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Biomedical subjects

R Myllylä

Publications and source records attributed to R Myllylä.

At least 19 recordsLinked to original sources
PubMed1992

Modification of vertebrate and algal prolyl 4-hydroxylases and vertebrate lysyl hydroxylase by diethyl pyrocarbonate. Evidence for histidine residues in the catalytic site of 2-oxoglutarate-coupled dioxygenases.

A search for conserved amino acid residues within the cDNA-derived amino acid sequences of 2-oxoglutarate-coupled dioxygenases revealed the presence of two distinct motifs, spaced 49-71 amino acids apart, toward the C-terminal regions of these proteins. Each of the two common motifs contains an invariant histidine residue at a conserved position. The 2-oxoglutarate-coupled dioxygenases function in diverse processes, including the post-translational hydroxylation of proline and lysine residues in vertebrate collagens and the biosynthesis of microbial cephalosporins, yet they have a common reaction mechanisms, which requires the binding of Fe2+, 2-oxoglutarate, O2 and ascorbate at the catalytic site. The two regions of homology, and specifically the identical histidines, potentially represent functionally important sites related to their catalytic activity. Modification of histidine residues by diethyl pyrocarbonate inactivated vertebrate and algal prolyl 4-hydroxylase and vertebrate lysyl hydroxylase, indicating that histidine residues function in the catalytic site of these 2-oxoglutarate-coupled dioxygenases. Inactivation was prevented by the presence of co-substrates, but not by the peptide substrate. It is proposed that the histidine residues in the conserved motifs may function as Fe(2+)-binding ligands.

Amino Acid Sequence↗
PubMed1992

Expression and site-directed mutagenesis of human protein disulfide isomerase in Escherichia coli. This multifunctional polypeptide has two independently acting catalytic sites for the isomerase activity.

Protein disulfide isomerase (PDI, EC 5.3.4.1) is a highly unusual multifunctional polypeptide, being identical to the beta subunit of prolyl 4-hydroxylase, a cellular thyroid hormone binding protein and a component of the microsomal triglyceride transfer protein complex, and highly similar to a polypeptide acting in vitro as a glycosylation site binding protein. It has two -Cys-Gly-His-Cys- sequences which, it has been proposed, act as catalytic sites for the isomerase activity, but few data have been available to indicate whether one or both of them do indeed act as catalytic sites and whether the two presumed catalytic sites act independently or cooperatively. We report here on the expression of human PDI in Escherichia coli with three different signal sequences. All three polypeptide variants were secreted into the periplasmic space as fully active enzymes. Oligonucleotide-directed mutagenesis was used to convert either one or both of the -Cys-Gly-His-Cys- sequences to -Ser-Gly-His-Cys-. The PDI activity of both polypeptides containing a single modified sequence was about 50% of that of the wild-type polypeptide, whereas the polypeptide with two modified sequences had no isomerase activity. It is thus concluded that both -Cys-Gly-His-Cys- sequences act as catalytic sites for the isomerase activity, and the two catalytic sites appear to operate independently of one another.

Amino Acid Sequence↗
PubMed1992

Minoxidil specifically decreases the expression of lysine hydroxylase in cultured human skin fibroblasts.

The levels of lysine hydroxylase protein and the levels of the mRNAs for lysine hydroxylase and the alpha- and beta-subunits of proline 4-hydroxylase were measured in cultured human skin fibroblasts treated with 1 mM-minoxidil. The data demonstrate that minoxidil decreases the amount of lysine hydroxylase protein, this being due to a decrease in the level of lysine hydroxylase mRNA. The effect of minoxidil appears to be highly specific, as no changes were observed in the amounts of mRNAs for the alpha- and beta-subunits of proline 4-hydroxylase.

Blotting, Northern↗
PubMed1992

Site-directed mutagenesis of human protein disulphide isomerase: effect on the assembly, activity and endoplasmic reticulum retention of human prolyl 4-hydroxylase in Spodoptera frugiperda insect cells.

