Human biotinidase: The 5' structure of the gene and its possible role in tissue-specific and intracellular localization.

摘要

Biotinidase is essential for recycling the vitamin biotin and is possibly required for other functions indicating that the enzyme may localize to various cellular and extracellular sites.; The biotinidase gene is contained in a GenBank (accession number AC027129) sequence identified by a BLAST search of NCBI databases. Intron 1 is 35 kb. RACE-PCR was used to extend the 5′ sequence of exon 1 by 90 bases and to identify three novel, alternatively spliced variants of biotinidase, 1a, 1b and 1c, in multiple human tissues. Exon 1c was only in testes and includes sequence originally designated as intron 1. Together these sequences complete the structure of the 5′ end of the human biotinidase gene. The 5′ splice variants, 1a and 1b, contain sequence motifs that suggest that biotinidase localizes to the mitochondria/nucleus and ER, respectively.; Biotinidase localizes to the cytoplasm, but not nucleus, of human fibroblasts and Hep G2 cells in immunohistochemical studies. Organelle separation by isopycnic gradient centrifugation of rat liver identified an 85 kDa biotinidase protein, which contained biotinyl-hydrolase and transferase activities, in fractions enriched for microsomal marker enzymes. These results confirmed earlier studies localizing biotinidase to microsomes. A 48 kDa protein, that also reacted with anti-biotinidase, co-localized to fractions enriched with mitochondrial marker enzyme activities. The 48 kDa protein is biotinylated, is in the inner mitochondrial matrix, and has no biotinyl-hydrolase or transferase activities. The 5′ splice variants and organelle fractionation studies suggest that biotinidase is directed to the mitochondria and the secretory pathway.; Because biotinidase deficiency is associated with specific neurological symptoms, such as sensorineural hearing loss and optic atrophy, immunohistochemical techniques were used to localize biotinidase in mouse brain. Biotinidase staining in the dorsal and ventral cochlear nuclei and vestibular nucleus indicates that biotinidase is concentrated in areas involved in vision and hearing. However, expression studies do not confirm discrete regions of up-regulation of the gene in rat or human brain. The localization of biotinidase in mouse brain supports a role for biotinidase in the CNS.; These studies identified the intracellular localization of biotinidase to microsomes and mitochondria and the localization of biotinidase to brain.