Molecular cloning and expression of a porcine chondrocyte nucleotide pyrophosphohydrolase.
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.
Masuda
I
I
Department of Medicine, Medical College of Wisconsin, Milwaukee 53226, USA. imasuda@post.its.mcw.edu
Halligan
B D
BD
Barbieri
J T
JT
Haas
A L
AL
Ryan
L M
LM
McCarty
D J
DJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D002358:Cartilage, Articular / Q000201:enzymology; D019902:Chondrocytes / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010003:Osteoarthritis; D014176:Protein Biosynthesis; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D013552:Swine; D014158:Transcription, Genetic
D000596:Amino Acids; D018076:DNA, Complementary; D012333:RNA, Messenger; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase