Lysyl hydroxylase 3-mediated glucosylation in type I collagen: molecular loci and biological significance

Recently, by employing the short hairpin RNA technology, we have generated MC3T3-E1 (MC)-derived clones stably suppressing lysyl hydroxylase 3 (LH3) (short hairpin (Sh) clones) and demonstrated the LH3 function as glucosyltransferase in type I collagen (Sricholpech, M., Perdivara, I., Nagaoka, H., Yokoyama, M., Tomer, K. B., and Yamauchi, M. (2011) Lysyl hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture. J. Biol. Chem. 286, 8846-8856). To further elucidate the biological significance of this modification, we characterized and compared type I collagen phenotypes produced by Sh clones and two control groups, MC and those transfected with empty vector. Mass spectrometric analysis identified five glycosylation sites in type I collagen (i.e. α1,2-87, α1,2-174, and α2-219. Of these, the predominant glycosylation site was α1-87, one of the major helical cross-linking sites. In Sh collagen, the abundance of glucosylgalactosylhydroxylysine was significantly decreased at all of the five sites with a concomitant increase in galactosylhydroxylysine at four of these sites. The collagen cross-links were significantly diminished in Sh clones, and, for the major cross-link, dihydroxylysinonorleucine (DHLNL), glucosylgalactosyl-DHLNL was diminished with a concomitant increase in galactosyl-DHLNL. When subjected to in vitro incubation, in Sh clones, the rate of decrease in DHLNL was lower, whereas the rate of increase in its maturational cross-link, pyridinoline, was comparable with controls. Furthermore, in Sh clones, the mean diameters of collagen fibrils were significantly larger, and the onset of mineralized nodule formation was delayed when compared with those of controls. These results indicate that the LH3-mediated glucosylation occurs at the specific molecular loci in the type I collagen molecule and plays critical roles in controlling collagen cross-linking, fibrillogenesis, and mineralization.     

Posttranslational Modifications in Type I Collagen from Different Tissues Extracted from Wild Type and Prolyl 3-Hydroxylase 1 Null Mice

Type I collagen extracted from tendon, skin, and bone of wild type and prolyl 3-hydroxylase 1 (P3H1) null mice shows distinct patterns of 3-hydroxylation and glycosylation of hydroxylysine residues. The A1 site (Pro-986) in the α1-chain of type I collagen is almost completely 3-hydroxylated in every tissue of the wild type mice. In contrast, no 3-hydroxylation of this proline residue was found in P3H1 null mice. Partial 3-hydroxylation of the A3 site (Pro-707) was present in tendon and bone, but absent in skin in both α-chains of the wild type animals. Type I collagen extracted from bone of P3H1 null mice shows a large reduction in 3-hydroxylation of the A3 site in both α-chains, whereas type I collagen extracted from tendon of P3H1 null mice shows little difference as compared with wild type. These results demonstrate that the A1 site in type I collagen is exclusively 3-hydroxylated by P3H1, and presumably, this enzyme is required for the 3-hydroxylation of the A3 site of both α-chains in bone but not in tendon. The increase in glycosylation of hydroxylysine in P3H1 null mice in bone was found to be due to an increased occupancy of normally glycosylated sites. Despite the severe disorganization of collagen fibrils in adult tissues, the D-period of the fibrils is unchanged. Tendon fibrils of newborn P3H1 null mice are well organized with only a slight increase in diameter. The absence of 3-hydroxyproline and/or the increased glycosylation of hydroxylysine in type I collagen disturbs the lateral growth of the fibrils.     

Maturation of Collagen Ketoimine Cross-links by an Alternative Mechanism to Pyridinoline Formation in Cartilage

The tensile strength of fibrillar collagens depends on stable intermolecular cross-links formed through the lysyl oxidase mechanism. Such cross-links based on hydroxylysine aldehydes are particularly important in cartilage, bone, and other skeletal tissues. In adult cartilages, the mature cross-linking structures are trivalent pyridinolines, which form spontaneously from the initial divalent ketoimines. We examined whether this was the complete story or whether other ketoimine maturation products also form, as the latter are known to disappear almost completely from mature tissues. Denatured, insoluble, bovine articular cartilage collagen was digested with trypsin, and cross-linked peptides were isolated by copper chelation chromatography, which selects for their histidine-containing sequence motifs. The results showed that in addition to the naturally fluorescent pyridinoline peptides, a second set of cross-linked peptides was recoverable at a high yield from mature articular cartilage. Sequencing and mass spectral analysis identified their origin from the same molecular sites as the initial ketoimine cross-links, but the latter peptides did not fluoresce and were nonreducible with NaBH₄. On the basis of their mass spectra, they were identical to their precursor ketoimine cross-linked peptides, but the cross-linking residue had an M+188 adduct. Considering the properties of an analogous adduct of identical added mass on a glycated lysine-containing peptide from type II collagen, we predicted that similar dihydroxyimidazolidine structures would form from their ketoimine groups by spontaneous oxidation and free arginine addition. We proposed the trivial name arginoline for the ketoimine cross-link derivative. Mature bovine articular cartilage contains about equimolar amounts of arginoline and hydroxylysyl pyridinoline based on peptide yields.     

