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Human Molecular Genetics, 2003, Vol. 12, No. 6 685-698
© 2003 Oxford University Press

PKHDL1, a homolog of the autosomal recessive polycystic kidney disease gene, encodes a receptor with inducible T lymphocyte expression

Marie C. Hogan, Matthew D. Griffin, Sandro Rossetti, Vicente E. Torres, Christopher J. Ward and Peter C. Harris*

Division of Nephrology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA

Received December 14, 2002; Accepted January 21, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Autosomal-recessive polycystic kidney disease (ARPKD) is caused by mutation to a large gene, PKHD1, encoding a putative receptor protein, fibrocystin. We have identified, through analysis of human genomic sequence, a PKHD1 homolog, PKHDL1, in chromosome region 8q23. The PKHDL1 transcript of 13081 bp was amplified as 16 fragments and sequenced; the sequence of the murine ortholog, Pkhdl1 (chromosome region 15B3) was also determined. PKHDL1 contains 78 exons, covers a genomic region of ~168 kb and encodes a large protein, fibrocystin-L. Screening PKHDL1 in ARPKD patients with no PKHD1 mutations revealed several sequence variants but no clear mutations, making it unlikely that it is ARPKD-associated. Human fibrocystin-L is predicted to be a large receptor protein (4243 aa; 466 kDa) with a signal peptide, single transmembrane domain and short cytoplasmic tail. Fibrocystin-L is homologous to fibrocystin throughout most of the extracellular region with overall identity of 25.0% and similarity of 41.5%. Fibrocystin-L has extracellular domains similar to fibrocystin with 14 copies of the TIG domain and two regions of significant homology to the protein TMEM2. Genomic sequence analysis identified no other full-length fibrocystin homologs in humans, mice or other sequenced organisms. The Fugu fish has a fibrocystin-L ortholog but no fibrocystin, suggesting that the newly identified protein may be the ancestral form. PKHDL1 and Pkhdl1 are widely expressed at a low level in most tissues but only detected in blood-derived cell-lines. Low level expression was detected in many primary immune cell subtypes but up-regulated specifically in T lymphocytes, following activation signals, suggesting a role in cellular immunity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Autosomal-recessive polycystic kidney disease (ARPKD) is an important renal cause of death in the perinatal period and of childhood renal failure. Neonatal disease presentation is typical, and characterized by greatly enlarged kidneys due to fusiform dilation of collecting ducts; congenital hepatic fibrosis is often a major complication in older patients (1,2). Progress toward understanding this complex disorder has recently been made by the identification of the disease-causing gene, PKHD1, in chromosome region 6p12 (3,4). PKHD1 is a very large gene (~470 kb), containing 67 exons and with a longest open reading frame (ORF) of 12 222 bp. The PKHD1 encoded protein, fibrocystin, is large (4074 aa) and predicted to be an integral membrane protein with a large extracellular region and a short cytoplasmic tail. The function of fibrocystin is unknown.

PKHD1 has a tissue-specific expression pattern with the highest levels in fetal and adult kidney and lower levels in liver, pancreas and lung (3). The murine ortholog, Pkhd1, has recently been described and a notable feature of both the human and murine genes is that multiple different splice forms may be generated (3–6). Visualization of PKHD1 transcripts by northern analysis has proved difficult with a smear of products often detected. These may represent multiple splice forms, unusual sensitivity of this transcript to degradation, or a combination of these factors (3–6). In situ hybridization of the murine transcript showed expression in the developing kidney and mature collecting ducts, plus ductal plate and bile ducts in the liver. Other sites of expression during development detected by in situ analysis were large vessels, testis, sympathetic ganglia, pancreas and trachea, with evidence that some sites of expression may be of specific splice forms (5).

Fibrocystin is not closely related to any other characterized protein, although it contains multiple copies of a defined domain and has regions of homology to other proteins; it seems to represent the founder member of a new protein family (3,4). In analogous situations, where a large and complex novel protein is associated with a genetic disease, characterization of homologous proteins has greatly added the elucidation of structure and function. In the case of autosomal-dominant polycystic kidney (ADPKD), the first characterized disease-associated protein, polycystin-1, was large and novel with unknown function (7,8). Subsequently, a second related disease-associated protein was identified, polycystin-2 (9), and now a family of at least eight homologous proteins has been described (10–17). Study of these has indicated that the polycystins have a functional role in regulating intracellular calcium (18–20) and that cilia may be an important site of expression (21–23); a finding recently identified in other polycystic kidney diseases (24).

The only well characterized domain in fibrocystin is the TIG/IPT (immunoglobulin-like fold shared by plexins and transcription factors), which is also found in the hepatocyte growth factor receptor (HGFR), plexins and other receptor molecules (3,4,25). Although fibrocystin has many more copies of this domain than these other proteins, the presence of the TIG domain, along with the structure of the protein, suggested that it may also act as a receptor (3). The largest region of homology to fibrocystin (to the N-terminal half of the protein) is to an unpublished mouse protein, D86, described in the database as a novel protein secreted from lymphocytes. Here we describe the identification and cloning of the human D86 ortholog and show that it is homologous over almost its entire length to PKHD1. Expression analysis indicates that this protein may be an important receptor in the T-cell lineage.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification and cloning of PKHDL1 and Pkhdl1
To identify the human ortholog of D86, the 1945 aa protein sequence was analyzed against the human genomic sequence by BLAST. The likely human ortholog was identified by a strong hit in genomic sequence from chromosome region 8q23, in the BAC clone RP11-419L20. Comparison of the genomic sequence to full-length fibrocystin using Pustell protein/DNA sequence alignment and BLAST showed that the homology on chromosome 8 extended over most of the length of the disease-related protein, covering at least 150 kb of genomic DNA. This region not only contained homology to D86 but also matched the previously described cDNA, DKFZp586C1021, that is similar to the 3' region of PKHD1 (3), indicating that these cDNAs are part of the same large gene. To clone the full-length human D86 ortholog, that we called the PKHD-like1 gene (PKHDL1), we used an RT–PCR exon linking approach with primers located in exons strongly predicted by GenomeScan and NIX analysis, and by homology with fibrocystin. The full-length transcript was cloned as 16 overlapping fragments (see Table 1 for details) and the 5' and 3' ends of the mRNA identified and cloned by RACE strategies (see Methods for details). RNA from human lung and adrenal was used for the RT–PCR and all products were cloned and sequenced. There was some evidence of alternative splicing, but sequence from the largest amplified fragment in each case was assembled into a contig containing an ORF of 12 729 bp. PKHDL1 has a 5' untranslated region (UTR) of 104 bp and the putative start codon is the first in-frame ATG in the sequence. The start codon does not strongly match the Kozak consensus (26), but overall five of 13 residues, including +4 and -2, match the consensus. The 3' UTR is 248 bp and has a typical polyadenylation signal preceding the site of polyA addition by 21 bp. The total transcript is 13 081 bp.


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  		Table 1. Details of PKHDL1 cDNA clones
 
Comparison of the PKHDL1 transcript to the genomic sequence showed that the gene contains 78 exons (see Table 2 for details) and the total genomic size of the gene is 167 918 bp. Two splice donor sites (for IVS 8 and 67) have the non-canonical GC sequence rather than the typical GT. As is often found in the ~0.5% of splice donor sites that have a GC, the rest of the donor sequence (at both exons) closely matches the splice site consensus (27). The transcriptional start of PKHDL1 is associated with a CpG island.


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  		Table 2. Intron/exon structure of human and murine PKHDL1
 
To confirm the structure of PKHDL1 and determine if the D86 cDNA contained the entire mouse ortholog, the sequence of the mouse transcript was determined. The human transcript was compared with murine genomic sequence by BLAST and the NW000106 Mouse Supercontig was identified, indicating that Pkhdl1 is located in mouse chromosome region 15B3. Strong similarity between PKHDL1 and the murine genomic sequence (and also using the D86 cDNA) enabled the full-length Pkhdl1 ORF of 12 747 bp to be predicted. The intron/exon structure of Pkhdl1 is the same as its human counterpart with 78 exons and all coding regions of exons the same size, except for exons 32 and 77 (see Table 2 for details). The atypical GC splice donor to exon 67 is conserved in the mouse but the exon 8 donor is GT in this organism. The size of mouse gene cannot be completely determined as the genomic sequence of IVS 4 and IVS 49 is not yet complete. The murine Pkhdl1 gene is also associated with a CpG island.

Tissue expression of PKHDL1
Initially, PKHDL1 cDNAs of human and mouse were hybridized to multiple tissue northern blots, but no clear bands were visualized, only faint smears were seen in many lanes (Fig. 1A). The problem of visualizing PKHDL1 as a specific transcript by northern blotting appears similar to that seen with PKHD1 (3,4). Therefore, RT–PCR was used to examine the tissue expression of this gene. Analysis of human adult material showed expression in most tissues (data not shown). Analysis of a fuller range of tissues was possible in mouse and this also showed that expression levels were low, with the product found in most tissues, both newborn and adult, after multiple cycle PCR (Fig. 1B). PKHDL1/Pkhdl1 therefore appear to be expressed at a low level in most tissue types.



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  		Figure 1. Expression analysis of PKHDL1/Pkhdl1. (A) Murine, multiple tissue (adult) northern blot hybridized with Pkhdl1 showing weak smears in most lanes. RT–PCR analysis of: (B) newborn and adult murine tissues with Pkhdl1 and ß-actin control showing widespread low level expression of Pkhdl1; (C) human cell-lines – SW13, adrenal carcinoma; ACHN, renal adenocarcinoma; HEK293, embryonic kidney; HT29, colonic adenocarcinoma; G-CCM, astrocytoma; Fib, skin fibroblasts; G-401, renal Wilm's tumor; Hep3B, hepatoma, HeLa, cervical carcinoma; K562, erythroleukemia; B-lymph, EBV-transformed B-lymphocytes and MCF-7, breast cancer, with PKHDL1 and GAPDH; and (D) various murine leukocyte populations as indicated (see Methods for details) with 30 and 40 cycles of PCR for Pkhdl1, and ß-actin control. NK=natural killer cells and DCs=dendritic cells.

