Polycystin-L is a calcium-regulated cation channel permeable to calcium ions.

Polycystic kidney diseases are genetic disorders in which the renal parenchyma is progressively replaced by fluid-filled cysts. Two members of the polycystin family (polycystin-1 and -2) are mutated in autosomal dominant polycystic kidney disease (ADPKD), and polycystin-L is deleted in mice with renal and retinal defects. Polycystins are membrane proteins that share significant sequence homology, especially polycystin-2 and -L (50% identity and 71% similarity). The functions of the polycystins remain unknown. Here we show that polycystin-L is a calcium-modulated nonselective cation channel that is permeable to sodium, potassium and calcium ions. Patch-clamp experiments revealed single-channel activity with a unitary conductance of 137 pS. Channel activity was substantially increased when either the extracellular or intracellular calcium-ion concentration was raised, indicating that polycystin-L may act as a transducer of calcium-mediated signalling in vivo. Its large single-channel conductance and regulation by calcium ions distinguish it from other structurally related cation channels.

Identification of PKDL, a novel polycystic kidney disease 2-like gene whose murine homologue is deleted in mice with kidney and retinal defects.

Polycystin-1 and polycystin-2 are the products of PKD1 and PKD2, genes that are mutated in most cases of autosomal dominant polycystic kidney disease. Polycystin-2 shares approximately 46% homology with pore-forming domains of a number of cation channels. It has been suggested that polycystin-2 may function as a subunit of an ion channel whose activity is regulated by polycystin-1. Here we report the identification of a human gene, PKDL, which encodes a new member of the polycystin protein family designated polycystin-L. Polycystin-L has 50% amino acid sequence identity and 71% homology to polycystin-2 and has striking sequence and structural resemblance to the pore-forming alpha1 subunits of Ca2+ channels, suggesting that polycystin-L may function as a subunit of an ion channel. The full-length transcript of PKDL is expressed at high levels in fetal tissues, including kidney and liver, and down-regulated in adult tissues. PKDL was assigned to 10q24 by fluorescence in situ hybridization and is linked to D10S603 by radiation hybrid mapping. There is no evidence of linkage to PKDL in six ADPKD families that are unlinked to PKD1 or PKD2. The mouse homologue of PKDL is deleted in Krd mice, a deletion mutant with defects in the kidney and eye. We propose that PKDL is an excellent candidate for as yet unmapped cystic diseases in man and animals.

Distribution and developmentally regulated expression of murine polycystin.

PKD1, the gene that is mutated in approximately 85% of autosomal dominant polycystic kidney disease (ADPKD) cases in humans, has recently been identified (Eur. PKD Consortium. Cell 77: 881-894, 1994; also, erratum in Cell 78: 1994). The longest open-reading frame of PKD1 encodes polycystin, a novel approximately 460-kDa protein that contains a series of NH2-terminal adhesive domains (J. Hughes, C. J. Ward, B. Peral, R. Aspinwall, K. Clark, J. San Millan, V. Gamble, and P. C. Harris. Nat. Genet. 10: 151-160, 1995; and Int. PKD Consortium. Cell 81: 289-298, 1995) and several putative transmembrane segments. To extend studies of polycystin to an experimentally accessible animal, we have isolated a cDNA clone encoding the 3' end of Pkd1, the mouse homologue of PKD1, and raised a specific antibody to recombinant murine polycystin. This antibody was used to determine the subcellular localization and tissue distribution of the protein by Western analysis and immunocytochemistry. In the mouse, polycystin is an approximately 400-kDa molecule that is predominantly found in membrane fractions of tissue and cell extracts. It is expressed in many tissues including kidney, liver, pancreas, heart, intestine, lung, and brain. Renal expression, which is confined to tubular epithelia, is highest in late fetal and early neonatal life and drops 20-fold by the third postnatal week, maintaining this level into adulthood. Thus the temporal profile of polycystin expression coincides with kidney tubule differentiation and maturation.

Identification and localization of polycystin, the PKD1 gene product.