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.     

The covalent structure of cartilage collagen. Amino acid sequence of residues 552–661 of bovine α1(II) chains

The covalent structure of the first 111 residues from the N-terminus of peptide α1(II)-CB10 from bovine nasal-cartilage collagen is presented. This region comprises residues 552–661 of the α1(II) chain. The sequence was determined by automated Edman degradation of peptide α1(II)-CB10 and of peptides produced by cleavage with trypsin and hydroxylamine. Comparison of this region of the α1(II) chain with the homologous segment of the α1(I) chain indicated a homology level of 85%, slightly higher than that of 81% reported for the N-terminal region of the α1(II) chain (Butler, Miller & Finch (1976) Biochemistry 15, 3000–3006). The occurrence of two residues of glycosylated hydroxylysine was established at positions 564 and 603, the first present exclusively as galactosylhydroxylysine and the latter as a mixture of galactosylhydroxylysine and glucosylgalactosylhydroxylysine. Also, two residues at positions 648 and 657 were tentatively identified as glycosylated hydroxylysines. The amino acid sequences adjacent to the hydroxylysine residues so far identified in the α1(II) chain were compared with the homologous regions of the α1(I) and α2 chains, but no obvious prerequisite for hydroxylation could be seen. From comparison with the homologous sequence of the α1(I) chain, it appears that the α1(II)-chain sequence presented here contains three more amino acids than that reported for the α1(I) chain. This triplet would be interposed between residues 63 and 64 of the reported sequence of peptide α1(I)-CB7 from calf skin collagen. Data on the purification of the subpeptides and their amino acid compositions have been deposited as Supplementary Publication SUP 50087 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Fibroblast behavior on gels of type I, III, and IV human placental collagens

Various collagens were extracted and purified from human placenta after partial pepsin digestion. We prepared type III + I (57:43), enriched type I, type III, and type IV collagens on an industrial level, and studied their biological properties with MRC5 fibroblast cells. Using the process of contraction of a hydrated collagen lattice described by Bell, we found tha the contraction rate was dependent on collagen type composition. The contraction was faster and more pronounced with pepsinized type I collagen than with pepsinized type III + I (57:43) collagen; the lowest rate was obtained with the pepsinized type III collagen. Using a new technique of collagen cross-linking, a gel was made with type IV collagen. This cross-linking procedure, based on partial oxidation of sugar residues and hydroxylysine by periodic acid, followed by neutralization, resulted in an increased number of natural cross-link bridges between oxidized and nonoxidized collagen molecules, without internal toxic residues. The fibroblasts were unable to contract type IV/IVox collagen gels. The type IV/IVox collagen gel was transparent and its amorphous ultrastructure lacked any visible striated fibrils. Fibroblast cells exhibited atypical behavior in these type IV/IVox collagen gels as evidenced by optical and electron microscopy. The penetration of fibroblasts could be measured. Fibroblasts penetrated faster in type IV/IVox collagen gels than in untreated type III + I collagen gels. The lowest rate of penetration was obtained with cross-linked type III + I gels. Fibroblast proliferation was similar on untreated or cross-linked type III + I collagen gels and slightly increased on type IV/IVox collagen gels, suggesting that this cross-linking procedure was not toxic.     

Assembly of collagen types II, IX and XI into nascent hetero-fibrils by a rat chondrocyte cell line.

The cell line, RCS-LTC (derived from the Swarm rat chondrosarcoma), deposits a copious extracellular matrix in which the collagen component is primarily a polymer of partially processed type II N-procollagen molecules. Transmission electron microscopy of the matrix shows no obvious fibrils, only a mass of thin unbanded filaments. We have used this cell system to show that the type II N-procollagen polymer nevertheless is stabilized by pyridinoline cross-links at molecular sites (mediated by N- and C-telopeptide domains) found in collagen II fibrils processed normally. Retention of the N-propeptide therefore does not appear to interfere with the interactions needed to form cross-links and mature them into trivalent pyridinoline residues. In addition, using antibodies that recognize specific cross-linking domains, it was shown that types IX and XI collagens, also abundantly deposited into the matrix by this cell line, become covalently cross-linked to the type II N-procollagen. The results indicate that the assembly and intertype cross-linking of the cartilage type II collagen heteropolymer is an integral, early process in fibril assembly and can occur efficiently prior to the removal of the collagen II N-propeptides.     

Glycosylation Modulates Melanoma Cell α2β1 and α3β1 Integrin Interactions with Type IV Collagen

Although type IV collagen is heavily glycosylated, the influence of this post-translational modification on integrin binding has not been investigated. In the present study, galactosylated and nongalactosylated triple-helical peptides have been constructed containing the α1(IV)382–393 and α1(IV)531–543 sequences, which are binding sites for the α2β1 and α3β1 integrins, respectively. All peptides had triple-helical stabilities of 37 °C or greater. The galactosylation of Hyl³⁹³ in α1(IV)382–393 and Hyl⁵⁴⁰ and Hyl⁵⁴³ in α1(IV)531–543 had a dose-dependent influence on melanoma cell adhesion that was much more pronounced in the case of α3β1 integrin binding. Molecular modeling indicated that galactosylation occurred on the periphery of α2β1 integrin interaction with α1(IV)382–393 but right in the middle of α3β1 integrin interaction with α1(IV)531–543. The possibility of extracellular deglycosylation of type IV collagen was investigated, but no β-galactosidase-like activity capable of collagen modification was found. Thus, glycosylation of collagen can modulate integrin binding, and levels of glycosylation could be altered by reduction in expression of glycosylation enzymes but most likely not by extracellular deglycosylation activity.     

Minoxidil exerts different inhibitory effects on gene expression of lysyl hydroxylase 1, 2, and 3: implications for collagen cross-linking and treatment of fibrosis.

Collagen deposits in fibrotic lesions often display elevated levels of hydroxyallysine (pyridinoline) cross-links. The relation between the occurrence of pyridinoline cross-links and the irreversibility of fibrosis suggests that these cross-links contribute to the aberrant accumulation of collagen. Based on its inhibitory effect on lysyl hydroxylase activity minoxidil has been postulated to possess anti-fibrotic properties by limiting the hydroxylysine supply for hydroxyallysine cross-linking. However, to interfere with hydroxyallysine cross-linking specifically lysyl hydroxylation of the collagen telopeptide should be inhibited, a reaction predominantly catalysed by lysyl hydroxylase (LH) 2b. In this study, we demonstrate that minoxidil treatment of cultured fibroblasts reduces LH1>LH2b>LH3 mRNA levels dose-and time-dependently, but has essentially no effect on the total number of pyridinoline cross-links in the collagen matrix. Still the collagen produced in the presence of minoxidil displays some remarkable features: hydroxylation of triple helical lysine residues is reduced to 50% and lysylpyridinoline cross-linking is increased at the expense of hydroxylysylpyridinoline cross-linking. These observations can be explained by our finding that LH1 mRNA levels are the most sensitive to minoxidil treatment, corroborating that LH1 has a preference for triple helical lysine residues as substrate. In addition, the non-proportional increase in cross-links (20-fold) with respect to the decrease in lysyl hydroxylation state of the triple helix (2-fold) even suggests that LH1 preferentially hydroxylates triple helical lysine residues at the cross-link positions. We conclude that minoxidil is unlikely to serve as an anti-fibroticum, but confers features to the collagen matrix, which provide insight into the substrate specificity of LH1.     

Identification of cyanogen bromide peptides involved in intermolecular cross-linking of bovine type III collagen.

Cyanogn bromide peptides derived from bovine type III collagen and containing reducible cross-links were isolated and identified. Two peptides, alpha 1 (III)CB7 and alpha 1 (III)CB9B, from within the helical portion of the molecule were shown to contain the 'amino donor' residues cross-linked to non-helical 'aldehyde donor' residues in the formation of cross-links. This information, in conjunction with previously published data for the order of the cyanogen bromide peptides [Fietzek, Allman, Rauterberg & Wachter (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 84-86], suggests that in type III collagen intermolecular cross-links are located in the end-overlap regions, so as to stabilize a quarter-stagger arrangement of molecules within the fibre in a similar manner to that proposed for type I and type II collagens.     

