Polyclonal and monoclonal antibodies to human lysyl hydroxylase and studies on the molecular heterogeneity of the enzyme.
Human placental lysyl hydroxylase gave two bands in SDS/polyacrylamide-slab-gel electrophoresis: a broad, diffuse, major band corresponding to an apparent Mr of 80,000-85,000, and a sharp minor band with Mr 78,000. Mouse and chick-embryo lysyl hydroxylases gave only the broad, diffuse band, whereas the sharp band could not be detected. Polyclonal antibodies were prepared to the two bands of the human enzyme separately, and monoclonal antibodies were prepared to the whole purified enzyme preparation. Both types of polyclonal antibody inhibited and precipitated the enzyme activity, and both stained the two polypeptide bands in immunoblotting after SDS/polyacrylamide-gel electrophoresis. Only one out of five monoclonal antibodies inhibited the enzyme activity, whereas they all precipitated the activity when studied with antibody coupled to Sepharose. All five monoclonal antibodies stained the whole broad band in immunoblotting, and at least three of them also stained the sharp band. Peptide maps produced from the two polypeptide species by digestion with Staphylococcus aureus V8 protease were highly similar. Experiments with endoglycosidase H demonstrated that the Mr-80,000-85,000 polypeptide contains asparagine-linked carbohydrate units, which are required for maximal lysyl hydroxylase activity. The data suggest that the lysyl hydroxylase dimer consists of only one type of monomer, the heterogeneity of which is due to differences in glycosylation.
D000118:Acetylglucosaminidase; D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D002642:Chick Embryo; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007122:Immunoelectrophoresis; D046911:Macromolecular Substances; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D051379:Mice; D006899:Mixed Function Oxygenases / Q000276:immunology; D009844:Oligosaccharides / Q000494:pharmacology; D010449:Peptide Mapping; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D009844:Oligosaccharides; D006899:Mixed Function Oxygenases; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase