Molecular insights into prolyl and lysyl hydroxylation of fibrillar collagens in health and disease

Collagen is a macromolecule that has versatile roles in physiology, ranging from structural support to mediating cell signaling. Formation of mature collagen fibrils out of procollagen α-chains requires a variety of enzymes and chaperones in a complex process spanning both intracellular and extracellular post-translational modifications. These processes include modifications of amino acids, folding of procollagen α-chains into a triple-helical configuration and subsequent stabilization, facilitation of transportation out of the cell, cleavage of propeptides, aggregation, cross-link formation, and finally the formation of mature fibrils. Disruption of any of the proteins involved in these biosynthesis steps potentially result in a variety of connective tissue diseases because of a destabilized extracellular matrix. In this review, we give a revised overview of the enzymes and chaperones currently known to be relevant to the conversion of lysine and proline into hydroxyproline and hydroxylysine, respectively, and the O-glycosylation of hydroxylysine and give insights into the consequences when these steps are disrupted.     

Subcellular localization of procollagen I and prolyl 4-hydroxylase in corneal endothelial cells

To investigate the molecular mechanism of intracellular degradation of type I collagen in normal corneal endothelial cells (CEC), we studied the role of prolyl 4-hydroxylase (P4-H) and protein disulfide-isomerase (PDI; the beta subunit of P4-H) during procollagen I biosynthesis. When the subcellular localization of P4-H and PDI was determined, P4-H demonstrated a characteristic diffuse endoplasmic reticulum (ER) pattern, whereas PDI showed a slightly more restricted distribution within the ER. When colocalization of procollagen I with the enzymes was examined, procollagen I and PDI showed a large degree of colocalization. P4-H and procollagen I were predominantly colocalized at the perinuclear site. When colocalization of type IV collagen with PDI and P4-H was examined, type IV collagen was largely colocalized with PDI, which showed a wider distribution than type IV collagen. Type IV collagen is similarly colocalized with P4-H, except in some perinuclear sites. The colocalization profiles of procollagen I with both PDI and P4-H were not altered in cells treated with alpha,alpha'-dipyridyl compared to those of the untreated cells. The underhydroxylated type IV collagen demonstrated a colocalization profile with PDI similar to that observed with procollagen I, while the underhydroxylated type IV collagen was predominantly colocalized with P4-H at the perinuclear sites. Immunoblot analysis showed no real differences in the amounts of the beta subunit/PDI and the catalytic alpha subunit of P4-H in CEC compared to those of corneal stromal fibroblasts (CSF). When protein-protein association was determined, procollagen I was associated with PDI much more in CEC than it was in CSF, whereas type IV collagen showed no differential association specificity to PDI in both cells. Limited proteolysis of the newly synthesized intracellular procollagen I with pepsin showed that procollagen I in CEC was degraded by pepsin, whereas CSF contained type I collagen composed of alpha1(I) and alpha2(I). These findings suggest that procollagen I synthesized in CEC is not in triple helical conformation and that the improperly folded procollagen I may be preferentially associated with PDI before targeting to the intracellular degradation.     

Lysine hydroxylation of collagen in a fibroblast cell culture system

The lysine (Lys) hydroxylation pattern of type I collagen produced by human fibroblasts in culture was analyzed and compared. Fibroblasts were cultured from normal human skin (NSF), keloid (KDF), fetal skin (FDF), and skin tissues of Ehlers-Danlos syndrome type VIA and VIB patients (EDS-VIA and -VIB). The type I collagen alpha chains with or without non-helical telopeptides were purified from the insoluble matrix and analyzed. In comparison with NSFs, KDF and FDF showed significantly higher Lys hydroxylation, particularly in the telopeptide domains of both alpha chains. Both EDS-VIA and -VIB showed markedly lower Lys hydroxylation in the helical domains of both alpha chains whereas that in the telopeptides was comparable with those of NSFs. A similar profile was observed in the tissue sample of the EDS-VIB patient. These results demonstrate that the Lys hydroxylation pattern is domain-specific within the collagen molecule and that this method is useful to characterize the cell phenotypes in normal/pathological connective tissues.     

Identification of prolyl hydroxylation modifications in mammalian cell proteins

Prolyl hydroxylation is a PTM that plays an important role in the formation of collagen fibrils and in the oxygen‐dependent regulation of hypoxia inducible factor‐α (HIF‐α). While this modification has been well characterized in the context of these proteins, it remains unclear to what extent it occurs in the remaining mammalian proteome. We explored this question using MS to analyze cellular extracts subjected to various fractionation strategies. In one strategy, we employed the von Hippel Lindau tumor suppressor protein, which recognizes prolyl hydroxylated HIF‐α, as a scaffold for generating hydroxyproline capture reagents. We report novel sites of prolyl hydroxylation within five proteins: FK506‐binding protein 10, myosin heavy chain 10, hexokinase 2, pyruvate kinase, and C‐1 Tetrahydrofolate synthase. Furthermore, we show that identification of prolyl hydroxylation presents a significant technical challenge owing to widespread isobaric methionine oxidation, and that manual inspection of spectra of modified peptides in this context is critical for validation.     

Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the hydroxylation of -X-Pro-Gly- sequences and plays a central role in the synthesis of all collagens. The [alpha(I)]2beta2 type I enzyme is effectively inhibited by poly(L-proline), whereas the [alpha(II)]2beta2 type II enzyme is not. We report here that the poly(L-proline) and (Pro-Pro-Gly)10 peptide substrate-binding domain of prolyl 4-hydroxylase is distinct from the catalytic domain and consists of approximately 100 amino acids. Peptides of 10-19 kDa beginning around residue 140 in the 517 residue alpha(I) subunit remained bound to poly(L-proline) agarose after limited proteolysis of the human type I enzyme tetramer. A recombinant polypeptide corresponding to the alpha(I) subunit residues 138-244 and expressed in Escherichia coli was soluble, became effectively bound to poly(L-proline) agarose and could be eluted with (Pro-Pro-Gly)10. This polypeptide is distinct from the SH3 and WW domains, and from profilin, and thus represents a new type of proline-rich peptide-binding module. Studies with enzyme tetramers containing mutated alpha subunits demonstrated that the presence of a glutamate and a glutamine in the alpha(II) subunit in the positions corresponding to Ile182 and Tyr233 in the alpha(I) subunit explains most of the lack of poly(L-proline) binding of the type II prolyl 4-hydroxylase. Keywords: collagen/dioxygenases/peptide-binding domain/ proline-rich/prolyl hydroxylase     

Differential and Reciprocal Regulation between Hypoxia-inducible Factor-α Subunits and Their Prolyl Hydroxylases in Pulmonary Arteries of Rat with Hypoxia-induced Hypertension

Hypoxia-inducible factor (HIF)-α subunits (HIF-1α,HIF-2α and HIF-3α),which play a pivotalrole during the development of hypoxia-induced pulmonary hypertension (HPH),are regulated through post-U'anslational hydroxylation by their three prolyl hydroxylase domain-containing proteins (PHD 1,PHD2 and PHD3).PHDs could also be regulated by HIF.But differential and reciprocal regulation between HIF-α and PHDs duringthe development of HPH remains unclear.To investigate this problem,a rat HPH model was established.Meanpulmonary arterial pressure increased significantly after 7 d of hypoxia.Pulmonary artery remodeling indexand right ventricular hypertrophy became evident after 14 d of hypoxia.HIF-1α and HIF-2α mRNA increasedslightly after 7 d of hypoxia,but HIF-3α increased significantly after 3 d of hypoxia.The protein expressionlevels of all three HIF-α were markedly upregulated after exposure to hypoxia.PHD2 mRNA and proteinexpression levels were upregulated after 3 d of hypoxia;PHD 1 protein declined after 14 d of hypoxia withoutsignificant mRNA changes.PHD3 mRNA and protein were markedly upregulated after 3 d of hypoxia,then themRNA remained at a high level,but the protein declined after 14 d of hypoxia.In hypoxic animals,HIF-lotproteins negatively correlated with PHD2 proteins,whereas HIF-2α and HIF-3α proteins showed negativecorrelations with PHD3 and PHD 1 proteins,respectively.All three HIF-α proteins were positively correlatedwith PHD2 and PHD3 mRNA.In the present study,HIF-α subunits and PHDs showed differential andreciprocal regulation,and this might play a key pathogenesis role in hypoxia-induced pulmonary hypertension.     

Prolyl and lysyl hydroxylase activation and cofactor specificity in young and senescent WI-38 fibroblast cultures.

The activation of prolyl hydroxylase and lysyl hydroxylase by ascorbate was studied in young and senescent WI-38 fibroblast cultures using a tritium-release assay. Prolyl hydroxylase activity could be increased 3–4 fold in young cultures but remained unchanged in senescent cultures when these cultures underwent a two-hour preincubation in medium containing 0.2mM sodium ascorbate. Lysyl hydroxylase levels were unaffected both in young and senescent cultures. In another series of experiments, ascorbate was replaced with several other compounds in the tritium-release assay demonstrating that this reducing agent is not a specific cofactor of the partially purified enzymes from WI-38 cultures.     