Enzymatic and nonenzymatic cross-linking of collagen and elastin.

Knowledge regarding the steps and mechanisms related to the intra- and interchain cross-linking of collagen and elastin has evolved steadily during the past 30 years. Recently, effort has been directed at identifying the location and types of cross-links that are found in collagen and elastin. There are two major groups of cross-links: those initiated by the enzyme lysyl oxidase and those derived from nonenzymatically glycated lysine and hydroxylysine residues. The formation of enzymatic cross-links depends on specific enzymes, amino acid sequences, and quaternary structural arrangements. The cross-links that are derived nonenzymatically occur more adventitiously and are important to pathobiological processes. Considerable progress has been made in elucidating the pathways of synthesis for several of the enzymatically mediated cross-links, as well as possible mechanisms regulating the specificity of cross-linking. Although less is known about the chemistry of cross-links arising from nonenzymatically glycated residues, recent progress has also been made in understanding possible biosynthetic pathways and control mechanisms. This review focuses on such progress and hopes to underscore the biological importance of collagen and elastin cross-linking.     

The effects of different crossing-linking conditions of genipin on type I collagen scaffolds: an in vitro evaluation

The purpose of this paper is to analyze the properties of fabricating rat tail type I collagen scaffolds cross-linked with genipin under different conditions. The porous genipin cross-linked scaffolds are obtained through a two step freeze-drying process. To find out the optimal cross-link condition, we used different genipin concentrations and various cross-linked temperatures to prepare the scaffolds in this study. The morphologies of the scaffolds were characterized by scanning electron microscope, and the mechanical properties of the scaffolds were evaluated under dynamic compression. Additionally, the cross-linking degree was assessed by ninhydrin assay. To investigate the swelling ratio and the in vitro degradation of the collagen scaffold, the tests were also carried out by immersion of the scaffolds in a PBS solution or digestion in a type I collagenase respectively. The morphologies of the non-cross-linked scaffolds presented a lattice-like structure while the cross-linked ones displayed a sheet-like framework. The morphology of the genipin cross-linked scaffolds could be significantly changed by either increasing genipin concentration or the temperature. The swelling ratio of each cross-linked scaffold was much lower than that of the control (non-cross-linked).The ninhydrin assay demonstrated that the higher temperature and genipin concentration could obviously increase the cross-linking efficiency. The in vitro degradation studies indicated that genipin cross-linking can effectively elevate the biostability of the scaffolds. The biocompatibility and cytotoxicity of the scaffolds was evaluated by culturing rat chondrocytes on the scaffold in vitro and by MTT. The results of MTT and the fact that the chondrocytes adhered well to the scaffolds demonstrated that genipin cross-linked scaffolds possessed an excellent biocompatibility and low cytotoxicity. Based on these results, 0.3 % genipin concentrations and 37 °C cross-linked temperatures are recommended.     

Intermolecular cross-linking and stereospecific molecular packing in type I collagen fibrils of the periodontal ligament

A trypsin digest of denatured NaB3H4-reduced native bovine periodontal ligament was prepared and fractionated by gel filtration and cellulose ion-exchange column chromatography. Prior to trypsin digestion, a complete acid hydrolysate was subjected to analyses for nonreducible stable and reducible intermolecular cross-links. Minute amounts of the former and significant amounts of the reduced cross-links dihydroxylysinonorleucine (1.1 mol/mol of collagen), hydroxylysinonorleucine (0.9 mol/mol of collagen), and histidinohydroxymerodesmosine (0.6 mol/mol of collagen) were found. The covalent intermolecular cross-linked two-chained peptides that were isolated were subjected to amino acid and sequence analyses. The structures for the different two-chained linked peptides were alpha 1CB4-5(76-90)[Hyl-87] X alpha 1CB6-(993-22c)[Lysald-16c], alpha 1CB4-5(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Hylald-16c], alpha 2CB4(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Lysald-16c], and alpha 2CB4(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Hylald-16c]. The cross-link in each peptide was glycosylated. This is the first characterization by sequence analysis of a cross-link involving Hyl-87 in an alpha 2 chain in collagen. A stoichiometric conversion of residue 16c aldehyde to an intermolecular cross-link in each of the COOH-terminal nonhelical peptide regions of both alpha 1 chains in a molecule of type I collagen was found. The ratio of alpha 1 to alpha 2 intermolecularly cross-linked chains involved was 3.3:1, indicating a stereospecific three-dimensional molecular packing of type I collagen molecules in bovine periodontal ligament.     

