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K Tryggvason

Publications and source records attributed to K Tryggvason.

At least 145 records · Page 8Linked to original sources
PubMed1993

COL4A5 splice site mutation and alpha 5(IV) collagen mRNA in Alport syndrome.

Mutations affecting the COL4A5 gene encoding the alpha 5 chain of type IV collagen, are involved in the pathogenesis of X-linked Alport syndrome. We used denaturing gradient gel electrophoresis (DGGE) to screen PCR amplified exons of COL4A5 for point mutations in a set of 18 Alport patients previously characterized by Southern blotting. One sequence variant was identified in the exon 38 region of a male Alport patient. Sequence analysis revealed a G to C transversion in the 5' intron splice donor site downstream from exon 38 (GT to CT). To determine the effect of the mutation on mRNA splicing, alpha 5(IV) cDNA was generated from total RNA of peripheral blood lymphocytes. Subsequent cDNA PCR yielded a product 81 base pairs shorter in the affected Alport patient, compared to normal controls. The absence of exon 38 from the alpha 5(IV) cDNA was confirmed by sequence analysis. The results demonstrated that the mutation leads to skipping of exon 38 in the processing of alpha 5(IV) pre-mRNA. The shortened transcript lacked 27 codons encoding a Gly-X-Y-repeat sequence with a preserved reading frame, enabling the translation of codons further downstream. Clinically, the patient presented with juvenile onset Alport syndrome, end-stage renal failure, and deafness. He had no ocular lesions. Typical ultrastructural changes of the glomerular basement membrane (GBM) were shown on electron microscopy. The patient developed anti-GBM antibodies after renal transplantation, however, renal function deteriorated only moderately.

Adult↗
PubMed1993

Molecular genetics of Alport syndrome.

Alport syndrome is a progressive hereditary kidney disease characterized by hematuria, sensorineural hearing loss and ocular lesions with structural defects in the glomerular basement membrane (GBM). The gene frequency has been estimated to be 1:5000. The disease is primarily X chromosome-linked, but autosomal forms have also been reported. The X-linked form has been shown to be caused by mutations in a recently identified alpha 5(IV) collagen chain gene (COL4A5). We have isolated cDNA clones for providing the entire primary structure of the human alpha 5(IV) chain. The gene has been located to the Xq22 region. Using antibodies against synthetic peptides, the alpha 5(IV) chain was shown to be located in the kidney only in the glomerular basement membrane. Thus far, the exon-intron structure has been determined for a large portion of the gene which probably has a size of over 200 kb. Numerous different mutations have been identified in the COL4A5 gene. The mutations include single base mutations, large deletions and other major rearrangements such as inversion and duplication. The consequences of the mutations observed can be considered sufficient to cause structural and functional defects in the type IV collagen molecule and, therefore, also the GBM network. This, in turn can explain the disruption of the GBM and hematuria occurring in these Alport patients. Alport syndrome is the first genetic basement membrane and kidney disease whose gene has been cloned. These recent results have enabled the development of antibodies and DNA probes for accurate diagnosis of Alport syndrome.

Chromosome Mapping↗
PubMed1993

Expression of 72 kilodalton type IV collagenase (gelatinase A) in benign and malignant ovarian tumors.