Protein disulphide isomerase (PDI) is a highly unusual multifunctional polypeptide, identical to the beta-subunit of prolyl 4-hydroxylase. It has two -Cys-Gly-His-Cys- sequences which represent two independently acting catalytic sites of PDI activity. We report here on the expression in baculovirus vectors of various mutant PDI/beta-subunits together with a wild-type alpha-subunit of the human prolyl 4-hydroxylase alpha 2 beta 2 tetramer in Spodoptera frugiperda insect cells. When either one or both of the -Cys-Gly-His-Cys- sequences was converted to -Ser-Gly-His-Cys-, a tetramer was formed as with wild-type PDI/beta-subunit. This tetramer was fully active prolyl 4-hydroxylase. The data demonstrate that PDI activity of the PDI/beta-subunit is not required for tetramer assembly or for the prolyl 4-hydroxylase activity of the tetramer, and thus other sequences of the PDI/beta-subunit may be critical for keeping the alpha-subunits in a catalytically active, non-aggregated conformation. Measurements of the PDI activities of tetramers containing the various mutant PDI/beta-subunits demonstrated that the activity of the wild-type tetramer is almost exclusively due to the C-terminal PDI catalytic sites, which explains the finding that the PDI activity of the PDI/beta-subunit present in the tetramer is about half that in the free polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Cloning of human lysyl hydroxylase: complete cDNA-derived amino acid sequence and assignment of the gene (PLOD) to chromosome 1p36.3----p36.2.

Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 dimer, catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in peptide linkages. A deficiency in this enzyme activity is known to exist in patients with the type VI variant of the Ehlers-Danlos syndrome, but no amino acid sequence data have been available for the wildtype or mutated human enzyme from any source. We report the isolation and characterization of cDNA clones for lysyl hydroxylase from a human placenta lambda gt11 cDNA library. The cDNA clones cover almost all of the 3.2-kb mRNA, including all the coding sequences. These clones encode a polypeptide of 709 amino acid residues and a signal peptide of 18 amino acids. The human coding sequences are 72% identical to the recently reported chick sequences at the nucleotide level and 76% identical at the amino acid level. The C-terminal region is especially well conserved, a 139-amino-acid region, residues 588-727 (C-terminus), being 94% identical between the two species and a 76-amino-acid region, residues 639-715, 99% identical. These comparisons, together with other recent data, suggest that lysyl hydroxylase may contain functionally significant sequences especially in its C-terminal region. The human lysyl hydroxylase gene (PLOD) was mapped to chromosome 1 by Southern blot analysis of human-mouse somatic cell hybrids, to the 1p34----1pter region by using cell hybrids that contain various translocations of human chromosome 1, and by in situ hybridization to 1p36.2----1p36.3. This gene is thus not physically linked to those for the alpha and beta subunits of prolyl 4-hydroxylase, which are located on chromosomes 10 and 17, respectively.

Amino Acid Sequence↗
PubMed1992

A homozygous stop codon in the lysyl hydroxylase gene in two siblings with Ehlers-Danlos syndrome type VI.

Ehlers-Danlos syndrome (EDS) is characterized by joint hypermobility, alterations in the skin and additional signs of connective tissue involvement. EDS type VI was the first connective tissue disorder for which a specific defect in collagen metabolism was identified, namely a deficiency of lysyl hydroxylase activity. We now report a homozygous single basepair substitution converting the CGA codon (Arg319) to a TGA termination codon in two siblings with EDS type VI. The healthy parents, who are first cousins, and two of the three healthy siblings of the patients are heterozygous. The mutation leads to an almost complete absence of lysyl hydroxylase activity in extracts derived from fibroblasts of the patients.

Adolescent↗
PubMed1991

Molecular cloning of chick lysyl hydroxylase. Little homology in primary structure to the two types of subunit of prolyl 4-hydroxylase.

Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 dimer, catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in X-Lys-Gly sequences. We report here on the isolation of cDNA clones coding for the enzyme from a chick embryo lambda gt11 library. Several overlapping clones covering all the coding sequences of the 4-kilobase mRNA and virtually all the noncoding sequences were characterized. These clones encode a polypeptide of 710 amino acid residues and a signal peptide of 20 amino acids. The polypeptide has four potential attachment sites for asparagine-linked oligosaccharides and 9 cysteine residues, at least one of which is likely to be involved in the binding of the Fe2+ atom to a catalytic site. A surprising finding was that no significant homology was found between the primary structures of lysyl hydroxylase and prolyl 4-hydroxylase in spite of the marked similarities in kinetic properties between these two enzymes. A computer-assisted comparison indicated only an 18% identity between lysyl hydroxylase and the alpha-subunit of prolyl 4-hydroxylase and a 19% identity between lysyl hydroxylase and the beta-subunit of prolyl 4-hydroxylase. Visual inspection of the most homologous areas nevertheless indicated the presence of several regions of 20-40 amino acids in which the identity between lysyl hydroxylase and one of the prolyl 4-hydroxylase subunits exceeded 30% or similarity exceeded 40%. Southern blot analyses of chick genomic DNA indicated the presence of only one gene coding for lysyl hydroxylase.