 
Are mutations to PKHDL1 associated with ARPKD?
Previously, mutation analysis of PKHD1 revealed a population of clinically well-characterized ARPKD patients in which no mutation was identified (3,4). The homology of PKHDL1 to PKHD1 and expression in kidney and liver suggested that it could be a candidate as a second ARPKD gene. To test this hypothesis, ARPKD patients from seven families without definite PKHD1 mutations were screened for base-pair changes throughout the gene. The gene was partially screened in a further 13 PKHD1 mutation negative ARPKD patients (see Methods for details). The 78 PKHDL1 coding exons and flanking intronic sequences were amplified from genomic DNA and analyzed for base-pair mismatches by denaturing high-performance liquid chromatography (DHPLC; see Methods for details). This analysis revealed 17 exonic changes (see Table 3 for details), including silent changes at the amino acid level and conservative and non-conservative substitutions, but no nonsense or deletion/insertion mutations were found. Seven non-conservative changes were screened in normal controls; five of these were found in that population (Table 3) but two, Q/P702 and L/S1199, were not detected. Analysis to determine whether these two substitutions segregated with the disease was uninformative. The L/S1199 change, however, is probably not ARPKD associated as the family in which this change was detected (M52) also has the PKHD1 substitution, T36M (3). Although initially the significance of T36M was unclear, finding this change in other ARPKD families (4,28; manuscript submitted Rossetti S., Torra R., Coto E., Consugar M., Kubly V., Málaga S., Narvarro M., El-Youssef M., Torres V.E., Harris PC) showed that M52 is a typical (PKHD1 mutated) ARPKD family. Q/P702 is conserved in the mouse ortholog, but not fibrocystin (where it is aspartic acid), and the pathogenic significance of this change remains unclear (see Discussion).


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  		Table 3. Detected variants in PKHDL1
 
Cellular expression of PKHDL1
To determine the cell types that express PKHDL1, tissue-specific cell lines were analyzed by RT–PCR (Fig. 1C). These showed the highest level of expression in K562, an erythroleukemia cell line and stimulated T-cells (data not shown), with the only other expressing cells being EBV transformed lymphoblasts. The detection of PKHDL1 in cell-lines of bone marrow origin is consistent with the database description of D86 as a lymphocyte-secreted protein. Furthermore, the tissue origin of described murine Pkhdl1 ESTs is in thymus (n=5) and lymph node (n=2), as well as adrenal (n=2).

Detection of the PKHDL1 transcript in organs that are composed entirely of immune cell subtypes (spleen and thymus) as well as in activated T-cells and B lymphoblasts suggested that expression of PKHDL1 may be important within cells of the immune system. To determine whether the expression is confined to specific immune cell populations or to states of immune activity, flow cytometric sorting from murine lymphoid organs and in vitro activation protocols were carried out (see Methods for details). RT–PCR analysis of RNA isolated from these cells resulted in detection of Pkhdl1 at low cycle number only in activated bulk T-cells and purified CD4+ve (helper) and CD8+ve (cytotoxic) T-cells (Fig. 1D). Strong in vitro stimulation of B-cells and inflammatory macrophages did not result in high-level expression. At high cycle number expression was also detectable in CD4+ve, CD8+ve (double positive) thymocytes, resting naive and memory B-cells, unstimulated and stimulated peritoneal macrophages, resting CD4+ve and CD8+ve T-cells, NKT-cells, and both CD8+ve and CD8-ve dendritic cells.

The structure of fibrocystin-L
Analysis of the longest ORF of the PKHDL1 sequence revealed the structure of the corresponding protein, which we have termed fibrocystin-like (fibrocystin-L). Fibrocystin-L is predicted to be larger than fibrocystin, with 4243 aa and a calculated unglycosylated molecular mass of 466 kDa. A signal peptide is predicted at the N-terminal end with cleavage at the sequence CAA-DF (Fig. 2A). Analysis of likely transmembrane regions in fibrocystin-L gave conflicting results but the most likely structure (predicted by SOSUI) is of a single transmembrane domain, from 4213 to 4235 aa, leaving a short, 8 aa, cytoplasmic tail. The predicted topology is therefore similar to fibrocystin with a large, 4212 aa, extracellular region and single transmembrane pass (Fig. 3). The extracellular region contains 56 putative N-linked glycosylation sites indicating that this region may be highly glycosylated. A single potential protein kinase C phosphorylation site is found in the C-terminal tail at position 4239 aa.






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  		Figure 2. Alignment of fibrocystin-L sequence. (A) Clustal W alignment of human fibrocystin (fib) and human fibrocystin-L (fibL) from their N-termini to positions 3849 and 4185 aa, respectively. Black boxes show identities and shaded boxes similarities. Conserved domains (TIG; thin lines) and defined regions of homology (TMEM; thick lines) are indicated in red (fibrocystin-L) and blue (fibrocystin). The predicted signal peptide cleavage sites are indicated with arrowheads.

