DNPEP is not the only peptidase that produces SPAK fragments in kidney

Abstract

SPAK (STE20/SPS1-related proline/alanine-rich kinase) regulates Na⁺ and Cl^- reabsorption in the distal convoluted tubule, and possibly in the thick ascending limb of Henle. This kinase phosphorylates and activates the apical Na-Cl cotransporter in the DCT. Western blot analysis reveals that SPAK in kidney exists as a full-length protein as well as shorter fragments that might affect NKCC2 function in the TAL. Recently, we showed that kidney lysates exerts proteolytic activity towards SPAK, resulting in the formation of multiple SPAK fragments with possible inhibitory effects on the kinase. The proteolytic activity is mediated by a Zn²⁺ metalloprotease inhibited by 1,10-phenanthroline, DTT, and EDTA. Size exclusion chromatography demonstrated that the protease was a high-molecular-weight protein. Protein identification by mass-spectrometry analysis after ion exchange and size exclusion chromatography identified multiple proteases as possible candidates and aspartyl aminopeptidase, DNPEP, shared all the properties of the kidney lysate activity. Furthermore, recombinant GST-DNPEP produced similar proteolytic pattern. No mouse knockout model was, however, available to be used as negative control. In this study, we used a DNPEP-mutant mouse generated by EUCOMM as well as a novel CRISPR/cas9 mouse knockout to assess the activity of their kidney lysates towards SPAK. Two mouse models had to be used because different anti-DNPEP antibodies provided conflicting data on whether the EUCOMM mouse resulted in a true knockout. We show that in the absence of DNPEP, the kidney lysates retain their ability to cleave SPAK, indicating that DNPEP might have been misidentified as the protease behind the kidney lysate activity, or that the aspartyl aminopeptidase might not be the only protease cleaving SPAK in kidney.

Keywords: Antibody specificity; CRISPR/cas9; DNPEP knockout; Ste20p‐like kinases; mouse model validation; proteolytic cleavage.

Figure 1

Design of the EUCOMM Dnpep mouse. (A) The Dnpep gene consists of 15 exons spaced within a relatively small 10 kb genomic stretch of mouse chromosome 1. Many exons (numbered in red font) are multiple of 3 or cassette exons. Their removal does not affect the open reading frame of the protein. (B) The construct consisted of 5.6 kb of 5′ arm of recombination, β‐galactosidase and neomycin‐resistance gene cassettes flanked by Frt recombination sites, followed by two loxP sites surrounding exons 9–11, and a smaller 3.4 kb 3′ arm of recombination. (C) Structure of the mutant allele in the Dnpep‐targeted mouse. Note the missing loxP site, confirmed by sequencing using primers DNP1 and DNP2. The location of the genotyping PCR primers DNP7, DNP4, and CF1 is indicated by arrows. Frt: Flippase recognition target; En2‐SA: splice acceptor from En‐2 gene; IRES: Internal Ribosomal Entry Site; LAC Z: β‐galactosidase open reading frame; pA: poly adenylation signal; loxP: bacteriophage P1 original sequence recognized by CRE recombinase; hBactIP: autonomous promoter; neo: neomycin resistance open reading frame.

Figure 2

DNPEP expression and cleavage activity in Dnpep homozygous mice. (A) PCR genotyping of 7 pups of Dnpep heterozygous cross. Top picture represents the mutant allele (CF1‐DNP4 primers) and bottom picture represents the wild‐type allele (DNP7‐DNP4 primers). Sizes of PCR fragments are 226 bp (mutant) and 324 bp (wild‐type), respectively. All three anticipated genotypes are observed. Homozygote mutant are highlighted in red box. (B) Western blot analysis with increasing amount of kidney protein lysate from wild‐type and EUCOMM (EU) mutant mouse using an anti‐DNPEP antibody from Abcam. (C) Comparison between Abcam and Abgent antibodies on protein samples isolated from wild‐type (+/+), heterozygote EUCOMM (±), and EUCOMM homozygote (‐/‐) mouse kidneys. D. GST‐SPAK (100 kDa) fusion protein (FP) is cleaved into smaller bands with both wild‐type (lane 4) and Dnpep EUCOMM mutant (lane 5) kidney lysates. The intense ~50 kDa band does not come from kidney lysate as identical lysate samples are loaded in lanes 1 and 2.

Figure 3

Design of a CRISPR/Cas9 Dnpep knockout mouse. (A) Design of the guide RNA around amino acids 18–24, which are encoded by exon 2. The PAM sequence consists of the Arg25 codon (CGG). Cas9 and the break site are indicated. (B) Sequence of the guide RNA with DNPEP‐specific target sequence followed by trace RNA. (C) Several mutations indicated in red font in the upper strand are created in the core repair DNA to introduce a stop codon (Lys21Ter) and a BspHI restriction site, and prevent Cas9 to recognize the repair DNA. The repair DNA consists of a 191 base single‐stranded oligonucleotide with a 15 bp core repair sequence flanked by 88 base arms of recombination.

Figure 4

Generation of the CRISPR/Cas9 Dnpep knockout mouse (A) Agarose gel showing genotyping of 10 CRISPR/cas9 offsping. An aliquot of the PCR reaction was digested with BspHI. Note the full‐length PCR fragment (lower band), digested products in samples labeled with a star, and absence of full‐length product in sample #24, indicating homozygosity. (B) Sequence of the two alleles of five mice showing mutations in red. The designed allele with stop codon (TAG) and BspHI restriction site is found in four of these mice. (C) Chromatogram of one heterozygous offspring showing germline transmission with one wild‐type sequence and one mutant sequence.

Figure 5

Western blot analysis of DNPEP wild‐type and knockout kidneys. (A) Presence of a band at ~50 kDa in kidneys from wild‐type mice but not Dnpep knockout mice is seen with the anti‐Dnpep Abcam antibody. Actin labeling demonstrated equal labeling. (B) Identical experiment was performed using anti‐Dnpep antibody from Abgent. While the signal was somewhat weaker in the knockout, there was still very significant signal at the ~50 kDa molecular size. Actin signal confirmed equal loading. (C). The GST‐SPAK fusion protein (100 kDa, lane 3) is cleaved into smaller bands with both wild‐type (lanes 4, 6) and Dnpep knockout (lane 5, 7) kidney lysates. (D) Western blot analysis of SPAK in kidneys isolated from wild‐type and DNPEP knockout mice. FP, Fusion Protein; w.t., samples from wild‐type mice; KO, samples from CRISPR/cas9‐generated DNPEP knockout mice.