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K Tryggvason

Publications and source records attributed to K Tryggvason.

At least 91 records · Page 5Linked to original sources
PubMed1997

Loss of type IV collagen alpha 5 and alpha 6 chains in human invasive prostate carcinomas.

Type IV collagen, a major component of basement membranes, is organized in a network responsible for the mechanical resistance of the basement membranes. It also plays a key role in epithelial cell adhesion to basement membranes. This study was designed to investigate the distribution of type IV collagen alpha-chains in normal, preneoplastic, and malignant prostate basement membranes. For this purpose, immunohistochemistry using specific antibodies raised against the different alpha-chains of type IV collagen was performed in eight normal samples, six prostatic intraepithelial neoplasia, and 20 malignant lesions of the prostate. Our results demonstrate the presence of the "novel" alpha 5 (IV) and alpha 6 (IV) chains along with the "classical" alpha 1 (IV)/alpha 2 (IV) chains in the basement membrane of the normal prostate gland. The alpha 3 (IV) chain was never detected in any prostate specimen. Prostatic intraepithelial neoplasia showed a similar immunostaining pattern to that found in normal glands. In cancer gland basement membranes, we demonstrate for the first time a specific loss of the alpha 5 (IV) and alpha 6 (IV) chains, whereas the classical alpha 1 (IV) and alpha 2 (IV) chains were consistently exhibited. Additionally, type VII collagen colocalized with alpha 5 (IV) collagen chain, and these two proteins, which were always observed in normal and prostatic intraepithelial neoplasia gland basement membranes, were lost in invasive carcinoma basement membranes. This observation raises questions about the possible association or cooperation between alpha 5 (IV)/alpha 6 (IV) chains and anchoring fibrils in prostate glands basement membrane.

Adenocarcinoma↗
PubMed1996

Structure of the human laminin alpha2-chain gene (LAMA2), which is affected in congenital muscular dystrophy.

We have determined the structure and complete exon size pattern of the human laminin alpha2-chain gene (LAMA2), which has been shown to be affected in congenital muscular dystrophy (Helbling-Leclerc, A., Zhang, X., Topaloglu, H., Cruaud, C., Tesson, F., Weissenbach, J., Tomé, F. M. S., Schwartz, K., Fardeau, M., Tryggvason, K., and Guicheney, P. (1995) Nat. Genet. 11, 216-218). The gene is over 260, 000 base pairs and contains 64 exons. The sequence of all exon-intron borders was determined. Two of the exons, i.e. exons 43 and 52, are extremely small in size, 6 and 12 base pairs, respectively. Comparison of the exon pattern of the human LAMA2 gene with that of the Drosophila LAMA gene revealed that only 2 of 63 intron locations in the 5'-end of the human gene match the intron locations in the Drosophila gene, which contains 14 introns.

Amino Acid Sequence↗
PubMed1996

Expression and characterization of human perlecan domains I and II synthesized by baculovirus-infected insect cells.

We present the in vitro expression and purification of N-terminal fragments of human perlecan in insect cells. Three tailored fragments of human perlecan cDNA were introduced into the polyhedrin locus of baculovirus expression vectors (BEVs) encoding amino acids 1-196 (domain I), 1-404 (domain I + IIa) and 1-506 (domain I + IIab). The integrity of the BEVs was checked by DNA sequencing, polymerase chain reaction, restriction enzyme analysis and Southern blotting. Northern hybridization and metabolic labeling with [35S]methionine showed that expression of the perlecan-(1-404)- and the -(1-506)- peptide was successful, but in the case of the perlecan-(1-196)-peptide no recombinant protein was produced. Immunoblotting showed that both the (1-404)-peptide and (1-506)-peptide are recognized by 95J10, a monoclonal antibody that was previously raised against perlecan-(24-404)-peptide expressed in Escherichia coli. Gel permeation and anion-exchange chromatography were applied to purify the recombinant proteins. Glycosaminoglycans were demonstrated to be present. Deglycosylation with chondroitinase ABC showed that the perlecan-(1-404)-peptide was glycosylated with chondroitin sulfate residues. Consistent with these results, glycosaminoglycans isolated from the perlecan-(1-404)-peptide were identified as chondroitin sulfate by agarose gel electrophoresis. Furthermore the perlecan-(1-404)-peptide showed affinity to immobilized basic fibroblast growth factor. The availability of baculovirus-derived recombinant perlecan fragments will facilitate domain-specific investigation of the structural and functional properties of perlecan in the future.

