HUMAN SERUM BIOTINIDASE - CDNA CLONING, SEQUENCE, AND CHARACTERIZATION

Biotinidase (EC 3.5.1.12) catalyzes the hydrolysis of biocytin, the pr oduct of biotin-dependent carboxylase degradation, to biotin and lysin e. Biotinidase deficiency is an inherited metabolic disorder of biotin recycling that is characterized by neurological and cutaneous abnorma lities, and can be successfully treated with biotin supplementation. S equences of tryptic peptides of the purified human serum enzyme were u sed to design oligonucleotide primers for polymerase chain reaction am plification from human hepatic total RNA to generate putative biotinid ase cDNA fragments. Sequence analysis of a cDNA isolated from a human liver library by plaque hybridization with the largest cDNA probe reve aled an open reading frame of 1629 bases encoding a protein of 543 ami no acid residues, including 41 amino acids of a potential signal pepti de. Comparison of the open reading frame with the known biotinidase tr yptic peptides and recognition of the expressed protein encoded by thi s cDNA by monoclonal antibodies prepared against purified biotinidase demonstrated the identity of this cDNA. Southern analyses suggested th at biotinidase is a single copy gene and revealed that human cDNA prob es hybridized to genomic DNA from mammals, but not from chicken or yea st. Northern analysis indicated the presence of biotinidase mRNA in hu man heart, brain, placenta, liver lung, skeletal muscle, kidney, and p ancreas.