Subcellular fractionation of WI-38 fibroblasts: Compatison between young and old cells

WI-38 fibroblasts cultivated in vitro were homogenized and their subcellular organelles analysed by the techniques of differential centrifugation and isopycnic equilibration in density gradient. In these experiments, the assayed enzymes were known to be specifically associated with subcellular components in other cells types. In most cases, their behaviour and properties corresponded with observations made in earlier studies and we could consider them as being representative of the specific subcellular organelles.Some significant differences were observed between young and old fibroblasts. The specific activity of alkaline phosphodiesterase was lower in the old cells whereas for the other enzymes it was identical or higher, especially for the 5′-nucleotidase; also the particulate fractions obtained by differential centrifugation contained more material. After equilibration in density gradient, the average density of the 5′-nucleotidase, alkaline phosphodiesterase and $\text{N-}\text{acetyl}\text{-β-}\text{D}\text{-glucosaminidase}$ was less in the old than in the young cells, whereas that of the galactosyltransferase of Golgi apparatus was greater. For mitochondria, endolasmic reticulum and peroxisomes, the differences observed were small.     

Inhibition of prolyl 4-hydroxylase by oxaproline tetrapeptidesin vitro and mass analysis for the enzymatic reaction products

A series of 5-oxaproline peptide derivatives was synthesized and evaluated for its ability to inhibit the prolyl 4-hydroxylasein vitro. Structure-activity studies show that the 5-oxaproline sequences, prepared by the 1,3-dipolar cycloaddition of the C-methoxycarbonyl-N-mannosyl nitrone in the presence of the ethylene, are more active than the corresponding proline derivatives. Prolyl 4-hydroxylase belongs to a family of Fe²⁺-dependent dioxygenase, which catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in-Gly-Xaa-Pro-Gly- of procollagen chains. In this paper we discover the more selective N-Cbz-Gly-Phe-Pro-Gly-OEt (K _m=520 μM) sequences which are showed stronger binding than othersin vitro. Therefore, we set out to investigate constrained tetrapeptide that was designed to mimic the proline structure of peptides for the development of prolyl 4-hydroxylase inhibitor. From this result, we found that the most potent inhibitor is N-Dansyl-Gly-Phe-5-oxaPro-Gly-OEt (K _i=1.6 μM). This has prompted attempts to develop drugs which inhibit collagen synthesis. Prolyl 4-hydroxylase would seem a particularly suitable target for antifibrotic therapy.     

Neuronal apoptosis by prolyl hydroxylation: implication in nervous system tumours and the Warburg conundrum

Oxygen-sensing mechanisms are often dysfunctional in tumours. Oxygen sensing is mediated partly via prolyl hydroxylation. The EglN prolyl hydroxylases are well characterized in regulating the hypoxia inducible factor α (HIF-α) hypoxic response, but also are implicated in HIF-independent processes. EglN3 executes apoptosis in neural precursors during development and failure of EglN3 developmental apoptosis can lead to certain forms of sympathetic nervous system tumours. Mutations in metabolic/mitochondrial enzymes (SDH, FH, IDH) impair EglN activity and predisposes to certain cancers. This is because the EglNs not only require molecular oxygen to execute hydroxylation, but also equally require the electron donor α-ketoglutarate, a metabolite from the Krebs cycle. Therefore EglN enzymes are considered oxygen, and also, metabolic sensors. α-Ketoglutarate is crucial for EglN hydroxylation activity, whereas the metabolites succinate and fumarate are inhibitors of the EglN enzymes. Since EglN activity is dependent upon metabolites that take part in the Krebs cycle, these enzymes are directly tied into the cellular metabolic network. Cancer cells tend to convert most glucose to lactate regardless of whether oxygen is present (aerobic glycolysis), an observation that was first made by Otto Warburg in 1924. Despite the striking difference in ATP production, cancer cells might favour aerobic glycolysis to escape from EglN hydroxylation, resulting in the accumulation of oncogenic HIFα and/or resistance to EglN3-mediated apoptosis.     

Regulation of fibroblast proliferation in three-dimensional collagen gel matrix

Summary Fibroblastsin vivo reside in a three-dimensional (3-D) matrix. The 3-D culture method using collagen gels provides valuable information, but is also has some practical difficulties. In particular, the changes caused by the contraction of gels and the occasional abrupt detachment from the underlying surface have made extended culture difficult. In this study, the 3-D culture method was modified in order to observe the cells with minimal change of substrata for longer periods. The proliferation characteristics of fibroblasts cultured in gels in response to fetal calf serum (FCS), to two defined growth factors, insulin and platelet-derived growth factor (PDGF), and to a growth inhibitory factor, prostaglandin E₂ (PGE₂), were evaluated with this system in comparison with monolayer cultured fibroblasts. The DNA content of fibroblasts cultured both in gels and on dishes increased in response to FCS in a concentration-dependent manner. The proliferation of gel-cultured fibroblasts, however, was lower than that of dish-cultured cells, and higher concentrations of serum were necessary for proliferation. The response of gel-cultured cells to PDGF was also less than that of dish-cultured cells. In addition, fibroblasts cultured in gel culture did not respond to insulin, while the fibroblasts on dishes responded to insulin in a concentration-dependent manner. In contrast to the reduced response to growth stimulators, PGE₂ inhibited proliferation in gel culture and in monolayer culture similarly. The reduced responsiveness to growth stimulation but equivalent response to growth inhibition may account for reduced proliferation of fibroblasts in 3-D culture.     

