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.

Immunological characterization of lysyl hydroxylase, an enzyme of collagen synthesis.

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.