 


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  		Figure 3. Diagram comparing the proposed structures of fibrocystin and fibrocystin-L. See key for details.

 
The protein most similar to fibrocystin-L is fibrocystin, which is homologous from the N-terminal end to 4185 aa (Fig. 2A). In this region the two proteins show homology of 25.0% and similarity of 41.5%. There is no significant homology between the two proteins in the transmembrane or short cytoplasmic regions. As with fibrocystin, the most clearly recognized protein domain in fibrocystin-L is the TIG/IPT domain. Analysis by Pfam indicated that fibrocystin-L contains 14 copies of this immunoglobulin-like fold, with three immediately after the signal peptide and the remaining 11 in tandem from 1067 to 2177 aa, with a gap between 1470 and 1566 aa; almost one-third of the protein is in TIG domains (Figs 2A and 3). Figure 2B shows that all the TIG domains closely match the TIG consensus and are similar to the corresponding domains of fibrocystin, the HGFR and members of the plexin family. In all the fibrocystin-L TIG domains, apart from TIG10, the cysteine residues, that are important to stabilize the domain through the formation of a disulfide bond, are present.

A second region of significant homology, that was also noted with fibrocystin, is to a protein of unknown function, TMEM2, and two related proteins. We have defined two TMEM regions of homology with the fibrocystins: TMEM domain-A (2180–2375 aa) and -B (3032–3376 aa; Fig. 2A and C). The size of TMEM-B has been extended further N-terminal compared with the area of homology that was previously described with fibrocystin (3). Interestingly, this homology is not only with the previously described proteins, TMEM2 and XP051860, but also to a newly described hypothetical protein from the thermorphilic, filamentous, photosynthetic bacteria, Chloroflexus aurantiacus. Indeed, the bacterial protein is more clearly related to the TMEM domains of fibrocystin and fibrocystin-L than either of the other human proteins as it does not require to be gapped to match the sequence (Fig. 2C).

Fibrocystin-L orthologs and homologs
Murine fibrocystin-L is predicted to have 4249 aa with an overall identity of 81.9% and similarity of 90.0% to the human protein. This is higher than the corresponding figures of 72.6 and 83.1% for human and murine fibrocystin (5). The murine fibrocystin-L is predicted to have a signal peptide with cleavage at the corresponding position to the human protein and a similarly located single transmembrane domain, leaving a six residue C-terminal tail. Murine fibrocystin-L is also predicted to have 14 TIG domains and similar TMEM homology. Fifty-six N-linked glycosylation sites are predicted in the extracellular region of the protein but the C-terminal tail does not contain a PKC site.

BLAST analysis for related proteins in other species where the complete genomic sequence is available showed a fibrocystin-L ortholog in the fish Takifugu rubripes. Strong similarity is seen with several predicted proteins and the corresponding genome sequence, Scaffold 2621. The Fugu PKHDL1 ortholog is encoded by a genomic region of ~30 kb. Interestingly, analysis of Fugu genomic sequence with fibrocystin only identified the PKHDL1 ortholog, but with a much lower score and E value than with fibrocystin-L. This indicates that Fugu has only PKHDL1 and no PKHD1 ortholog. Analysis of available genomic sequence from other eukaryotes and prokaryotes revealed no clear full-length orthologs of PKHD1 or PKHDL1. However, other significant regions of homology were detected in these species. The strongest homology was with the TMEM domain in C. aurantiacus, as described above. The next most significant region was with a conserved hypothetical protein from the bacterium Thermoanaerobacter tengeongensis that has 11 TIG domains, two from 246 to 332 aa and nine tandemly arranged from 580 aa. This protein also has a fibronectin type III domain, and a signal peptide indicating that it is a secreted protein. Other high scoring homologies were with other TIG domain proteins, most notably to plexin-like proteins, that typically have four such domains.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have described here the identification, cloning, sequence analysis and expression studies of a homolog of the ARPKD gene, PKHDL1. The level of identity and similarity of the newly described fibrocystin-L to fibrocystin is low but highly significant as it extends over the entire length of the protein, except the extreme C-terminal region containing the predicted transmembrane domain and cytoplasmic tail. This level of homology is greater than that seen between polycystin homologs (13), because of the length of homology, and similar to other protein families, establishing the fibrocystins as a new protein family.

Many PKHDL1 exons were identified by gene prediction programs and GenomeScan defined the human and murine gene as three and two different genes, respectively (see Methods for details). Of the 78 PKHDL1 exons, 53 were predicted correctly, three exons had one different splice junction (one associated with the GC splice donor) and 22 exons were not predicted. A further six exons were predicted that we did not find in the final transcript. Therefore, although these prediction programs are helpful to identify exons, RT–PCR and sequencing were required to define the most likely gene sequence. In this case the availability of the D86 murine cDNA of Pkhdl1 (exons 1–38) and human cDNA DKFZp586C1021 (exons 69–78) helped determine the structure of the gene.

