Characterization of Mouse Carboxypeptidase N Small Active Subunit Gene Structure

Abstract

Carboxypeptidase N (CPN) is a plasma zinc metalloprotease comprised of two small subunits that have enzymatic activity, and two large subunits, which protect the enzyme from degradation. CPN cleaves the carboxyl-terminal amino acids arginine and lysine from biologically active peptides such as complement anaphylatoxins, kinins, and fibrinopeptides. To delineate the murine CPN small subunit coding region, gene structure, and chromosome location, cDNA and genomic clones were isolated, characterized, and used in Northern and fluorescence in situ hybridization analyses. The results from this study demonstrate that the murine CPN small subunit gene is a single copy gene of ∼29 kb that is transcribed in the liver into a 1793-bp mRNA with an open reading frame of 1371 nucleotides encoding 457 aa. The gene contains nine exons ranging in size from 455 bp (exon 1) to 100 bp (exon 7), and eight introns ranging in size from 6.2 kb (intron 2) to 1.4 kb (intron 4). All intron/exon junctions follow the normal consensus rule. The mouse CPN small subunit gene localized to chromosomal band 19D2, which is syntenic to human chromosome 10q23–25. Primer extension experiments using mouse liver mRNA indicate one major transcriptional initiation site and three minor sites. Sequence analysis of the 5′-flanking region indicated a TATA-less promoter and numerous transcription factor binding sites, which may confer liver-specific expression of the CPN small subunit gene.

Targeted Disruption of the Gene Encoding the Murine Small Subunit of Carboxypeptidase N (CPN1) Causes Susceptibility to C5a Anaphylatoxin-Mediated Shock

“…The murine CPN1 gene was disrupted by replacing exon 3 (15), which participates in substrate binding, with a neomycin resistance cassette as described in Materials and Methods (Fig. 1 A ).…”

“…A targeting vector was designed to replace exon 3 of the murine cpn1 gene (15) with a neomycin-resistance gene from pKO SelectNeo V800 (Lexicon Genetics) (see Fig. 1 A ).…”

Whole-genome sequencing study of serum peptide levels: the Atherosclerosis Risk in Communities study

“…It is doubtful that this mechanism explains the effect of CPN1 on DSGEGDFXAEGGGV, however, because fibrinopeptide A is cleaved from fibrinogen prior to the formation of fibrin clots. Although carboxypeptide N may cleave fibrinopeptides (44), it is equally unlikely that carboxypeptidase N cleaves the N-terminal alanine from fibrinopeptide A to form DSGEGDFXAEGGGV, since carboxypeptidase N specifically cleaves C-terminal arginine and lysine. Our additional analyses do not support an effect on conversion since the lead variant was related to increased levels of both DSGEGDFXAEGGGV and fibrinopeptide A, and these associations were more significant than the association with the ratio of DSGEGDFXAEGGGVR to fibrinopeptide A.…”