Amino Acid Sequence↗
PubMed1991

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)

Animals↗
PubMed1991

Markers of collagen and basement membrane metabolism in sera of patients with progressive systemic sclerosis.

The concentrations of the amino terminal propeptide of type III procollagen, the 7S domain of type IV collagen, and the fragment P1 of laminin (PIIINP, 7S, and P1 respectively) and the activity of galactosylhydroxylysyl glucosyltransferase (GGT) in serum were evaluated as indicators of disease activity in a cross sectional study of 84 patients with progressive systemic sclerosis. The mean values of PIIINP, P1, and GGT were raised in progressive systemic sclerosis, 19-32% of patients having abnormal values of the various tests. PIIINP, measured with two different assays, and P1 were associated with active, acute, or subacute disease. GGT also correlated positively with some acute phase proteins in the whole group, without a clear association with the course of the disease. Arthritis was associated with increased PIIINP concentrations as well as with an increased activity of GGT. Kidney disease led to raised concentrations of the degradation products of PIIINP. Raynaud's phenomenon in the hands was related to increased PIIINP concentrations.

Adult↗
PubMed1991

[Changes in collagen biosynthesis in patients with hip joint replacement surgery and reoperation].

The authors investigated the intensity of collagen synthesis in patients subjected to operation or re-operation of a total endoprosthesis of the hip joint. The patients suffered from osteoarthritis, rheumatoid arthritis and ankylosing spondylitis. The authors assessed the activity of collagen glucosyl transferase (S-GGT) and the concentration of the N-terminal propeptide procollagen type III by two methods (S-Pro III-N-P and S-Fab) in serum before and after operation. A significant rise of S-GGT and S-Fab, as compared with controls, occurred only after operation while S-Pro III-N-P was elevated already before operation. S-GGT did not differ before and after operation, while N-propeptide concentration rose when either method was used.

Adult↗
PubMed1990

Purification and characterization of protein disulphide-isomerase from the unicellular green alga Chlamydomonas reinhardii. A 120 kDa dimer antigenically distinct from the vertebrate enzyme.

Protein disulphide-isomerase (PDI) has been isolated from the unicellular green alga Chlamydomonas reinhardii and purified by (NH4)2SO4 precipitation, gel filtration and DEAE-Sephacel, hydroxyapatite and f.p.l.c. chromatography. The active algal enzyme is a 120 kDa dimer with a subunit molecular mass of 60 kDa when determined by SDS/PAGE. Although similar in size to the previously isolated vertebrate PDIs, the algal enzyme is antigenically distinct, polyclonal antibodies against the algal PDI showing no cross-reactivity with the vertebrate enzyme on immunoblots, and vice versa. The anti-(algal PDI) antiserum did not inhibit algal PDI activity, and C. reinhardii PDI could be immobilized on anti-PDI-Protein A-Sepharose in active form. In contrast with the situation in vertebrates, where PDI functions as a subunit of prolyl 4-hydroxylase, the C. reinhardii PDI is not associated with the algal prolyl 4-hydroxylase.

Chlamydomonas↗
PubMed1989

The catalytic mechanism of the hydroxylation reaction of peptidyl proline and lysine does not require protein disulphide-isomerase activity.

Prolyl 4-hydroxylase, an alpha 2 beta 2 tetramer, catalyses the formation of 4-hydroxyproline in collagens. The beta subunit is known to be identical with the enzyme protein disulphide-isomerase and to possess disulphide-isomerase activity even when present in the prolyl 4-hydroxylase tetramer. We here report that lysyl hydroxylase, a homodimer, and algal prolyl 4-hydroxylase, a monomer, do not contain detectable protein disulphide-isomerase activity. Since the hydroxylase reaction mechanisms are similar, the data suggest that the protein disulphide-isomerase activity of the vertebrate prolyl 4-hydroxylase beta subunit is unlikely to be involved in the catalytic mechanism of the hydroxylation reaction.

Animals↗
PubMed1989

Markers of collagen metabolism in sera of patients with various rheumatic diseases.

The activity of galactosylhydroxylysyl glucosyltransferase (S-GGT) and concentration of the amino-terminal propeptide of type III procollagen, measured with two different radioimmunoassays, S-Pro(III)-N-P and S-Fab, were determined in the sera of 209 patients with various rheumatic diseases. The mean values for all these three assays were elevated in the patients with rheumatoid arthritis, whereas only the mean value for S-GGT was elevated in osteoarthrosis. The mean S-GGT but not S-Pro(III)-N-P or S-Fab was also elevated in psoriatic arthritis, ankylosing spondylitis and systemic lupus erythematosus. S-GGT correlated significantly both with S-Pro(III)-N-P and S-Fab in the pooled group of all the patients and in the cases of rheumatoid arthritis and psoriatic arthritis, and also with S-Fab in the cases of osteoarthrosis. S-Pro(III)-N-P and S-Fab correlated with each other in every disease group. The ratio S-Fab:S-Pro(III)-N-P was significantly higher in the patients with osteoarthrosis, ankylosing spondylitis and systemic lupus erythematosus than in those with rheumatoid arthritis. The data indicate that definite changes can be seen in the values of serum markers of collagen metabolism in rheumatoid arthritis, psoriatic arthritis, osteoarthrosis, ankylosing spondylitis and systemic lupus erythematosus, the most sensitive indicator being S-GGT.

Adult↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1988

Syncatalytic inactivation of prolyl 4-hydroxylase by synthetic peptides containing the unphysiologic amino acid 5-oxaproline.

Peptides containing the unphysiological amino acid 5-oxaproline (Opr) in the sequence R1-Xaa-Opr-Gly-OR2 were found to inactivate prolyl 4-hydroxylase from chick and human origins. Of the substances investigated, compounds with aromatic substituents R1 and R2 were particularly effective when compared with those with an aliphatic group or without a C-terminal blocking group. Both affinity of the individual peptides for the enzyme and partition ratio contributed to the differences in efficiency. Benzylcarbonyl-Phe-Opr-Gly-benzyl ester was the most effective substance tested, its concentration giving 50% inactivation in 1 h being 0.8 microM. Inactivation was only observed in the presence of 2-oxoglutarate and Fe2+. The Opr peptides enhanced the decarboxylation of 2-oxoglutarate by prolyl 4-hydroxylase, the Vmax values obtained with the individual peptides being positively correlated with their inactivating efficiency. Inactivation was prevented by high concentrations of peptide substrate and ascorbate. Lineweaver-Burk kinetics experiments suggested noncompetitive inhibition with respect to peptide substrate and ascorbate. Lysyl hydroxylase was not affected by Opr peptides in concentrations of up to 1.5 mM in either the presence or absence of prolyl 4-hydroxylase. The results suggest that the oxaproline compounds are specific syncatalytic inactivators of prolyl 4-hydroxylase.

Animals↗
PubMed1988

Prolyl 4-hydroxylase from Volvox carteri. A low-Mr enzyme antigenically related to the alpha subunit of the vertebrate enzyme.

Prolyl 4-hydroxylase was isolated in a highly purified form from a multi-cellular green alga, Volvox carteri, by a procedure consisting of ion-exchange chromatography and affinity chromatography on poly(L-hydroxyproline) coupled to Sepharose. Two other affinity-column procedures were also developed, one involving 3,4-dihydroxyphenylacetate and the other 3,4-dihydroxyphenylpropionate linked to Sepharose. The Km values of the Volvox enzyme for the co-substrates and the peptide substrate, as well as the inhibition constants for selected 2-oxoglutarate analogues, were similar to those of the enzyme from Chlamydomonas reinhardii, except that the Km for 2-oxoglutarate with the Volvox enzyme was 6-fold greater. The temperature optimum of the Volvox enzyme was also 10 degrees C higher. The apparent Mr of the Volvox enzyme by gel filtration was about 40,000, being similar to that reported for the Chlamydomonas enzyme but markedly lower than that of the vertebrate enzymes. A similar apparent Mr of about 40,000 was also found for prolyl 4-hydroxylase from the green alga Enteromorpha intestinalis, whereas the enzyme from various vascular plants gave an apparent Mr greater than 300,000. SDS/polyacrylamide-gel electrophoresis demonstrated in the highly purified Volvox enzyme the presence of a major protein band doublet with a Mr of about 65,000 and a minor doublet of Mr about 55,000-57,000. A polyclonal antiserum, prepared against the Mr-65,000 doublet, stained in immunoblotting the Mr-65,000 doublet as well as the alpha subunit, but not the beta subunit, of the vertebrate prolyl 4-hydroxylase. An antiserum against the beta subunit of the vertebrate enzyme stained in immunoblotting a Mr-50,000 polypeptide in a partially purified Volvox enzyme preparation, but did not stain either the Mr-65,000 or the Mr-55,000-57,000 doublet of the highly purified enzyme. The data thus suggest that the active Volvox carteri prolyl 4-hydroxylase is an enzyme monomer antigenically related to the alpha subunit of the vertebrate enzyme.

Chlorophyta↗