Desmosine biosynthesis: nature of inhibition by D-penicillamine.

Administration of D-penicillamine and lathyrogens such as beta-amino-propionitrile to animals markedly alters connective tissue by preventing the normal cross-linkage of elastin and collagen. It had been shown that beta-aminopropionitrile blocks the cross-linkage of elastin and collagen by preventing the initial step in cross-linkage: the conversion of lysine in peptide linkage to alpha-amino adipic-delta-semialdehyde. We show that penicillamine acts after the initial step, causing the accumulation of an elastin rich in alpha-amino adipic-delta-semialdehyde.

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

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

A substitution at a non-glycine position in the triple-helical domain of pro alpha 2(I) collagen chains present in an individual with a variant of the Marfan syndrome.

A substitution for a highly conserved non-glycine residue in the triple-helical domain of the pro alpha 2(I) collagen molecule was found in an individual with a variant of the Marfan syndrome. A single base change resulted in substitution of arginine618 by glutamine at the Y position of a Gly-X-Y repeat, and is responsible for the decreased migration in SDS-polyacrylamide gels of some pro alpha 2(I) chains of type I collagen synthesized by dermal fibroblasts from this individual. Family studies suggest that this substitution was inherited from the individual's father who also produces abnormally migrating pro alpha 2(I) collagen chains and shares some of the abnormal skeletal features. This single base change creates a new Bsu36 I (Sau I, Mst II) restriction site detectable in genomic DNA by Southern blot analysis when probed with a COL1A2 fragment. The analysis of 52 control individuals (103 chromosomes) was negative for the new Bsu36 I site, suggesting that the substitution is not a common polymorphism.

A patient with Ehlers-Danlos syndrome type VI is a compound heterozygote for mutations in the lysyl hydroxylase gene.

In the present study, we have isolated and sequenced the complementary DNAs of two mutant alleles for lysyl hydroxylase (LH) in fibroblasts from one patient (AT750) with Ehlers-Danlos syndrome type VI (EDS VI). We have identified a putative mutation in each allele which may be responsible for the patient's decreased LH (normalized to prolyl hydroxylase) activity (24% of normal). Intermediate levels of LH activity were measured in the patient's parents, who are clinically normal (father 52%; mother 86%). After the cloning of cDNAs and amplification by PCR, sequence analysis revealed two equally distributed populations of cDNAs for LH in the AT750 cell line. Each allele revealed different but significant changes from the normal sequence. In one allele (allele 1), the most striking change was a triple base deletion that would result in the loss of residue Glu532. The most significant difference in the other allele (allele 2) was a G-->A change which would produce a Gly678-->Arg codon change in a highly conserved region of the enzyme. Restriction analysis identified that allele 1 was inherited from the proband's mother and allele 2 from the father. This study represents the first example of compound heterozygosity for the LH gene in an EDS VI patient, and it appears that there is an additive effect of each mutant allele on clinical expression in this patient.

Urinary excretion of hydroxylysine and its glycosides as an index of collagen degradation.

Urimary excretion of hydroxyprolin (Hyp) is one index of total collagen degradation, from all sources. Since some of the Hyp released from collagen may be further metabolized before it is excreted, other markers are necessary to measure collagen breakdown. Excretion of the glycosides of hydroxylysine (Hyl), glucosyl galactosyl hydroxylysine (Hy1[Gl)cGa1]), and galactosyl hydroxylysine (Hyl[Ga)]), more accurately reflects collagen metabolism since these products occur in specificratios in different tissue collagens and are themselves metabolized only to a minor degree. The ratios of total Hy1/Hyp and Hyl(GlcGal)/Hyl(Ga1) were measured in the urine of norma. subjects and of patients with Paget's disease of bone, hyperphosphatasia, and extensive thermal burns. In patients with extensive thermal burns the pattern of urinary Hy1 and its glycosides was consistent with degradation of collagen in dermis and fascia. When bone collagen degradation was dominant, the pattern of urinary metabolites reflected that source. Pagetic bone collagen has an amino acid composition similar to normal bone and Hy1(G1cGa1/Hyl(G1) of 0.396-0.743,vs. normal of 0.474+/-0.088. In untreated patients with severe Paget's disease of bone or hyperphosphatasia (urinary Hyp greater than 2.0 micronmol/mg creatinine) urinary Hyl/Hyp averaged 0.052+/-0.042 (0.042+/-0.009 in normal bone) and Hy1(G1cGa1)/Hy1(Ga1) 0.601+/-0.017 (0.47+/-0.009 in normal bone). When bone resorption was decreased sufficiently with calcitonin or disodium etidronate in these patients, both the urinary ratios of Hy1/Hyp and Hy1(G1cGa1)/Hyl(Gal) rose. In normal subjects treated with calcitonin and excreting relatively little Hyp, the ratio of Hy1/H)P approached 0.7 and Hy1(G1ycGa1)/Hy1(Ga1) approached 3.5. There increased ratios reveal the existence of a source of collagen breakdown other than skin or bone. The first subcompoent of complement, Clq, which has collagen-like sequences, relatively high amounts of Hy1, and most of the glycosylated Hy1 as Hy1(G1cGa1), could be the source of these metabolites.

Ascorbic acid stimulates collagen production without altering intracellular degradation in cultured human skin fibroblasts.

The influence of ascorbic acid on intracellular degradation of collagen synthesized by cultured human-skin fibroblasts was examined. In confluent cells maintained in 0.5% serum-supplemented medium, ascorbic acid had no significant effect on collagen degradation measured with hydroxyproline as the marker. Similar results were obtained when collagen degradation was measured with the marker hydroxylysine, the cellular synthesis of which is independent of ascorbic acid. The stimulatory effects of ascorbic acid on collagen production therefore cannot be explained by a change in the rate of degradation. Ascorbic acid acts at some as yet undetermined level to increase the rate of collagen synthesis.

Inhibition of lysyl hydroxylase by catechol analogs.

Catechol analogs inhibit the activity of lysyl hydroxylase (peptidyllysine, 2-oxyglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4), a microsomal enzyme which catalyzes the transformation of certain lysyl residues in collagen to hydroxylysine. Chick embryo lysyl hydroxylase activity was measured by specific tritium release as tritiated water from an L-[4,5-3H]lysine-labelled unhydroxylated collagen substrate prepared from chick calvaria. Catechol analogs did not bind irreversibly to either enzyme or substrate, as full activity was restored with dialysis. Addition of excess cofactor, Fe2+, ascorbic acid, or alpha-ketoglutarate, did not affect inhibition. Kinetic analysis revealed that with respect to collagen substrate, catechol demonstrated a noncompetitive type of inhibition with a Ki of 15 muM.

Inhibition of collagen hydroxylation by lithospermic acid magnesium salt, a novel compound isolated from Salviae miltiorrhizae Radix.

We have screened several chinese medicinal herbs for the presence of antifibrotic agents. An aqueous extract of Salviae miltorrhizae Radix was found to inhibit collagen secretion by human skin fibroblasts without affecting DNA or noncollagen protein synthesis. We have subsequently purified the material exhibiting the inhibitory activity and identified it as magnesium lithospermate. From its chemical structure this compound was predicted to be an inhibitor of the post-translational modifying enzymes prolyl and lysyl hydroxylases in collagen biosynthesis. Accordingly, it decreased the extent of prolyl and lysyl hydroxylations in collagen by approx. 50%. Added to cell extracts it inhibited both prolyl and lysyl hydroxylase activities, but only lysyl hydroxylase activity when added to intact cells. Oral administration of this compound to mice led to a significant reduction of prolyl hydroxylation in newly-synthesized skin collagen. This naturally-occurring compound thus offers a potential means for treating fibrotic diseases, such as systemic scleroderma and keloid.

