Transfer of the α5(IV) Collagen Chain Gene to Smooth Muscle Restores in Vivo Expression of the α6(IV) Collagen Chain in a Canine Model of Alport Syndrome
2003; Elsevier BV; Volume: 162; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)63883-7
ISSN1525-2191
AutoresScott J. Harvey, Keqin Zheng, Barbara Jefferson, Peter Moak, Yoshikazu Sado, Ichiro Naito, Yoshifumi Ninomiya, Robert M. J. Jacobs, Paul S. Thorner,
Tópico(s)Osteoarthritis Treatment and Mechanisms
ResumoX-linked Alport syndrome is a progressive renal disease caused by mutations in the COL4A5 gene, which encodes the α5(IV) collagen chain. As an initial step toward gene therapy for Alport syndrome, we report on the expression of recombinant α5(IV) collagen in vitro and in vivo. A full-length cDNA-encoding canine α5(IV) collagen was cloned and expressed in vitro by transfection of HEK293 cells that synthesize the α1(IV) and α2(IV), but not the α3(IV) to α6(IV) collagen chains. By Northern blotting, an α5(IV) mRNA transcript of 5.2 kb was expressed and the recombinant protein was detected by immunocytochemistry. The chain was secreted into the medium as a 190-kd monomer; no triple helical species were detected. Transfected cells synthesized an extracellular matrix containing the α1(IV) and α2(IV) chains but the recombinant α5(IV) chain was not incorporated. These findings are consistent with the concept that the α5(IV) chain requires one or more of the α3(IV), α4(IV), or α6(IV) chains for triple helical assembly. In vivo studies were performed in dogs with X-linked Alport syndrome. An adenoviral vector containing the α5(IV) transgene was injected into bladder smooth muscle that lacks both the α5(IV) and α6(IV) chains in these animals. At 5 weeks after injection, there was expression of both the α5(IV) and α6(IV) chains by smooth muscle cells at the injection site in a basement membrane distribution. Thus, this recombinant α5(IV) chain is capable of restoring expression of a second α(IV) chain that requires the presence of the α5(IV) chain for incorporation into collagen trimers. This vector will serve as a useful tool to further explore gene therapy for Alport syndrome. X-linked Alport syndrome is a progressive renal disease caused by mutations in the COL4A5 gene, which encodes the α5(IV) collagen chain. As an initial step toward gene therapy for Alport syndrome, we report on the expression of recombinant α5(IV) collagen in vitro and in vivo. A full-length cDNA-encoding canine α5(IV) collagen was cloned and expressed in vitro by transfection of HEK293 cells that synthesize the α1(IV) and α2(IV), but not the α3(IV) to α6(IV) collagen chains. By Northern blotting, an α5(IV) mRNA transcript of 5.2 kb was expressed and the recombinant protein was detected by immunocytochemistry. The chain was secreted into the medium as a 190-kd monomer; no triple helical species were detected. Transfected cells synthesized an extracellular matrix containing the α1(IV) and α2(IV) chains but the recombinant α5(IV) chain was not incorporated. These findings are consistent with the concept that the α5(IV) chain requires one or more of the α3(IV), α4(IV), or α6(IV) chains for triple helical assembly. In vivo studies were performed in dogs with X-linked Alport syndrome. An adenoviral vector containing the α5(IV) transgene was injected into bladder smooth muscle that lacks both the α5(IV) and α6(IV) chains in these animals. At 5 weeks after injection, there was expression of both the α5(IV) and α6(IV) chains by smooth muscle cells at the injection site in a basement membrane distribution. Thus, this recombinant α5(IV) chain is capable of restoring expression of a second α(IV) chain that requires the presence of the α5(IV) chain for incorporation into collagen