BACKGROUND: 72 Kilodalton (kd) type IV collagenase is a matrix metalloproteinase that specifically cleaves type IV collagen molecules. The enzyme has been postulated to have an important role in the invasion and spread of malignant tumors. EXPERIMENTAL DESIGN: In situ hybridization was used to study the expression of the 72 kd type IV collagenase mRNA in 24 benign, 2 semimalignant, and 15 malignant ovarian tumors and in 5 metastases of ovarian serous adenocarcinomas. The results were correlated with the expression of the mRNA for the alpha 1(IV) chain of type IV collagen and with the corresponding immunohistochemical distribution of the enzyme. RESULTS: The results showed that the more malignant an ovarian tumor was, the more clearly mRNA expressions for both 72 kd type IV collagenase and the alpha 1(IV) chain could be detected in tumor cells. The expression of both types of mRNAs was localized within the cells of tumor stroma and occurred mainly in fibroblasts and vascular endothelial cells. Epithelial tumor cells only rarely expressed these mRNAs. Immunohistochemical stainings localized the 72 kd collagenase as well to the stromal cells as to the epithelial cells of both benign and malignant tumors. CONCLUSIONS: The findings indicate that genes for the 72 kd type IV collagenase and for its substrate are simultaneously active in the same cells of the tumor stroma. The difference in the in situ hybridization and immunohistochemical findings could be explained by a possible variation in the metabolic balance between synthesis and accumulation of the protein in different cell types. It can also be proposed that the activity of the 72 kd type IV collagenase would be mediated through a receptor-like mechanism present on epithelial cells which could bind the 72 kd type IV collagenase synthesized elsewhere. There is also a possibility that the gelatinolytic activity of the mesenchymally synthesized 72 kd type IV collagenase would be consumed to degrade extracellular matrix proteins other than basement membranes.

Adenocarcinoma↗
PubMed1993

Messenger RNA for two type IV collagenases is located in stromal cells in human colon cancer.

The gene expression of two type IV collagen-degrading enzymes (72-kd and 92-kd type IV collagenases) was investigated in human colon adenocarcinomas by in situ hybridization. In all cases (18 out of 18), messenger RNA for the 72-kd type IV collagenase was present and located in numerous fibroblasts in the stroma surrounding the invasive cancer tissue. In normal-appearing colonic mucosa distant from the cancer tissue, either no expression or only very weak expression of this enzyme was detected. Also the 92-kd type IV collagenase was found in all samples investigated (10 out of 10), exclusively expressed by tissue macrophages. A very strong hybridization signal for messenger RNA for the 92-kd enzyme was found in a subpopulation of tissue macrophages surrounding invading malignant epithelium. In normal-appearing colon tissue, a markedly weaker hybridization signal was observed in macrophages contained in Peyer's patches. No hybridization signals for either of the two type IV collagenases were detected in cancer cells. Together with previous findings on expression of components of the plasminogen activation system, these results indicate that several nonepithelial cell types in the tumor stroma are involved in production of factors involved in extracellular proteolysis during colon cancer invasion.

Adenocarcinoma↗
PubMed1992

Recombinant laminin B1 chains exhibit intact short-arm domains but do not form oligomeric molecules.

The human laminin B1 chain has been produced in the baculovirus expression system in sufficient amounts for biochemical and functional studies. A full-length cDNA, which was constructed of four partially overlapping clones and verified by in vitro transcription and translation to be functional, was cloned into the transfer vector pVL1392 behind the polyhedrin promoter. The recombinant construct was incorporated by in vivo homologous recombination into the genome of the wild-type baculovirus, Autographa californica nuclear polyhedrosis virus. Infection of Spodoptera frugiperda cells (Sf9) with the recombinant virus resulted in the expression of the recombinant B1 chain (recB1) in these insect cells. The recB1 was found to be synthesized in two forms with apparent molecular masses of 220 kDa and 200 kDa. The 220-kDa form is an N-glycosylated form of recB1, because it was not present in cultures containing tunicamycin, an inhibitor of N-linked glycosylation. The recB1 accumulated inside the cell and only a small portion of it was secreted into the culture medium. Thus purification had to be started from the cell extract in order to obtain reasonable amounts of the protein. About 500 micrograms was obtained from a 500-ml culture with three steps of chromatography, concavalin A, DEAE-Sepharose and Mono Q anion-exchange chromatography. Only the glycosylated form was purified. The recB1 was found to be sensitive to degradation during the purification, because two proteolytic forms of about 180 kDa were present in every preparation. The accumulation of recB1 inside the cell was possibly due to the lack of correct assembly. Electron microscopy studies showed that the short arm part had a native or near-native structure, but the C-terminal heptad repeat domain had not folded correctly and did not exist in an alpha-helical structure, as it does in native laminin. Electron microscopy and cross-linking studies further revealed that recB1 was a monomeric protein. It was also shown to be unable to oligomerize in vitro, suggesting that the B1 chain is not designed to form homo-oligomers. Finally, cell attachment assays were carried out, but the native recB1 appeared to be inactive in these assays.