Expression of p53 mRNA is proliferation-dependent in the human fibroblast cell line WI-38

Abstract. Although alterations in the p53 tumour suppressor gene are one of the most frequent genetic lesions occurring in human cancers, the exact function and mechanism of action of normally regulated p53 in the control of cell cycle is unclear. To clanfy further the possible role of this gene in the control of cell proliferation, the cellular level of p53-specific mRNA and its changes during density-dependent growth, and in different proliferation states induced by serum starvation and subsequent serum-stimulation, were followed in WI-38 cells, a normal human diploid fibroblast cell line. Marked differences in the expression of p53 mRNA could be observed in the different proliferation states tested. The pattern of p53 expression proved to be inversely proportional to the growth-rate of the cultures. mRNA was considerably more abundant when cells reached confluency or were arrested by serum deprivation while serum-stimulation caused the opposite effect. These results support the hypothesis that the p53 gene plays a role in G₁ control of normal cell proliferation.     

Comparative metabolic effects of fructose and glucose in human fibroblast cultures

Summary The comparative metabolic effects of fructose and glucose were determined in human fibroblast cultures. Cells were grown in four different media containing 5.5 and 27.5 mM of glucose and fructose, respectively. For these two hexoses, we compared their uptake, consumption, and conversion into¹⁴CO₂ and¹⁴C-lipids. D-Fructose was taken up in fibroblasts by an unsaturable process and its consumption was much smaller than that ofD-glucose. Whatever the experimental procedure, the glycogen content of cells grown in fructose media was significantly lower than of those grown in glucose media. Labeling of fructose and glucose with¹⁴C showed that more carbon from fructose than from glucose was incorporated into CO₂ and glycerolipids. The relative distribution of¹⁴C in the different lipid fractions was similar for both hexoses. These results indicated that the pathways of intermediary metabolism in fibroblast cultures were influenced by the nature of the carbohydrate present in the culture medium and that fructose was a better lipogenic substrate than glucose in human fibroblast cultures. This work was supported by grants for the Institut National de la Santé et de la Recherche Medicale (ATP 82-79-114).     

IGF-1 modulation of rat cardiac fibroblast behavior and gene expression is age-dependent

The collagenous extracellular matrix (ECM) forms a stress-tolerant network that is essential for proper function of the vertebrate heart. Profound changes have been detected in the interstitial ECM concurrent with developmental and disease processes of the heart. These alterations in either the organization or accumulation of ECM components markedly affect myocardial function. Studies have shown that a number of biochemical factors, including angiotensin II, transforming growth factor-β, and insulin-like growth factors, modulate collagen expression by heart fibroblasts, however, few studies have examined the differential effects of these factors on fibroblasts from animals of different physiological backgrounds. The present studies were carried out to determine whether cardiac fibroblasts isolated from different aged animals (fetal, neonatal, and adult) have diverse responses to insulin-like growth factor-1 (IGF-1). Fibroblasts isolated from fetal, neonatal, and adult rat hearts were treated with IGF-1, and several downstream responses were measured, including collagen gel contraction, adhesion to ECM, and expression of interstitial collagen and integrins. IGF-1 affected these parameters to different degrees, depending on the age of the animal from which the fibroblasts were isolated. These experiments indicate that IGF-1 is a potent modulator of fibroblast behavior in general; however, significant differences are apparent in the responsiveness of cells to this growth factor depending on the age of the animal of origin. Future experiments will be directed at determining how the in vivo chemical and biomechanical environment affects the response of heart fibroblasts to growth factors such as IGF-1.     

The purification of plasma membranes from WI-38 fibroblasts Effects of ageing on their composition

A three-step method for the purification of plasma membranes from WI-38 fibroblasts was developed thus allowing the recovery of 36–44% of the plasma membrane. Except in the case of galactosyltransferase, the activity of the contaminating enzymes was very low. Morphological observations confirm the presence of a homogeneous population of vesicles.Preparations obtained from young and old cell cultures were compared for their enzymatic and protein contents. With ageing the activity of 5′-nucleotidase significantly increases whereas that of alkaline phosphodiesterase I decreases. Out of the 26 components detected after sodium dodecyl sulphate polyacrylamide gel electrophoresis, four decreased but only one increased. Cellular ageing seems to fulfil a specific and localized effect on the plasma membrane.