Animals↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1996

Structure of the human laminin gamma 2 chain gene (LAMC2): alternative splicing with different tissue distribution of two transcripts.

We have determined the structure of the human laminin gamma 2 chain gene (LAMC2), which is mutated in some patients with junctional epidermolysis bullosa. Eight lambda phage clones isolated from a genomic library and three subgenomic lambda phage clones made from a plasmid artificial chromosome clone spanned 75 kb, including the 55-kb gene. The LAMC2 gene contains 23 exons and is structurally highly homologous with the 28-exon LAMC1 gene (Kallunki et al., 1991, J. Biol. Chem. 266: 221-228), with 16 exons having identical sizes in the two genes. The gene analysis demonstrated that two previously described different size gamma 2 chain cDNAs (Kallunki et al., 1992, J. Cell Biol. 119: 679-693) are the result of alternative splicing. The longer gamma 2 chain is formed by using the coding sequence of the last exon 23, while the shorter gamma 2* chain is formed by using only 22 exons, together with part of the 5' end of intron 22. The two mRNAs were shown to have different expression patterns in 17-week-old human embryonic tissues, with the longer gamma 2 chain transcript strongly expressed in epithelia of all tissues studied, while distinct expression of the shorter gamma 2* chain mRNA was observed only in the cerebral cortex, in lung, and in distal tubules of the kidney.

Alternative Splicing↗
PubMed1996

Expression of the laminin gamma 2 chain in pancreatic adenocarcinoma.

Forty-two pancreatic adenocarcinomas were investigated immunohistochemically and by in situ hybridization for the expression of the laminin gamma 2 chain. In 41 cases, intracytoplasmic immunoreactivity for the gamma 2 chain was seen. Positive tumour cells were located especially at the epithelial-stromal interface of the tumour cell islands. In 22 cases, diffuse laminin gamma 2 chain immunoreactivity could also be seen in stroma and in seven cases, occasional positivity was detected in the neoplastic basement membranes. Signals for laminin gamma 2 chain mRNA in tumour cells displayed a distribution similar to that observed on immunohistochemistry. There were significantly more cases with less than 20 per cent of laminin gamma 2 chain-positive tumour cells in tumours extending to peripancreatic tissues and/or tumours with regional or distant metastases (p = 0.029). A corresponding statistical significance could also be noted in the mRNA level (P = 0.025). The results show that pancreatic adenocarcinomas display a high activity of laminin gamma 2 chain synthesis. Tumours with a strong laminin gamma 2 chain synthesis show a lower invasive and metastatic potential than tumours with a weak or moderate laminin gamma 2 chain expression.

Adenocarcinoma↗
PubMed1996

Identification of post-transplant anti-alpha 5 (IV) collagen alloantibodies in X-linked Alport syndrome.

X-linked Alport syndrome (AS) is a heritable disorder which is associated with mutations in the type IV collagen alpha 5 (IV) chain gene (COL4A5) located on chromosome X. Following renal transplantation, an average of 6% of male AS patients develop anti-GBM nephritis. We studied the specificity of the antibodies against type IV collagen in the serum of a patient with COL4A5 partial deletion. The specificity of these alloantibodies was determined against collagenase-digested GBM, as well as against recombinant non-collagenous (NC1) domains of the type IV collagen alpha 1(IV)-alpha 6(IV) chains expressed in escherichia coli. Immunoblotting and ELISA demonstrated that these antibodies bound specifically to the NC1 domain of alpha 5(IV) collagen. There was no binding to the NC1 domain of the other chains, including the Goodpasture antigen. Competitive ELISA confirmed the results obtained by ELISA and immunoblotting. This patient developed alloantibodies directed against antigens present in the grafted kidney, but absent from his Alport kidney. The pathogenesis of post-transplantation glomerulonephritis in the Alport patient studied is thus similar to that of Goodpasture syndrome, with the exception that the pathogenic antibodies are targeted to another alpha chain of type IV collagen.

