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R Myllylä

Publications and source records attributed to R Myllylä.

At least 37 records · Page 2Linked to original sources
PubMed1997

Cloning and characterization of a novel human lysyl hydroxylase isoform highly expressed in pancreas and muscle.

We report the isolation and characterization of cDNA clones for a novel isoform of lysyl hydroxylase (lysyl hydroxylase 2), a posttranslational enzyme of collagen biosynthesis. The open reading frame predicted a protein of 737 amino acids, including an amino-terminal signal peptide. The amino acid sequence has overall similarity of over 75% to the lysyl hydroxylase (lysyl hydroxylase 1) characterized earlier. This similarity is even higher in the carboxyl-terminal end of the molecules. Lysyl hydroxylase 2 contains nine cysteine residues, which are conserved in lysyl hydroxylase 1. Furthermore, the conserved histidines and aspartate residues required for lysyl hydroxylase activity are present in the sequence. Northern analysis identified a transcript of 4.2 kilobases, which was highly expressed in pancreas and muscle tissues. Expression of cDNA in insect cells using a baculovirus vector yielded proteins with lysyl hydroxylase activity and an antiserum against a synthetic peptide of the deduced amino acid sequence recognized proteins with molecular weights of 88 and 97 kDa in homogenates of the transfected cells.

Amino Acid Sequence↗
PubMed1997

Protein disulfide isomerase and newly synthesized procollagen chains form higher-order structures in the lumen of the endoplasmic reticulum.

A number of proteins that act as necessary catalysts for correct protein folding and oligomerization in the endoplasmic reticulum (ER) are known to be retained in the organelle via the KDEL-receptor mediated retrieval mechanism. However, a complementary system that may help to retain these proteins in the organelle lumen has been suggested to exist and likely involves physical protein-protein interactions at the level of endoplasmic reticulum (ER) itself. In this report, we provide both morphological and biochemical evidence in support of this proposal. We show that in collagen-secreting human skin fibroblasts, protein disulfide isomerase and newly synthesized procollagen chains exist predominantly in an "aggregated" state, and form a reticular-like matrix in the ER lumen in vivo. The size of the aggregates was found to be variable, and may exceed 1.5 million Da. Aggregate formation appeared to be transient and to involve multiple types of protein-protein interactions, including formation of aberrant disulfide bonds. Association of protein disulfide isomerase, on the other hand, was found to require at least partly function-related disulfide bonds. These results support the existence of a reticular-like matrix in the ER lumen, and suggest that aggregation may be part of the normal maturation pathway during collagen biosynthesis.

Antibodies, Monoclonal↗
PubMed1997

Duplication of seven exons in the lysyl hydroxylase gene is associated with longer forms of a repetitive sequence within the gene and is a common cause for the type VI variant of Ehlers-Danlos syndrome.

The type VI variant of the Ehlers-Danlos syndrome (EDS) is a recessively inherited connective tissue disorder which, in most families, is due to a deficiency in lysyl hydroxylase activity. We have recently characterized a homozygous duplication of 8.9 kb in the lysyl hydroxylase gene (PLOD) in two EDS VI families. The duplication is caused by a homologous recombination of Alu sequences in introns 9 and 16. Using PCR, we have analyzed 26 additional EDS VI families from various countries and found that 7 of them have this duplication. Our data has shown a frequency of 19.1% for this mutant allele among 35 EDS VI families studied by us so far. Our haplotype analysis shows a variation in the sequence of DNA region surrounding the duplication. There is an association between a particular allele size class, the long form, at the dinucleotide repeat within intron 16 and the duplication mutation in PLOD. Screening of a general population revealed one positive finding among 582 alleles tested. An abnormal sequence in exon 17 of the gene, which generated a stop codon in the exon sequence and aberrant mRNA processing, was responsible for the nonfunctionality of the other allele in one of the compound heterozygous patients.

Alleles↗
PubMed1996

Site-directed mutagenesis of human lysyl hydroxylase expressed in insect cells. Identification of histidine residues and an aspartic acid residue critical for catalytic activity.

Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 homodimer, catalyzes the formation of hydroxylysine in collagens. We expressed here human lysyl hydroxylase in insect cells by baculovirus vectors. About 90% of the enzyme produced was soluble 32 h after infection, whereas only 10% was soluble at 72 h. Twelve histidines, five aspartates, and all four asparagines that may act as N-glycosylation sites were converted individually to serine, alanine, or glutamine, respectively, and the mutant enzymes were expressed in insect cells. Three histidine mutations and one aspartate mutation appeared to inactivate the enzyme completely. These and other data suggest that histidines 656 and 708 and aspartate 658 provide the three ligands required for the binding of Fe2+ to a catalytic site, whereas the role of the third critical histidine (residue 706) remains to be established. Three additional histidine mutations also had a major effect, although they did not inactivate the enzyme completely, whereas six further histidine mutations and four out of five aspartate mutations had a much more minor effect. Data on the four asparagine mutations suggested that only two of the potential N-glycosylation sites may be fully glycosylated in insect cells and that one of these carbohydrate units may be needed for full enzyme activity.

Amino Acid Sequence↗
PubMed1995

Demyelinating polyneuropathy in a patient with the tRNA(Leu)(UUR) mutation at base pair 3243 of the mitochondrial DNA.

A novel feature of demyelinating polyneuropathy was observed in a patient with the tRNA(Leu(UUR)) mutation at base pair 3243 of the mitochondrial DNA. Based on electrodiagnostic examination, the polyneuropathy was defined as being of the demyelinating, mixed (motor more than sensory) type. In a 1-year follow-up we observed approximately 7% reduction in both the motor and sensory conduction velocities. The other clinical features of the proband included a mild to moderate cognitive impairment and a combined hearing loss with a moderate sensorineural component. The proportion of the mutant genome found in the muscle of the proband was 29%, but the mutation was not found in his blood. A wide variability of the clinical phenotype was observed in the family of the proband. Heteroplasmic mutation was detected in the blood of most family members. The proportion of abnormal mitochondrial DNA was highest in the proband's brother, who had clinically definite mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome, while the mutant genome was less frequent or absent in the subjects with less severe phenotypes and in healthy individuals. The findings on this pedigree emphasize the need for studies of complete families in the search for new clinical phenotypes of mutations in mitochondrial DNA.

Adult↗
PubMed1994

Lysyl hydroxylase, a collagen processing enzyme, exemplifies a novel class of luminally-oriented peripheral membrane proteins in the endoplasmic reticulum.

Lysyl hydroxylase (LH), an enzyme required early during collagen biosynthesis, appears to be exceptional among proteins that are thought to be residents of the endoplasmic reticulum (ER). It is a homodimer and does not contain either of the two previously characterized ER-specific retention motifs (KDEL or the double lysine motif) in its primary structure. We now show that LH, nevertheless, resides in the lumen of the ER. In immunofluorescence experiments, LH co-localizes with a KDEL-containing protein, protein disulfide isomerase (PDI), and also co-sediments with it after fractionation of subcellular organelles by sucrose density gradient centrifugation. In addition, LH seems to be stress-inducible. In one respect, however, LH differs from PDI and other known luminal proteins in the organelle. It is found in situ only in association with the ER membranes. Our cell fractionation and Triton X-114 phase separation experiments suggest that it binds to the membranes via weak electrostatic interactions. LH can thus be regarded as a first luminally-oriented "peripheral membrane" protein which has been characterized in the ER. The results suggest a novel possibility by which ER lumen can acquire its specific protein components from the bulk flow.

Amino Acid Sequence↗
PubMed1994

Structure and expression of the human lysyl hydroxylase gene (PLOD): introns 9 and 16 contain Alu sequences at the sites of recombination in Ehlers-Danlos syndrome type VI patients.

Lysyl hydroxylase (EC 1.14.11.4) catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in peptide linkages. This enzyme activity is known to be reduced in patients with the type VI variant of the Ehlers-Danlos syndrome, and the first mutations in the lysyl hydroxylase gene (PLOD) have recently been identified. We have now isolated genomic clones for human lysyl hydroxylase and determined the complete structure of the gene, which contains 19 exons and a 5' flanking region with characteristics shared by housekeeping genes. The constitutive expression of the gene in different tissues further suggests that lysyl hydroxylase has an essential function. We have sequenced the introns of the gene in the region where many mutations and rearrangements analyzed to date are concentrated. Intron 9 and intron 16 show extensive homology resulting from the many Alu sequences found in these introns. Intron 9 contains five and intron 16 eight Alu sequences. The high homology and many short identical or complementary sequences in these introns generate many potential recombination sites with the gene. The delineation of the structure of the lysyl hydroxylase gene contributes significantly to our understanding of the rearrangements in the genome of Ehlers-Danlos type VI patients.

