Deletion of the gene encoding proprotein convertase 5/6 causes early embryonic lethality in the mouse

Abstract

PC5 belongs to the proprotein convertase family and activates precursor proteins by cleavage at basic sites during their transit through the secretory pathway and/or at the cell surface. These precursors include prohormones, proreceptors, growth factors, adhesion molecules, and viral glycoproteins. The Pcsk5 gene encodes two alternatively spliced isoforms, the soluble PC5A and transmembrane PC5B. We have carefully analyzed the expression of PC5 in the mouse during development and in adulthood by in situ hybridization, as well as in mouse tissues and various cell lines by quantitative reverse transcription-PCR. The data show that adrenal cortex and intestine are the richest sources of PC5A and PC5B, respectively. To better define the specific physiological roles of PC5, we have generated a mouse Pcsk5(Delta4)-deficient allele missing exon 4 that encodes the catalytic Asp173. While Delta4/+ heterozygotes were healthy and fertile, genotyping of progeny obtained from Delta4/+ interbreeding indicated that Delta4/Delta4 embryos died between embryonic days 4.5 and 7.5. These data demonstrate that Pcsk5 is an essential gene.

FIG. 1.

Isoforms PC5A and PC5B and their processing. Exons 1 to 20 of the mouse Pcsk5 gene encode the signal peptide (SP; amino acids [aa] 1 to 34), prosegment (aa 35 to 116), catalytic domain (aa 117 to 458), P domain (aa 459 to 600), and N-terminal part of the Cys-rich domain (aa 638 to 877). Exon 21A, specific to PC5A, encodes its last 38 residues, while the 18 additional exons of PC5B (21B to 38) extend the Cys-rich domain up to a transmembrane domain (TMD; aa 1769 to 1789) followed by an 88-aa-long cytosolic tail (CT). The residues of the catalytic triad comprise Asp₁₇₃ (boxed D), encoded by exon 4; His₂₁₄ (H); and Ser₃₈₈ (S). The position of the oxyanion hole Asn₃₁₅ (N) is also shown. Arrows in the prosegment indicate autocatalytic cleavage sites, and the arrowhead points to the intracellular cleavage site, generating an ∼68-kDa PC5A-ΔC form. White ellipses emphasize the putative N-glycosylation sites (Asn 227, 383, 667, 754, 804, 854, 951, 1016, 1220, 1317, 1523, 1711, and 1733).

FIG. 2.

PC5 expression during mouse development and in adulhood. Distribution of PC5 transcripts was obtained by ISH of cryosections with a ³²P-labeled specific probe. Embryos at E9.5, E10.5, and E11.5 are twice enlarged compared to the others. In the E15.5 inset, a longer exposure of sections hybridized with either the control sense (S) or antisense (AS) probe is shown. The main sites of expression are indicated as follows: ad, adrenal; ad p, adrenal primordium; cart p, cartilage primordium; cx, cortex; ep, epididymis; k, kidney; fut d raphé n, future dorsal raphe nucleus; hip, hippocampus; i, intestine; liv, liver; lg, lung; lym nd, lymphatic node; ov, otic vesicle; panc p, pancreas primordium; pv, portal vein; pulm, pulmonary; som, somite; tg, tongue; umb cord, umbilical cord.

FIG. 3.

PC5 expression in tissues and cell lines. Using QPCR, mRNAs of isoforms A (white bars) and B (black bars) of PC5 were independently quantified in tissues (primers in exons 20/21 and 37/38, respectively), while a global estimation of A and B isoforms (primers in exons 19/20) was performed for cell lines. For the last six tissues in which PC5 levels were low, an inset was added showing an enlargement of the levels of PC5A and PC5B. Note that a linear scale was used for tissues (left panel) and a logarithmic one for cell lines (right panel).

FIG. 4.

Pcsk5 knockout strategy. Three genomic DNA fragments covering 12 kb of the Pcsk5 locus were subcloned into the targeting vector, in which the exon 4 is flanked with two loxP sites (open triangles) and a PGK-neo cassette. ES cells were transfected with the NotI-KpnI insert (solid line). The expected neo allele is shown. Mating of heterozygotes bearing a Pcsk5^neo recombinant allele with transgenic mice expressing the Cre recombinase generated progeny in which Cre was expressed and excised the sequences between the loxP sites. The generated Pcsk5^Δ4-inactivated allele misses ∼4.5 kb of genomic DNA, including exon 4. E, EcoRI; RV, EcoRV; Ecl, Ecl136II; H, HindIII; K, KpnI; N, NotI; X, XhoI.

FIG. 5.

Genomic analysis of 5B10-derived mice. Genomic DNA (6 μg) from C57BL/6 (+/+), 5B10-derived (neo/+), and Δ4/+ heterozygotes issued from mating of 5B10-derived mice to Cre-expressing ones was digested and analyzed by Southern blotting. Using a 3′ external probe and HindIII digestion, wild-type and neo recombinant alleles generate 7.2- and 9.2-kb fragments, respectively. Using an internal probe and BstEII digestion, wild-type and Δ4 alleles generate 4.2- and 11-kb fragments, respectively. The lanes marked “1 kb” and “λ/HindIII” contain specific ladders.

FIG. 6.

PC5Δ4 expression and activity. (A) QPCR analysis of PC5 mRNA from +/+ and Δ4/+ livers using primers located in exons 3/5 revealed a unique 239 bp product in +/+ liver and an additional 95-bp one in Δ4/+ liver resulting from an altered splicing connecting exons 3 and 5. (B) HEK293 cells were transfected with the pIRES2-EGFP vector empty (V) or expressing wild-type PC5A or PC5Δ4, both C-terminally V5 tagged. Cell extracts (20 μg) and media (40 μl out of 1 ml) were analyzed by anti-V5 Western blotting (WB) prior (left panel) or after (right panel) immunoprecipitation (IP) with a prosegment antibody (anti-ppPC5). (C) Online PC5 activity in relative fluorescence units (RFU) was measured by incubation of 60 μl of medium with the fluorogenic substrate Pyr-RTKR-AMC. Note the absence of activity in PC5Δ4 and control V media. (D) HEK293 cells were cotransfected with 0.1 μg of a vector expressing PDGF-A carrying a C-terminal V5 tag and 0.9 μg of a vector expressing either no product (V), α1-PDX, the prosegment of furin (ppFur), the prosegment of PC5 (ppPC5), or PC5Δ4. Media (2 μl out of 1 ml) were analyzed by Western blotting using V5 antibodies.