Adolescent↗
PubMed1996

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.

Adult↗
PubMed1996

Haplotype analysis of congenital nephrotic syndrome of the Finnish type in non-Finnish families.

Congenital nephrotic syndrome of the Finnish type (CNF) has an estimated incidence of 1 in 8000 newborns in the genetically isolated population of Finland. Although the disease is most common in Finland, it occurs throughout the world in families without known Finnish origin. In the past, these authors recently localized the CNF gene to the chromosome 19q13.1 region and observed strong linkage disequilibrium to the genetic markers D19S610, D19S608, D19S224, and D19S220 in Finnish families. In these Finnish families, four main CNF haplotype categories have been observed. In the study presented here, haplotype analysis was applied to several non-Finnish CNF families to determine whether the same genetic locus is involved in these families. The results of the haplotype analysis suggest linkage to the 19q13.1 chromosomal region. It was also observed that, in most cases, alleles typically found on CNF chromosomes of Finnish families are also found on CNF chromosomes of non-Finnish families from North America and Europe. In these families, the strongest association was found with marker D19S608. These findings suggest that Finnish and many non-Finnish CNF cases share the same disease locus.

Alleles↗
PubMed1996

Identification of 17 mutations in ten exons in the COL4A5 collagen gene, but no mutations found in four exons in COL4A6: a study of 250 patients with hematuria and suspected of having Alport syndrome.

Conditions for polymerase chain-reaction amplification of ten exon regions (Exons 3, 7, 11 through 13, and 15 through 19) of the collagen COL4A5 gene and four exon regions (Exons 2, and 12 through 14) of the COL4A6 gene were sequenced and established in this study. These Type IV collagen genes contain 51 and 48 exons, respectively. The sequences of these exons were determined in the two genes in 250 male patients with hematuria and suspected Alport syndrome. Seventeen mutations were found in nine of the ten exons studied in the COL4A5 gene in 17 patients, whereas no mutations were identified in COL4A6. One mutation was identical in two patients known to be unrelated. The results indicate that mutations in COL4A5 that leading to renal failure are more frequent than those involved in classic Alport syndrome, and also that mutations in COL4A6 are not likely to cause this disease. Furthermore, mutations in COL4A5 are distributed quite randomly and no "hot spots" were found.

Adolescent↗
PubMed1996

Substitution of a conserved cysteine-996 in a cysteine-rich motif of the laminin alpha2-chain in congenital muscular dystrophy with partial deficiency of the protein.

Congenital muscular dystrophies (CMDs) are autosomal recessive muscle disorders of early onset. Approximately half of CMD patients present laminin alpha2-chain (merosin) deficiency in muscle biopsies, and the disease locus has been mapped to the region of the LAMA2 gene (6q22-23) in several families. Recently, two nonsense mutations in the laminin alpha2-chain gene were identified in CMD patients exhibiting complete deficiency of the laminin alpha2-chain in muscle biopsies. However, a subset of CMD patients with linkage to LAMA2 show only partial absence of the laminin alpha2-chain around muscle fibers, by immunocytochemical analysis. In the present study we have identified a homozygous missense mutation in the alpha2-chain gene of a consanguineous Turkish family with partial laminin alpha2-chain deficiency. The T-->C transition at position 3035 in the cDNA sequence results in a Cys996-->Arg substitution. The mutation that affects one of the conserved cysteine-rich repeats in the short arm of the laminin alpha2-chain should result in normal synthesis of the chain and in formation and secretion of a heterotrimeric laminin molecule. Muscular dysfunction is possibly caused either by abnormal disulfide cross-links and folding of the laminin repeat, leading to the disturbance of an as yet unknown binding function of the laminin alpha2-chain and to shorter half-life of the muscle-specific laminin-2 and laminin-4 isoforms, or by increased proteolytic sensitivity, leading to truncation of the short arm.