Regulation of collagen synthesis.

Collagen synthesis is a complex orchestration of intracellular and extracellular events. In addition to synthesis of the polypeptide chains more than a dozen modifications of the molecule occur; most of these are enzymatic and specific for collagen. Regulational control of collagen synthesis promises to be equally complex. Examples are described to 4 specific regulatory influences. Ascorbic acid markedly stimulates collagen synthesis without affecting synthesis of other proteins. This effect appears to be unrelated to its cofactor roles for hydroxylation of lysine and proline. Glucocorticoids at microM concentration specifically inhibit collagen synthesis. Tissues treated with glucocorticoids have diminished levels of mRNA for collagen. During collagen synthesis the aminoterminal propeptide of procollagen is cleaved by a specific protease. This peptide appears to be a feedback inhibitor of collagen synthesis. This effect can be demonstrated in cells and in cell-free synthesizing systems. A membrane receptor system may permit the peptide to be recognized and subsequently act as a translational control mechanism. Viral transformation of fibroblasts results in selectively decreased synthesis of collagen. Levels of cytoplasmic and nuclear mRNA are likewise selectively diminished consistent with transcriptional control.

Molecular defects in the Ehlers-Danlos syndrome.

Several abnormalities in collagen biosynthesis have been described in patients with Ehlers-Danlos syndrome. Examples of collagen structural mutations as well as post-translational enzymatic defects have been detected. Patients with hydroxylysine-deficient collagen disease (Ehlers-Danlos type VI) have diminished lysyl hydroxylase activity. One mutant enzyme has been characterized which is thermally labile and had an altered affinity for ascorbate. Another mutant enzyme had a normal requirement for cofactors but activity was diminished. Type VII Ehlers-Danlos syndrome is associated with altered processing of procollagen to collagen. Most often the disorder is associated with deficient procollagen aminoprotease activity. One patient appears to represent a structural mutation of pro alpha 2 (I) resulting in incomplete cleavage of the amino terminal propeptide. One family with x-linked Ehlers-Danlos syndrome (type V) has been described with altered lysyl oxidase activity. Other patients with this disorder have had normal lysyl oxidase activity. The ecchymotic form of Ehlers-Danlos syndrome (type IV) has defective type III collagen-synthesis. Patients have been described with absent synthesis, diminished synthesis and diminished secretion.

Collagen synthesis by human skin fibroblasts in culture: studies of fibroblasts explanted from papillary and reticular dermis.

Matched human skin fibroblast cultures were established from papillary and reticular dermis. Papillary dermal fibroblasts exhibited increased plating efficiency, exponential growth, and confluent density when compared with their matched reticular dermal cultures. Collagen synthesis by these cells, however, was essentially similar regardless of their origin. Relative collagen synthesis was similar at confluent densities. No differences in type specific collagen synthesis could be detected; relative amounts of types I and III collagens in culture media and types I, III, and AB collagens in the cellular pellet were similar. Type I pC collagen was consistently elevated in culture media from reticular dermal fibroblasts when compared to papillary dermal fibroblasts. The significance of this difference in procollagen processing is unknown.

Prolyl and lysyl hydroxylase activities of human skin fibroblasts: effect of donor age and ascorbate.

Prolyl and lysyl hydroxylase activities in cultures of human skin fibroblasts from fetal to 94-yr-old donors were measured. In contrast to earlier studies with whole skin, neither prolyl nor lysyl hydroxylase activity was found related to donor age. Prolyl hydroxylase activity increased 3- to 6-fold when cell extracts were incubated with ascorbate and other hydroxylation cofactors before assay. A similar increase in prolyl hydroxylase activity occurred when cells were incubated with ascorbate. Lysyl hydroxylase activity remained unaltered under these conditions.

A familial cutis laxa syndrome with ultrastructural abnormalities of collagen and elastin.