trimers. This vector will serve as a useful tool to further explore gene therapy for Alport syndrome. Alport syndrome is a hereditary disorder characterized by progressive nephropathy and ultrastructural abnormalities of the glomerular basement membrane (GBM) that is often associated with sensorineural deafness and ocular lesions.1Grunfeld J-P The clinical spectrum of hereditary nephritis.Kidney Int. 1985; 27: 83-92Crossref PubMed Scopus (88) Google Scholar, 2Habib R Gubler M-C Hinglais N Noël L-H Droz D Levy M Mahieu P Foidart J-M Perrin D Bois E Grünfeld J-P Alport's syndrome: experience at Hôpital Necker.Kidney Int. 1982; 21: S20-S28PubMed Google Scholar, 3Kashtan C Michael A Alport syndrome.Kidney Int. 1996; 50: 1445-1463Crossref PubMed Scopus (168) Google Scholar, 4Jais J Knebelmann B Giatras I De Marchi M Rizzoni G Renieri A Weber M Gross O Netzer K-O Flinter F Pirson Y Verellen C Wieslander J Persson U Tryggvason K Martin P Hertz J Schroder C Sanak M Krejcova S Carvalho M Saus J Antignac C Smeets H Gubler M X-linked Alport syndrome: natural history in 195 families and genotype-phenotype correlations in males.J Am Soc Nephrol. 2000; 11: 649-657Crossref PubMed Google Scholar In affected males, progression to end-stage renal disease usually occurs by the third decade of life, whereas females express variable phenotypes. The pathogenesis of Alport syndrome has been linked to defects of type IV collagen, which is a major structural component of basement membranes. Type IV collagen exists as a family of triple helical isoforms assembled from three α-chains. In humans, six genetically distinct α-chains, designated α1(IV) to α6(IV), have been identified and each is encoded by a separate gene designated COL4A1 to COL4A6, respectively.5Hudson B Reeders S Tryggvason K Type IV collagen: structure, gene organization and role in human diseases: molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis.J Biol Chem. 1993; 268: 26033-26036Abstract Full Text PDF PubMed Google Scholar From their amino to carboxyl termini, mature collagen α(IV) chains are characterized by a minor noncollagenous domain (∼15 residues), a minor collagenous domain (∼130 residues, 7S domain), a major collagenous domain (∼1400 residues) containing 15 to 20 noncollagenous interruptions, and a carboxyl terminal noncollagenous domain (∼230 residues, NC1 domain).6Timpl R Structure and biological activity of basement membrane proteins.Eur J Biochem. 1989; 180: 487-502Crossref PubMed Scopus (814) Google Scholar Triple helices are formed intracellularly and secreted, whereupon they self-assemble to form dimers through their NC1 domains and tetramers through their 7S domains. To date, more than 300 different mutations in the COL4A5 gene have been identified in families with the more common X-linked form of Alport syndrome,4Jais J Knebelmann B Giatras I De Marchi M Rizzoni G Renieri A Weber M Gross O Netzer K-O Flinter F Pirson Y Verellen C Wieslander J Persson U Tryggvason K Martin P Hertz J Schroder C Sanak M Krejcova S Carvalho M Saus J Antignac C Smeets H Gubler M X-linked Alport syndrome: natural history in 195 families and genotype-phenotype correlations in males.J Am Soc Nephrol. 2000; 11: 649-657Crossref PubMed Google Scholar, 7Knebelmann B Breillat C Forestier L Arrondel C Jacassier D Giatras I Drouot L Deschênes G Grünfeld J-P Broyer M Gubler M-C Antignac C Spectrum of mutations in the COL4A5 collagen gene in X-linked Alport syndrome.Am J Hum Genet. 1996; 59: 1221-1232PubMed Google Scholar whereas autosomal forms of the disease are associated with mutations in the COL4A3 and COL4A4 genes.8Mochizuki T Lemmink H Mariyama M Antignac C Gubler M-C Pirson Y Verellen-Dumoulin C Chan B Schröder C Smeets H Reeders S Identification of mutations in the α3(IV) and α4(IV) collagen genes in autosomal recessive Alport syndrome.Nat Genet. 