Hydroxylation Of Proline Residues Research Articles

The structure and function of surfactant protein A. Hydroxyproline- and carbohydrate-deficient mutant proteins.

Pulmonary surfactant protein A (SP-A) regulates the uptake and secretion of phospholipid by alveolar type II cells and is an important component of surfactant lipid aggregates. In an attempt to understand how specific structural domains of SP-A relate to the function of the protein, we used site-directed mutagenesis of the cDNA for SP-A and heterologous expression with baculovirus vectors. Synthesis of the wild type SP-A in insect cells resulted in a form of the protein in which proline residues were not hydroxylated and that is denoted SP-Ahyp. Three mutant SP-As with substitutions in the consensus sequences for glycosylation (SP-Ahyp,glc) to prevent N-linked oligosaccharide attachment at Asn1 and Asn187 were produced, individually and in tandem. The SP-Ahyp was glycosylated at both the Asn1 and Asn187 positions, demonstrated partial sulfhydryl-dependent oligomerization, and formed incomplete oligomers in solution. The SP-Ahyp and SP-Ahyp,glc bound to immobilized carbohydrate and to phospholipid liposomes and partially competed for occupancy of a plasma-membrane receptor for SP-A. The SP-Ahyp and the SP-Ahyp,glc were equally effective inhibitors of the secretion of surfactant lipids from isolated type II cells (IC50 = 0.5 microgram/ml) and aggregated phospholipid liposomes at 20 degrees C. All of the recombinant SP-As demonstrated markedly reduced aggregation of lipid at 37 degrees C. We conclude that the hydroxylation of proline residues is required for perfect oligomerization of SP-A and for thermal stability in the interaction with lipid. Furthermore, recombinant SP-A is able to inhibit the secretion of phospholipid from isolated type II cells and to aggregate lipid vesicles independent of the presence of N-linked carbohydrate or the site of glycosylation.

Prolyl 4-hydroxylase and its role in collagen synthesis.

Excessive accumulation of collagen in the extracellular matrix has a crucial role in fibrosis. Thus pharmacological inhibition of collagen deposition is likely to be beneficial for patients suffering from fibrotic disorders such as liver cirrhosis. Prolyl 4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens and other proteins with collagen-like amino acid sequences by the hydroxylation of proline residues in -X-Pro-Gly- sequences. The reaction products, 4-hydroxyproline residues, serve to stabilize the collagen triple helices under physiological conditions. Conversely, collagen chains that contain no 4-hydroxyproline cannot fold into triple helical molecules that are stable at body temperature. The prolyl 4-hydroxylase reaction therefore seems to be a particularly suitable target for the pharmological regulation of excessive collagen formation. The reaction catalyzed by prolyl 4-hydroxylase requires Fe2+, 2-oxoglutarate, O2 and ascorbate and involves an oxidative decarboxylation of 2-oxoglutarate. The active enzyme is an alpha 2 beta 2 tetramer that consists of two types of inactive monomer and has two catalytic sites. Some parts of the catalytic sites may be built up cooperatively of both the alpha and beta subunits, but the alpha subunit appears to contribute the major part. The beta subunit contains the carboxyl-terminal tetrapeptide sequence -Lys-Asp-Glu-Leu which is essential for the retention of a polypeptide within the lumen of the endoplasmic reticulum. Since the alpha subunit lacks the carboxyl-terminal retention signal, one function of the beta subunit in the prolyl 4-hydroxylase tetramer may be to retain the enzyme within the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional assignment of the human gene coding for a multifunctional polypeptide (P4HB) acting as the beta-subunit of prolyl 4-hydroxylase and the enzyme protein disulfide isomerase to 17q25.

Prolyl 4-hydroxylase, an alpha 2 beta 2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens and related proteins by hydroxylating proline residues in peptide linkages. The beta-subunit of prolyl 4-hydroxylase (P4HB) is a highly unusual multifunctional polypeptide that is identical to the enzyme protein disulfide isomerase and a major cellular thyroid hormone-binding protein and is highly similar to a glycosylation site-binding polypeptide of oligosaccharyl transferase. We report here the regional assignment of the gene for this multifunctional polypeptide. In situ hybridization mapped the gene to 17q25. Southern blot analyses of restricted DNA from a chromosome-mediated gene transfer transfectant panel suggested that the P4HB gene is located distal to the gene for thymidine kinase, either between the genes for thymidine kinase and galactokinase or on the telomeric side of both these genes.

Increases in mRNA concentrations of the alpha and beta subunits of prolyl 4-hydroxylase accompany increased gene expression of type IV collagen during differentiation of mouse F9 cells.

Mouse F9 teratocarcinoma stem cells differentiate in monolayer cultures in the presence of retinoic acid, dibutyryl cAMP, and isobutyl methylxanthine. This differentiation is associated with a marked increase in the synthesis rates and mRNA concentrations of basement membrane proteins such as type IV collagen. We report here that the differentiation also involves an increase of up to 50-fold in the concentrations of the mRNAs for the alpha and beta subunits of prolyl 4-hydroxylase, the enzyme required for the cotranslational and post-translational hydroxylation of proline residues in collagens. The time courses and magnitudes of increases in these two mRNA concentrations were similar to those observed in the same experiments for the mRNA of the alpha chain of type IV collagen. In the differentiated F9 cells the concentration of the alpha subunit mRNA was about 30% of the beta subunit mRNA concentration. Northern blot analyses indicated that the sizes of the alpha and beta subunit mRNAs in the differentiated mouse F9 cells are similar to those in human skin fibroblasts. The F9 cell differentiation system appears to provide a useful model for studies on the regulation of prolyl 4-hydroxylase synthesis.

Protein Disulphide Isomerase, A Multifunctional Endoplasmic Reticulum Protein

Protein disulphide isomerase (E.C. 5.3.4.1) has been' purified, cloned, and sequenced from a variety of vertebrate tissues. The enzyme and its isoforms have been assigned a role in four functional activities: (1) hydroxylation of proline residues in procollagen; (2) disulphide bond oxidation, isomerization, and reduction; (3) the major non-nuclear binding protein of the thyroid hormone 3,3',5-triiodo-L-thryonine; and (4) a component of oligosaccharide transferase. The concentration of the enzyme has been shown to be positively correlated with an endoplasmic reticulum network which is active in secreting disulphide-bonded polypeptides. The enzyme is directed into the endoplasmic reticulum by virtue of a 19 residue N-terminal signal peptide; a four amino acid C-terminal KDEL sequence prevents the enzyme from being secreted. Careful inspection of the sequence data of the isoforms from human tissues reveals a 97% similarity; whereas, analyses of the data from chick tissues reveals only a 80% level of similarity. Chromosomal localizations using human cDNA probes against different human isoforms have assigned the gene(s) to opposite ends of the long arm of chromosome 17. The compiled data suggest the presence of a family of related polypeptides, all of which reside within the lumen of the endoplasmic reticulum.

Molecular cloning of the alpha-subunit of human prolyl 4-hydroxylase: the complete cDNA-derived amino acid sequence and evidence for alternative splicing of RNA transcripts.

Prolyl 4-hydroxylase [procollagen-proline, 2-oxyglutarate 4-dioxygenase; procollagen-L-proline, 2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2], an alpha 2 beta 2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here on the isolation of cDNA clones encoding the alpha-subunit of the enzyme from human tumor HT-1080, placenta, and fibroblast cDNA libraries. Eight overlapping clones covering almost all of the corresponding 3000-nucleotide mRNA, including all the coding sequences, were characterized. These clones encode a polypeptide of 517 amino acid residues and a signal peptide of 17 amino acids. Previous characterization of cDNA clones for the beta-subunit of prolyl 4-hydroxylase has indicated that its C terminus has the amino acid sequence Lys-Asp-Glu-Leu, which, it has been suggested, is necessary for the retention of a polypeptide within the lumen of the endoplasmic reticulum. The alpha-subunit does not have this C-terminal sequence, and thus one function of the beta-subunit in the prolyl 4-hydroxylase tetramer appears to be to retain the enzyme within this cell organelle. Interestingly, three of the cDNA clones for the alpha-subunit contained a 64-nucleotide sequence homologous but not identical to the corresponding 64-nucleotide sequence found in four other cDNA clones. Nuclease S1 mapping experiments demonstrated that this difference was due to the existence of two types of mRNA present in approximately equal amounts. Southern blot analyses of human genomic DNA with a cDNA probe for the alpha-subunit suggested the presence of only one gene encoding the two types of mRNA, which appear to result from mutually exclusive alternative splicing of primary transcripts of one gene.

