Differential effects of ascorbate depletion and α,α′-dipyridyl treatment on the stability,but not on the secretion,of type IV collagen in differentiated F9 cells

Ascorbic acid stimulates secretion of type I collagen because of its role in 4-hydroxyproline synthesis, but there is some controversy as to whether secretion of type IV collagen is similarly affected. This question was examined in differentiated F9 cells, which produce only type IV collagen, by labeling proteins with [¹⁴C]proline and measuring collagen synthesis and secretion. Hydroxylation of proline residues in collagen was inhibited to a greater extent in cells treated with the iron chelator α,α′-dipyridyl (97.7%) than in cells incubated without ascorbate (63.1%), but both conditions completely inhibited the rate of collagen secretion after 2–4 h, respectively. Neither treatment affected laminin secretion. Collagen synthesis was not stimulated by ascorbate even after treatment for 2 days. On SDS polyacrylamide gels, collagen produced by α,α′-dipyridyl-treated cells consisted mainly of a single band that migrated faster than either fully (+ ascorbate) or partially (− ascorbate) hydroxylated α1(IV) or α2(IV) chains. It did not contain interchain disulfide bonds or asn-linked glycosyl groups, and was completely digested by pepsin at 15°C. These results suggested that it was a degraded product lacking the 7 S domain and that it could not form a triple helical structure. In contrast, the partially hydroxylated molecule contained interchain disulfide bonds and it was cleaved by pepsin to collagenous fragments similar in size to those obtained from the fully hydroxylated molecule, but at a faster rate. Kinetic experiments and monensin treatment suggested that completely unhydroxylated type IV collagen was degraded intracellularly in the endoplasmic reticulum or cis Golgi. These studies indicate that partial hydroxylation of type IV collagen confers sufficient helical structure to allow interchain disulfide bond formation and resistance to pepsin and intracellular degradation, but not sufficient for optimal secretion. J Cell. Biochem. 67:338–352, 1997. Published 1997 Wiley-Liss, Inc.     

The stimulation of collagen secretion by ascorbate as a result of increased proline hydroxylation in chick embryo fibroblasts.

Collagen secretion by chick embryo fibroblasts was measured by incorporating [¹⁴C]proline into proteins and then analyzing the amount of collagen in the cell and medium separately by using purified bacterial collagenase. In order to produce varying levels of hydroxylation, cells were incubated with varying concentrations of ascorbate or with varying concentrations of α,α′-dipyridyl in the presence of saturating ascorbate. Ascorbate stimulated both the hydroxylation of proline in collagen and the secretion of collagen; the concentration of ascorbate required for half-maximal stimulation of both proesses was approximately 4.5 × 10^?7, m. Since the cells could concentrate ascorbate 10-fold, this K_M for proline hydroxylation is 100-fold lower than values reported for purified prolyl hydroxylase (Abbot, M. T., and Udenfriend, S. (1974) in Molecular Mechanisms of Oxygen Activation (Hayaishi, O., ed.), p. 173, Academic Press New York; Kivirikko K. I., et al. (1968) Biochim. Biophys. Acta, 151, 558–567). Conversely, α,ga′-dipyridyl inhibited both proline hydroxylation and collagen secretion; half-maximal inhibition of both processes was observed at 7 × 10^?5, m. The results of the two types of experiments show that the secretion of collagen becomes directly proportional to proline hydroxylation when approximately 30% of the proline residues in collagen have been hydroxylated compared to maximal hydroxylation of 50%. Since the stability of triple-helical collagen at 37 °C has been shown to be dependent on the hydroxyproline content of the molecule (Rosenbloom, J., et al. (1973) Arch. Biochem. Biophys., 158, 478–484), we suggest that the observed proportionality between secretion and hydroxylation is a reflection of the increased amount of stable triple helical collagen at 37 °C. When the cells were incubated with a concentration of ascorbate that was saturating for secretion and hydroxylation, there was no significant activation of prolyl hydroxylase as measured in a cell-free extract. These experiments suggest that ascorbate effects collagen secretion by acting at the site of proline hydroxylation but not by increasing the activity of prolyl hydroxylase.     

