The intracellular location of the glycosylation of hydroxylysine of collagen.

The subcellular location at which hydroxylysine residues of collagen are glycosylated was studied in chick embryo fibroblasts. Ribosomes were isolated from ¹⁴C-lysine pulse-labeled cells in tissue culture. Alkaline hydrolysis followed by amino acid analysis and scintillation counting of the effluent showed that glucosylgalactosyl hydroxylysine and galactosyl hydroxylysine as well as hydroxylysine and lysine were the major ¹⁴C-labeled components. Acid hydrolysis destroyed the glycoconjugates and yielded only free ¹⁴C-hydroxylysine and ¹⁴C-lysine. These data indicate that glycosylation of peptide-bound hydroxylysine is initiated while the polypeptide chain is still in the stages of assembly on the ribosome.     

Hydroxylysine glycosides in the collagen of normal and scarred rabbit corneas

Rabbit corneal scar collagen contains a lower content of glucosylgalactosylhydroxylysine than normal corneal collagen. Although the ratio of lysine to total hydroxylysine in normal and scar collagen is approximately the same, the relative proportion of glycosylated hydroxylysine to non-glycosylated hydroxylysine is different. These observations relate to the disposition of glycoside residues in scar and normal collagen and to the ultrastructural characteristics of the collagen fibrils.     

Effect of Hydroxylysine on the Biosynthesis of Lysine in Streptococcus faecalis

We were able to show that two lysine-independent mutants of Streptococcus faecalis ATCC 8043 contained the enzymes for the usual bacterial pathway for lysine biosynthesis. Because of this synthetic capacity, one mutant, the Lys(+)OHLys(s) strain, could not grow in the presence of hydroxylysine without a lysine supplement. Both lysine and hydroxylysine inhibited the first enzyme of the pathway, aspartokinase. Unlike the Escherichia coli enzyme, S. faecalis dihydrodipicolinic acid synthetase was not inhibited by either lysine or hydroxylysine. Both amino acids caused the repression of dihydrodipicolinic acid synthetase and diaminopimelic acid decarboxylase. Failure of Lys(+)OHLys(s) strain to grow in hydroxylysine-supplemented medium was caused by the mimicking of lysine control by hydroxylysine. Because hydroxylysine could not completely substitute for lysine and lysine could not be synthesized, the organism did not grow. We tested three lysine analogues and found that they prevented lysine-depletion lysis in the Lsy(-)OHLys(s) strain, as did hydroxylysine. Each analogue seemed to support cell wall mucopeptide synthesis, although ornithine did not. Preliminary data indicated that these analogues like hydroxylysine, have growth-inhibitory action on the Lys(+)OHLys(s) strain, but not the Lys(+)OHLys(r) strain. The nature of the specificity of the lysine-adding enzyme for cell wall mucopeptide synthesis is discussed.     

Recombinant human type II collagens with low and high levels of hydroxylysine and its glycosylated forms show marked differences in fibrillogenesis in vitro

Type II collagen is the main structural component of hyaline cartilages where it forms networks of thin fibrils that differ in morphology from the much thicker fibrils of type I collagen. We studied here in vitro the formation of fibrils of pepsin-treated recombinant human type II collagen produced in insect cells. Two kinds of type II collagen preparation were used: low hydroxylysine collagen having 2.0 hydroxylysine residues/1,000 amino acids, including 1.3 glycosylated hydroxylysines; and high hydroxylysine collagen having 19 hydroxylysines/1,000 amino acids, including 8.9 glycosylated hydroxylysines. A marked difference in fibril formation was found between these two kinds of collagen preparation, in that the maximal turbidity of the former was reached within 5 min under the standard assay conditions, whereas the absorbance of the latter increased until about 600 min. The critical concentration with the latter was about 10-fold, and the absorbance/microgram collagen incorporated into the fibrils was about one-sixth. The morphology of the fibrils was also different, in that the high hydroxylysine collagen formed thin fibrils with essentially no interfibril interaction or aggregation, whereas the low hydroxylysine collagen formed thick fibrils on a background of thin ones. The data thus indicate that regulation of the extents of lysine hydroxylation and hydroxylysine glycosylation may play a major role in the regulation of collagen fibril formation and the morphology of the fibrils.     

Effect of Hydroxylysine on the Biosynthesis of Lysine in Saccharomyces

Hydroxylysine acts as a growth inhibitor of Saccharomyces for a certain period of time. The inhibition is concentration-dependent and is reversed by a small amount of lysine in the medium. After the growth-inhibitory period, the wild-type cells are able to grow rapidly even in the presence of hydroxylysine. Both lysine auxotrophs and wild-type cells are unable to utilize hydroxylysine in place of lysine. Hydroxylysine, mimicking lysine, controls the biosynthesis of lysine and thereby limits the availability of biosynthetic lysine to the cells. Hydroxylysine affects the biosynthesis of lysine at a number of enzymatic steps. Accumulation of homocitric acid, the first intermediate of lysine biosynthesis, in the mutant strains 19B and A B9 is reduced significantly in the presence of hydroxylysine. Hydroxylysine, like lysine, exerts a significant inhibition in vitro on the homocitric acid-synthesizing activity. Enzymes following the alpha-aminoadipic acid step respond in a noncoordinate fashion to hydroxylysine. Level of the enzyme saccharopine reductase, but not of alpha-aminoadipic acid reductase or saccharopine dehydrogenase, is reduced significantly. These regulatory effects of hydroxylysine are similar to those observed for lysine.     

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.     

Replacement of Lysine by Hydroxylysine and Its Effects on Cell Lysis in Streptococcus faecalis.

Shockman, Gerald D. (Temple University, Philadelphia, Pa.), J. Stuart Thompson, and Margaret J. Conover. Replacement of lysine by hydroxylysine and its effects on cell lysis in Streptococcus faecalis. J. Bacteriol. 90:575-588. 1965.-Hydroxylysine was not significantly incorporated by Streptococcus faecalis ATCC 9790 or 8043 until exponential growth ceased as a result of lysine exhaustion. Uptake was then rapid and virtually complete within 1 hr. Lysine absence, rather than physiological age, seemed to be the governing factor. Hydroxylysine uptake rapidly reached a peak in the acid-soluble fraction, suggesting a precursor role for substances in this fraction. Substitution of hydroxylysine for lysine was much more efficient in mucopeptide synthesis than in protein synthesis. In wall medium, less than 1% of the incorporated hydroxylysine was found in the protein fraction. Addition of lysine to both growth and wall media inhibited both further hydroxylysine uptake and transfer of hydroxylysine from acid-soluble to mucopeptide or protein fractions. Hydroxylysine resulted in decreased penicillin susceptibility only after it was postexponentially incorporated. This effect was physiologically similar to that seen after threonine deprivation or chloramphenicol treatment. Hydroxylysine incorporation increased resistance to autolysis, but failed to decrease lysozyme susceptibility when measured after heat inactivation of autolysis. Electron microscopy of negatively stained cells showed increased thickness of cell walls containing hydroxylysine. Thus, most of the effects of replacement of lysine by hydroxylysine resemble those seen after deprivation of a nonwall amino acid (e.g., threonine or valine) or after chloramphenicol treatment. Each of these conditions results in inhibition of protein synthesis while permitting cell-wall synthesis to continue, resulting in autolysis-resistant, thick-walled cells.     

Studies on the rat glomerular basement membrane: Age-related changes in composition

To examine the effect of age on the glomerular basement membrane, compositional analyses were performed on membranes isolated in highly purified form from rats at different stages of their growth (35 to 200 days old). Substantial age-related changes were observed in the amino acid composition of the basement membranes. A significant correlation with age (P < 0.01) was evident in the contents of 3- and 4-hydroxyproline, threonine, serine, alanine, valine, half-cystine, hydroxylysine, and lysine. Of these amino acids, hydroxylysine and both isomers of hydroxyproline demonstrated a progressive increase with age, while the others were found to decline. The direct relationship of hydroxylysine content with age (P < 0.001) was associated with an inverse correlation of lysine with age (P < 0.001) so that the ratio of hydroxylysine to lysine increased in a highly significant manner from 0.92 at 35 days to 1.33 at 200 days. This elevation in the hydroxylysine content was accompanied by an augmentation in the number of saccharide units linked to it so that the percentage glycosylation of this amino acid was not significantly affected by age. The relative differences in the hydroxylysine and lysine levels between young and older rats were maintained in sodium dodecyl sulfateextracted membranes. These results suggest that the compositional changes observed during the aging process reflect an alteration in the subunit makeup of the basement membrane, possibly due to an increased synthesis or decreased degradation of the more collagen-like polypeptide components.     

Mimivirus collagen is modified by bifunctional lysyl hydroxylase and glycosyltransferase enzyme

Collagens, the most abundant proteins in animals, are modified by hydroxylation of proline and lysine residues and by glycosylation of hydroxylysine. Dedicated prolyl hydroxylase, lysyl hydroxylase, and collagen glycosyltransferase enzymes localized in the endoplasmic reticulum mediate these modifications prior to the formation of the collagen triple helix. Whereas collagen-like proteins have been described in some fungi, bacteria, and viruses, the post-translational machinery modifying collagens has never been described outside of animals. We demonstrate that the L230 open reading frame of the giant virus Acanthamoeba polyphaga mimivirus encodes an enzyme that has distinct lysyl hydroxylase and collagen glycosyltransferase domains. We show that mimivirus L230 is capable of hydroxylating lysine and glycosylating the resulting hydroxylysine residues in a native mimivirus collagen acceptor substrate. Whereas in animals from sponges to humans the transfer of galactose to hydroxylysine in collagen is conserved, the mimivirus L230 enzyme transfers glucose to hydroxylysine, thereby defining a novel type of collagen glycosylation in nature. The presence of hydroxylysine in mimivirus proteins was confirmed by amino acid analysis of mimivirus recovered from A. polyphaga cultures. This work shows for the first time that collagen post-translational modifications are not confined to the domains of life. The utilization of glucose instead of the galactose found throughout animals as well as a bifunctional enzyme rather than two separate enzymes may represent a parallel evolutionary track in collagen biology. These results suggest that giant viruses may have contributed to the evolution of collagen biology.     

