Heath Cole$, Thomas R. Reynolds$, Jean M. Lockyern, Gregory A. Buck% 'I'd Denson%. J. Edward Spence$ll**, Jeanne HymesS, and Barry WolfsIISS.
THEJOURNALOF BIOL~CICALCHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, h .
Vol. 269, No. 9, Issue of March 4, pp. 6566-6570, 1994 Printed in U.S.A.
Human Serum Biotinidase cDNA CLONING,
SEQUENCE, AND CHARACTERIZATION* (Received for publication, August 16, 1993, and in revised form, November 11, 1993)
Heath Cole$, Thomas R. Reynolds$, Jean M. Lockyern, GregoryA. Buck%’I’d Denson% J. Edward Spence$ll**,Jeanne HymesS, and Barry WolfsIISS From the Departments of $Human Genetics, $Microbiology and Immunology, and lpediatrics, Medical College of Virginia! Virginia Commonwealth University, Richmond, Virginia23298 and the IDepartment of Medicine, lhlane University School of Medicine, New Orleans, Louisiana 70112
Biotinidase (EC 3.5.1.12) catalyzes the hydrolysis of normal activity) include seizures, hypotonia, skin rash, alopebiocytin, the product of biotin-dependent carboxylase cia, developmental delay, conjunctivitis, visual problems, heardegradation, to biotin andlysine. Biotinidase deficiency ing loss, metabolic ketolactic acidosis, organic acidemia, and is an inherited metabolic disorder of biotin recycling hyperammonemia (1).There is variability inthe expression of that is characterized by neurological and cutaneous ab-these features and in the age of onset of symptoms, even within normalities, and can be successfully treated with biotin the same family (3). Because biotin therapy, when initiated supplementation. Sequences of tryptic peptides of the early, prevents many clinical and biochemical symptomsof this purified human serum enzyme were used to design oli- disorder (l), newborn screening for biotinidase deficiency has gonucleotide primers for polymerase chain reaction am- been implemented in manystates in the United Statesand i n plification from human hepatic total RNA to generate many countries (4). putative biotinidase cDNA fragments. Sequence analyWe now describe the cloning and sequencing of the cDNA sis of a cDNA isolated from a human liver library by encoding normal human biotinidase.The tissue distribution of plaque hybridization with the largest cDNA probe revealed an open reading frameof 1629 bases encodinga the biotinidase mRNA, copy number, and preliminary analysis protein of 543 amino acid residues, including41 amino of conservation of t h e genomic gene for biotinidase were deteracids of a potential signal peptide. Comparison of the mined. open reading framewith the known biotinidase tryptic EXPERIMENTALPROCEDURES peptides and recognition of the expressed protein enGeneral Methods-Standard methods wereperformed (51, except coded by this cDNA by monoclonal antibodies prepared against purified biotinidase demonstratedthe identity where noted. Restriction endonucleases and DNA modifying enzymes prehybridof this cDNA Southernanalyses suggested that biotini- were obtained from Life Technologies, Inc.. Membranes were ized in 5 x SSC, 5 x Denhardt’s reagent (0.1% Ficoll, 0.1% polyvinyldase is a single copygeneandrevealedthathuman pyrrolidone, 0.1%bovine serum albumin), 2% SDS, and 100 pg/ml sonicDNA probes hybridized to genomic DNAfrommamcatedsalmon sperm DNA at 60“C for 4 h. Hybridizations were mals, but not from chicken or yeast. Northern analysis performed in fresh prehybridization solution with 10%dextran sulfate indicated the presence of biotinidase mRNA in human at 60 “C for 14-18 h. The final stringency of washing conditions was 0.2 heart, brain, placenta, liver, lung, skeletal muscle, kid- x SSC, 1%SDS at 60 “C for 30 min, except where noted. Filters were dried, coveredin plastic wrap and exposed to X-Omat AR scientific ney, and pancreas. imaging film (Kodak) with Cronex intensifying screens (DuPont) at -70 “C for 24 h, unless otherwise noted. Oligonucleotides used in polymerase chain reactions (PCR)’ and sequencing procedures weresynBiotinidase (EC 3.5.1.12) catalyzes the release of biotin, an thesized by the Louisiana State University Core Facility (New Orleans, essential B-complex vitamin, from biocytin, the degradative L A ) and the Nucleic Acid Synthesis and Analysis Laboratory (Medical College of VirginiaNirginia Commonwealth University (MCVNCU), product of the four biotin-dependent holocarboxylases, pyruDuvate carboxylase, acetyl-coA carboxylase, propionyl-CoA car- Richmond, VA). Radiolabeled deoxynucleotides were obtained from Pont NEN (Boston, MA). boxylase a n d p-methylcrotonyl-CoA carboxylase,and from procDNA Probes-BTD400 was liberated from pCRlOOO upon digestion (1).Mammals cannot teolyticallydegradeddietaryproteins with EcoRI and HindIII. BTD2000 was excised from pBluescript SK synthesize biotin and, therefore, must obtain the vitamin fromwith BamHI and ApaI. Inserts were isolated from a 1.3% low melting their diet and from recycling endogenous biotin. point agarose gel, purified by extraction with pheno1:chloroform and Biotinidase deficiency, an autosomal recessive disorder, re- recoveredby ethanol precipitation. The p-actin cDNAwas obtained from Clontech (Palo Alto, CAI. cDNAs were radiolabeled with sults in a secondary biotin deficiency that leads to multiple [CI-~*PI~CTP (3000 Ci/mmol) (DuPont NEN) using an oligolabeling kit carboxylase deficiency (2). Clinical features of untreated indi(Pharmacia LKB Biotechnology Inc.). viduals with profound biotinidase deficiency of mean Preparation of Ryptic Peptides of Biotinidase-Enzymatically active biotinidase was purified 22,000-fold from poolednormal human serum * This work was supported in part by Grant DK33022 from the Na- (6).Asingle amino acidN terminuswas foundon analysis of the protein. A single silver-staining protein was observed on SDS and native polytional Institutes of Health (to B. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This acrylamide gel electrophoresis (PAGE). Polyclonaland monoclonal anarticle must therefore be hereby marked “advertisement”in accordance tibodies prepared against the purified serum enzyme detected a single, with 18 U.S.C. Section 1734 solely to indicate this fact. approximately 74-kDa protein in serum on immunoblots of SDS-PAGE The nucleotide sequence(s) reported in this paper has been submitted (7). Using these antibodies, we have identified several individuals with to the GenRankTM/EMBLDataRank with accession number(s) U03274. profound biotinidase deficiency who lack cross-reactingmaterial to an** Present address: Dept. of Pediatrics, Carolinas Medical Center, Charlotte, NC 28232. The abbreviations used are: PCR, polymerase chain reaction; PAGE, $.$ Towhom reprint requests should be addressed: Dept. of Human Genetics, Medical College of Virginia, P. 0. Box 33, MCV Station, Rich- polyacrylamide gelelectrophoresis;bp, base pair(s);kb, kilobase(s1; Tricine, N-tris(hydroxymethy1)methylglycine. mond, VA 23298. Tel.: 804-786-9632; Fax: 804-786-3760.
(