Recombinant expression of hydroxylated human collagen in Escherichia coli

Collagen is the most abundant protein in the human body and thereby a structural protein of considerable biotechnological interest. The complex maturation process of collagen, including essential post-translational modifications such as prolyl and lysyl hydroxylation, has precluded large-scale production of recombinant collagen featuring the biophysical properties of endogenous collagen. The characterization of new prolyl and lysyl hydroxylase genes encoded by the giant virus mimivirus reveals a method for production of hydroxylated collagen. The coexpression of a human collagen type III construct together with mimivirus prolyl and lysyl hydroxylases in Escherichia coli yielded up to 90 mg of hydroxylated collagen per liter culture. The respective levels of prolyl and lysyl hydroxylation reaching 25 % and 26 % were similar to the hydroxylation levels of native human collagen type III. The distribution of hydroxyproline and hydroxylysine along recombinant collagen was also similar to that of native collagen as determined by mass spectrometric analysis of tryptic peptides. The triple helix signature of recombinant hydroxylated collagen was confirmed by circular dichroism, which also showed that hydroxylation increased the thermal stability of the recombinant collagen construct. Recombinant hydroxylated collagen produced in E. coli supported the growth of human umbilical endothelial cells, underlining the biocompatibility of the recombinant protein as extracellular matrix. The high yield of recombinant protein expression and the extensive level of prolyl and lysyl hydroxylation achieved indicate that recombinant hydroxylated collagen can be produced at large scale for biomaterials engineering in the context of biomedical applications.     

A post-translational modification, unrelated to hydroxylation, in the collagenous domain of nonhelical pro-alpha 2(I) procollagen chains secreted by chemically transformed hamster fibroblasts.

Transformed Syrian hamster embryo (NQT-SHE) fibroblasts do not synthesize the pro-alpha 1 subunit of type I procollagen, but secrete two modified forms of the pro-alpha 2(I) subunit that migrate more slowly than the normal chain during gel electrophoresis (Peterkofsky, B., and Prather, W. (1986) J. Biol. Chem. 261, 16818-16826). By electrophoretic analysis of cyanogen bromide and V8 protease-derived peptides from the collagenous domains of intra- and extracellular pro-alpha 2(I) chains, we find that the modification occurs almost exclusively in secreted molecules, is located in the region spanned by the cyanogen bromide peptide CB3,5, and persists when hydroxylation is inhibited. Thus, modification is due to a post-translational reaction other than hydroxylation. The modified chains appear to be secreted in the denatured state since: 1) helical structures formed at 4 degrees C under acidic conditions were unstable under neutral conditions at 37 degrees C; 2) conditions that destabilize the type I procollagen helix and thus inhibit its secretion, i.e. inhibition of proline hydroxylation or incorporation of the proline analog cis-hydroxyproline, did not affect secretion of the modified chains. The time courses for secretion of nonhelical modified chains from NQT-SHE and of hydroxylated helical procollagen I from control cells, as a proportion of total collagen synthesized, were similar. Although cis-hydroxyproline did not inhibit the secretion of the modified chains, it induced their rapid intracellular degradation.     

Structural Aspects of Hydroxyproline-Containing Proteins

Abstract

The occurrence of hydroxyproline (Hyp) in collagen, Clq and acetylcholinesterase (AChE) raises important questions concerning the role of this unusual imino acid in the structure and function of these proteins. Available data on collagen indicate that Hyp is necessary for the normal secretion of the protein after its synthesis and for the integrity of the triple-helical conformation. Studies from our laboratory have dealt with the structural aspects of the posttranslational conversion of proline to hydroxyproline in collagen mediated by prolyl hydroxylase. We proposed that the β-turn conformation at the Pro-Gly segments in the nascent procollagen molecule are the sites of the enzymatic hydroxylation and that this conformation changes over to the collagen-like helix as a result of the hydroxylation process. Recently, we have provided additional experimental support to our proposal by a) synthesizing specific β-turn oligopeptides containing the Pro-Gly as well as Pro-Ala and Pro-DAla sequences and showing that these act as inhibitors of the enzymatic hydroxylation of a synthetic substrate and b) demonstrating, by circular dichroism spectroscopy, the occurrence of a conformational change leading to the triple-helix as a direct consequence of proline hydroxylation in a non-helical polypeptide substrate. We have also observed that the acquisition of hydroxylation results in a significant enhancement of the rate of folding of the polypeptide chain from the unfolded to the triple-helical conformation. We believe that our observations on proline hydroxylation in collagen should also be applicable to Clq and acetylcholineesterase both of which share the general structural and functional properties of collagen in their “tail” regions. Using the techniques employed in collagen studies, one should be able to assess the role of hydroxyproline in the folding, structural stabilities and functions of Clq and AChE. This would also involve the study of the unhydroxylated and hydroxylated precursors of these proteins which may share common structural features with their collagen counterparts. Finally, a systematic study of hydroxyproline-containing peptides and polypeptides has been initiated by us so as to understand the exact manner in which Hyp participates in the formation and stability of the triple-helical conformation in the proteins in which it occurs.     

Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs. Evidence for the formation and degradation of a partially hydroxylated collagen

1. After the administration of l-[G-(3)H]proline to guinea pigs deprived of ascorbic acid for increasing periods of time, the specific radioactivities of proline and hydroxyproline in skin collagen and aortic elastin were determined at various time-intervals after administration of the labelled compound with a view to studying the formation and degradation of collagen and elastin both deficient in hydroxyproline. 2. As judged from the incorporation of radioactivity into elastin proline, elastin synthesis was not decreased in the ascorbic acid-deficient animals. There was however, a rapid decline in the specific radioactivity of elastin hydroxyproline. The proline/hydroxyproline specific-radioactivity ratio was approx. 1.5:1 after 6 days and 20:1 after 12 days of ascorbic acid deprivation, in contrast with the ratio of 1:1 in controls. The results suggested that the effect of ascorbic acid deficiency on elastin biosynthesis could be regarded as simply an elimination of hydroxylation of elastin proline with the formation and retention of a polymer increasingly deficient in hydroxyproline. 3. Collagen proline and hydroxyproline specific radioactivities were derived from material that was soluble in hot trichloroacetic acid, non-diffusible and collagenase-degradable. In contrast with elastin, there was a rapid decline in the specific radioactivity of proline as well as hydroxyproline in collagen from the ascorbic acid-deficient animals. However, the proline/hydroxyproline specific-radioactivity ratio in all samples from scorbutic animals was consistently slightly above 1:1. The results suggest the appearance in place of collagen, but in rapidly diminishing amounts, of a partially hydroxylated collagen in which the degree of hydroxylation may be decreased only by approx. 10%. 4. Incorporation of radioactivity into the diffusible hydroxyproline in skin remained relatively high despite the rapid decline in the incorporation of radioactivity into collagen. This observation is interpreted as indicative of an increasing degree of degradation of partially hydroxylated collagen to diffusible peptides. An alternative explanation might be that partially hydroxylated peptides are released to an increasing extent from ribosomes before they attain a length at least sufficient to render them non-diffusible. In either case it implies the accumulation in scurvy of low-molecular-weight peptides enriched in proline and deficient in hydroxyproline and could explain the failure to accumulate a high-molecular-weight collagen deficient in hydroxyproline. 5. It is thought, however, that, in addition, an inhibition of ribosomal amino acid incorporation leading to decreased synthesis of partially hydroxylated collagen may also occur, perhaps secondarily to impaired hydroxylation.     