The similarity of fibrocystin-L to fibrocystin, evidence of expression in kidney and liver and data from ARPKD families, that in a significant minority of families no mutation had been identified in PKHD1, led us to explore the possibility that PKHDL1 may be an ARPKD-associated gene (3,4). Analysis of PKHDL1 in a group of ARPKD patients without detected PKHD1 mutations revealed a number of missense changes but no inactivating mutations (Table 3). Although one non-conservative change was found only in an ARPKD family, overall the data did not provide compelling evidence associating this gene with ARPKD, even if this possibility cannot be entirely excluded. The lack of association of PKHDL1 with ARPKD is consistent with the major sites of expression of this gene in blood cell lineages.

The widespread, but low level, expression of PKHDL1/Pkhdl1 at the tissue level is in sharp contrast to PKHD1, which is expressed in just a few tissues, most notably kidney, but also liver, pancreas and lung (with other specific cell types detected by in situ hybridization). The widespread low level of expression of PKHDL1 may reflect the association of a CpG island with the promoter of this gene, which are often associated with more widely expressed genes, in contrast to the lack of a CpG island at PKHD1. However, more limited expression in cell-lines suggests that the widespread tissue expression is due to its major site of expression in blood cell lineages. A preliminary analysis of highly purified cell populations from murine lymphoid organs raises the intriguing possibility that fibrocystin-L is up-regulated in T-cells following activation and, therefore, may serve a specific function in cellular immunity. Increased expression of mRNA in purified helper (CD4+ve) and cytotoxic (CD8+ve) T-cells could be detected following activating stimuli delivered by lectins (data not shown), allogeneic antigen presenting cells (APCs), and immobilized antibodies to the T-cell receptor and the co-stimulatory receptor CD28. In contrast, strong activation stimuli failed to induce up-regulation of Pkhdl1 in memory phenotype B-cells and inflammatory macrophages. The genetic programs that are initiated by T-cell interactions with APCs bearing cognate antigen regulate a host of specialized functions including T-cell proliferation, cytokine production, migration patterns, cytotoxicity and cell survival (29,30). Coordination of these functions is essential for elimination of infection, immune surveillance against neoplasia and generation of memory responses (29–32). Furthermore, aberrant T-cell activation underlies the pathogenesis of autoimmunity and rejection of transplanted organs and tissues (33). The predicted fibrocystin-L protein may be secreted or cell surface-bound when expressed in activated T-cells. As a large receptor that is not closely related to other characterized T-cell protein families, its functional profile cannot be accurately predicted based on structure alone. However, analogies with other large, highly glycosylated proteins suggest possible roles in regulation of the T-cell-APC interface structure or in adhesion and migration patterns (34,35). The high level to which the murine immune system has been characterized and compared with human immunity will allow this aspect of fibrocystin-L function to be rigorously explored.

One similarity between PKHDL1 and PKHD1 is the difficulty visualizing the transcripts by northern blotting. In the case of PKHDL1 the problems of resolving the transcript as a discrete fragment are compounded by its low level of expression. Northerns generally revealed faint smears and this may reflect particular sensitivity of this transcript to degradation or, as suggested for PKHD1, multiple alternatively spliced transcripts (4). There is evidence of possible alternative splicing of PKHDL1 with several RT–PCR reactions generating more than one product. The GC splice donor found at two exon junctions is also often associated with alternative splicing (36). Furthermore, one human adrenal gland cDNA clone, ADBBEB10, that we fully sequenced, extends exon 18 a further 886 bp and leads to a novel 3' UTR with an atypical, ATTAAA, polyadenylation signal (37) shortly before the site of polyA addition. D86, the originally described transcript of the 5' part of Pkhdl1, has an extension of exon 38 into IVS38, producing a 3' UTR of 62 bp, although no clear polyadenylation site is present in this sequence and we have not confirmed this product by 3' RACE. It therefore seems likely that PKHDL1, like PKHD1, will generate many alternatively spliced transcripts, some predicted to produce secreted proteins (such as ADBBEB10 and D86), as well as the membrane-bound form indicated by the longest ORF. There are significant regions of breakdown in homology between the two proteins (Fig. 2A) and it remains to be seen whether alternative splice forms of these proteins will better match the other homolog.

The description of a second member of the fibrocystin protein family has helped to refine the likely structure of these proteins. Fibrocystin-L is predicted to have 14 TIG domains, far more than the seven predicted in fibrocystin by Pfam and other programs (3,4). This supports the possibility, as we have previously suggested, that fibrocystin may have further TIG-like domains with similar three-dimensional structure (3). Inspection of sequence alignments of the two proteins suggests that fibrocystin may have further TIG-like domains C-terminal to those defined previously (Figs 2A and 3). A second important region of homology that is present twice in the fibrocystins is with TMEM2 and related proteins. TMEM2, XP051860 and the newly described sequence in the filamentous bacteria C. aurantiacus, have a single copy of the TMEM repeat; in the first two proteins is it interrupted by additional sequence (Fig. 2C). In the TMEM2, XP051860 and C. aurantiacus proteins, as in the fibrocystins, this region is predicted to be extracellular. As C. aurantiacus is the only sequenced prokaryote with this protein domain, it appears likely that this may be an example of horizontal gene transfer. This simple organism may provide an opportunity to better understand the structure and function of this domain.

