Molecular cloning and expression of a porcine chondrocyte nucleotide pyrophosphohydrolase. Gene 1997 Sep 15;197(1-2):277-87
Date
10/23/1997Pubmed ID
9332376DOI
10.1016/s0378-1119(97)00272-2Scopus ID
2-s2.0-0030845712 (requires institutional sign-in at Scopus site) 25 CitationsAbstract
The porcine 127-kDa nucleotide pyrophosphohydrolase (NTPPHase) had been previously purified from the conditioned culture media of porcine articular cartilage. Protein sequencing of an internal 61-kDa proteolytic fragment of NTPPHase (61-kDa NTPPHase) determined the 26 N-terminal amino acids. This sequence was used to amplify a DNA fragment, which was used as a probe to clone the gene encoding the 61-kDa NTPPHase from a porcine chondrocyte cDNA library. DNA sequence analysis showed the cDNA insert to be 2509 bp, corresponding to a predicted open reading frame (ORF) encoding 599 amino acids. The 26 N-terminal amino acids of the 61-kDa NTPPHase were located within the ORF immediately downstream of a putative protease recognition region, RRKRR. This is consistent with this cDNA insert representing an internal proteolytic fragment of the full length 127-kDa NTPPHase. BLAST and FASTA analysis confirmed that the deduced amino acid sequence of 61-kDa NTPPHase was unique and did not possess a high degree of homology to sequence in the non-redundant protein and nucleotide databases. Proteins that possess limited homology (< 17%) with the 61-kDa NTTPPHase include several prokaryotic and eukaryotic ATP pyrophosphate-lyases (adenylate cyclase). Northern blot analysis of porcine chondrocyte RNA showed that the DNA encoding the 61-kDa NTPPHase hybridized to a single 4.0-kb RNA transcript. This DNA probe also hybridized to a single species of human chondrocyte RNA. Expression of a 61-kDa protein was detected by coupled in-vitro transcription/translation. Western blot analysis of this in-vitro transcription/translation reaction detected a 61-kDa protein, using an antibody raised against the peptide sequence that was originally used to clone the 61-kDa NTPPHase. These data indicate the successful in-vitro cloning and expression of the porcine chondrocyte 61-kDa NTPPHase. Future studies that utilize the gene encoding the 61-kDa NTPPHase may allow the characterization of the role of NTPPHase in calcium pyrophosphate dihydrate (CPPD) crystal deposition disease.
Author List
Masuda I, Halligan BD, Barbieri JT, Haas AL, Ryan LM, McCarty DJAuthor
Joseph T. Barbieri PhD Professor in the Microbiology and Immunology department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Amino Acid SequenceAmino Acids
Animals
Base Sequence
Cartilage, Articular
Chondrocytes
Cloning, Molecular
DNA, Complementary
Gene Expression Regulation, Enzymologic
Gene Library
Humans
Molecular Sequence Data
Molecular Weight
Osteoarthritis
Protein Biosynthesis
Pyrophosphatases
RNA, Messenger
Sequence Analysis, DNA
Species Specificity
Swine
Transcription, Genetic