Polycystin, the product of autosomal dominant polycystic kidney disease (ADPKD) 1 gene (PKD1) is the cardinal member of a novel class of proteins. As a first step towards elucidating the function of polycystin and the pathogenesis of ADPKD, three types of information were collected in the current study: the subcellular localization of polycystin, the spatial and temporal distribution of the protein within normal tissues and the effects of ADPKD mutations on the pattern of expression in affected tissues. Antisera directed against a synthetic peptide and two recombinant proteins of different domains of polycystin revealed the presence of an approximately 400-kD protein (polycystin) in the membrane fractions of normal fetal, adult, and ADPKD kidneys. Immunohistological studies localized polycystin to renal tubular epithelia, hepatic bile ductules, and pancreatic ducts, all sites of cystic changes in ADPKD, as well as in tissues such as skin that are not known to be affected in ADPKD. By electron microscopy, polycystin was predominantly associated with plasma membranes. Polycystin was significantly less abundant in adult than in fetal epithelia. In contrast, polycystin was overexpressed in most, but not all, cysts in ADPKD kidneys.

A gene similar to PKD1 maps to chromosome 4q22: a candidate gene for PKD2.

We report a partial cDNA sequence that encodes a protein, dubbed "polycystwin," with 21% identify and 46% similarity to amino acids 3688-4109 of the carboxyl terminus of polycystin, the gene product of the autosomal dominant polycystic kidney disease locus located on chromosome 16 at band p13 (PKD1). Northern analysis demonstrates that the R48321 gene is expressed in all tissues examined, including both adult and fetal kidneys. Finally, in situ hybridization studies localize this novel gene to 4q22, where PKD2, the second most common locus for ADPKD, is known to map. Therefore, R48321 is an excellent candidate gene for PKD2.

Sera from patients with anti-GBM nephritis including goodpasture syndrome show heterogenous reactivity to recombinant NC1 domain of type IV collagen alpha chains.

BACKGROUND: Goodpasture (GP) syndrome is defined by the clinical association of pulmonary haemorrhage with rapidly progressive glomerulonephritis. The disease is caused by pathogenic autoantibodies directed against type IV collagen, which is a major structural component of glomerular basement membranes (GBM). METHODS: The non-collagenous domains (NC1) of all six human type IV collagen alpha chains was produced in E. coli as recombinant fusion proteins with glutathione-S transferase. Sera from 10 patients with different types of anti-GBM nephritis, including GP syndrome, were tested for reactivity with the six proteins using immunoblotting of denatured and reduced proteins and ELISA without reduction. RESULTS: All 10 sera reacted with the alpha 3 (IV) collagen chain by immunoblotting and ELISA. One serum also recognized the alpha 2(IV), alpha 4(IV), alpha 5(IV) and alpha 6(IV) chains by immunoblotting. ELISA measurements revealed reactivity of several other sera with alpha 2(IV), alpha 4(IV) or alpha 6(IV) but not with alpha 5(IV) collagen chains. No reactivity was observed with the alpha 1(IV) chain. CONCLUSION: Autoantibodies in anti-GBM nephritis may not be directed only against the alpha 3(IV) collagen chain and they frequently recognize conformational epitopes.

Structure of the human type IV collagen COL4A6 gene, which is mutated in Alport syndrome-associated leiomyomatosis.

Basement membrane (type IV) collagen, a subfamily of the collagen protein family, is encoded by six distinct genes in mammals. Three of those, COL4A3, COL4A4, and COL4A5, are linked with Alport syndrome (hereditary nephritis). Patients with leimoyomatosis associated with Alport syndrome have been shown to have deletions in the 5' end of the COL4A6 gene, in addition to having deletions in COL4A5 (Zhou et al., Science 261: 1167-1169, 1993). The human COL4A6 gene is reported to be 425 kb as determined by mapping of overlapping YAC clones by probes for its 5' and 3' ends. In the present study we describe the complete exon/intron size pattern of the human COL4A6 gene. The 12 lambda phage clones characterized in the study spanned a total of 110 kb, including 85 kb of the actual gene and 25 kb of flanking sequences. The overlapping clones contained all 46 exons of the gene and all introns, except for intron 2. Since the total size of the exons and all introns except for intron 2 is about 85 kb, intron 2 must be about 340 kb. All exons of the gene were assigned to EcoRI restriction fragments to facilitate analysis of the gene in patients with leiomyomatosis associated with Alport syndrome. The exon size pattern of COL4A6 is highly homologous with that of the human and mouse COL4A2 genes, with 27 of the 46 exons of COL4A6 being identical in size between the genes.

Structural motifs of the PKD1 protein.

The complete sequence of the polycystic kidney disease gene (PKD1) and its transcript have been described. The predicted protein is not a member of a previously described gene family, but contains several structural motifs that are present in proteins of known function. Most of these domains are present in the extracellular parts of proteins involved in interactions with other proteins and carbohydrates. The PKD1 gene product also contains potential transmembrane sequences. The molecule is likely to be involved in cell-cell or cell-matrix interactions, which is consistent with the different manifestations of polycystic kidney disease.