A notable difference between fibrocystin-L and fibrocystin is the length of the predicted cytoplasmic tail. In fibrocystin-L it is only 8 aa, while in fibrocystin it contains 192 aa and has several possible PKA, PKC and casein kinase phosphorylation sites; we have suggested it may signal through one or more of these sites (3). Although the short fibrocystin-L tail in humans has a single potential PKC site, this is not conserved in the mouse and it is questionable whether this protein will signal in the same way.

Analysis of available human and murine genomic sequences, plus that of other eukaryotes and prokaryotes, indicated that fibrocystin and fibrocystin-L are likely to be the only full-length members of this family. One of the most interesting findings from this study is that fibrocystin-L is probably the ancestral fibrocystin protein. There is significantly greater similarity between the murine and human fibrocystin-Ls than the corresponding fibrocystin orthologs and most telling (if the Fugu sequence is complete) (38); only a fibrocystin-L ortholog appears to be present in that species. This indicates that PKHD1 is a relatively recently evolved gene that arose after the divergence of fish and mammals, although analysis of other species will be required to determine if this is a universal finding in the teleosts. The recent evolution of fibrocystin (with its role in regulating renal and hepatic tubule maturation) from fibrocystin-L, with its possible role as a receptor in blood cell development, seems reminiscent of the adaption of the polycystins from roles in mating behavior and fertilization (15,16,20) to maintaining the tubular architecture in the kidney and liver. Identification of fibrocystin-L should greatly aid the understanding of the structure and function of the fibrocystin protein family.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Preparation of resting and stimulated immune cell sub-populations
Spleens, thymuses and subcutaneous lymph nodes were dissected from B6 and BALB/C mice under sterile conditions. Cell suspensions were prepared by disruption of the organs in DMEM/10% FCS and passage through 45 µm nylon mesh. For spleen and thymus suspensions, erythrocytes were lysed by 5 min incubation in ACK buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2). For flow cytometric sorting, surface staining with a panel of fluorochrome-labeled monoclonal antibodies (BD Pharmingen, San Diego, CA, USA) was carried out by 1 h incubation of cells in DMEM/10% FCS at 4°C. Labeled cell suspensions were washed and re-suspended in DMEM/10% FCS at 4–8x106 cells/ml and flow sorted using a FACS Vantage sorter (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). The antibody combinations were as follows: Splenic T-cell sub-populations: anti-mouse CD4-FITC (RM4-4), and anti-mouse CD8{alpha}-PE (53-6.7). Sorted populations: CD4+ve/CD8-ve (CD4+ T-cells) and CD4-ve/CD8+ve (CD8+ T-cells). Splenic dendritic cell sub-populations: anti-mouse CD11c-FITC (HL3) and anti-mouse CD8{alpha}-PE. Sorted populations: CD11c+ve/CD8-ve (myeloid) and CD11c+ve/CD8+ve (lymphoid). Thymocyte sub-populations: anti-mouse CD4-FITC, anti-mouse CD8{alpha}-PE. Sorted populations: CD4-ve/CD8-ve, CD4+ve/CD8+ve, CD4+ve/CD8-ve, and CD4-ve/CD8+ve. Splenic NK and NKT cells: anti-mouse CD3{varepsilon}-FITC (145-2C11) and anti-NK 1.1-PE (PK136). Sorted population were: NK1.1+ve/CD3{varepsilon}-ve (NK-cells) and NK1.1+ve/CD3{varepsilon}+ve (NKT-cells). Splenic B-cell sub-population were: anti-mouse IgD-FITC (11-26c.2a) and anti-mouse CD19-PE (1D3). Sorted population were: CD19+ve/IgD+ve (naïve B-cells) and CD19+ve/IgD-ve (memory B-cells). An aliquot of the memory B-cells was stimulated for 96 h (activated memory B-cells) with plate-bound goat-anti-mouse IgG (ICN Biomedicals Inc., Aurora, OH, USA), 25 ng/ml lipopolysaccharide (LPS, Sigma Aldrich, St Louis, MO, USA), and 2.5 µg/ml purified anti-mouse CD40 (HM40-3, BD Pharmingen). Murine CD4+ve and CD8+ve lymph node T-cells were purified by nylon wool column and complement-mediated depletion and activated for 72 h in tissue culture plates coated with a combination of hamster anti-mouse CD3{varepsilon} (145-2C11) and hamster anti-mouse CD28 (PV-1), as previously described (39); or stimulated for 96 h by co-culture with irradiated allogeneic (B6) bone marrow-derived dendritic cells (allo-stimulated T-cells).

Murine peritoneal inflammatory macrophages were generated from B6 mice by intraperitoneal injection (1 ml/animal) of sterile 3% thioglycollate (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA). After 7 days the animals were sacrificed, cells extracted by peritoneal lavage using sterile DMEM/10% FCS, washed and an aliquot retained for RNA preparation (fresh peritoneal inflammatory cells). The remainder were re-suspended in DMEM/10% FCS and allowed to adhere to tissue culture flasks at 37°C for 2 h. Non-adherent cells were removed by washing with sterile PBS and individual cell layers were exposed for 1 h at 37°C to PBS alone (unstimulated macrophages) or to PBS containing LPS 2 µg/ml (stimulated macrophages). These solutions were then removed, the cell layers washed with PBS and culture medium was re-applied for 24 h.

Human lymphocytes were isolated from whole blood using 54% Percoll (Amersham Biosciences, Piscataway, NJ, USA) and the resulting PBS washed and pelleted cells incubated in RPMI/10% FCS with concanavalin A (10 µg/ml) for 72 h at 37°C.

RNA analysis
RNA was isolated from snap frozen human tissues (adrenal, breast, colon, heart, kidney, liver, lung, pancreas, placenta and uterus, obtained as surgical waste), mouse tissues, cell-lines and the leukocyte populations described above (see Fig. 1) using the Trizol method (Invitrogen, Carlsbad, CA, USA) or with the NucleoSpin (BD Biosciences, San Jose, CA, USA) column system. The isolated RNA (1–5 µg) was used to make cDNA with the Clontech PowerscriptTM Reverse Transcriptase cDNA Synthesis Kit (BD Biosystems, San Jose, CA, USA) and 250 µg random primers (Invitrogen, Carlsbad, CA, USA). PKHDL1 (Pkhdl1) expression was analyzed by RT–PCR using 50 ng cDNA and equalized by amplification of the control ß-actin (mouse: F5'-CTGGCACCACACCTTCTACAATGAGCTG-3': R-5'-GCACAGCTTCTCTTTGATGTCACGCACGATTTC-3' 395 bp product) or GAPDH (human: F5'-GACCACAGTCCATGCCATCACT-3': R5'-TCCACCACCC TGTTGCTGTA-3'; 453 bp product). For analysis of murine Pkhdl1 a 258 bp region from exons 76–77 (12 429–12 686 nt; F5'-TCCATTTAGCACCTGTTGGGC-3'; R5'-AGTCTTCC TACAAGGCACGCTG-3') was amplified and for human PKHDL1 a 247 bp region from exons 35–36 (4232–4478 nt; F5'-CACCAGTCCTAATGTGTCTGTGG-3'; R5'-TGGAGAAAAATGGAGTGAGCCTC-3') was assayed. PCR conditions were as follows: 0.125U AmpliTaq Gold (Applied Biosystems, Foster City, CA, USA) in the supplied buffer, 2.0 mM MgCl2, 0.2 mM each nucleotide, 4 pM each primer and 50 ng cDNA, and PCR conditions of: 4 min at 94°C; 60 s at 94°C, 30 s at 56–64°C and 30–60 s at 72°C for 30–40 cycles; and finally 10 min at 72°C. We assayed the products on 2.0% agarose gels, visualized by ethidium bromide staining. Multi-tissue northern blots (BD Biosciences, San Jose, CA, USA) were hybridized and washed using standard methods with the probes: human PKHDL1, exons 1–12, 979 bp (9–986 nt) and exons 38–41, 1248 bp (5065–6312 nt) and murine Pkhdl1, exons 32–38, 1048 bp (3788–4835 nt).

Cloning PKHDL1
The positions of likely human PKHDL1 exons were determined by comparison of genomic DNA (AC021001) to the mouse D86 cDNA sequence, PKHD1, Genome Scan putative genes (Hs 8205 30 21 2; Hs 8205 30 23 1 and Hs 8205 30 23 2) and using the NIX suite of programs (www.hgmp.mrc.ac.uk). The transcript was amplified as 16 overlapping fragments with primers positioned in the most strongly predicted exons (Table 1) using PCR conditions as described above and human lung or adrenal cDNA. Fragments were cloned into specific restriction sites of pZERO using amplification primers with matching sites, or unmodified product was cloned into terminal transferase (New England Biolabs, Beverly, MA, USA) treated EcoRV cut vector using the Rapid DNA Ligation Kit (Roche Applied Science, Indianapolis, IN, USA) and grown in E. coli XL-2MRF' (Stratagene, LaJolla, CA, USA). The 5' and 3' regions were amplified and cloned using RACE strategies with the SMART RACE cDNA Amplification Kit (BD Biosciences, San Jose, CA, USA). For the 5' RACE human adrenal RNA was reverse-transcribed with Powerscript RT and amplified with the 5' RACE CDS and SMART-II primers using touchdown PCR and nested gene specific primers. At the 3' end cDNA synthesis was with the tailed and anchored oligo (dT) primer, 3'-CDS, and amplified as above with nested gene specific primers. Sequencing employed the Big-Dye Terminator Kit (Applied Biosystems, Foster City, CA, USA) and were analyzed on ABI377 Sequencers. The sequence was assembled into a contig using the Sequencer 4.1.2 program.