Amino Acid Sequence↗
PubMed1996

X-linked Alport syndrome: an SSCP-based mutation survey over all 51 exons of the COL4A5 gene.

The COL4A5 gene encodes the alpha5 (type IV) collagen chain and is defective in X-linked Alport syndrome (AS). Here, we report the first systematic analysis of all 51 exons of COL4A5 gene in a series of 201 Italian AS patients. We have previously reported nine major rearrangements, as well as 18 small mutations identified in the same patient series by SSCP analysis of several exons. After systematic analysis of all 51 exons of COL4A5, we have now identified 30 different mutations: 10 glycine substitutions in the triple helical domain of the protein, 9 frameshift mutations, 4 in-frame deletions, 1 start codon, 1 nonsense, and 5 splice-site mutations. These mutations were either unique or found in two unrelated families, thus excluding the presence of a common mutation in the coding part of the gene. Overall, mutations were detected in only 45% of individuals with a certain or likely diagnosis of X-linked AS. This finding suggests that mutations in noncoding segments of COL4A5 account for a high number of X-linked AS cases. An alternative hypothesis is the presence of locus heterogeneity, even within the X-linked form of the disease. A genotype/phenotype comparison enabled us to better substantiate a significant correlation between the degree of predicted disruption of the alpha5 chain and the severity of phenotype in affected male individuals. Our study has significant implications in the diagnosis and follow-up of AS patients.

Adult↗
PubMed1996

Adenovirus-mediated gene transfer into kidney glomeruli using an ex vivo and in vivo kidney perfusion system - first steps towards gene therapy of Alport syndrome.

In order to develop gene therapy for Alport syndrome, we have examined the efficiency of adenovirus-mediated transfer of the beta-galactosidase gene into cultured cells and intact glomeruli in vitro, and developed an organ perfusion system for gene transfer into kidney ex vivo and in vivo. Human endothelial and mesangial cells, as well as isolated human glomeruli, were readily infected and exhibited expression of the reporter gene. Single or multiple injections of the virus solution into the renal artery of pig in vivo did not lead to significant gene transfer and expression of the reporter gene in kidney cells. To increase the exposure time of kidney cells to the virus we perfused kidneys ex vivo and in vivo for up to 12 and 2 h, respectively. The perfusion system consisted of a perfusate container, a peristaltic pump and an artificial membrane lung gassed with carbogen. Using this system, intense expression of the reporter gene could be achieved in up to about 85% of the glomeruli after perfusion of an explanted kidney ex vivo and about the same efficiency of gene transfer could be obtained in glomerular cells after 2-h perfusion in vivo. Some expression was observed in other vascular endothelial cells following the perfusion, but no expression was observed in cells of the Bowman's capsule or epithelial cells of the tubuli. The X chromosome-linked form of Alport syndrome is caused by defects in the gene for the type IV collagen of alpha5 chain, which is primarily expressed in the kidney in glomerular cells. The present results demonstrated that efficient gene transfer can be achieved into glomerular cells, a prerequisite for gene therapy of this disease. The organ perfusion method developed in this study might also be applicable for gene therapy of other diseases.

Adenoviruses, Human↗
PubMed1995

Laminin-5 is a marker of invading cancer cells in some human carcinomas and is coexpressed with the receptor for urokinase plasminogen activator in budding cancer cells in colon adenocarcinomas.