Sequence analysis of the human hTg737 gene and its polymorphic sites in patients with autosomal recessive polycystic kidney disease.

DNA sequence analysis of the human Tg737 gene was performed in 36 patients with the autosomal recessive form of polycystic kidney disease (ARPKD). Coding exons and their adjacent splice sites were screened for mutations. Pathogenic exon or splice region mutations were not identified although one exonic and two intronic polymorphic sites were discovered. These results are in agreement with another study that has recently reported linkage to Chromosome (Chr) 6p21-cen in a set of 16 ARPKD families. STS mapping has localized the gene to a YAC contig that includes D13S175 on chromosome 13q12.1. The polymorphisms found in the htG737 gene will permit its future evaluation as a candidate gene for other recessive cystic renal diseases and as a modifier gene in human PKD.

A new human gene located in the PKD1 region of chromosome 16 is a functional homologue to ERV1 of yeast.

A new human gene has been identified on chromosome 16 in the interval containing the locus for polycystic kidney disease (PKD1) by analysis of a genomic cosmid clone and cDNAs. The gene contains at least one intron and is actively transcribed in tissues from kidney and brain. The putative gene product is predicted to be homologous to the yeast scERV1 protein by virtue of the high degree of identity (42%) over the entire length of the polypeptides. In former studies the yeast scERV1 gene was found to be essential for oxidative phosphorylation, the maintenance of mitochondrial genomes, and the cell-division cycle. In this study a yeast expression vector with a chimeric reading frame coding for the first 21 amino acids of the yeast protein and the terminal 100 amino acid residues of the human factor was transformed into yeast mutants with two different defects for scERV1. The chimeric human gene product was able to complement the yeast mutants and restored near normal viability. This identifies the human gene as a structural and functional homologue of the scERV1 gene.

Characterization of the human homologue of the mouse Tg737 candidate polycystic kidney disease gene.

We previously identified a gene from the mutant locus in a new mouse mutation that causes recessive polycystic kidney disease. Here we describe the cloning, characterization and mapping of the homologous human gene. The human and mouse genes are 95% identical at the predicted amino acid sequence level, and both genes encode a putative protein that contains a tetratricopeptide repeat motif. The human gene, called hTg737, is expressed with a broad tissue distribution that includes the the kidney and liver, and gives rise to a 2.9 kb mRNA. The gene contains 26 exons and spans a genomic region greater than 100 kb. Chromosome mapping experiments revealed that the hTg737 gene maps near the centromere on the long arm of human chromosome 13, at position 13q12.1. While this gene does not map to the primary locus that has been identified for ARPKD in humans, it may represent a candidate gene for other recessive renal disorders that have yet to be mapped.

A novel cyclin gene (CCNF) in the region of the polycystic kidney disease gene (PKD1).

The major locus for autosomal dominant polycystic kidney disease (PKD1) is located in a gene-rich region on chromosome 16p13.3. Recently the identification of the gene responsible for PKD1 has been described. While searching for candidate genes in this region, we isolated a new member of the cyclin family. We have characterized the transcript by sequencing, determination of the exon intron boundaries, and Northern blot analysis. Cyclin F is related to A- and B-type cyclins by sequence, but its function is unknown.

Complete primary structure of the human type IV collagen alpha 4(IV) chain. Comparison with structure and expression of the other alpha (IV) chains.

The entire sequence of the human alpha 4(IV) collagen chain was determined from cDNA clones and polymerase chain reaction-amplified DNAs. The complete translation product has 1,690 amino acid residues and the processed alpha 4(IV) chain proper 1,652 residues. There is a 38-residue putative signal peptide, a 1,421-residue collagenous domain starting with a 23-residue noncollagenous sequence, and a 231-residue NC1 domain. The Gly-Xaa-Yaa-repeat sequence of the collagenous domain is interrupted at 26 locations by noncollagenous sequences of 1-12 residues in length. The alpha 4(IV) chain contains 31 cysteine residues of which 18 are conserved in the other type IV collagen alpha chains. The calculated molecular weight of the mature alpha 4(IV) chain is 164,123. Analysis of the primary structure showed that the alpha 4(IV) chain belongs to the alpha 2-like type IV collagen chains together with alpha 2(IV) and alpha 6(IV). Northern analyses with RNA from several human fetal tissues revealed quite similar expression patterns for the alpha 4(IV) and alpha 3(IV) chains, but there were also distinct differences in some tissues. The expression patterns of alpha 5(IV) and alpha 6(IV) differed extensively between each other and they also differed from those of alpha 3(IV) and alpha 4(IV).