Structural aspects of hydroxyproline-containing proteins

The occurrence of hydroxyproline (Hyp) in collagen, C1q and acetylcholineesterase (AChE) raises important questions concerning the role of this unusual imino acid in the structure and function of these proteins. Available data on collagen indicate that Hyp is necessary for the normal secretion of the protein after its synthesis and for the integrity of the triple-helical conformation. Studies from our laboratory have dealt with the structural aspects of the posttranslational conversion of proline to hydroxyproline in collagen mediated by prolyl hydroxylase. We proposed that the beta-turn conformation at the Pro-Gly segments in the nascent procollagen molecule are the sites of the enzymatic hydroxylation and that this conformation changes over to the collagen-like helix as a result of the hydroxylation process. Recently, we have provided additional experimental support to our proposal by a) synthesizing specific beta-turn oligopeptides containing the Pro-Gly as well as Pro-Ala and Pro-DAla sequences and showing that these act as inhibitors of the enzymatic hydroxylation of a synthetic substrate and b) demonstrating, by circular dichroism spectroscopy, the occurrence of a conformational change leading to the triple-helix as a direct consequence of proline hydroxylation in a non-helical polypeptide substrate. We have also observed that the acquisition of hydroxylation results in a significant enhancement of the rate of folding of the polypeptide chain from the unfolded to the triple-helical conformation. We believe that our observations on proline hydroxylation in collagen should also be applicable to C1q and acetylcholineesterase both of which share the general structural and functional properties of collagen in their "tail" regions. Using the techniques employed in collagen studies, one should be able to assess the role of hydroxyproline in the folding, structural stabilities and functions of C1q and AChE. This would also involve the study of the unhydroxylated and hydroxylated precursors of these proteins which may share common structural features with their collagen counterparts. Finally, a systematic study of hydroxyproline-containing peptides and polypeptides has been initiated by us so as to understand the exact manner in which Hyp participates in the formation and stability of the triple-helical conformation in the proteins in which it occurs.     

Hydroxylation of Human Type III Collagen Alpha Chain by Recombinant Coexpression with a Viral Prolyl 4-Hydroxylase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

High-level expression of recombinant collagen by genetic engineering is urgently required. Recombinant collagen is different from natural collagen in its hydroxyproline (Hyp) content and thermal stability. To obtain hydroxylated collagen for applications in biomedicine and biomaterials, the human collagen α1(III) chain was co-expressed with the viral prolyl 4-hydroxylase A085R in Escherichia coli. Unlike previous reports using human prolyl 4-hydroxylase, this study examined the hydroxylation of full-length human collagen α1(III) chain (COL3A1) by viral prolyl 4-hydroxylase. The genes encoding these two proteins were controlled by different promoters, Ptac and PRPL, on a recombinant pKK223-3 plasmid. The sequencing results verified that the target genes were successfully inserted into the recombinant vector. Based on quantitative PCR, SDS–PAGE, and western blotting, successful expression by E. coli BL21(DE3) was detected at the mRNA and protein levels for both loci. Liquid chromatography–mass spectrometry (LC–MS/MS) results suggested that the highest Hyp yield was obtained when the two proteins were induced with 0.5 mM IPTG and heat-shock treatment at 50?°C, corresponding to high enzyme expression and low human collagen α1(III) chain expression levels. A biological activity analysis indicated that the recombinant collagen with the highest hydroxylation level supported the growth of baby hamster kidney cells, similar to observations for native collagen. The production of hydroxylated collagen in this study establishes a new method for collagen hydroxylation and provides a basis for the application of recombinant collagen expressed in E. coli.     

The metabolic requirements for transcellular movement and secretion of collagen.

Cultures of chick tendon fibroblasts were capable of normal ATP production and protein synthetic activity even though the normally high rate of glycolysis was markedly reduced by substitution of pyruvate for glucose. Iodoacetate and 2-deoxyglucose reduced ATP levels and protein synthesis even in the presence of pyruvate. Under these conditions, both inhibitors were shown to have effects on the energy metabolism of cells which were apparently unrelated to an inhibition of glycolysis. Selective inhibition of either glycolysis, by incubation in glucose-free medium, or of oxidative phosphorylation, by incubation with an uncoupler, was shown to have little effect on cellular ATP levels or intracellular transport and secretion of collagen. However, inhibition of both glycolysis and oxidative phosphorylation resulted in decreased cellular ATP levels and an inhibition of collagen secretion. This effect was not due to a requirement for continued protein synthesis, since inhibition of protein synthesis with cycloheximide or puromycin had little effect on collagen secretion. The ATP requirement for intracellular transport and secretion is discussed in relation to the secretory pathway for collagen.     

Conformational requirement for lysine hydroxylation in collagen. Structural studies on synthetic peptide substrates of lysyl hydroxylase.

