ABSTRACT
Lysyl hydroxylase is an enzyme involved in collagen biosynthesis, catalyzing the hydroxylation of lysyl residues as a post-translational event. Three isoforms have been characterized so far (LH1, LH2, LH3). Our recent findings indicate that LH3 possesses, not only lysyl hydroxylase activity, but also galactosylhydroxylysyl glucosyltransferase activity [Heikkinen et al., J. Biol. Chem. 275 (2000) 36158-36163]. We report here the characterization of mouse LH2 (Plod2) and LH3/glucosyltransferase (Plod3) genes. Plod2 spans approximately 50 kb of the genomic DNA, and is organized in 20 exons, one of the exons being alternatively spliced in the RNA processing. Plod3 spans approximately 10 kb of the genomic DNA, and contains 19 exons. Analysis of the 5' flanking region with many transcription start sites reveals the lack of a TATAA box in both genes. Sequence analysis indicated many retroposon-like elements within the Plod3 gene. A comparison was carried out among the LH1, LH2 and LH3 gene structures characterized so far from different species.
Subject(s)
Alternative Splicing , Glucosyltransferases/genetics , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/genetics , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary , Humans , Mice , Molecular Sequence Data , Protein Isoforms/genetics , Sequence Homology, Nucleic Acid , Tissue DistributionABSTRACT
Lysyl hydroxylase (EC ) and glucosyltransferase (EC ) 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.
Subject(s)
Glucosyltransferases/metabolism , Multienzyme Complexes/metabolism , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/metabolism , Alternative Splicing , Animals , Antibodies/pharmacology , Cell Line , Chickens , Collagen/biosynthesis , Glucosyltransferases/antagonists & inhibitors , Glucosyltransferases/genetics , Glucosyltransferases/isolation & purification , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Isoenzymes/metabolism , Kinetics , Microscopy, Fluorescence , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/genetics , Multienzyme Complexes/isolation & purification , Mutation/genetics , Precipitin Tests , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/antagonists & inhibitors , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/genetics , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/isolation & purification , Protein Biosynthesis , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Spodoptera , TransfectionABSTRACT
Lysyl hydroxylase is the enzyme catalyzing the formation of hydroxylysyl residues in collagens. Large differences in the extent of hydroxylysyl residues are found among collagen types. Three lysyl hydroxylase isoenzymes (LH1, LH2, LH3) have recently been characterized from human and mouse tissues. Nothing is known about the distribution of these isoforms within cells or whether they exhibit collagen type specificity. We measured mRNA levels of the three isoforms, as well as the mRNAs of the main collagen types I, III, IV, and V and the alpha subunit of prolyl 4-hydroxylase, another enzyme involved in collagen biosynthesis, in different human cell lines. Large variations were found in mRNA expression of LH1 and LH2 but not LH3. Immunoblotting was utilized to confirm the results of Northern hybridization. The levels of mRNA of LH1, LH2, and the alpha subunit of prolyl 4-hydroxylase showed significant correlations with each other. The LH3 mRNA levels did not correlate with those of LH1, LH2, or the alpa subunit of prolyl 4-hydroxylase, clearly indicating a difference in the regulation of LH3. No correlation was observed between LH isoforms and individual collagen types, indicating a lack of collagen type specificity for lysyl hydroxylase isoforms. Our observations suggest that LH1, LH2, and the alpha subunit of prolyl 4-hydroxylase are coregulated together with total collagen synthesis but not with the specific collagen types and indicate that LH3 behaves differently from LH1 and LH2, implying a difference in their substrates. These observations set the basis for further studies to define the functions of lysyl hydroxylase isoforms.
Subject(s)
Collagen/metabolism , Isoenzymes/metabolism , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/metabolism , Animals , Cell Line , Collagen/chemistry , Collagen/genetics , Gene Expression , HeLa Cells , Humans , Isoenzymes/genetics , Mice , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/genetics , Procollagen-Proline Dioxygenase/chemistry , Procollagen-Proline Dioxygenase/genetics , Procollagen-Proline Dioxygenase/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Substrate SpecificityABSTRACT
We report characterization of a novel isoform of lysyl hydroxylase (lysyl hydroxylase 3, LH3). The cDNA clones encode a polypeptide of 738 amino acids, including a signal peptide. The amino acid sequence has a high overall identity with LH1 and LH2, the isoforms characterized earlier. Conserved regions are present in the carboxyl-terminal portion of the isoforms and also in the central part of the molecules. Histidine and asparagine residues, which are conserved in the other isoforms and are known to be required for enzymatic activity, are also conserved in the novel isoform. The gene for LH3 (PLOD3) has been assigned to human chromosome 7q36 and rat chromosome 12. Gene expression of LH3 is highly regulated in adult human tissues. A strong hybridization signal, corresponding to an mRNA 2.75 kilobases in size, is obtained in heart, placenta and pancreas on multiple tissue RNA blots. Expression of the cDNA in vitro results in the synthesis of a protein that hydroxylates lysyl residues in collagenous sequences in a non-triple helical conformation.
Subject(s)
Chromosomes, Human, Pair 7 , Isoenzymes/metabolism , Protein-Lysine 6-Oxidase/metabolism , Adult , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Cloning, Molecular , DNA, Complementary , Humans , Hydrolysis , Isoenzymes/chemistry , Isoenzymes/genetics , Lysine/metabolism , Molecular Sequence Data , Protein-Lysine 6-Oxidase/chemistry , Protein-Lysine 6-Oxidase/genetics , Rats , Sequence Homology, Amino AcidABSTRACT
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.