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.     

Effect of amino acids on collagen biosynthesis

Summary The effect of various amino acids on collagen biosynthesis was studied in organ cultures of chicken embryo tibiae. Competitive interrelationship between selected amino acids influences independently the uptake of proline and lysine, precursors of hydroxyproline and hydroxylysine, respectively, which are the two amino acids characteristics of collagen. On the basis of these influences, the possibility of biosynthesis of anomalous collagens is stressed. Parallel studies of biosynthesis of hydroxyproline and hydroxylysine are necessary.     

Rapid assay for lysyl-protocollagen hydroxylase activity

A method for the assay of lysyl-protocollagen hydroxylase activity is described. This method depends upon the formation of tritiated water when lysine residues of 4,5-³H-lysyl-protocollagen are hydroxylated. The labeled protocollagen was prepared from carrageenan induced guinea pig granuloma tissue. There was a linear relationship between the amount of tritiated water and tritiated hydroxylysine formed in this assay. The assay is reproducible and more rapid than previously described assays for lysyl-protocollagen hydroxylase. Use of this method will facilitate further studies on collagen hydroxylysine formation.     

Hydroxylation of peptide-bound proline and lysine before and after chain completion of the polypeptide chains of procollagen

The synthesis of procollagen hydroxyproline and hydroxylysine was examined in matrix-free cells which were isolated from embryonic tendon by controlled enzymic digestion and then incubated in suspension. After the cells were labeled with [¹⁴C]proline for 2 min, or about one-third the synthesis time for a Pro-α chain, [¹⁴C]hydroxyproline was found in short peptides considerably smaller than the Pro-α chains of procollagen. The results, therefore, confirmed previous reports indicating that the hydroxylation of proline can begin on nascent chains. In similar experiments in which the cells were labeled with [¹⁴C]lysine, [¹⁴C]hydroxylysine was found in short, newly synthesized peptides, providing the first evidence that the hydroxylation of lysine can also begin on nascent peptides. However, further experiments demonstrated that the synthesis of hydroxyproline and hydroxylysine continues until some time after assembly of the polypeptide chains is completed.     

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.     

Post-translational modifications of collagen upon BMP-induced osteoblast differentiation

The pattern of collagen cross-linking is tissue specific primarily determined by the extent of hydroxylation and oxidation of specific lysine residues in the molecule. In this study, murine pre-myoblast cell line, C2C12 cells, were transdifferentiated into osteoblastic cells by bone morphogenetic protein (BMP)-2 treatment, and the gene expression of lysyl hydroxylases (LH1, 2a/b, and 3) and lysyl oxidase (LOX)/lysyl oxidase-like proteins (LOXL1-4), and the extent of hydroxylysine were analyzed. After 24 h of treatment, the expression of most isoforms were upregulated up to 96 h whereas LH2a and LOXL2 decreased with time. In the treated cells, both hydroxyproline and hydroxylysine were detected at day 7 and increased at day 14. The ratio of hydroxylysine to hydroxyproline was significantly increased at day 14. The results indicate that LHs and LOX/LOXLs are differentially responsive to BMP-induced osteoblast differentiation that may eventually lead to the specific collagen cross-linking pattern seen in bone.     

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.     

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.     

Characterization of N-glycosylated type I collagen in streptozotocin-induced diabetes.

The N epsilon-glycosylation of lysine and hydroxylysine residues in collagen from streptozotocin-induced-diabetic rats was confirmed and the stability of the complex shown to be due to an Amadori rearrangement. The studies also demonstrate the relative specificities of glucose, galactose and mannose in their reaction with collagen. The glycosylation of lysine in vitro occurs with glucose and galactose, but not with mannose, whereas only gucose reacts with hydroxylysine to any significant extent. Glycosylation of collagen occurs slowly during normal aging, but in contrast with reports suggesting accelerated aging of collagen in diabetic animals, we clearly demonstrated that the apparent increased stability is not due to an acceleration of the normal maturation process involving the reducible cross-links.     

Mature collagen crosslinks

During incubation with physiological buffers at 37°, as well as during $\text{in vivo}$ maturation, native collagen fibers display a progressive increase in tensile strength and insolubility. This is paralleled by a progressive loss of reducible, intermolecular crosslinks. The experiments described in this paper indicate that nucleophilic addition of lysine and/or hydroxylysine residues to the electrophilic double bond of the reducible crosslinks transforms them into more stable, non-reducible crosslinks. Indeed, modification of lysine/hydroxylysine residues completely blocks this transformation, while modification of his, arg, glu and asp is without effect. On the basis of these and other experiments, tentative structures are proposed for the stable crosslinks.     

Stimulation of hepatic biogenesis of sterols on administration of adenosine compounds.

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.