Recombinant human gamma 2 chain of laminin-5 was expressed in Escherichia coli, and used to generate specific polyclonal antibodies which were used to study the distribution of the protein in human cancers. A total of 72 biopsies of human cancers were stained, including 23 cases of colon adenocarcinomas, 16 ductal breast carcinomas, 9 malignant melanomas, 14 squamous cell carcinomas of the skin and cervix, and 10 sarcomas. As a control for the specificity of the antibodies, we performed in situ hybridization on adjacent sections of a number of the cases, and in all of these cases the localization of the gamma 2 chain protein and mRNA was identical. We found gamma 2 chain immunoreactivity in cancer cells in all cases of colon adenocarcinomas and squamous cell carcinomas but not in any of the sarcomas, supporting the view that the laminin-5 protein is specific for cells of epithelial origin. Notably, in all of the cases of colon adenocarcinomas, the positive staining was invariably associated with budding cancer cells located at the tip of invading malignant epithelium, whereas the cancer cells deeper in the tumors were most often negative. The staining was cytoplasmic in all cases and only in one case did we see additional extracellular immunoreactivity, indicating that this laminin isoform in cancer tissue is not laid down in the extracellular matrix but probably exerts its function at the cell surface or in its immediate vicinity. Using in situ hybridization to analyze the coexpression of laminin-5 and components of the plasminogen activation system, we found that the histological distribution of laminin-5-positive budding cancer cells at the invasion front in colon adenocarcinomas was identical to that of the receptor for urokinase-type plasminogen activator. These findings suggest that laminin-5 is a marker of invading cancer cells in at least some human malignancies, and that it therefore might represent a valuable marker for the invasive potential of these cancers. The colocalization of laminin-5 and urokinase-type plasminogen activator receptor in a subset of cancer cells in colon cancer also suggests that a controlled up-regulation of a number of gene products is a characteristic of budding colon cancer cells, and that these gene products serve functions crucial for the invasive phenotype of these cancer cells.

Adenocarcinoma↗
PubMed1995

Primary structure and expression of a novel human laminin alpha 4 chain.

The complete primary structure of a novel human laminin alpha 4 chain was derived from cDNA clones. The translation product contains a 24-residue signal peptide preceding the mature alpha 4 chain of 1792 residues. The domain structure is similar to that of the recently described alpha 3 chain [Ryan, Tizard, Van Devanter and Carter (1994) J. Biol. Chem. 269, 22779-22787]. Northern analysis of RNA from human fetal and adult tissues revealed developmental regulation of expression. In adult, strong expression was observed in heart as well as lung, ovary, small and large intestines, placenta and liver, whereas weak or no expression was detected in skeletal muscle, kidney, pancreas, testis, prostate or brain. In contrast, fetal lung and kidney revealed high expression. In situ hybridization analysis of human fetal and newborn tissues showed expression of the laminin in alpha 4 chain in certain mesenchymal cells in tissues such as smooth muscle and dermis.

Adult↗
PubMed1995

Autolytic activation of recombinant human 72 kilodalton type IV collagenase.

Human 72 kDa type IV collagenase (gelatinase A, MMP-2) was expressed in a baculovirus/insect cell system. The enzyme was produced in the wild-type form and in two mutant forms, where the active site Glu375 was substituted by Asp or Gln. The mutated proteins had strongly reduced or no detectable activity, respectively, allowing detailed analysis of rapid autoactivation reactions. MMP-2 was readily degraded to a proenzyme form lacking the first four amino acid residues. This cleavage was shown to be an autolytic process, although enzyme activity was apparently not affected by this truncation. Conversion to the active enzyme form was achieved without external activator in a concentration-dependent manner at 37 degrees C. The activation of MMP-2 was shown to be a stepwise process, probably via a delta 1-50 form as a highly unstable intermediate. The C-terminal hemopexin-like domain is removed rather early at two cleavage sites, and degradation within the Zn-binding site inactivates the enzyme. The fibronectin- and hemopexin-like domains are stable, although the autodegradation pattern did not show any sequence specificity, except for charged residues in the P1' position. The results indicate that a specific activator may not be essential for MMP-2.

Amino Acid Sequence↗