Complete primary structure of the human alpha 3(IV) collagen chain. Coexpression of the alpha 3(IV) and alpha 4(IV) collagen chains in human tissues.

We report the entire primary structure of the human alpha 3(IV) collagen chain determined from cDNA clones and polymerase chain reaction-amplified DNAs. The deduced amino acid sequence demonstrates that the complete translation product consists of 1670 amino acid residues and the mature alpha 3(IV) chain contains 1642 residues with a corresponding calculated molecular mass of 161,753. The full-length translated polypeptide has a signal peptide of 28 amino acids, a 1410-residue collagenous domain starting with a 14-residue noncollagenous sequence, and a 232-residue NC1 domain. There are 23 noncollagenous interruptions in the Gly-X-Y repeat sequence of the collagenous domain. The major transcription start site of the alpha 3(IV) chain gene was also determined from genomic DNA by primer extension and S1 nuclease protection assays. Northern analysis revealed coexpression of the alpha 3(IV) and alpha 4(IV) chains in tissues where expression was observed such as in kidney, muscle, and lung.

Mutations in the type IV collagen alpha 3 (COL4A3) gene in autosomal recessive Alport syndrome.

A group of 22 unrelated patients with sporadic or non-X-linked Alport syndrome were screened for mutations in the non-collagenous domain of the type IV collagen alpha 3 (COL4A3) chain gene. The five 3'-exons of this gene, located on chromosome 2qter, were tested by single strand conformation polymorphism analysis and direct sequencing. One patient was heterozygous and another homozygous (Mochizuki et al., Nature Genetics, in press) for a deletion of five nucleotides. A third patient appeared to be a compound heterozygote for two different nonsense mutations. In two patients and the father of a deceased patient we found a heterozygous substitution of an evolutionary conserved leucine by proline. However, segregation data of the mutation and a COL4A3/COL4A4 CA-repeat marker in their families argued against a causative role of the missense mutation. Even drastic changes of strongly conserved amino acids, as in the Leu36Pro case, may not be significant. Autosomal recessive inheritance due to pathogenic COL4A3 mutations accounts for at least 13% of Alport syndrome cases in this sample. It is concluded that COL4A3 is a major gene in the genetically and clinically heterogeneous Alport syndrome.

Linkage disequilibrium in the region of the autosomal dominant polycystic kidney disease gene (PKD1).

The gene for autosomal dominant polycystic kidney disease (PKD1) is located on chromosome 16p, between the flanking markers D16S84 and D16S125 (26.6prox). This region is 750 kb long and has been cloned. We have looked at the association of 10 polymorphic markers from the region, with the disease and with each other. This was done in a set of Scottish families that had previously shown association with D16S94, a marker proximal to the PKD1 region. We report significant association between two CA repeat markers and the disease but have not found evidence for a single founder haplotype in these families, indicating the presence of several mutations in this population. Our results favor a location of the PKD1 gene in the proximal part of the candidate region.

Complete primary structure of the sixth chain of human basement membrane collagen, alpha 6(IV). Isolation of the cDNAs for alpha 6(IV) and comparison with five other type IV collagen chains.

Basement membranes were previously believed to contain five distinct type IV collagen subunits. We have recently isolated part of the cDNA for a novel type IV collagen, alpha 6(IV), and shown that COL4A6, the gene encoding this new chain, is deleted in Alport syndrome-associated leiomyomatosis (Zhou, J., Mochizuki, T., Smeets, H., Antignac, C., Laurila, P., de Paepe, A., Tryggvason, K., and Reeders, S. T. (1993) Science 261, 1167-1169). Here, we describe the entire human alpha 6(IV) cDNA and show that the gene encodes a classical type IV collagen with homology throughout its length to all the other five chains. There is a 21-residue signal peptide, a 1417-residue collagenous domain interrupted at 25 points, and a 228-residue carboxyl-terminal non-collagenous domain. When the complete primary structure of this new chain was compared with all the other known chains, it became clear that alpha 6(IV) has the most resemblance to alpha 2(IV) and alpha 4(IV). The evolution of the six chains was deduced, allowing a new classification of the type IV collagen family. The alpha 6(IV) chain is a candidate gene for X-linked Alport syndrome; knowledge of the complete structure of the chain will permit us to screen systematically for mutations in patients and to generate recombinant proteins and synthetic peptides for further study of cell-matrix interactions involving the alpha 6(IV) chain.