An attempt has been made to understand the conformational determinants that govern the hydroxylation of selected lysyl residues in the nascent collagen molecule by lysyl hydroxylase (EC 1.14.11.4). A series of peptide substrates of the enzyme, ranging in length from 3 to 12 residues, were synthesized. These included: tert-butyloxylcarbonyl (t-Boc)-Ile-Lys-Gly; Boc-Ala-Lys-Gly; N-acetyl-Ala-Lys-Gly-Ser; Hyp-Gly-Pro-Lys-Gly-Glu; Leu-Hyp-Gly-Ala-Lys-Gly-Glu; Gly-Phe-Hyp-Gly-Leu-Hyp-Gly-Ala-Lys-Gly-Glu; (Hyp-Gly-Pro-Lys-Gly-Glu)2; and Ala-Arg-Gly-Ile-Lys-Gly-Ile-Arg-Gly-Phe-Ser-Gly. The conformational features of these peptides were studied by spectroscopic methods so as to relate this information with the kinetic parameters for the interaction of these peptides with purified lysyl hydroxylase. Spectroscopic data, supported by conformational energy calculations, indicated that the tripeptides t-Boc-Ile-Lys-Gly and t-Boc-Ala-Lys-Gly adopt a gamma-turn structure in water and trifluoroethanol with Lys in the second position of the turn. In the tetra- and larger peptides two structures, the beta-turn and a polyproline-II (PP-II) type extended conformation, were identified. The proportions of these two structures in a given peptide depended on the polarity of the solvent. All of the peptides were hydroxylated by lysyl hydroxylase isolated from chicken embryos. In contrast, a control peptide, t-Boc-Ala-Gly-Lys which adopted a beta-turn with Lys at the end of the turn, was not hydroxylated. Competitive inhibition of the hydroxylation of protocollagen by some of the peptides showed a common binding site for these substrates in the enzyme's active site. Kinetic data on the peptides indicated improved hydroxylation rate (higher Vmax) in peptides having relatively higher beta-turn content and improved binding (lower Km) in peptides with higher content of the PP-II structure. The efficacy of the substrate was also governed by its chain length. These data suggest that the conformational criterion for lysine hydroxylation in collagen-related peptides is the presence of a "bent" structure, such as the gamma- or beta-turn at the catalytic site of lysyl hydroxylase and an "extended" PP-II type structure at the binding site(s) of the enzyme's active site. This suggestion also provides a conformational rationale for earlier observations on the substrate specificity of lysyl hydroxylase.     

Ascorbate-independent proline hydroxylation resulting from viral transformation of Balb 3T3 cells and unaffected by dibutyryl cAMP treatment.

Collagen synthesis, hydroxylation of proline in collagen, and collagen secretion were studied in the contact-inhibited mouse fibroblast line, Balb 3T3; the Kirsten virus transformed line, Ki-3T3; and dibutyryl cAMP (dbcAMP)-treated Ki-3T3 cells, during the various phases of the growth cycle. Transformed cells in both logarithmic and stationary phase produced lower levels of collagen than the parent line but 85-90% of the theoretically possible hydroxyproline residues of the collagen were formed even when ascorbic acid was not added to the culture medium. Moreover, the transformed cells showed only about a 20% increase of collagen secretion upon addition of ascorbate. This was in contrast to the ascorbate requirement for maximal proline hydroxylation and the 2-3 fold stimulation of collagen secretion by ascorbate in the parent Balb 3T3 cells. Although dbcAMP treatment caused Ki-3T3 cells to assume a more normal morphology and increased the relative rate of collagen synthesis to levels similar to that of 3T3, such treatment did not restore an ascorbate requirement for proline hydroxylation or collagen secretion. The specific activity of the enzyme prolyl hydroxylase also was not affected by dbcAMP treatment although collagen synthesis was increased by such treatment. In addition, it was found that ascorbic acid was not effective in activating prolyl hydroxylase derived from Ki-3T3 or dbcAMP-treated Ki-3T3 cell cultures either in logarithmic phase or stationary phase. Ki-3T3 cultures did not accumulate ascorbic acid in cells or medium nor was ascorbic acid synthesized from the precursor 14C-glucuronate in cell homogenates. The results suggest that virally transformed Balb 3T3 cells acquire the capacity to synthesize a reducing cofactor for prolyl hydroxylase and that this function may be related to the increased glycolytic metabolism of these cells since neither cellular metabolism nor ascrobate-independent hydroxylation was altered by treatment with dbcAMP.     