A familial cutis laxa syndrome is reported in a mother and son. In addition, the son had Klippel-Tranaunay-Weber syndrome, which may be related to his underlying disorder. Ultrastructural examination of skin revealed previously recognized abnormalities of elastic tissue. In addition, abnormalities in collagen structure are demonstrated. The underlying defect in this disorder appears to result in abnormalities in both elastic tissue and collagen structure.

Isolation and characterization of a human pro alpha 2(I) collagen gene segment.

Over 20 kilobase pairs of the human pro alpha 2(I) collagen gene have been isolated and characterized by restriction endonuclease mapping, cell-free translation of hybrid-selected RNA, and DNA sequence analysis. We have sequenced an exon and determined its length to be 108 base pairs (bp). This is consistent with the organization of chick and sheep collagen genes in that exons are multiples of 9 bp in length, frequently being 54 and 108 bp. The sequenced exon was bordered by a GT (guanine-thymine) at its 3' end and an AT (adenine-thymine) at its 5' end. This pattern has been found at all normal intron-exon junctions in eukaryotic cells. The amino acid sequence derived from DNA sequencing of this 108 bp exon revealed 88% homology compared to the amino acid sequence of bovine pro alpha 2(I). The bases encoded 12 Gly-X-Y triplets characteristic of the helical portion of collagen. A unique sequence Gly-Gly-Lys-Gly-Glu-Lys identified this fragment as alpha 2(I) collagen.

In vitro inhibition of collagen cross links by catechol analogs.

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

Effects of ascorbic acid on proliferation and collagen synthesis in relation to the donor age of human dermal fibroblasts.

Several events are associated with cellular aging: alterations in the extracellular matrix, loss of the cell's proliferative capacity, and decreased responsiveness to growth factors. In skin, a major component of the extracellular matrix is collagen; an important regulator of collagen synthesis is ascorbic acid, which may also have growth factor-like properties. To investigate the relationship of the extracellular matrix and proliferative capacity to aging, we examined the effects of ascorbic acid on cell proliferation and collagen expression in dermal fibroblasts from donors of two age classes, newborn (3-8 d old) and elderly (78-93 years old). In the absence of ascorbic acid (control) proliferative capacities were inversely related to age; newborn cell lines proliferated faster and reached greater densities than elderly cell lines. However, in the presence of ascorbic acid both newborn and elderly cells proliferated at a faster rate and reached higher densities than controls. To determine whether there are age-related differences in extracellular matrix production and ascorbic acid responsiveness we examined and found that collagen biosynthesis (collagenase-digestible protein) was inversely related to age, but the stimulation by ascorbic acid appeared age independent. The increase in collagen synthesis was reflected by coordinate increases in steady-state pro alpha 1(I) and pro alpha 1(III) collagen mRNAs, suggesting a pretranslational mechanism. Ascorbic acid appears capable of overcoming the reduced proliferative capacity of elderly dermal fibroblasts, as well as increasing collagen synthesis in elderly cells by similar degrees as in newborn cells even though basal levels of collagen synthesis are age dependent.

Procollagen I synthesis in human skin fibroblasts: effect on culture conditions on biosynthesis.

Human skin fibroblast culture promises to be a useful system for the investigation of the regulation of collagen biosynthesis and the study of abnormalities in collagen biosynthesis in connective tissue disorders. The effect of culture conditions on procollagen I biosynthesis has been determined. Optimal conditions for collagen biosynthesis were: 10% dialyzed heat-inactivated fetal calf serum, 0.15 mM ascorbate, and 0.078 mM beta-aminopropionitrile. Newly synthesized procollagen I accumulated in the medium at a linear rate for 18 hr. Preincubation of cells in labelling media for 4 hr before adding radioactive proline enhanced synthesis. Collagenase digestion was used to study overall collagen biosynthesis. 94% of all collagen synthesized was found in the medium, and 6% in the cell pellet. Under optimal conditions, collagen comprised 24% of all protein in the medium, and 14% of protein produced by the whole culture.