1994; 8: 77-82Crossref PubMed Scopus (441) Google Scholar, 9Heidet L Arrondel C Forestier L Cohen-Solal L Mollet G Gutierrez B Stavrou C Gubler M Antignac C Structure of the human type IV collagen gene COL4A3 and mutations in autosomal Alport syndrome.J Am Soc Nephrol. 2001; 12: 97-106PubMed Google Scholar These mutations lead to the assembly of a GBM that is abnormal with respect to morphology and composition. Whereas normal GBM contains the α1(IV) to α5(IV) chains, for most Alport patients, the GBM contains only the α1(IV) and α2(IV) chains and lacks the α3(IV) to α5(IV) chains.10Ninomiya Y Kagawa M Iyama K Naito I Kishiro Y Seyer J Sugimoto M Oohashi T Sado Y Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies.J Cell Biol. 1995; 130: 1219-1229Crossref PubMed Scopus (266) Google Scholar, 11Peissel B Geng L Kalluri R Kashtan C Rennke H Gallo G Yoshioka K Sun M Hudson B Neilson E Zhou J Comparative distribution of the α1(IV), α5(IV), and α6(IV) collagen chains in normal human adult and fetal tissues and in kidneys from X-linked Alport syndrome patients.J Clin Invest. 1995; 96: 1948-1957Crossref PubMed Scopus (121) Google Scholar, 12Yoshioka K Hino S Takemura T Maki S Wieslander J Takekoshi Y Makino H Kagawa M Sado Y Kashtan C Type IV collagen α5 chain: normal distribution and abnormalities in X-linked Alport syndrome revealed by monoclonal antibody.Am J Pathol. 1994; 144: 986-996PubMed Google Scholar, 13Kleppel M Fan W Cheong H Michael A Evidence for separate networks of classical and novel basement membrane collagen. Characterization of α3(IV)-Alport antigen heterodimer.J Biol Chem. 1992; 267: 4137-4142Abstract Full Text PDF PubMed Google Scholar Normal GBM contains two networks, the first consisting of the α1(IV)/α2(IV) chains and the second of the α3(IV)/α4(IV)/α5(IV) chains.14Borza D-B Bondar O Todd P Sundaramoorthy M Sado Y Ninomiya Y Hudson B Quaternary organization of the Goodpasture autoantigen, the α3(IV) collagen chain: sequestration of two cryptic autoepitopes by intra-protomer interactions with the α4 and α5 NC1 domains.J Biol Chem. 2002; 277: 40075-40083Crossref PubMed Scopus (70) Google Scholar, 15Gunwar S Ballester F Noelken M Sado Y Ninomiya Y Hudson B Identification of a novel disulfide-cross-linked network of α3, α4 and α5 chains of type IV collagen and its implications for the pathogenesis of Alport syndrome.J Biol Chem. 1998; 273: 8767-8775Crossref PubMed Scopus (176) Google Scholar The existence of an α3(IV)/α4(IV)/α5(IV) network provides an explanation for the absence of each of these chains in Alport syndrome in the setting of COL4A3, COL4A4, or COL4A5 mutations, in that all three chains are required for the assembly of this network.15Gunwar S Ballester F Noelken M Sado Y Ninomiya Y Hudson B Identification of a novel disulfide-cross-linked network of α3, α4 and α5 chains of type IV collagen and its implications for the pathogenesis of Alport syndrome.J Biol Chem. 1998; 273: 8767-8775Crossref PubMed Scopus (176) Google Scholar, 16Borza D Bondar O Ninomiya Y Sado Y Naito I Todd P Hudson B The NC1 domain of collagen IV encodes a novel network composed of the alpha 1, alpha 2, alpha 5, and alpha 6 chains in smooth muscle basement membranes.J Biol Chem. 2001; 276: 28532-28540Crossref PubMed Scopus (123) Google Scholar There is no conservative or curative treatment currently available for those patients with Alport syndrome who have progressed to renal failure. However, as a result of advances in molecular genetic research, gene therapy is emerging as a possible treatment for some renal diseases,17Lien Y-H Lai L-W Gene therapy for renal diseases.Kidney Int. 1997; 52: S85-S88Google Scholar, 18Kitamura M Strategic gene transfer into the kidney: current status and prospects.Clin Exp Nephrol. 