Protein hydroxylation: prolyl 4‐hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in X-Pro-Gly sequences. The reaction requires Fe2+, 2-oxoglutarate, O2, and ascorbate and involves an oxidative decarboxylation of 2-oxoglutarate. Ascorbate is not consumed during most catalytic cycles, but the enzyme also catalyzes decarboxylation of 2-oxoglutarate without subsequent hydroxylation, and ascorbate is required as a specific alternative oxygen acceptor in such uncoupled reaction cycles. A number of compounds inhibit prolyl 4-hydroxylase competitively with respect to some of its cosubstrates or the peptide substrate, and recently many suicide inactivators have also been described. Such inhibitors and inactivators are of considerable interest, because the prolyl 4-hydroxylase reaction would seem a particularly suitable target for chemical regulation of the excessive collagen formation found in patients with various fibrotic diseases. The active prolyl 4-hydroxylase is an alpha 2 beta 2 tetramer, consisting of two different types of inactive monomer and probably containing two catalytic sites per tetramer. The large catalytic site may be cooperatively built up of both the alpha and beta subunits, but the alpha subunit appears to contribute the major part. The beta subunit has been found to be identical to the enzyme protein disulfide isomerase and a major cellular thyroid hormone-binding protein and shows partial homology with a phosphoinositide-specific phospholipase C, thioredoxins, and the estrogen-binding domain of the estrogen receptor. The COOH-terminus of this beta subunit has the amino acid sequence Lys-Asp-Glu-Leu, which was recently suggested to be necessary for the retention of a polypeptide within the lumen of the endoplasmic reticulum. The alpha subunit does not have this COOH-terminal sequence, and thus one function of the beta subunit in the prolyl 4-hydroxylase tetramer appears to be to retain the enzyme within this cell organelle.

HYPERPURIFICATION OF PAIRED HELICAL FILAMENTS REVEALS ELEVATIONS IN HYDROXYPROLINE CONTENT AND A CORE STRUCTURE RELATED PEPTIDE FRAGMENT

We have subjected conventionally-purified Alzheimer's paired helical filaments (PHF) to electrophoresis in a Tris/borate/SDS buffer and obtained the separation of PHF core protein(s) (PHFi) from solubilized PHF-associated proteins (PHFs). Electron microscopy revealed intact PHF structures before and after this separation, but no evidence of any other structures in the PHFi fraction. The percent mass of hydroxyproline and glycine increased in the PHFi fraction after 4.5 hr of electrophoresis to account for 5.8% and 13.6% of the total mass, respectively. ELISA data confirmed that our PHFi and PHFs fractions were reactive with several putative PHF-specific antibodies. These data suggest that inappropriate hydroxylation of proline residues occurs in precursor PHF protein(s), resulting in the polymerization and subsequent insolubility of PHF in brain regions affected with Alzheimer's disease. Neurofibrillary tangles (NFT), one of the primary neuropathological features of Alzheimer's disease, are comprised of cytosolic bundles of uniform proteins which microscopically appear to be paired helical filaments (PHF). PHF are thought to be responsible for the cellular necrosis associated with the clinical symptoms of Alzheimer's disease (Dayan, 1970; Hirano and Zimmerman, 1962). Optical reconstruction of PHF has recently indicated that the true structure is more accurately described as a twisted ribbon of 30 A in the axial direction (Wischik et al., 1988). Immunological studies have suggested that tubulin (Grundke-Iqbal et al., 1979), microtubule associated proteins (Grundke-Iqbal et al., 1986; Kosik et al., 1986; Wood et al., 1986; Ksiezak-Reding et al., 1987), intermediate filaments (Yen et al., 1983), neurofilaments (Anderton et al., 1982), and ubiquitin (Mori et al., 1987; Perry et al., 1987), form part of the PHF core protein. To date, however, no study has been able to definitively show that any one of these purported PHF components is contained in the PHF core structure, rather than being non-covalently bound proteins or co-purifying contaminants. The present study extends previously published purification procedures, resulting in an isolated stable PHF core structure which is indistinguishable from previously published descriptions of PHF. The amino acid composition (Bidlingmeyer et al., 1984) of the PHF core structure appears to include hydroxyproline, an amino acid not commonly found in cytosolic proteins. We also demonstrate that some monoclonal antibodies previously raised against semi-purified PHF recognize determinants which are not related to the PHF core structure but rather recognize PHF-associated proteins or contaminants of the purification process.

Molecular cloning of the beta-subunit of human prolyl 4-hydroxylase. This subunit and protein disulphide isomerase are products of the same gene.

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyses the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here the isolation of cDNA clones coding for the beta-subunit of prolyl 4-hydroxylase from a human hepatoma lambda gt11 library and a corresponding human placenta library. Five overlapping clones covering all the coding sequences and almost all the non-coding sequences were characterized. The size of the mRNA hybridizing with these clones in Northern blotting is approximately 2.5 kb. The clones encode a polypeptide of 508 amino acid residues, including a signal peptide of 17 amino acids. These human sequences were found to be very similar to those recently reported for rat protein disulphide isomerase (EC 5.3.4.1). The degree of homology between these two proteins was 84% at the level of nucleotide sequences or 94% at the level of amino acid sequences. Southern blot analyses of human genomic DNA with a cDNA probe for the beta-subunit indicated the presence of only one gene containing these sequences. The product of a single gene thus appears to possess two different enzymatic functions depending on whether it is present in cells in monomer form or in the prolyl 4-hydroxylase tetramer.

Procollagen secretion meets the minimum requirements for the rate-controlling step in the ascorbate induction of procollagen synthesis.

Ascorbate addition to primary avian tendon cells has been shown previously to cause a approximately 6-fold increase in procollagen translation that is first observable after 4 h and reaches a maximum level after 48 h. Similarly, procollagen mRNA has been shown to increase after ascorbate addition by approximately 6-fold starting at 12 h and reaching a maximum level by 72 h. The rate constant for procollagen secretion is now shown to also react to ascorbate by a 6-fold change. This results in a drop in the half-life of procollagen within the cell from 120 to 20 min. In sharp contrast to the other steps in the procollagen pathway, the change in the secretion rate constant is extremely fast occurring in less than 30 min. Moreover, after ascorbate addition, greater than 80% of the internal procollagen can be secreted at the fast rate. Since this change results from an increase in hydroxylation of proline residues and since the hydroxylation reaction has been localized to the endoplasmic reticulum, this evidence strongly supports the model that the slow step in the secretion pathway is transport out of the endoplasmic reticulum. Further support for this comes from electron microscope autoradiography of [3H]proline-labeled cells where the labeled procollagen pool within the cells was highly localized to the endoplasmic reticulum.

Decreased procollagen production in cultured fibroblasts from patients with Lowe's syndrome.

The oculo-cerebro-renal syndrome described by Lowe is a catastrophic disease in children characterized by progressive eye, central nervous system and kidney degeneration. We determined procollagen production in cultured skin fibroblasts originating from patients with Lowe's syndrome as well as normal individuals after incubation of cells with [14C]proline for 1,4 and 20 h. Using [14C]hydroxyproline formation, in relation to cell protein or DNA, as an index of procollagen production, we found that cultured cells from patients synthesized collagenous protein at a substantially reduced level. This decrease in synthesis in cultured fibroblasts from patients could not be accounted for by differences in the specific radioactivity of the free intracellular proline; moreover, it could not be attributed to differences in the kinetics of growth between normal and affected cell lines. The degree of hydroxylation of proline residues in collagen was the same in both normal and affected cells.

Influence of cell density on collagen biosynthesis in fibroblast cultures.

Characteristic features of collagen metabolism in human skin fibroblasts were studied in relation to cell density. Measuring peptide-bound hydroxyproline we found that collagen synthesis per cell decreased when cultures approached confluency. On the other hand, the relative rate of collagen synthesis (collagen/total protein) was higher in quiescent than in proliferating cultures. With increasing cell density the proportion of type III collagen in comparison with type I was found to be slightly increased. In addition, in low-density cultures [alpha I(I)]3 collagen trimers were produced in considerable amounts, whereas they were no longer detected in cultures with a high cell density. Although hydroxylation of proline residues was normal in all cell stages, conversion of procollagen into collagen was found to depend strongly on the density at which the cells were investigated. Almost no cleavage of procollagen peptides was observed in rapidly growing cells, whereas highly confluent cell cultures converted most of the newly synthesized procollagen molecules.

Interaction of polypeptide models of elastin with prolyl hydroxylase

Significant amounts of hydroxyproline occur in the structural proteins collagen and elastin which usually exist in close association and are presumably synthesized by the same cells. Hydroxyproline in collagen is synthesized by the hydroxylation of proline residues in -X-Pro-Glysequences, by prolyl hydroxylase (EC 1.14.11.2) in a concerted reaction in which an obligatory co-substrate, o-ketoglutarate is oxidatively decarboxylated (reviewed in [I]). Much of the proline in elastin occurs in -Pro-Glysequences and therefore is potentially hydroxylatable by prolyl hydroxylase. Both collagen and elastin contain repeating sequences. We have examined the interaction of synthetic polymeric models of elastin with prolyl hydroxylase. Our studies suggest that proline in elastin-like polymers is efticiently hydroxylated under conditions where these polymers exhibit preferred secondary and tertiary structures. These studies explain the presence of hydroxyproline in the non-polar polymeric regions of elastin [2].

Self-assembly of collagen.

AbstractThe structure and self‐assembly of collagen and procollagen molecules are reviewed. The registration peptides of procollagen have specific recognition properties which assure (1) selection of component polypeptide chains and (2) registration of their N‐termini, facilitating orderly folding into a collagen helix. The stability of this helix relative to body temperature is critically altered by post‐ribosomal hydroxylation of proline residues. The registration peptides of procollagen may have additional functions such as preventing intracellular fiber formation.

Collagen biosynthesis: synthesis and secretion of a high molecular weight collagen precursor (procollagen).

Sodium dodecyl sulfate-acrylamide gel electrophoresis and molecular-sieve chromatography on 8% agarose demonstrate the existence of a very high molecular weight (500,000-600,000), proline-rich protein in cultured 3T6 fibroblasts that appears to be the precursor molecule (procollagen) of collagen. The kinetics of [(3)H]-proline uptake indicate that this precursor is synthesized at a different rate than are other cell proteins and is secreted apparently unchanged into the medium, where it undergoes modification; it then precipitates around the cells as collagen fibrils that contain the characteristic tropocollagen polypeptide chains. The solubility of this precursor in hot 5% Cl(3)CCOOH, its hydroxyproline to proline ratio, and its sensitivity to highly-purified bacterial collagenase all indicate that this molecule is of collagenous nature, but that it has considerable regions of noncollagen peptide (about half of the molecule is collagenase sensitive, and it has half of the normal amount of hydroxylated proline residues). These results support the concept of a procollagen molecule, of molecular weight about 500,000-600,000, that contains large regions of noncollagen peptides, which might allow the collagen alpha-chain regions to associate in register and that also can provide the correct chain composition for tropocollagen. Upon secretion of the procollagen molecule, the intercollagen-peptide regions are cleaved and the finished tropocollagen molecule then polymerizes into typical intercellular fibrils.

