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Cloning and characterization of a third human lysyl hydroxylase isoform.

ABSTRACT: Lysyl hydroxylase (EC 1.14.11.4), a homodimer, catalyzes the formation of hydroxylysine in collagens. Recently, an isoenzyme termed lysyl hydroxylase 2 has been cloned from human sources [M. Valtavaara, H. Papponen, A.-M. Pirttilä, K. Hiltunen, H. Helander and R. Myllylä (1997) J. Biol. Chem. 272, 6831-6834]. We report here on the cloning of a third human lysyl hydroxylase isoenzyme, termed lysyl hydroxylase 3. The cDNA clones encode a 738 amino acid polypeptide, including a signal peptide of 24 residues. The overall amino acid sequence identity between the processed human lysyl hydroxylase 3 and 1 polypeptides is 59%, and that between the processed lysyl hydroxylase 3 and 2 polypeptides is 57%, whereas the identity to the processed Caenorhabditis elegans polypeptide is only 45%. All four recently identified critical residues at the catalytic site, two histidines, one aspartate, and one arginine, are conserved in all these polypeptides. The mRNA for lysyl hydroxylase 3 was found to be expressed in a variety of tissues, but distinct differences appear to exist in the expression patterns of the three isoenzyme mRNAs. Recombinant lysyl hydroxylase 3 expressed in insect cells by means of a baculovirus vector was found to be more soluble than lysyl hydroxylase 1 expressed in the same cell type. No differences in catalytic properties were found between the recombinant lysyl hydroxylase 3 and 1 isoenzymes. Deficiency in lysyl hydroxylase 1 activity is known to cause the type VI variant of the Ehlers-Danlos syndrome, and it is therefore possible that deficiency in lysyl hydroxylase 3 activity may lead to some other variant of this syndrome or to some other heritable connective tissue disorder.

SUBMITTER: Passoja K

PROVIDER: S-EPMC27920 | biostudies-literature | 1998 Sep

REPOSITORIES: biostudies-literature

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Cloning and characterization of a third human lysyl hydroxylase isoform.

Passoja K K Rautavuoma K K Ala-Kokko L L Kosonen T T Kivirikko K I KI

Proceedings of the National Academy of Sciences of the United States of America 19980901 18

Lysyl hydroxylase (EC 1.14.11.4), a homodimer, catalyzes the formation of hydroxylysine in collagens. Recently, an isoenzyme termed lysyl hydroxylase 2 has been cloned from human sources [M. Valtavaara, H. Papponen, A.-M. Pirttilä, K. Hiltunen, H. Helander and R. Myllylä (1997) J. Biol. Chem. 272, 6831-6834]. We report here on the cloning of a third human lysyl hydroxylase isoenzyme, termed lysyl hydroxylase 3. The cDNA clones encode a 738 amino acid polypeptide, including a signal peptide of 24 ...[more]

PMID: 9724729

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Project description:Antibodies to pure lysyl hydroxylase from whole chick embryos were prepared in rabbits and used for immunological characterization of this enzyme of collagen biosynthesis. In double immunodiffusion a single precipitation line was seen between the antiserum and crude or pure chick-embryo lysyl hydroxylase. The antiserum effectively inhibited chick-embryo lysyl hydroxylase activity, whether measured with the biologically prepared protocollagen substrate or a synthetic peptide consisting of only 12 amino acids. This suggests that the antigenic determinant was located near the active site of the enzyme molecule. Essentially identical amounts of the antiserum were required for 40% inhibition of the same amount of lysyl hydroxylase activity units from different chick-embryo tissues synthesizing various genetically distinct collagen types. In double immunodiffusion a single precipitation line of complete identity was found between the antiserum and the purified enzyme from whole chick embryos and the crude enzymes from chick-embryo tendon, cartilage and kidneys. These results do not support the hypothesis that lysyl hydroxylase has collagen-type-specific or tissue-specific isoenzymes with markedly different specific activities or immunological properties. The antibodies to chick-embryo lysyl hydroxylase showed a considerable degree of species specificity when examined either by activity-inhibition assay or by double immuno-diffusion. Nevertheless, a distinct, although weak, cross-reactivity was found between the chick-embryo enzyme and those from all mammalian tissues tested. The antiserum showed no cross-reactivity against prolyl 3-hydroxylase, hydroxylysyl galactosyl-transferase or galactosylhydroxylysyl glucosyltransferase in activity-inhibition assays, whereas a distinct cross-reactivity was found against prolyl 4-hydroxylase. Furthermore, antiserum to pure prolyl 4-hydroxylase inhibited lysyl hydroxylase activity. These findings suggest that there are structural similarities between these two enzymes, possibly close to or at their active sites.

Dataset Information

Cloning and characterization of a third human lysyl hydroxylase isoform.

Publications

Cloning and characterization of a third human lysyl hydroxylase isoform.

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