1997; 1: 157-178Crossref Google Scholar including Alport syndrome.19Tryggvason K Heikkilä P Pettersson E Tibell A Thorner P Can Alport syndrome be treated by gene therapy?.Kidney Int. 1997; 51: 1493-1499Crossref PubMed Scopus (25) Google Scholar, 20Heikkila P Tibell A Morita T Chen Y Wu G Sado Y Ninomiya Y Pettersson E Tryggvason K Adenovirus-mediated transfer of type IV collagen α5 chain cDNA into swine kidney in vivo: deposition of the protein into the glomerular basement membrane.Gene Therapy. 2001; 8: 882-890Crossref PubMed Scopus (55) Google Scholar An experimental model is essential for testing the feasibility of gene therapy for the treatment of Alport syndrome in humans. The model of X-linked Alport syndrome in Samoyed dogs mimics the human disease at the clinical, ultrastructural, and protein chemistry levels.21Jansen B Thorner P Baumal R Valli V Maxie M Singh A Samoyed hereditary glomerulopathy (SHG): evolution of splitting of glomerular capillary basement membranes.Am J Pathol. 1986; 125: 536-545PubMed Google Scholar, 22Jansen B Valli V Thorner P Baumal R Lumsden J Samoyed hereditary glomerulopathy (SHG): serial clinical and laboratory (urine, serum biochemistry and hematology) studies.Can J Vet Res. 1987; 51: 387-393PubMed Google Scholar, 23Thorner P Jansen B Baumal R Valli V Goldberger A Samoyed hereditary glomerulopathy. Immunohistochemical staining of basement membranes of kidney for laminin, collagen type IV, fibronectin, and Goodpasture antigen, and correlation with electron microscopy of glomerular capillary basement membranes.Lab Invest. 1987; 56: 435-443PubMed Google Scholar The canine nephropathy arises because of a nonsense mutation in the COL4A5 gene, resulting in ∼90% reduction in the level of α5(IV) mRNA.24Zheng K Thorner P Marrano P Baumal R McInnes R Canine X chromosome-linked hereditary nephritis: a genetic model for human X-linked hereditary nephritis resulting from a single base mutation in the gene encoding the α5 chain of collagen type IV.Proc Acad Natl Sci USA. 1994; 91: 3989-3993Crossref PubMed Scopus (103) Google Scholar The GBM of affected male dogs lacks the α3(IV), α4(IV), and α5(IV) chains.25Harvey S Zheng K Sado Y Naito I Ninomiya Y Jacobs R Hudson B Thorner P The role of distinct type IV collagen networks in glomerular development and function.Kidney Int. 1998; 54: 1857-1866Crossref PubMed Scopus (97) Google Scholar, 26Thorner P Zheng K Kalluri R Jacobs R Hudson B Coordinate gene expression of the α3, α4 and α5 chains of collagen type IV: evidence from a canine model of X-linked nephritis with a COL4A5 gene mutation.J Biol Chem. 1996; 271: 13821-13828Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar As an initial step toward developing gene therapy for X-linked Alport syndrome, we report on the cloning of a full-length cDNA encoding canine α5(IV) collagen and its expression in vitro and in vivo using this canine model. Total RNA was extracted from normal canine testis using Trizol reagent (Invitrogen, Carlsbad, CA), then purified using the Straight A's mRNA isolation system (Novagen, Madison, MI). First- and second-strand cDNA was synthesized with the Marathon cDNA Amplification kit (Clontech, Palo Alto, CA). The full-length construct (Figure 1A) was generated as two overlapping fragments obtained by 5′ and 3′ rapid amplification of cDNA ends (RACE) and nested polymerase chain reaction (PCR). The primers were based on human α5(IV) sequence27Zhou J Hertz J Leinonen A Tryggvason K Complete amino acid sequence of the human α5(IV) collagen chain and identification of a single-base mutation in exon 23 converting glycine 251 