MOLECULAR-CLONING AND EXPRESSION OF A PORCINE CHONDROCYTE NUCLEOTIDE PYROPHOSPHOHYDROLASE

The porcine 127-kDa nucleotide pyrophosphohydrolase (NTPPHase) had bee n previously purified from the conditioned culture media of porcine ar ticular cartilage. Protein sequencing of an internal 61-kDa proteolyti c fragment of NTPPHase (61-kDa NTPPHase) determined the 26 N-terminal amino acids. This sequence was used to amplify a DNA fragment, which w as 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 f rame (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-redu ndant 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 RN A. Expression of a 61-kDa protein was detected by coupled in-vitro tra nscription/translation. Western blot analysis of this in-vitro transcr iption/translation reaction detected a 61-kDa protein, using an antibo dy raised against the peptide sequence that was originally used to clo ne the 61-kDa NTPPHase. These data indicate the successful in-vitro cl oning and expression of the porcine chondrocyte 61-kDa NTPPHase. Futur e 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. (C) 1997 Elsevier Scienc e B.V.