of hydroxylysine-glycosides per molecule, respectively. The percentage of hydroxylysine residues glycosylated in each of these chains is relatively low (on average 38%). 4. Neither the high percentage of hydroxylysine residues glycosylated nor the high values for the ratios of disaccharides to monosaccharides in the subcomponent C1q resembles that in alpha 1(I), alpha 2, alpha 1(II) and alpha 1(III). 5. Similarities between the extent of glycosylation of hydroxylysine residues in collagen-like regions in the subcomponent C1q molecule and that of the collagenous constituents of human glomerular basement membranes, aortic intima, skin A- and B-chains and of bovine anterior lens capsule are discussed.

Animals↗
PubMed1976

Peptide-bound hydroxylysine and large polypeptides related to collagen synthesis.

About 10% of the urinary hydroxylysine is linked to peptides. There is no significant difference between the excretion of peptide-bound hydroxylysine and hydroxylysine measured in the non-dialysable fraction of the urine. Non-dialysable hydroxylysine is highly glycosylated and contains mainly glucosyl-galactosyl-hydroxylysine. It is concluded that the urinary peptide-bound hydroxylysine is present in the non-dialysable collagen polypeptides. Their unusual carbohydrate composition is discussed.

Collagen↗
PubMed1979

Collagen synthesis by cultured skin fibroblasts from siblings with hydroxylysine-deficient collagen.

It has been previously shown that dermis from subjects with hydroxylysine-deficient collagen contains approximately 5% of normal levels of hydroxylysine and sonicates of skin fibroblasts contain less than 15% of normal levels of collagen lysyl hydroxylase activity. However, cultures of dermal fibroblasts from two siblings with hydroxylysine-deficient collagen (Ehlers-Danlos Syndrome Type VI) compared to fibroblasts from normal subjects synthesize collagen containing approximately 50% of normal amounts of hydroxylysine. The lysyl hydroxylase deficient cultures synthesize both Type I and Type III collagen in the same proportion as control cultures. Both alpha 1(I) and alpha 2 chains are similarly reduced in hydroxylysine content. Collagen prolyl hydroxylation by normal collagen lysyl hydroxylation is the same with or without ascorbate supplementation. In mutant cells the rate of prolyl hydroxylation measured after release of inhibition by alpha, alpha'-dipyridyl is the same as in control cells. The rate of lysyl hydroxylation is reduced in mutant cells but only to approximately 50% of normal.

Ascorbic Acid↗
PubMed1987

Subfractionation of the dansylated derivatives of glucosyl galactosyl hydroxylysine by liquid chromatography and its application to a specific alpha-1,2-glucosidase assay.

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.

Animals↗
PubMed1993

High-performance liquid chromatographic method for measuring hydroxylysine glycosides and their ratio in urine as a possible marker of human bone collagen breakdown.

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.

Adult↗
PubMed1998

Expression and characterization of recombinant human type II collagens with low and high contents of hydroxylysine and its glycosylated forms.

Insect cells coinfected with two baculoviruses, one coding for the pro alpha chains of human type II procollagen and the other for both the alpha and beta subunits of human prolyl 4-hydroxylase, produced the cartilage-specific type II collagen with a stable triple helix. The highest expression levels, up to 50 mg/l of type II collagen, were obtained in suspension culture using a modified construct in which sequences coding for the signal peptide and N propeptide of type II procollagen had been replaced by those for type III procollagen. The type III N propeptide artificially generated into type II procollagen was found to be cleaved at a much higher rate than the wild-type type II N propeptide, probably because the former interacted poorly with the triple-helical domain of type II procollagen. The amino acid composition of the recombinant type II collagen was very similar to that of the non-recombinant protein, but the hydroxylysine content was only 17% and that of glycosylated hydroxylysines was equally low. The hydroxylysine content was increased to the level found in the non-recombinant collagen by using an additional baculovirus coding for lysyl hydroxylase, and a substantial increase was also found in the glycosylated hydroxylysine content. No difference in thermal stability was found between the low- and high-hydroxylysine collagens.

Animals↗
PubMed1977

Determination of hydroxylysine in urine.

A new method for colorimetric determination of urinary hydroxylysine is described. Approximately one-hundredth of human urine collected for 24 hr was diluted to 25 ml and titrated to pH 2.0 with 2 hcl, and then subjected to column chromatography on Dowex 50 X 4 (H+ form). Amino acids were eluted from the column with 1.5 N NH4OH. Hydroxylysine in the elute with 1.5 N NH4OH was separated from the other amino acids, especially from serine and threonine, by preparative paper chromatography. The paper corresponding to hydroxylysine was cut and eluted with water. An aliquot of the eluate with water was oxidized by sodium metaperiodate, and formaldehyde liberated from hydroxylysine was assayed by chromotropic acid reagents, after removing periodate and iodate with Dowex 1 X 8 (formate form). Excretion rate of hydroxylysine in urine of adults was shown to be approximately 110 mumoles per day.

Chromatography↗
PubMed1982

Glycosylation of human glomerular basement membrane collagen: increased content of hexose in ketoamine linkage and unaltered hydroxylysine-O-glycosides in patients with diabetes.

To study the glycosylation of glomerular basement membrane collagen (GBMC) in diabetes, kidneys were obtained at autopsy from 5 patients with insulin-requiring diabetes of long duration and diabetic complications, and from 5 control subjects. Glomeruli were prepared by sieving and collagen was isolated by limited pepsin proteolysis followed by salt precipitations. Amino acid analyses of the collagen preparations, after acid hydrolysis, indicated a composition consistent with that of type IV collagen. No differences in the relative contents of various amino acids, and in particular, 3-hydroxyproline, 4-hydroxyproline and hydroxylysine, were noted between diabetic and control samples. Non-enzymatic glucosylation was assessed by measuring hexose in ketoamine linkage with thiobarbituric acid after conversion to 5-hydroxymethylfurfural. In 4 of the 5 patients studied, glucosylation values exceeded the mean +2 S.D. of the controls; in the fifth subject glucosylation was in the high normal range. No correlation between the severity of diabetes and hexose content of GBMC was noted, however. In further studies, enzymatic glycosylation of GBMC was assayed after alkaline hydrolysis by separation of glucosylgalactosyl-O-hydroxylysine, galactosyl-O-hydroxylysine, and unsubstituted hydroxylysine in an amino acid analyzer. No differences in the relative contents of hydroxylysine-O-glycosides were evident between diabetic and control GBMC. The results suggest that non-enzymatic glucosylation, but not glycosylation catalyzed by collagen glucosyl and galactosyl transferases, is increased in diabetes. The increased carbohydrate content of collagen may lead to decreased turnover and/or excessive accumulations of basement membrane collagen thus contributing to the vascular complications of diabetes.

Adult↗
PubMed1997

Ovariectomy in the rat induces a rapid increase in the urinary excretion of hydroxylysine glycosides and non-reducible crosslink residues.

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.

Amino Acids↗
PubMed1980

Regulation of the glycosylations of collagen hydroxylysine in chick embryo tendon and cartilage cells.