in the collagenous domain to cysteine in an Alport syndrome patient.J Biol Chem. 1992; 267: 12475-12481Abstract Full Text PDF PubMed Google Scholar (primers II and VI) and canine α5(IV) sequence24Zheng K Thorner P Marrano P Baumal R McInnes R Canine X chromosome-linked hereditary nephritis: a genetic model for human X-linked hereditary nephritis resulting from a single base mutation in the gene encoding the α5 chain of collagen type IV.Proc Acad Natl Sci USA. 1994; 91: 3989-3993Crossref PubMed Scopus (103) Google Scholar (primers I, III, IV, and V): I, 5′ CTG GGT CTC CAG GCA AAC CCT GGT 3′; II, 5′ TTC GTG CGG GTG CTG AAG GA 3′; III, 5′ ATA CCT GGT AAG CCA GGG TCC CC 3′; IV, 5′ GGT TTG CAG GGT CAG CCA GGA CCT 3′; V, 5′ GGT GGT AAA GGA GAG CCT GGC CT 3′; VI, 5′ TTA TGT CCT CTT CAT GCA CAC TT 3′. 5′ and 3′ RACE PCR was performed using the cDNA generated above as template, an adapter primer (5′ CCA TCC TAA TAC GAC TCA CTA TAG GGC 3′, Clontech) and primers I and IV, respectively. PCR was performed using rTth DNA polymerase-XL (Perkin Elmer, Norwalk, CT) under the following conditions: 94°C for 1 minute (1×); 94°C for 30 seconds, 68°C for 4 minutes (35×); 72°C for 10 minutes (1×). Nested PCR was performed under the same conditions using aliquots of the amplified mixtures above as template and primer sets II and III and V and VI. The full-length cDNA was generated on subcloning of overlapping 5′ and 3′ halves at a common BglII restriction site in pBluescript II vector (Stratagene, La Jolla, CA), and its nucleotide sequence was determined by automated sequencing using primers providing at least twofold coverage of the entire cDNA. For expression studies, this construct was subcloned into the mammalian expression vector pcDNA3.1 (Invitrogen). All cell culture and transfection reagents were obtained from Invitrogen. HEK293 human embryonic kidney cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained in α-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B) by weekly subcultivation using 0.25% trypsin and 1 mmol/L ethylenediaminetetraacetic acid. Primary cultures of smooth muscle cells (SMCs) from normal and affected male dog bladder were established according to the protocol described by Baskin and colleagues.28Baskin L Howard P Duckett J Snyder H Macarak E Bladder smooth muscle cells in culture: i. Identification and characterization.J Urol. 1993; 149: 190-197PubMed Google Scholar SMCs (passage ≤5) were cultured on a substrate of type I collagen (1 μg/cm2) (Sigma, St. Louis, MO) in M199 medium containing 10% fetal bovine serum, antibiotics, 100 μmol/L α-MEM amino acids, and 1× α-MEM vitamins. Cells were transiently transfected using lipofectamine and transgene expression was evaluated at 36 to 72 hours after transfection. For experiments examining the influence of ascorbate on collagen synthesis, cells were cultured and fed daily with Dulbecco's modified Eagle's medium containing 5% fetal bovine serum and antibiotics with or without 2 mmol/L of l-ascorbate. Total RNA was extracted by lysing cells in Trizol reagent (Invitrogen). Samples (10 μg) were separated by electrophoresis on 1% agarose-formaldehyde gels and transferred to Hybond membranes (Amersham, Cleveland, OH). The probes for α1(IV) to α6(IV) mRNAs were cDNAs for the respective canine NC1 domains generated as described previously24Zheng K Thorner P Marrano P Baumal R McInnes R Canine X chromosome-linked hereditary nephritis: a genetic model for human X-linked hereditary nephritis resulting from a single base mutation in the gene encoding the α5 chain of collagen type IV.Proc Acad Natl Sci USA. 1994; 91: 3989-3993Crossref PubMed Scopus (103) Google Scholar and labeled with 32P-dCTP using the random primed DNA labeling system (Roche, Laval, Quebec, Canada). Detection was performed at −70°C using Kodak Biomax film with an intensifying screen. The rat monoclonal antibodies H11, H22, H52, and H53 to the human α1(IV), α2(IV), and α5(IV) collagen chains and B66 to the bovine α6(IV) chain were used. Their specificity and reactive epitopes have been described previously.10Ninomiya Y Kagawa M Iyama K Naito I Kishiro Y Seyer J Sugimoto M Oohashi T Sado Y Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies.J Cell Biol. 1995; 130: 1219-1229Crossref PubMed Scopus (266) Google Scholar, 29Sado Y Kagawa M Kishiro Y Sugihara K Naito I Seyer J Sugimoto M Oohashi T Ninomiya Y Establishment by the rat lymph node method of epitope-defined monoclonal antibodies recognizing the six different α chains of human type IV collagen.Histochem Cell Biol. 1995; 104: 267-275Crossref PubMed Scopus (205) Google Scholar The mouse monoclonal antibodies mAb 1 and mAb 5 recognizing the human α1(IV) and α5(IV) NC1 domains, respectively, were obtained from Weislab AB (Lund, Sweden). For smooth muscle actin staining, a monoclonal mouse anti-human α-smooth muscle actin antibody was used (DAKO, Carpinteria, CA). Cells were seeded and transfected on glass coverslips, washed in phosphate-buffered saline (PBS), and fixed in acetone for 5 minutes at 4°C. Cryosections (5 μm) were fixed in acetone for 10 minutes at 4°C and then pretreated in a 100 mmol/L acid-KCl solution (pH 1.5) for 10 minutes to expose epitopes. The procedure used for immunoperoxidase staining has been described previously.30Harvey S Mount R Sado Y Naito I Ninomiya Y Jefferson B Jacobs R Thorner P The inner ear of dogs with X-linked nephritis provides clues to the pathogenesis of hearing loss in X-linked Alport syndrome.Am J Pathol. 2001; 159: 1097-1104Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar For histochemical detection of β-galactosidase, cryosections were fixed in 2% formaldehyde/0.2% glutaraldehyde for 5 minutes, then washed in PBS and incubated in PBS containing 5 mmol/L K3Fe(CN)6, 5 mmol/L K4Fe(CN)6, 2 mmol/L MgCl2, and 2 mmol/L X-gal (5-bromo-4-chloro-3-indolyl β-d-galactopyranoside) at 37°C for color development (1 to 16 hours). Counterstaining was performed with nuclear fast red. For dual-immunofluorescent detection of the α5(IV) and α6(IV) chains, cryosections were fixed as described above, and then denatured in 6 mol/L urea, 0.1 mol/L glycine, pH 3.5, for 1.5 hours at 4°C. Sections were blocked in PBS containing 1.5% normal donkey and rabbit sera (Vector Laboratories, Burlingame, CA) and then incubated simultaneously with mAb 5 (1:50 dilution) and B66 (1:10 dilution) antibodies for 1 hour. Detection was performed using an fluorescein isothiocyanate-conjugated donkey anti-mouse antibody (1:100 dilution; Jackson Immunoresearch, West Grove, PA) and a biotinylated rabbit anti-rat antibody (1:200 dilution, Vector Laboratories) for 1 hour, followed by tetramethyl-rhodamine isothiocyanate-conjugated streptavidin (1:50 dilution, Vector Laboratories) for 30 minutes. The mouse monoclonal antibodies mAb 1, mAb 5, or anti-human α-smooth muscle actin (50 μl) were incubated with 30 μl of protein G agarose (Sigma) in 0.5 ml of PBS for 2 hours at 4°C. The beads were washed twice in PBS then resuspended in 10 μl of 10% bovine serum albumin. Serum-free medium (1 ml) from transfected cells was buffered by the addition of HEPES to 25 mmol/L, then added to the beads and incubated with gentle mixing for 16 hours at 4°C. Immune complexes were collected by centrifugation and washed in 50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% Nonidet P-40 and 0.5% sodium deoxycholate (4 × 5 minutes), then in the same buffer adjusted to 0.5 mol/L NaCl, 0.05% sodium deoxycholate (2 × 5 minutes), and