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Title: Molecular cloning and chromosomal localization of human holocarboxylase synthetase, a gene responsible for biotin dependency

Abstract

Holocarboxylase synthetase (HCS) catalyzes biotin incorporation into various carboxylases that require biotin as a prosthetic group. They are acetyl-CoA carboxylase, a rate-limiting enzyme of fatty acid synthesis; pyruvate carboxylase, a key enzyme of gluconeogenesis; propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase, enzymes involved in amino acid catabolism. HCS is therefore involved in various metabolic processes and is a key enzyme for biotin utilization by mammalian cells. Deficiency of HCS in man is known to cause biotin-responsive multiple carboxylase deficiency. Isolation of cDNA clones for the enzyme is essential to understand HCS and its deficiency at the molecular level. We purified bovine liver HCS and sequenced its proteolytic peptides. Degenerative oligonucleotide primers were synthesized from the two peptide sequences and used to amplify a putative HCS cDNA fragment from human liver by PCR. Using the amplified DNA fragment as a probe, we screened {lambda}gt10 human liver cDNA library and isolated 12 positive clones. The isolated cDNAs encoded a protein of 726 amino acids with molecular mass of 80,759. The protein contained several sequences identical or similar to those of peptides derived from the bovine liver HCS. The predicted protein had a homologous region with BirA which acts as both a biotin-[acetyl-CoA-carboxylase] ligase andmore » a biotin repressor in E. coli, suggesting a functional relationship between the two proteins. We expressed the protein using pET3 a vector in E. coli (BL21 strain) and raised antiserum against the expressed protein. The antiserum immunoprecipitated HCS activities of human lymphoblasts and bovine liver. A one-base deletion and a missense mutation were found in cells from siblings with HCS deficiency. The human HCS gene was assigned to chromosome 21, region 21q22.1 by fluorescence in situ hybridization analysis.« less

Authors:
; ;  [1]
  1. Tohoku Univ. School of Medicine, Sedai (Japan); and others
Publication Date:
OSTI Identifier:
134337
Report Number(s):
CONF-941009-
Journal ID: AJHGAG; ISSN 0002-9297; TRN: 95:005313-1070
Resource Type:
Journal Article
Journal Name:
American Journal of Human Genetics
Additional Journal Information:
Journal Volume: 55; Journal Issue: Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; GENES; GENE MUTATIONS; DNA SEQUENCING; DNA-CLONING; PATIENTS; METABOLIC DISEASES; HUMAN CHROMOSOME 21; GENETIC MAPPING; AMINO ACIDS; CATABOLISM; CARBOXYLASE; LIGASES; BIOTIN; BIOSYNTHESIS; PROTEINS; CODONS; OLIGONUCLEOTIDES; POLYMERASE CHAIN REACTION; PROBES; DNA HYBRIDIZATION; FLUORESCENCE; ESCHERICHIA COLI

Citation Formats

Suzuki, Y, Aoki, Y, and Ishida, Y. Molecular cloning and chromosomal localization of human holocarboxylase synthetase, a gene responsible for biotin dependency. United States: N. p., 1994. Web.
Suzuki, Y, Aoki, Y, & Ishida, Y. Molecular cloning and chromosomal localization of human holocarboxylase synthetase, a gene responsible for biotin dependency. United States.
Suzuki, Y, Aoki, Y, and Ishida, Y. 1994. "Molecular cloning and chromosomal localization of human holocarboxylase synthetase, a gene responsible for biotin dependency". United States.
@article{osti_134337,
title = {Molecular cloning and chromosomal localization of human holocarboxylase synthetase, a gene responsible for biotin dependency},
author = {Suzuki, Y and Aoki, Y and Ishida, Y},
abstractNote = {Holocarboxylase synthetase (HCS) catalyzes biotin incorporation into various carboxylases that require biotin as a prosthetic group. They are acetyl-CoA carboxylase, a rate-limiting enzyme of fatty acid synthesis; pyruvate carboxylase, a key enzyme of gluconeogenesis; propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase, enzymes involved in amino acid catabolism. HCS is therefore involved in various metabolic processes and is a key enzyme for biotin utilization by mammalian cells. Deficiency of HCS in man is known to cause biotin-responsive multiple carboxylase deficiency. Isolation of cDNA clones for the enzyme is essential to understand HCS and its deficiency at the molecular level. We purified bovine liver HCS and sequenced its proteolytic peptides. Degenerative oligonucleotide primers were synthesized from the two peptide sequences and used to amplify a putative HCS cDNA fragment from human liver by PCR. Using the amplified DNA fragment as a probe, we screened {lambda}gt10 human liver cDNA library and isolated 12 positive clones. The isolated cDNAs encoded a protein of 726 amino acids with molecular mass of 80,759. The protein contained several sequences identical or similar to those of peptides derived from the bovine liver HCS. The predicted protein had a homologous region with BirA which acts as both a biotin-[acetyl-CoA-carboxylase] ligase and a biotin repressor in E. coli, suggesting a functional relationship between the two proteins. We expressed the protein using pET3 a vector in E. coli (BL21 strain) and raised antiserum against the expressed protein. The antiserum immunoprecipitated HCS activities of human lymphoblasts and bovine liver. A one-base deletion and a missense mutation were found in cells from siblings with HCS deficiency. The human HCS gene was assigned to chromosome 21, region 21q22.1 by fluorescence in situ hybridization analysis.},
doi = {},
url = {https://www.osti.gov/biblio/134337}, journal = {American Journal of Human Genetics},
number = Suppl.3,
volume = 55,
place = {United States},
year = {1994},
month = {9}
}