The regulation of the glycosylations of hydroxylysine was studied in isolated chick-embryo cells by labelling with a [14C]lysine pulse. The course of the procollagen lysyl modifications was compared in tendon and cartilage cells, and the effect on the gycosylations of the degree of lysyl hydroxylation and the concentration of Mn2+ and Fe2+ were also studied, in tendon cells. Procollagen triple helix formation was inhibited in most experiments in order to eliminate the effect of this process on the continuation of the reactions. Both in the tendon and cartilage cells the intracellular lysyl modifications proceeded in a biphasic fashion. After an initial sharp linear increase, the reactions did not cease but were protracted at a slower but constant rate. Lysyl hydroxylation was followed by rapid galactosylation in both cell types and this was followed almost immediately by rapid glucosylation, suggesting a close association of the corresponding enzymes. The data further suggest that other factors must also exist, in addition to the differences in the timing of triple helix formation and the actual hydroxylysine content, which are responsible for the different amounts of galactose in the collagens synthesized by these cell types. The amount of glucosylgalactosylhydroxylysine nevertheless seemed to be determined by the available acceptor sites, i.e., the amount of galactosylhydroxylysine. In further experiments with tendon cells the oxygen participating in lysyl hydroxylation was displaced by nitrogen at various points in time. When the degree of lysyl hydroxylation was reduced to less than one-third of the original, the total amounts of glycosylated residues decreased correspondingly, but their proportion relative to total hydroxylysine remained unchanged. Extra Mn2+ increased the proportion of galactosylated hydroxylysine, suggesting that the activity of hydrosylysyl galactosyltransferase is not saturating in respect of the catalyzed reaction. Experiments on the addition of Fe2+ or its chelation by alpha, alpha'-dipyridyl gave indications that the presence of this co-factor is not required for either glycosylation reaction in isolated tendon cells.

Animals↗
PubMed1997

Pre-column derivatisation method for the measurement of glycosylated hydroxylysines of collagenous proteins.

Measurement of the glycosylated hydroxylysines galactosyl- and glucosylgalactosylhydroxylysine (GH and GGH) in combination with other amino acids has been based on ion-exchange chromatography followed by reaction with ninhydrin. Here, a rapid and sensitive high-performance liquid chromatographic method with fluorimetric detection has been developed and employed to determine the glycosylated hydroxylysine residues in alkaline collagen hydrolysates. After hydrolysis, amino acids were derivatised with 9-fluorenylmethyl chloroformate and separated on a Micropak ODS-80TM reversed-phase column (150x4.6 mm). With a multistep gradient system all amino acids were separated in less than 30 min, including the collagen-specific hydroxylysine, hydroxyproline and the glycosylated hydroxylysines. The method was used to evaluate the glycosylation levels of human articular cartilage derived from femoral head, femoral condyle, tibial plateau and ankle. GGH was highest in cartilage from femoral head and ankle; GH showed no differences between the different sources of cartilage.

Animals↗
PubMed1977

Further studies on the effect of the collagen triple-helix formation on the hydroxylation of lysine and the glycosylations of hydroxylysine in chick-embryo tendon and cartilage cells.

The hydroxylation of lysine and glycosylations of hydroxylysine were studied in isolated chick-embryo tendon and cartilage cells under conditions in which collagen triple-helix formation was either inhibited or accelerated. The former situation was obtained by incubating the tendon cells with 0.6mm-dithiothreitol, thus decreasing their proline hydroxylase activity by about 99%. After labelling with [(14)C]proline, the formation of hydroxy[(14)C]proline was found to have declined by about 95%. Since the hydroxylation of a relatively large number of proline residues is required for triple-helix formation at 37 degrees C, the pro-alpha-chains synthesized under these conditions apparently cannot form triple-helical molecules. Labelling experiments with [(14)C]lysine indicated that the degree of hydroxylation of the lysine residues in the collagen synthesized was slightly increased and the degree of the glycosylations of the hydroxylysine residues more than doubled, the largest increase being in the content of glucosylgalactosylhydroxylysine. Recovery of chick-embryo cartilage cells from temporary anoxia was used to obtain accelerated triple-helix formation. A marked decrease was found in the extent of hydroxylation of the lysine residues in the collagen synthesized under these conditions, and an even larger decrease occurred in the glycosylations of the hydroxylysine residues. The results support the previous suggestion that the triple-helix formation of the pro-alpha-chains prevents further hydroxylation of lysine residues and glycosylations of hydroxylysine residues during collagen biosynthesis.

Animals↗
PubMed1982

Collagen structural microheterogeneity and a possible role for glycosylated hydroxylysine in type I collagen.