finally in the latter buffer lacking NaCl (1 × 5 minutes). Samples were solubilized in Laemmli buffer and analyzed by Western blotting. For analysis of secreted proteins, cells were cultured in serum-free medium after transfection. Protein was precipitated with ethanol at −20°C then resuspended in 1% SDS. For collagenase digestion, samples were resuspended in 100 mmol/L Tris-HCl, pH 7.5, 10 mmol/L CaCl2 containing 50 U/ml of purified bacterial collagenase (Worthington Biochemical, Freehold, NJ) and digested for 1 hour at 37°C, then precipitated as above. For analysis of the extracellular matrix, cells were washed in PBS, then incubated in PBS containing 0.5% Triton X-100 until detachment of the cell monolayer occurred. After extensive washes with PBS, the extracellular matrix remaining adherent to the culture dish was digested with collagenase then recovered as described above. Protein was quantified by DC protein assay (Bio-Rad, Hercules, CA). Native or reduced (100 mmol/L dithiothreitol) samples were subjected to SDS-PAGE using 4.5 to 12% linear gels as required. As a blotting control, type IV collagen NC1 domain was prepared by collagenase digestion of GBM isolated from the kidneys of normal mixed breed dogs as described previously.31Thorner P Baumal R Valli V Marrano P Production of anti-NC1 antibody by affected male dogs with X-linked hereditary nephritis: a probe for assessing the NC1 domain of collagen type IV in dogs and humans with hereditary nephritis.Virchows Arch [A]. 1992; 421: 467-475Crossref Scopus (6) Google Scholar After electrophoresis, gels were transferred to Immobilon-P membranes and the blots were blocked in 10 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 0.1% Tween-20, 3% bovine serum albumin. Blots were incubated with primary antibodies (1:500 dilution) for 1.5 hours, followed by biotinylated rabbit anti-rat secondary antibody (1:1000 dilution, Vector Laboratories) for 1 hour, and then a peroxidase-conjugated avidin-biotin complex (Vector Laboratories) for 30 minutes. Detection was performed using LumiGLO chemiluminescent substrate (KPL Laboratories, Gaithersburg, MD). A recombinant adenovirus that expresses canine α5(IV) collagen (designated Adα5) was constructed using the Adeno-Quest expression system (Quantum Biotechnology, Laval, Quebec, Canada). The vector contains an expression cassette at the E1 deletion site consisting of the full-length α5(IV) cDNA flanked by the major late promoter of adenovirus type 2 and human growth hormone polyadenylation sequences (Figure 1B). The α5(IV) cDNA was subcloned into the transfer plasmid pAdBM5PAG, then ClaI-linearized and co-transfected with Ad5CMVLacZΔE1/ΔE3 DNA (9.4 to 100 map units) into HEK293 cells by calcium-phosphate precipitation. After transfection, cells were overlaid with α-MEM containing 10% fetal bovine serum, antibiotics, and 0.8% agar and cultured until plaques formed. Plaques were picked and eluted in α-MEM for 24 hours at 37°C. Viral eluates were amplified on HEK293 cells until a complete cytopathic effect was reached, at which point the medium was recovered and used for a second round of amplification. A multiplex PCR-based strategy was used to screen for recombinant viruses of the desired genotype. Primers were chosen that generated 960- and 496-bp amplimers spanning the 5′ and 3′ junctions of the α5(IV) cDNA with the transfer plasmid, respectively, as well as a 308-bp amplimer within the adenoviral hexon gene32Allard A Girones R Juto P Wadell G Polymerase chain reaction for detection of adenoviruses in stool samples.J Clin Microbiol. 1990; 28: 2659-2667PubMed Google Scholar that served as a positive internal control (Figure 1C). Viral DNA was extracted from infected cells using the protocol described by Brown and colleagues.33Brown M Petric M Middleton P Diagnosis of fastidious enteric adenoviruses 40 and 41 in stool specimens.J Clin Microbiol. 