Effects of ultraviolet-A and riboflavin on the interaction of collagen and proteoglycans during corneal cross-linking

Corneal cross-linking using riboflavin and ultraviolet-A (RFUVA) is a clinical treatment targeting the stroma in progressive keratoconus. The stroma contains keratocan, lumican, mimecan, and decorin, core proteins of major proteoglycans (PGs) that bind collagen fibrils, playing important roles in stromal transparency. Here, a model reaction system using purified, non-glycosylated PG core proteins in solution in vitro has been compared with reactions inside an intact cornea, ex vivo, revealing effects of RFUVA on interactions between PGs and collagen cross-linking. Irradiation with UVA and riboflavin cross-links collagen α and β chains into larger polymers. In addition, RFUVA cross-links PG core proteins, forming higher molecular weight polymers. When collagen type I is mixed with individual purified, non-glycosylated PG core proteins in solution in vitro and subjected to RFUVA, both keratocan and lumican strongly inhibit collagen cross-linking. However, mimecan and decorin do not inhibit but instead form cross-links with collagen, forming new high molecular weight polymers. In contrast, corneal glycosaminoglycans, keratan sulfate and chondroitin sulfate, in isolation from their core proteins, are not cross-linked by RFUVA and do not form cross-links with collagen. Significantly, when RFUVA is conducted on intact corneas ex vivo, both keratocan and lumican, in their natively glycosylated form, do form cross-links with collagen. Thus, RFUVA causes cross-linking of collagen molecules among themselves and PG core proteins among themselves, together with limited linkages between collagen and keratocan, lumican, mimecan, and decorin. RFUVA as a diagnostic tool reveals that keratocan and lumican core proteins interact with collagen very differently than do mimecan and decorin.     

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

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

Stable, nonreducible cross-links of mature collagen.

During in vivo maturation, and also during in vitro incubation with physiological buffers, native collagen fibers display a progressive increase in tensile strength and insolubility. Paralleling these physiologically important changes is a progressive loss of the reducible cross-links which initially join the triple-chained subunits of collagen fibers. Although there is evidence suggesting that the reducible cross-links are gradually transformed into more stable, nonreducible cross-links during maturation, the nature of the transformation process and the structure of the stable "mature" cross-links has remained a mystery. In order to test the possibility that cross-link transformation involves addition of a nucleophilic amino acid residue to the reducible cross-links, histidine, arginine, glutamate, aspartate, lysine, and hydroxylysine residues were chemically modified, and the effect of each modification procedure on the in vitro transformation of reducible cross-links was ascertained. The results of these experiments indicated that destruction of histidine, arginine, glutamate, and aspartate residues has no measurable effect on the rate and extent of reducible cross-link transformation in hard tissue collagens. In contrast, modification of lysine and hydrocylysine residues with a wide variety of specific reagents completely blocks the transformation of reducible cross-links. Removal of the reversible blocking groups from lysine and hydroxlylysine residues then allows the transformation to proceed normally. These results indicate that collagen maturation involves nucleophilic addition of lysine and/or hydroxylysine residues to the electrophilic double bond of the reducible cross-links, yielding derivatives which are not only more stable but also capable of cross-linking more collagen molecules than their reducible precursors.     

Role of decorin on in vitro fibrillogenesis of type I collagen

Tendon and corneal decorins are differently iduronated dermatan sulphate/proteoglycan (DS/PG) and the biochemical parameter that differentiates type I collagens is the hydroxylysine glycoside content. We have examined the effect of tendon and corneal decorins on the individual phases (tlag, dA/dt) of differently glycosylated type I collagens fibril formation, at molar ratios PG:collagen monomer ranging from 0.15 : 1 to 0.45 : 1. The results obtained indicate that decorins exert a different effect on the individual phases of fibril formation, correlated to the degree of glycosylation of collagen: at the same PG:collagen ratio the fibril formation of highly glycosylated corneal collagen is more efficiently inhibited than that of the poorly glycosylated one (tendon). Moreover tendon and corneal decorins exert a higher control on the fibrillogenesis of homologous collagen with respect to the heterologous one. These data suggest a possible tissue-specificity of the interaction decorin/type I collagen correlated to the structure of the PG and collagen present in extracellular matrices. This revised version was published online in November 2006 with corrections to the Cover Date.     

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.     

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.     

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.