A three-chained peptide from type I collagen, crosslinked by hydroxyaldolhistidine, has been isolated from a tryptic digest of 5 M guanidine.HCl-insoluble bovine skin collagen (a small but as yet unknown percentage of the total collagen in whole skin). OsO(4)/NaIO(4) specifically cleaved the crosslink at its double bond into a two-chained crosslink peptide and a single peptide. The sequence of the two-chained peptide containing the bifunctional crosslink was determined after amino acid analysis of the separated peptides. The crosslink consists of an aldehyde derived from hydroxylysine-87 in the aldehyde-containing cyanogen bromide fragment alpha1CB5(ald) and an aldehyde derived from the lysine in the COOH-terminal nonhelical region of the alpha1CB6(ald) fragment. The alpha1CB6(ald) portion of the peptide exhibited structural microheterogeneity, containing the inverted sequence Ala-Lys-His instead of the normal sequence Lys-Ala-His. This indicates that another structural gene exists for alpha1(I) chain. The original three-chained peptide did not contain any glycosylated hydroxylysine or glycosylated hydroxyaldolhistidine. The lack of glycosylation of hydroxylysine-87 in alpha1CB5, which is usually glycosylated, allowed formation of the aldehyde, and this, coupled with the sequence inversion, may have allowed formation of the nonreducible crosslink hydroxyaldolhistidine. We suggest that the role of glycosylation, a posttranslational modification, of specific hydroxylysine residues is to prevent their oxidative deamination to aldehydes, thereby precluding formation of complex stable crosslinks. Complex crosslinks would decrease the rate of collagen turnover. The decrease, with time, would increase the population of stable crosslinked collagen molecules, which would eventually accumulate with age.

Amino Acid Sequence↗
PubMed1985

Phosphorylation of hydroxylysine residues in collagen synthesized by cultured aortic smooth muscle cells.

O5-Phosphohydroxylysine was chemically synthesized and techniques were established for its identification by combined use of cation-exchange chromatography, thin-layer electrophoresis at pH 1.9 and 3.5, and thin-layer chromatography. Clean separation of phosphohydroxylysine from the other phospho amino acids, phosphoethanolamine, and phosphocholine was achieved. Conditions were also determined to permit hydrolysis of proteins in 2 M HCl without loss of the phosphono group of phosphohydroxylysine residues. Experiments were then performed showing that 32P was incorporated into the hydroxylysine residues of cell-associated collagens when cultured calf aorta medial smooth muscle cells were incubated with [32P]orthophosphate. In other experiments, the cells incorporated [3H]lysine into hydroxylysine residues of cell-associated collagen and then 32P into phosphohydroxylysine residues. The doubly labeled phosphohydroxylysine subsequently isolated showed nearly 1:1 stoichiometry with respect to incorporation of precursor lysine and phosphorus. Finally, in preliminary experiments done with a cell-free extract of the smooth muscle cells, 32P was transferred from [gamma-32P]ATP to hydroxylysine residues in several kinds of collagenous substrates. Thus, this work shows that smooth muscle cells have the capacity to phosphorylate hydroxylysine residues in their cell-associated collagens and provides preliminary evidence that a protein kinase is involved.

Acids↗
PubMed1976

Comparative study of carbohydrate-protein complexes. II. Determination of hydroxylysine and its glycosides in human skin and scar collagens by an improved method.

A modification of the existing methods for measuring hydroxylysine, galactosylhydroxylysine, and glucosylgalactosylhydroxylysine is described. The method is based on analysis with an automated amino acid analyzer using a conventional separation system for basic amino acids. The prior removal of acidic and neutral amino acids was necessary. This was achieved by passing an alkaline hydrolysate of collagen through a column of Amberlite CG-120, Type II (H+) and washing the column with 8% aqueous pyridine. A basic fraction containing the hydroxylysine compounds was then recovered from the column by elution with 3 M NH4OH. Model experiments showed that hydroxylysine and its glycosides could be analyzed with an hour and that recoveries exceeded 90%. This method was applied to human tissues to investigate whether the dermal scar is different in collagen composition from normal skin. With the limited number of samples analyzed, the data suggested that long-standing scar tissues reverted to a composition similar to that of normal skin. The composition of hydroxylysine-linked carbohydrate units is also discussed on the basis of the age-related change.

Adolescent↗