1984; 20: 334-338PubMed Google Scholar PCR was performed under the following conditions: 95°C for 10 minutes (1×); 94°C for 1 minute, 50°C for 1 minute, 72°C for 2 minutes (35×); 72°C for 10 minutes (1×) using 200 ng of viral DNA as a template and the following primers: I, 5′ TTC ACC TGG CCC GAT CTG G 3′; II, 5′ TTC CTG GTG ACC GAG GGC CT 3′; III, 5′ GCC CTC CCA TAT GTC CTT CCG AGT GAG AG 3′; IV, 5′ GGC CAG AGC ATC CAG CCA TT 3′; V, 5′ GCC GCA GTG GTC TTA CAT G 3′; VI, 5′ CAG CAC GCC GCG GAT GTC 3′. Positive clones were tested for expression of the α5(IV) transgene by infection of HEK293 cells followed by Western blot analysis of the culture medium. A selected clone was subjected to three rounds of plaque purification, then amplified on HEK293 cells and purified by double CsCl2 equilibrium centrifugation. Viral titer was determined by plaque assay. Adα5 vector was injected into bladder smooth muscle of affected male dogs by direct visualization after general anesthesia and laparotomy. Before surgery and throughout the course of the study, dogs were placed on cyclosporin A (Neoral; Novartis Inc., Dorval, Quebec, Canada). Whole blood trough concentrations were maintained between 100 and 400 ng/ml as determined by weekly radioimmunoassay (Cyclo-Trac; Incstar, Stillwater, MN) using ethylenediaminetetraacetic acid-anti-coagulated whole blood. Injections (150 μl) consisted of AdCMVLacZ (2 × 108 pfu, Quantum Biotechnology), Adα5 (1 × 108 pfu), the same amount of Adα5 spiked with AdCMVLacZ (2 × 107 pfu), or vehicle (10 mmol/L Tris-HCl, pH 7.5, 1 mmol/L MgCl2, 5% sucrose). Each dog was administered a minimum of six injections that were marked with sutures. Dogs were euthanized and the injection sites were recovered and snap-frozen in OCT at various time points after injection ranging from 1 week (two dogs), 2 weeks (two dogs), 5 weeks (three dogs), to 7 weeks (one dog). Serial cryosections of bladder tissue were cut and transgene expression was evaluated by histochemical X-gal staining and immunostaining for type IV collagen. Sequence analysis of the canine α5(IV) cDNA revealed 20 bp of 5′-untranslated sequence followed by a 5076-bp open reading frame coding for 1691 amino acids (Figure 1A). (This sequence has been deposited in GenBank with the accession number AY078501. Four corrections to the previously published 3′ half of the canine α5(IV) collagen amino acid sequence24Zheng K Thorner P Marrano P Baumal R McInnes R Canine X chromosome-linked hereditary nephritis: a genetic model for human X-linked hereditary nephritis resulting from a single base mutation in the gene encoding the α5 chain of collagen type IV.Proc Acad Natl Sci USA. 1994; 91: 3989-3993Crossref PubMed Scopus (103) Google Scholar were identified; D1N, W234G, G340^PTGFQG (see text), and R645A that were confirmed by sequencing a minimum of three different cDNA clones.) The canine cDNA shares ≥92% identity at the nucleotide and amino acid levels to the human sequence. The deduced amino acid sequence predicts a protein of 162 kd with a 26-residue leucine-rich hydrophobic signal peptide as predicted using SignalP V1.1 software based on the method of Neilson and colleagues.34Neilson H Engelbrecht J Brunak S Von Heijne G Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites.Protein Eng. 1997; 10: 1-6Crossref Scopus (4942) Google Scholar There is a 15-residue noncollagenous domain at the amino terminus that contains four of four conserved cysteine residues. The collagenous domain spans 1421 residues with 22 noncollagenous interr
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