Raili Myllylä

Posttranslational enzymes in the biosynthesis of collagen: intracellular enzymes.

Publisher Summary This chapter describes the assay, purification, and properties of the enzymes catalyzing the unique intracellular modifications—i.e., the hydroxylases and hydroxylysyl glycosyltransferases. The main types of assays methods for hydroxylases

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 ascor‐bate 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 α2β2 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 α and β subunits, but the a subunit appears to contribute the major part. The β 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 phospho‐inositide‐specific phospholipase C, thioredoxins, and the estrogen‐binding domain of the estrogen receptor. The COOH‐terminus of this β 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 α subunit does not have this COOH‐terminal sequence, and thus one function of the β subunit in the prolyl 4‐hydroxylase tetramer appears to be to retain the enzyme within this cell organelle.— Kivirikko, K. I.; Myllyla, R.; Pihlajaniemi, T. Protein hydroxylation: prolyl 4‐hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit. FASEB J. 3: 1609‐1617; 1989.

The role of ascorbate in the prolyl hydroxylase reaction.

Abstract Ascorbate was not required for the binding of 2-oxoglutarate to pure prolyl hydroxylase, and the enzyme catalyzed hydroxylation in the absence of ascorbate at an essentially maximal rate for 5–10 s, corresponding to 15–30 reaction cycles. After about one min the reaction rate in the absence of ascorbate was very low, even though only 1–2 % of the free bivalent iron had become oxidized. These and additional data indicate that prolyl hydroxylase can catalyze a number of reaction cycles without ascorbate, but at some stage the hydroxylation ceases, probably due to oxidation of the enzyme-bound iron, and ascorbate is then required as a quite specific reductant to re-activate the enzyme.

Detection of Intracellular Bacteria in the Buds of Scotch Pine (Pinus sylvestris L.) by In Situ Hybridization

ABSTRACT Bacterial isolates were obtained from pine (Pinus sylvestris L.) tissue cultures and identified asMethylobacterium extorquens and Pseudomonas synxantha. The existence of bacteria in pine buds was investigated by 16S rRNA in situ hybridization. Bacteria inhabited the buds of every tree examined, primarily colonizing the cells of scale primordia and resin ducts.

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.

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.

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)

Lysyl Hydroxylase 3 Is a Multifunctional Protein Possessing Collagen Glucosyltransferase Activity

Lysyl hydroxylase (EC 1.14.11.4) and glucosyltransferase (EC 2.4.1.66) are enzymes involved in post-translational modifications during collagen biosynthesis. We reveal in this paper that the protein produced by the cDNA for human lysyl hydroxylase isoform 3 (LH3) has both lysyl hydroxylase and glucosyltransferase (GGT) activities. The other known lysyl hydroxylase isoforms, LH1, LH2a, and LH2b, have no GGT activity. Furthermore, antibodies recognizing the amino acid sequence of human LH3 and those against a highly purified chicken GGT partially inhibited the GGT activity. Similarly, a partial inhibition was observed when these antibodies were tested against GGT extracted from human skin fibroblasts. In vitro mutagenesis experiments demonstrate that the amino acids involved in the GGT active site differ from those required for LH3 activity.

The third activity for lysyl hydroxylase 3: galactosylation of hydroxylysyl residues in collagens in vitro.

Lysyl hydroxylase (LH, EC 1.14.11.4), galactosyltransferase (EC 2.4.1.50) and glucosyltransferase (EC 2.4.1.66) are enzymes involved in posttranslational modifications of collagens. They sequentially modify lysyl residues in specific positions to hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosyl hydroxylysyl residues. These structures are unique to collagens and essential for their functional activity. Lysines and hydroxylysines form collagen cross-links. Hydroxylysine derived cross-links, usually as glycosylated forms, occur especially in weight-bearing and mineralized tissues. The detailed functions of the hydroxylysyl and hydroxylysyl linked carbohydrate structures are not known, however. Hydroxylysine linked carbohydrates are found mainly in collagens, but recent reports indicate that these structures are also present and probably have an important function in other proteins. Earlier we have shown that human LH3, but not isoforms LH1, LH2a and LH2b, possesses both LH and glucosyltransferase activity (J. Biol. Chem. 275 (2000) 36158). In this paper we demonstrate that galactosyltransferase activity is also associated with the same gene product, thus indicating that one gene product can catalyze all three consecutive steps in hydroxylysine linked carbohydrate formation. In vitro mutagenesis experiments indicate that Cys(144) and aspartates in positions 187-191 of LH3 are important for the galactosyltransferase activity. Our results suggest that manipulation of the gene for LH3 can be used to selectively alter the glycosylation and hydroxylation reactions, and provides a new tool to clarify the functions of the unique hydroxylysine linked carbohydrates in collagens and other proteins.

Glycosylation catalyzed by lysyl hydroxylase 3 is essential for basement membranes

Lysyl hydroxylase 3 (LH3) is a multifunctional enzyme possessing lysyl hydroxylase (LH), hydroxylysyl galactosyltransferase (GT) and galactosylhydroxylysyl glucosyltransferase (GGT) activities in vitro. To investigate the in vivo importance of LH3-catalyzed lysine hydroxylation and hydroxylysine-linked glycosylations, three different LH3-manipulated mouse lines were generated. Mice with a mutation that blocked only the LH activity of LH3 developed normally, but showed defects in the structure of the basement membrane and in collagen fibril organization in newborn skin and lung. Analysis of a hypomorphic LH3 mouse line with the same mutation, however, demonstrated that the reduction of the GGT activity of LH3 disrupts the localization of type IV collagen, and thus the formation of basement membranes during mouse embryogenesis leading to lethality at embryonic day (E) 9.5-14.5. Strikingly, survival of hypomorphic embryos and the formation of the basement membrane were directly correlated with the level of GGT activity. In addition, an LH3-knockout mouse lacked GGT activity leading to lethality at E9.5. The results confirm that LH3 has LH and GGT activities in vivo, LH3 is the main molecule responsible for GGT activity and that the GGT activity, not the LH activity of LH3, is essential for the formation of the basement membrane. Together our results demonstrate for the first time the importance of hydroxylysine-linked glycosylation for collagens.