BASIC RESEARCH www.jasn.org Epitope-Tagged Pkhd1 Tracks the Processing, Secretion, and Localization of Fibrocystin Jason L. Bakeberg,* Rachaneekorn Tammachote,† John R. Woollard,* Marie C. Hogan,* ʈ Han-Fang Tuan,* Ming Li,‡ Jan M. van Deursen,‡ Yanhong Wu, Bing Q. Huang,§ Vicente E. Torres,* Peter C. Harris,* and Christopher J. Ward* *Division of Nephrology and Hypertension, Mayo Clinic, Rochester, Minnesota; †Department of Botany, Faculty of Science, Chulalongkorn University, Thailand; ‡Department of Pediatric and Adolescent Medicine and Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota; §Miles and Shirley Fiterman Center for ʈ Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota; Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota ABSTRACT Mutations in the PKHD1 gene, which encodes fibrocystin, cause autosomal recessive polycystic kidney disease (ARPKD). Unfortunately, the lack of specific antibodies to the mouse protein impairs the study of splicing, post-translational processing, shedding, and temporal and spatial expression of endogenous fibrocystin at the cellular and subcellular level. Here, we report using a knock-in strategy to generate a null Pkhd1 strain and a strain that expresses fibrocystin along with two SV5-Pk epitope tags engineered in-frame into the third exon, immediately C-terminal to the signal-peptide cleavage site in a poorly conserved region. By 6 mo of age, the Pkhd1-null mouse develops massive cystic hepatomegaly and proximal tubule dilation, whereas the mouse with epitope-tagged fibrocystin has histologically normal liver and kidneys at 14 mo. Although Pkhd1 was believed to generate many splice forms, our western analysis resolved fibrocystin as a 500 kD product without other forms in the 15–550 kD range. Western analysis also revealed that exosome-like vesicles (ELVs) secrete the bulk of fibrocystin in its mature cleaved form, and scanning electron microscopy identified that fibrocystin on ELVs attached to cilia. Furthermore, the addition of ELVs with epitope-tagged fibrocystin to wild-type cells showed that label transferred to primary cilia within 5 min. In summary, tagging of the endogenous Pkhd1 gene facilitates the study of the glycosylation, proteolytic cleavage, and shedding of fibrocystin. J Am Soc Nephrol 22: 2266–2277, 2011. doi: 10.1681/ASN.2010111173 Autosomal recessive polycystic kidney disease and small (100nm diameter) membrane bound (ARPKD MIM ID #263200) is characterized by di- particles which are shed into urine and bile, known lation of both the collecting ducts (CDs) in the kid- as PKD exosome–like vesicles (PKD–ELVs).6–10 It ney and hepatic fibrosis with or without nonobstruc- has been postulated that the mouse Pkhd1 and hu- tive biliary dilation, affecting 1:20,000 live births.1–3 The gene responsible for ARPKD is the polycystic kid- Received November 16, 2010. Accepted May 26, 2011. ney and hepatic disease gene (PKHD1: 472kb), which is encoded on chromosome 6p12.2, has 67 exons and Published online ahead of print. Publication date available at www.jasn.org. a full length mRNA of 16,235bp (mouse mRNA is 12,928bp).4 Fibrocystin/polyductin, the product of Correspondence: Dr. Christopher Ward, Division of Nephrol- ogy and Hypertension, Mayo Clinic, 703 Stabile Building, 200 the PKHD1 gene is a large type I membrane protein First Street SW, Rochester, MN 55905. Phone: 507-266-3050; of 4074 amino acids with a pro-protein convertase Fax: 507-266-9315; E-mail: ward.christopher@mayo. site between residues 3617..3620.5 Fibrocystin has edu been localized to the primary cilium, the basal body Copyright © 2011 by the American Society of Nephrology 2266 ISSN : 1046-6673/2211-2266 J Am Soc Nephrol 22: 2266–2277, 2011 www.jasn.org BASIC RESEARCH man PKHD1genes undergo extensive differential splicing and the possibility that the smaller mRNAs of 9, 7.7 and 7.5kb that many of the putative fibrocystin products are secreted as represent differentially spliced forms of Pkhd1, but RT–PCR they have a signal peptide but no membrane anchor.11–13 If on mouse kidney poly–A mRNA showed that overlapping RT– so, these splice forms could be partially functional or antago- PCR products from exons 1–21, 19–34, 32–52, 48–52 and nistic to full length fibrocystin function and so mutations af- 48–67 only produced products of the predicted size, with only fecting a subset of splice forms are also thought to be respon- very faint products of different molecular mass. As there is only sible for some of the phenotypic variation seen in human one Pkhd1 promoter,18 these data suggests that there is one ARPKD and the range of renal phenotypes in the available full–length form of Pkhd1 mRNA at 13kb and that the smaller mouse models.14–18 However, there is debate as to the extent of forms are the products of premature poly–adenylation. We PKHD1 and