Functional characterization of Narc 1, a novel proteinase related to proteinase K
Section snippets
Plasmid construction and mutagenesis
Full length cDNA clones encoding human and rat NARC 1 (GenBank Accession Nos. AX207686 and AX207690, respectively) were used as PCR templates. For mammalian expression constructs, the upstream PCR primer introduced a HindIII site, and the downstream primer introduced an XhoI site engineered to maintain the reading frame of vector-encoded COOH-terminal epitope tags. PCR amplification was with Supermix Hi Fidelity (Gibco-BRL). Cloning was into the expression vectors pcDNA3.1mycHis-C (Invitrogen)
Protein sequence analysis and Narc 1 structural modeling
Translation of the deposited nucleotide sequences for human, mouse, and rat NARC 1 (GenBank Accession Nos. AX207686, AX207688, and AX207690, respectively) reveals an apparent heterogeneity in N-terminal extension. Since some doubt exists whether the upstream in-frame AUGs of the rat and mouse mRNA sequences are, in fact, translation initiation sites [12], we chose to limit our functional analysis of the rat orthologue to that sequence aligning with the translated human sequence. Thus, in this
Discussion
Structural modeling predicts the overall conformation of the Narc 1 catalytic domain to be very similar to those of proteinase K [24], subtilisin [41], [42], and furin [43] especially in their active site regions. Despite the overall conformational similarities, some important structural differences exist, even between Narc 1 and proteinase K. The proteinase domain of Narc 1 contains six cysteine residues, predicted to form two disulfide bridges, compared with the five cysteines seen in other
Acknowledgements
We thank Ru Shen, Smita Kotnis, and Nivedita Ghosh for molecular biology support, Erik Wagner for help with Narc 1 secretion experiments, Diana Chen, Lan He, and Albert Uveges for plasmid sequencing, and Dr. Jovita Marcinkeviciene for a critical reading of the manuscript.
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- 1
Present address: Bristol-Myers Squibb, Pennington, NJ 08534, USA.
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Present address: Purdue Pharma LP, Cranbury, NJ 08512, USA.
- 3
Present address: Compound Therapeutics, Waltham, MA 02451, USA.
- 4
Present address: Elixir Pharmaceuticals, Inc., Cambridge, MA 02139, USA.