Lysyl hydroxylase 2 is a specific telopeptide hydroxylase, while all three isoenzymes hydroxylate collagenous sequences.

Lysyl hydroxylase (LH), with three isoenzymes in vertebrates, catalyzes the formation of hydroxylysine by acting on -X-Lys-Gly- triplets in the collagenous domains of proteins of the collagen superfamily and also in -X-Lys-Ala- or -X-Lys-Ser- sequences in the telopeptides located at the ends of the polypeptide chains in some fibril-forming collagens. The hydroxylysine residues are essential for the stability of collagen crosslinks and act as carbohydrate attachment sites. The extent of lysine hydroxylation varies between collagen types, between tissues in the same collagen type and in certain diseases, suggesting that the LH isoenzymes may have different substrate specificities. We studied here the hydroxylation of synthetic peptides representing various hydroxylation sites in type I and IV collagens by purified recombinant LHs in vitro and of a recombinant full-length type I procollagen chain coexpressed with each LH in insect cells. All three LHs hydroxylated peptides representing collagenous sequences of type I and IV collagens, although with different K(m) and V(max) values. Furthermore, all three hydroxylated the collagenous domain of the coexpressed type I procollagen chain to a similar extent. None of the isoenzymes hydroxylated peptides representing the N and C telopeptides of type I collagen, but LH2, unlike the other two isoenzymes, hydroxylated the N telopeptide in the coexpressed procollagen chain. Hydroxylation of the telopeptide lysines by LH2 thus occurs only in the context of a long peptide. These data provide the first direct evidence that LH2 is a specific telopeptide hydroxylase, while all three LHs act on collagenous sequences.     

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.     

Collagen prolyl hydroxylation in WI-38 fibroblast cultures: Action of hydralazine

Summary The action of hydralazine on collagen prolyl hydroxylation was studied in a cell culture system using WI-38 fibroblasts. The prolyl hydroxylation level was determined by a method involving the digestion of collagen by bacterial collagenase and the examination of specific peptides. The presence of low concentrations of hydralazine (0.2 mM) in both “young” and “old” fibroblast cultures strongly inhibited collagen prolyl hydroxylation. The degree of inhibition was greater in serum-deficient cultures. No significant improvement in the degree of hydroxylation was observed by increasing either ascorbate or iron levels in the hydralazine-containing cultures in which hydroxylation was inhibited. Some of the reported side effects of hydralazine seen in patients might be related to its inhibitory effects on mixed function oxidative (MFO) hydroxylation systems. While the ascorbate dependence of the prolyl hydroxylase system of WI-38 decreased with the “age” of the culture, hydralazine inhibition of hydroxylation was dramatic with cultures of all “ages”. This work was supported by NIH grants nos. AM15671, AM1675 and HD07376, and fellowship no. HD01998.     

Collagen synthesized and modified by aging fibroblasts in culture.

Collagen is produced by WI-38 diploid human fibroblast cultures throughout their life cycle. It is examined by a sensitive method based on the analysis of specific peptides obtained after digestion with bacterial collagenase. The production and hydroxylation of the collagen is strongly dependent upon the age (population doublings) of the culture and the presence of ascorbic acid. Young cultures (passage 26) produce large amounts of collagen in the absence of ascorbic acid, and this collagen is about 50% hydroxylated compared to that produced by young cultures in the presence of ascorbic acid. Ascorbic acid reduces to about one-half the amount of collagen produced by these young cultures. The young confluent cultures also depend strongly on ascorbic acid for hydroxylation of proline. The dependence declines rapidly with the age of the culture. The collagen produced by young cultures supplied with ascorbic acid is very similar to the type I collagen produced by normal individuals and has about the same degree of hydroxylation of its prolyl residues. The amount of collagen produced by "older" cultures is unaffected by ascorbic acid, but the degree of hydroxylation is normal only if ascorbic acid is present, and is decreased to about 60 to 70% in the absence of the vitamin. "Senescent" cultures showed little, if any, dependency on ascorbic acid, and the collagen produced, with and without the vitamine, is about 80% hydroxylated. The prolyl hydroxylation system of the WI-38 cells and the various controls on the system are age-dependent.     

Collagen polysomes: Site of hydroxylation of proline residues

The biosynthesis of collagen on polysomes has been studied by using a newly devised method for obtaining polysomes in high yield from stationary-phase mouse fibroblast (line 3T6; Goldberg &, Green, 1967). These polysomes were completely disaggregated to monosomes by brief exposure to ribonuclease and they lost most of their radioactivity to the top of the sucrose gradients as a result of a 30-minute chase with unlabeled proline. After a ten-minute pulse with [³H]proline, nascent collagen peptides could be identified in these polysomes on sucrose gradients. Most of the proline residues susceptible to hydroxylation by collagen proline hydroxylase were found, in most cases, to be already hydroxylated in these nascent peptides. The nascent nature of these peptides was confirmed by the observation that treatment of the polysomes with RNase transferred the radioactive collagen peptides to the monosome area and these peptides could subsequently be removed to the soluble material at the top of the gradient upon treatment with puromycin. These findings therefore, show clearly that the hydroxylation of proline residues is occurring, in vivo under normal conditions, on nascent collagen chains. In no case was the degree of hydroxylation of the released collagen chains higher than that on the nascent collagen peptides. It seems likely, therefore, that the major site of proline hydroxylation is the nascent collagen peptide.     

Hydroxylation of prolyl residues in type II procollagen in vitro and in cellulo. Lack of preferential hydroxylation of specific regions of the protein

[14C]Proline-labeled protocollagen, the unhydroxylated form of procollagen, was isolated from cartilage cells incubated with alpha, alpha'-dipyridyl. For examination of the initial steps in the hydroxylation of the protein, it was incubated in vitro with prolyl hydroxylase so that an average of 1.3-2.7 prolyl residues per chain was hydroxylated. The partially hydroxylated alpha chain were cleaved with cyanogen bromide, and the fragments were separated by polyacrylamide gel electrophoresis or column chromatography. The cyanogen bromide fragments were hydroxylated to the same degree. The results indicated, therefore, that in the initial hydroxylation of alpha chains in vitro, there was no preferential hydroxylation of any specific regions of the protein. In a second series of experiments, cartilage cells were incubated with [14C]proline and alpha, alpha'-dipyridyl so that prolyl hydroxylase in the cells was extensively, but not completely, inhibited. Partially hydroxylated alpha chains were isolated, and cyanogen bromide fragments of the alpha chains from the cells were assayed for hydroxy[14C]proline. The alpha chains contained an average of two residues of hydroxyproline per chain, and the cyanogen bromide fragments were hydroxylated to about the same degree. The results indicated, therefore, that when prolyl hydroxylase activity in cells is low relative to the rate at which pro alpha chains are synthesized, hydroxylation of prolyl residues occurs as it does in vitro, and there is no preferential hydroxylation of a specific region of the protein.     

Role of prolyl hydroxylation in oncogenically stabilized hypoxia-inducible factor-1alpha

Stabilization of the hypoxia-inducible factor-1 (HIF-1) protein is essential for its role as a regulator of gene expression under low oxygen conditions. Here, employing a novel hydroxylation-specific antibody, we directly show that proline 564 of HIF-1alpha and proline 531 of HIF-2alpha are hydroxylated under normoxia. Importantly, HIF-1alpha Pro-564 and HIF-2alpha Pro-531 hydroxylation is diminished with the treatment of hypoxia, cobalt chloride, desferrioxamine, or dimethyloxalyglycine, regardless of the E3 ubiquitin ligase activity of the von Hippel-Lindau (VHL) tumor suppressor gene. Furthermore, in VHL-deficient cells, HIF-1alpha Pro-564 and HIF-2alpha Pro-531 had detectable amounts of hydroxylation following transition to hypoxia, indicating that the post-translational modification is not reversible. The introduction of v-Src or RasV12 oncogenes resulted in the stabilization of normoxic HIF-1alpha and the loss of hydroxylated Pro-564, demonstrating that oncogene-induced stabilization of HIF-1alpha is signaled through the inhibition of prolyl hydroxylation. Conversely, a constitutively active Akt oncogene stabilized HIF-1alpha under normoxia independently of prolyl hydroxylation, suggesting an alternative mechanism for HIF-1alpha stabilization. Thus, these results indicate distinct pathways for HIF-1alpha stabilization by different oncogenes. More importantly, these findings link oncogenesis with normoxic HIF-1alpha expression through prolyl hydroxylation.     

Age-related variations in hydroxylation of lysine and proline in collagen

The effect of age on the extent of hydroxylation of lysine and proline both generally and at certain specific sites in collagens from bone, skin and tendon was examined in the chick from the 14-day embryo to the 18-month-old adult. For all collagens there was a marked fall in the overall extent of hydroxylation of lysine with increasing age in both alpha(1) and alpha(2) chains, this fall occurring mostly in a relatively short period immediately after hatching. Hydroxylation of lysine declined to a constant value which, as expected, differed appreciably for each collagen and was considered to be characteristic of the collagen according to its tissue of origin. Hydroxylation of lysine in the N-terminal, non-helical telopeptide region of both alpha(1) and alpha(2) chains, which is important with regard to cross-linking, was relatively high in embryonic collagens. There was, however, a rapid loss of hydroxylation at these sites in skin collagen, occurring both during development of the embryo and in the period immediately after hatching. In contrast some hydroxylation at these sites persisted in bone and tendon collagens and, as judged by examination of peptide alpha(1)-CB1, appeared to reach a constant value in time of about 33% in bone and about 15% in tendon collagen. The actual extent of hydroxylation of lysine in the N-terminal telopeptides and the size of the changes in these values with age appeared to be unrelated to the corresponding whole-chain values, and it is suggested therefore that hydroxylation of telopeptidyl lysine may be under separate enzymic control. The increased hydroxylation of lysine in the embryo was accompanied by only minimal changes in proline hydroxylation, which was very slightly increased in embryonic bone and tendon collagens. Increased hydroxylation of proline in the embryo was, however, readily observed in peptide alpha(1)-CB2 from the helical region of tendon collagen. This hydroxylation was close to the theoretical maximum, in contrast with that observed in post-embryonic tendon, where hydroxylation was incomplete, as in rat tendon (Bornstein, 1967), only four on average, of the six susceptible proline residues being hydroxylated.     

Characterization of recombinant human prolyl 3-hydroxylase isoenzyme 2, an enzyme modifying the basement membrane collagen IV

The single 3-hydroxyproline residue in the collagen I polypeptides is essential for proper fibril formation and bone development as its deficiency leads to recessive osteogenesis imperfecta. The vertebrate prolyl 3-hydroxylase (P3H) family consists of three members, P3H1 being responsible for the hydroxylation of collagen I. We expressed human P3H2 as an active recombinant protein in insect cells. Most of the recombinant polypeptide was insoluble, but small amounts were also present in the soluble fraction. P3H1 forms a complex with the cartilage-associated protein (CRTAP) that is required for prolyl 3-hydroxylation of fibrillar collagens. However, coexpression with CRTAP did not enhance the solubility or activity of the recombinant P3H2. A novel assay for P3H activity was developed based on that used for collagen prolyl 4-hydroxylases (C-P4H) and lysyl hydroxylases (LH). A large amount of P3H activity was found in the P3H2 samples with (Gly-Pro-4Hyp)5 as a substrate. The Km and Ki values of P3H2 for 2-oxoglutarate and its certain analogues resembled those of the LHs rather than the C-P4Hs. Unlike P3H1, P3H2 was strongly expressed in tissues rich in basement membranes, such as the kidney. P3H2 hydroxylated more effectively two synthetic peptides corresponding to sequences that are hydroxylated in collagen IV than a peptide corresponding to the 3-hydroxylation site in collagen I. These findings suggest that P3H2 is responsible for the hydroxylation of collagen IV, which has the highest 3-hydroxyproline content of all collagens. It is thus possible that P3H2 mutations may lead to a disease with changes in basement membranes.