Pkhd1 splicing, since Pkhd1 has a relatively simple sequenced all of the RT-PCR products and showed that they band pattern on northern blotting.4 Here we re-investigate the were 100% identical to the published full length cDNA se- splicing of Pkhd1 at an mRNA level. Furthermore, to investi- quence NM_153179 GI:126157465. There was no evidence of gate splicing at a protein level we developed mouse models of heterogeneity, deletions or insertions implying that there was ARPKD where the Pkhd1 gene is initially transcriptionally si- no differential splicing. However, it remains possible that there lenced by a STOP cassette flanked by two loxP sites, an LSL are rare minor splice forms not delineated by this RT-PCR Ϫ Ϫ module.19 Upon removal of the LSL (Pkhd1LSL( )/LSL( )) with analysis. Cre recombinase the Pkhd1 gene reactivates, producing the ϩ Pkhd1Pk( ) allele, and expresses a form of fibrocystin with two Generation of A Pkhd1 Null Mouse and An Epitope ϩ ϩ SV5–Pk tags on its extreme N–terminus. This Pkhd1Pk( )/Pk( ) Tagged Pkhd1Pk(؉)/Pk(؉) Mouse mouse is phenotypically wildtype and produces epitope tagged To investigate differential splicing at a protein level we gener- PKD–ELVs in its urine and bile. The epitope tags are inserted ated a Pkhd1 gene with an epitope tag at its extreme N–termi- into exon–3, an exon which has been shown to be present in 21 nus. To produce mice that were completely null for Pkhd1 of 22 of the putative splice forms described by Boddu.13 If mRNA we inserted a loxP flanked transcriptional STOP cas- differential splicing occurs after exon–3 and these events alter sette into intron–2. The loxP STOP loxP (LSL) cassette con- the length of the protein, these forms of fibrocystin will be tains a puromycin acetyltransferase (pac) gene and four copies detectable upon western blotting of kidney or urine. of an SV–40 viral transcriptional termination sequence that PKD–ELVs are thought to be shed from multivesicular terminates all Pkhd1 transcripts in intron 2.19 The LSL cassette bodies (MVB) and to interact with primary cilia in a rapid and can be removed by the action of the Cre recombinase, leaving specific manner.10 Such interactions have been observed in the an SbfI site and a SalI flanked loxP site in intron-2 (54bp in embryologic node by Tanaka and in the maturation of male length), 369bp 5Ј to the beginning of exon–3, allowing the gene germ cells in the epididymis, where ELVs fuse with the flagel- to restart. This results in the expression of an epitope tagged lum (modified cilium) of the maturing sperm cells.20,21 How- form of fibrocystin that has two SV5–Pk tags encoded in– ever, there is no simple assay for PKD–ELV/primary cilium frame in exon–3. These tags, 26 amino acids in length, were interactions. Here we show that Pkhd1Pk(ϩ)/Pk(ϩ) mouse urine inserted into one of the most evolutionary nonconserved re- can supply tagged PKD–ELVs that interact with WT primary gions of fibrocystin and, hence, were predicted not to influence cilia in vitro and be detected by immuno–scanning electron the function of the protein (Figure 2B). Furthermore, once the microscopy (ISEM). We further show that tagging of the en- signal peptide is cleaved, the two SV5–Pk tags are predicted to dogenous Pkhd1 gene allows the monitoring of fibrocystin in be on the extreme N–terminus of the protein, an ideal position its physiologically relevant context, in particular its glycosyla- from which to detect the native protein in tissue sections and tion, proteolytic cleavage and shedding of fibrocystin on PKD– monitor proteolytic processing, glycosylation and putative ELVs. splice forms, by western blotting (Figure 2D). Exon–3 was also chosen as it ought to be present in 95% of putative splice forms.13 RESULTS (Comparison of Pkhd1LSL(؊)/LSL(؊) and Pkhd1Pk(؉)/Pk(؉ Investigation of PKHD1 Splicing Mice Pkhd1 is highly expressed in the kidney,4,11 and resolves as a Northern blotting and RT–PCR analysis indicates that major 13kb mRNA with three smaller minor species of 9, 7.7 the Pkhd1LSL(Ϫ)/LSL(Ϫ) mouse makes no fibrocystin mRNA and 7.5kb when probed with probes encompassing exons (Figures 1 and 3). The Pkhd1LSL(Ϫ)/LSL(Ϫ) mouse, on an F10 3–13, and 22–32. A probe encompassing exons 44–50 detects inbred BALB/cJ or C57BL/6J background, develops cysts and fi- the 9kb product weakly and not the 7.7 and 7.5kb species. brosis in the liver, histologically visible at 1 mo of age, and females However, a probe encompassing exons 60–67 detects only the develop PT dilation at 6 mo of age, (Figures 4 and 5). Male mice longest 13kb product (Figure 1A). The 9, 7.7 and 7.5kb species are protected from kidney cyst development in a manner appear to be polyadenylated as they are detected on northern similar to the Pkhd1del2/del2 mice described by Woollard blots of poly–A selected mRNA (Figure 1B).