Hydroxylation of proline residues in collagen nascent chains

Ribosomes isolated and purified from guinea pig granuloma minces, which had been incubated with proline- 14C, were shown to contain both proline- 14C and hydroxyproline- 14C in a ratio of 10:1. The radioactive nascent chains could be released, in a cell-free amino acid-incorporating system, with puromycin- 3H. After release chromatography on G-100 Sephadex yielded polypeptides ranging in size from approximately 10,000 to 100,000, with an average molecular weight of approximately 50,000. The ratio of proline- 14C to hydroxyproline- 14C was also approximately 10:1 for the released nascent polypeptides. Upon digestion of the puromycin released nascent polypeptides with collagen-specific collagenase, approximately 30% of the proline- 14C, 75% of the hydroxyproline- 14C, and 40% of the puromycin- 3H was solubilized. This indicated that the puromycin was covalently linked to hydroxyproline-containing polypeptides which were previously present on ribosomes as nascent collagen chains. Proline- 14C and hydroxyproline- 14C analysis on the collagenase-solubilized portion of the puromycin-released peptides indicated that almost 40% of the hydroxylatable proline- 14C in the nascent polypeptides had been hydroxylated.

Biosynthesis of cell wall protein: Sequential hydroxylation of proline, glycosylation of hydroxyproline and secretion of the glycoprotein

The biosynthesis of the hydroxyproline-rich cell wall protein involves several discrete cytoplasmic steps: the assembly of the polypeptide chain; the modification of this polypeptide through the hydroxylation of proline residues; the glycosylation of most of these hydroxyproline residues with arabinose. The glycoprotein is then transported to the cell wall.

Development of protocollagen proline hydroxylase activity in the chick embryo

The activity of protocollagen proline hydroxylase, the enzyme hydroxylating proline residues in protocollagen, the proline-rich and hydroxyproline-deficient polypeptide precursor of collagen, was determined in the chick embryo during embryonic development. In whole chick embryos, the activity of this enzyme increased until the 14th day of embryonic development and thereafter declined. The activity of protocollagen proline hydroxylase was found to be present in all tissues tested, the highest activities being observed in bone and skin. These tissues showed the same type of development curve for this enzyme as whole embryos. In contrast, in the other tissues the enzyme activity did not change markedly during embryonic development. The changes in protocollagen proline hydroxylase activity correlated well with the changes observed in collagen synthesis in the developing chick embryo and thus support the earlier conclusions that the activity of this enzyme may be an indicator of collagen synthesis. Administration of ferrogluconate onto the chorioallantoic membrane led to a marked increase in protocollagen proline hydroxylase activity in whole embryo homogenates, whereas administration of ascorbate caused only a slight increase in activity.

Purification and partial characterization of the enzyme for the hydroxylation of proline in protocollagen

A 200-fold purification was obtained for the enzyme which hydroxylates proline residues in protocollagen, the polypeptide precursor of collagen. Essentially all of the enzymic activity was recovered in the soluble protein fraction of chick embryo homogenates. Ferrous iron, ascorbate, and α-ketoglutarate were cofactors for the reaction with purified enzyme. The enzyme did not hydroxylate free proline, and it did not hydroxylate either the tripeptide, glycyl- l-prolyl- l-proline, or poly- l-proline II with a molecular weight of about 15,000. The synthetic polytripeptide, ( 14C- l-prolylglycyl- l-prolyl) n , with a molecular weight of about 4000 was hydroxylated, and the results indicated that the enzyme is specific for proline in the second position after glycine, the position in which the hydroxyproline in collagen is found. A 30-fold purified preparation of the enzyme also hydroxylated lysine residues in protocollagen, and the cofactor requirements were similar to those for the hydroxylation of proline-labeled protocollagen. The question of whether a single enzyme performs both hydroxylations was not resolved, but the general characteristics of the enzyme or enzymes suggest that it might be appropriate to call them protocollagen hydroxylases.