Amino Acid Sequence↗
PubMed1994

Alu-Alu recombination results in a duplication of seven exons in the lysyl hydroxylase gene in a patient with the type VI variant of Ehlers-Danlos syndrome.

The type VI variant of the Ehlers-Danlos syndrome (EDS) is a recessively inherited connective-tissue disorder. The characteristic features of the variant are muscular hypotonia, kyphoscoliosis, ocular manifestations, joint hypermobility, skin fragility and hyperextensibility, and other signs of connective-tissue involvement. The biochemical defect in most but not all patients is a deficiency in lysyl hydroxylase activity. Lysyl hydroxylase is an enzyme that catalyzes the formation of hydroxylysine in collagens and other proteins with collagen-like amino acid sequences. We have recently reported an apparently homozygous large-duplication rearrangement in the gene for lysyl hydroxylase, leading to the type VI variant of EDS in two siblings. We now report an identical, apparently homozygous large duplication in an unrelated 49-year-old female originally analyzed by Sussman et al. Our simple-sequence-repeat-polymorphism analysis does not support uniparental isodisomy inheritance for either of the two duplications. Furthermore, we indicate in this study that the duplication in the lysyl hydroxylase gene is caused by an Alu-Alu recombination in both families. Cloning of the junction fragment of the duplication has allowed synthesis of appropriate primers for rapid screening for this rearrangement in other families with the type VI variant of EDS.

Base Sequence↗
PubMed1993

A large duplication in the gene for lysyl hydroxylase accounts for the type VI variant of Ehlers-Danlos syndrome in two siblings.

Ehlers-Danlos syndrome is a heterogeneous disorder characterized by joint hypermobility, skin hyperextensibility, fragility, and other signs of connective tissue involvement. In addition to these, the type VI variant of the disease has some special characteristics such as kyphoscoliosis and ocular abnormalities. The biochemical abnormality in most patients with this autosomal recessively inherited type VI variant is a deficiency in the activity of lysyl hydroxylase (EC 1.14.11.4), the enzyme catalyzing the formation of hydroxylysine in collagens and other proteins with collagen-like amino acid sequences. The type VI variant of Ehlers-Danlos syndrome was first identified in two sisters with a reduced amount of lysyl hydroxylase activity in their skin fibroblasts (S.R. Pinnell, S. M. Krane, J. E. Kenzora, and M. J. Glimcher (1972) N. Engl. J. Med. 286: 1013-1020). Our recent molecular cloning of lysyl hydroxylase has now made it possible to study the mutations leading to the deficiency in lysyl hydroxylase activity in these cells. Our data indicate that the mRNA for lysyl hydroxylase produced in the affected cells is about 4 kb in size, whereas it is 3.2 kb in the control cells. The sequencing of the cDNA for lysyl hydroxylase from the affected cells revealed an apparently homozygous duplication rearrangement of nucleotides 1176 to 1955, corresponding to amino acids 326 to 585 in the normal sequence. From Southern blotting data, the duplicated area in the gene equals about 6-9 kb and corresponds to seven exons.

Amino Acid Sequence↗
PubMed1993

Assessment of skin erythema by eye, laser Doppler flowmeter, spectroradiometer, two-channel erythema meter and Minolta chroma meter.

Visual grading, a laser Doppler flowmeter, a spectroradiometer, a two-channel erythema meter and a Minolta chroma meter were compared in the measurement of erythemas arising from immediate contact reactions produced either by 250 mM benzoic acid or 10 mM methyl nicotinate in petrolatum in an open application test or by ultraviolet irradiation. A good correlation between visual grading and objectively measured values was found for all the instruments, but the laser Doppler flowmeter gave proportionally lower values for the ultraviolet erythemas than the skin reflectance meters, suggesting that these differ from erythemas induced by benzoic acid or methyl nicotinate. The laser Doppler flowmeter gave less repeatable results than other meters when measuring moderate and pronounced erythemas produced by ultraviolet irradiation. Measuring both the blood flow and the actual erythema may give more information about the reaction than either measurement system alone.

Adult↗
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↗