Ascorbic acid specifically increases type I and type III procollagen messenger RNA levels in human skin fibroblast.

In cultured human skin fibroblasts, ascorbic acid stimulates collagen production with no apparent change in the intracellular degradation of newly synthesized procollagen. To understand the basis for this effect, we measured the steady-state levels of type I and type III procollagen mRNAs in cells treated with ascorbic acid. A three- to fourfold increase in collagen synthesis was associated with a two- to threefold increase in the levels of mRNAs for both type I and type III procollagens. These effects of ascorbic acid are explained by a translational control linked either to procollagen gene transcription or mRNA degradation.

Characterization of a partial cDNA for lysyl hydroxylase from human skin fibroblasts; lysyl hydroxylase mRNAs are regulated differently by minoxidil derivatives and hydralazine.

Lysyl hydroxylase (LH) is an essential enzyme in collagen biosynthesis that catalyzes the formation of hydroxylysine required for intermolecular crosslinking of collagen. We have isolated a partial (2.2-kb) cDNA for LH from human skin fibroblasts using PCR. DNA sequencing revealed 72% homology of the human coding sequence with the chick LH sequence at the nucleotide level and 76% homology predicted at the amino acid level. The LH cDNA hybridized strongly with two mRNA species of 2.4 and 3.4 kb on Northern blots of normal fibroblast RNA. Administration of minoxidil decreased both mRNA species without affecting levels of the mRNAs for the beta subunit of prolyl 4-hydroxylase (PH) or alpha 1(I) collagen. Two derivatives of minoxidil (3' hydroxyminoxidil and 4' hydroxyminoxidil) produced similar decreases in LH mRNAs. In contrast hydralazine increased the mRNAs for LH in parallel with its previously reported effect on the mRNA for the beta subunit of PH. This effect is accompanied by virtual elimination of the alpha 1(I) collagen mRNAs. These results on the action of minoxidil and hydralazine at the pretranslational level correlate well with their previously reported effect on enzyme activity and collagen biosynthesis and indicate that changes in steady-state mRNA levels can account directly for changes at the protein level. Moreover, the unique action of minoxidil in specifically decreasing LH mRNAs contrasts with the less specific stimulatory effects of hydralazine and suggests that these pharmaceuticals are regulating expression of LH at a pretranslational level by different mechanisms.

Construction of a full-length murine pro alpha 2(I) collagen cDNA by the polymerase chain reaction.

Construction of large collagen cDNA has been hindered by the relatively large size and high G-C content of processed mRNA. We describe here the development of a rapid and efficient method for obtaining large full-length collagen cDNA. A full-length (4.3 kb) murine pro alpha 2(I) collagen cDNA was constructed by synthesis of a first-strand cDNA library with use of poly-A RNA (MC3T3-E1) and the oligo-dT17-adapter primer described by Frohman et al (Proc Natl Acad Sci USA 85:8998, 1988). Pro alpha 2(I) collagen cDNA were specifically amplified by the polymerase chain reaction (PCR) with a pro alpha 2(I) specific primer as the 5' primer (20mer; corresponding to nucleotide positions 42-61 in the first exon of the murine pro alpha 2(I) collagen gene, COL1A2), and with the adapter sequence 5' to the dT17 as the 3' primer. The PCR conditions were optimized to allow amplification of the expected 4.0-5.0-kb product; a major 4.3-kb product was visualized by ethidium bromide, identified by in situ gel hybridization, and cloned. DNA sequencing determined that it contained the correct 5' sequence and the 3' end had a 68 basepair (bp) 3' untranslated region. The entire sequence that codes the amino-terminal propeptide domain has been determined and compared to the human sequence. The homology between human and mouse is less in the amino terminal propeptide than in the triple helical domain; exon 5 of murine COL1A2 codes for an additional six amino acids not found in human COL1A2.