Mutation analysis of PKHDL1
All patients in the study gave informed consent and the project was approved by the Institutional Review Board at Mayo Clinic. Five previously described ARPKD patients from families: M36, P244, M51, M52 and M55 (3), plus two additional typical ARPKD patients, in which no PKHD1 mutation was identified (n=4) or where only a single missense change was detected (M52, M52 and M55), were screened for PKHDL1 mutations. An additional 13 typical ARPKD patients with no detected PKHD1 mutation were screened for changes in 34 PKHDL1 exons, but as no likely disease causing mutations were identified, the screening of the gene was not completed.

To screen for PKHDL1 mutations all the 78 coding exons were amplified from genomic DNA as 85 fragments of 150–350 bp. Primers were typically positioned in the intron ~20 bp from the intron/exon boundary. We amplified the fragments using the following protocol: genomic DNA, 60 ng; primers, 8 pmol each; dNTPs, 200 µM each; MgCl2, 2.5 mM and 1 U of Taq Gold in the supplied buffer (Applied Biosystems, Foster City, CA, USA), in a total volume of 25 µl. The PCR program included: 120 s, 94°C; 35–40 cycles of 30 s at 94°C; 30 s at 50°–63°C; and 30 s at 72°C; and 10 min at 72°C. The PCR products were treated to form heteroduplexes and analyzed for base-pair changes using DHPLC on the WAVE Fragment Analysis System (Transgenomic, Omaha, NE, USA), as previously described (3). Briefly, crude PCR (300–500 ng) was injected into the chromatographic column (DNASep Cartridge, Transgenomic, Omaha, NE, USA) and eluted using calculated and empirically determined optimal conditions. Samples showing an abnormal chromatogram were further characterized by direct sequencing, as previously described (3). Potentially pathogenic changes were validated by DHPLC analysis of 25–100 normal controls (50–200 chromosomes). PCR primer sequences, amplification and DHPLC analysis conditions are available on request.

Sequence analysis
The intron/exon structure of PKHDL1 was determined by comparison with genomic sequence using MacVector 7.0 and SIM4 (pbil.univ-lyon1.fr/sim4.html). The sequence of the murine Pkhdl1 transcript was determined by comparison of human PKHDL1 sequence and the Pkhdl1 cDNA clone, D86, to mouse genomic sequence using MacVector. The genomic sequence was used as the authentic sequence for the human and murine transcripts and the numbering of the transcript was from the start codon.

BLAST was used to screen for homologous sequences in the GenBank database (www.ncbi.nlm.nih.gov/blast/Blast.cgi). Comparison between orthologs, and fibrocystin and fibrocystin-L, was made by BLAST2 and the Pustell protein/DNA sequence alignment tool of MacVector. Protein domains were defined using the Pfam database (pfam.wustl.edu). To analyze protein topology the programs SOSUI (sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html), TMHMM (v2.0; cbs.dtn.dk/services/TMHMM-2.0) and SignalP (v2.0) (www.cbs.dtn.dk/services/SignalP/) were used. Potential N-glycosylation sites and phosphorylation sites were identified with MacVector and alignments made with the ClustalW (v1.4) program, within MacVector.

GenBank accession numbers
Fibrocystin (human) AAL74290; (mouse) AAN05018; D86 (mouse), NP_619615; DKF2p586C1021, XP_488444; PKHDL1 cDNA clone ADBBEB10 (5'), AV706327; PKHDL1 genomic sequence (human) RP11-419L20, AC02001; (mouse) NW_000106 (44,650K-44,800K). Fibrocystin-L related proteins: HGFR (mouse), NP_032617; plexin 1 (mouse), NP_032901; TMEM2 (human), NP_037522; XP051860 (human) XP_051860; hypothetical protein from C. aurantiacus, ZP_00018581 and hypothetical protein from T. tengeongensis, NP_621862. GenomeScan and other predicted proteins similar to fibrocystin-L: Hs8 8205 30 32 2; Hs 8205 30231; Hs 8205 30 232 (human); LOC271264 (mouse) XP_194970. Fugu PKHDL1, genomic sequence Scaffold 2621, CAAB01002621. PKHDL1 cDNA sequences (human), AY219181 and Pkhdl1 (mouse), AY219182.


   ACKNOWLEDGEMENTS
 
We thank Shelly Whelan for animal studies, the Mayo Flow Cytometry and Molecular Biology Core Facilities and Ze-Guang Han for the ADBBEB10 cDNA clone. This work was supported by NIDDK grants DK58816, DK59597 and DK59505, the PKD Foundation (fellowships for M.C.H. and S.R.) and the Mayo Graduate School and Mayo Foundation CR75 program (M.D.G.).


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +1 5072660541; Fax: +1 5072669315; Email: harris.peter{at}mayo.edu Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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