Abstract
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.
Highlights
From the Collagen Research Unit, Biocenter,and Department of Medical Biochemistry, University of Oulu, SF90220 Oulu, Finland
Both enzymes act on non-hydroxylatedcollagens and collagen-likepolypeptides, andboth enzymesrequire Fez+,8-oxoglutarate, Oz,and ascorbate [1, 2, 8].The kinetic was found between the primary structures of lysyl constants of the enzymes for their cosubstrates and competihydroxylase and prolyl 4-hydroxylase in spite of the tiveinhibitorsare likewise very similar,andthe enzymes marked similarities in kinetic properties between tahpepseear tohave identical reaction mechanisms (1,2,8N).evertwo enzymes
Visual inspectionof the most acid sequences have recently been determined for both thephomologous areas indicated the presence subunit [9,10,11] and the a-subunit (12,13o)f human andchick of several regions of 20-40 amino acids in which the prolyl 4-hydroxylase, whereas no amino acid sequence data identity between lysyl hydroxylase and oofntehe pro- have been available for lysyl hydroxylase from any source
Summary
Peptidesobtainedfrom lysyl hydroxylaseby CNBr digestion [14] byimmunological screening were hybridized with oligonucleotide were isolated using a linear gradient of acetonitrile as above and a probes For this purposefour oligonucleotides were designedfrom the ProRPCTM HR5/2 (Pharmacia) reverse phase column for purifica- amino acid sequences of fragments of chick lysyl hydroxylase accordtion. Lysyl hydroxylase activity was assayed bymeasuring the formation High molecular weight genomic DNA was isolated [26] from culof radioactive hydroxylysine in Library (Clontech) wasscreened with polyclonal antibodies against purified chick embryo lysyl hydroxylase [21] For this purpose IgG was purified from rabbit antiserum by affinity chromatography on a protein A-Sepharose column (Pharmacia) as suggested by the man-
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