Animals↗
PubMed1992

Complete amino acid sequence of the human alpha 5 (IV) collagen chain and identification of a single-base mutation in exon 23 converting glycine 521 in the collagenous domain to cysteine in an Alport syndrome patient.

We have generated and characterized cDNA clones providing the complete amino acid sequence of the human type IV collagen chain whose gene has been shown to be mutated in X chromosome-linked Alport syndrome. The entire translation product has 1,685 amino acid residues. There is a 26-residue signal peptide, a 1,430-residue collagenous domain starting with a 14-residue noncollagenous sequence, and a Gly-Xaa-Yaa-repeat sequence interrupted at 22 locations, and a 229-residue carboxyl-terminal noncollagenous domain. The calculated molecular weight of the mature alpha 5(IV) chain is 158,303. Analysis of genomic DNA from members of a kindred with Alport syndrome revealed a new HindIII cleavage site within the coding sequence of one of the cDNA clones characterized. The proband had a new 1.25-kilobase HindIII fragment and a lack of a 1.35-kilobase fragment, and his mildly affected female cousin had both alleles. The mutation which was located to exon 23 was sequenced from a polymerase chain reaction-amplified product, and shown to be a G----T change in the coding strand. The mutation changed the GGT codon of glycine 521 to cysteine. The same mutation was found in one allele of the female cousin. The results were confirmed by allele-specific hybridization analyses.

Adolescent↗
PubMed1992

Molecular cloning of murine 72-kDa type IV collagenase and its expression during mouse development.

We report the isolation of a cDNA clone providing the first and complete sequence of mouse 72-kDa type IV collagenase. The clone contains 2800 nucleotides with a 1986-nucleotide open reading frame coding for 662 amino acids. The amino acid sequence includes a 29-residue signal peptide, an 80-residue propeptide, and a 553-residue enzyme proper. The sequence identity between the mouse and human enzymes is 96% with all cysteine residues conserved. The carboxyl-terminal domain of the mouse enzyme contains two more residues than the human enzyme. Northern hybridization analysis revealed considerable expression of the enzyme gene in newborn mouse lung, heart, kidney, and psoas muscle tissues, whereas only weak or no signals were observed in liver, spleen, and brain. Expression of the gene was substantially reduced in the same tissues of 3-month-old mice. In situ hybridization analysis of 72-kDa type IV collagenase expression in 10-15-day-old mouse embryos showed that the gene was intensely expressed in mesenchymal cells. Brain and surface ectoderm were completely negative. The epithelial tissue component of developing organs was negative with the exception of salivary gland. Although the expression varied somewhat between different mesenchymal tissues, no temporal or spatial changes could be associated with the advancement of epithelial branching morphogenesis. These findings together with our previous data on the expression of 72-kDa type IV collagenase in human tumors indicate that this enzyme has some very specific roles both in the physiological and pathological degradation of extracellular matrix. Furthermore, it has become clear that the closely related 92-kDa type IV collagenase differs completely with respect to expression pattern as well as gene regulation. The mouse cDNA clones reported in this study may provide important tools unraveling the actual roles of these enzymes in vivo.

3T3 Cells↗
PubMed1992

Localization of messenger RNA for Mr 72,000 and 92,000 type IV collagenases in human skin cancers by in situ hybridization.

We have examined the expression of 2 type IV collagen degrading enzymes (Mr 72,000 and 92,000 type IV collagenases) in human skin cancer by in situ hybridization. In all cases of infiltrating carcinomas of squamous cell (9 of 9) and basal cell (5 of 5) types, messenger RNA for the Mr 72,000 type IV collagenase was present in numerous fibroblasts. These were especially abundant in the stroma adjacent to the invasive tumor nodules. Malignant cells were negative for mRNA for the Mr 72,000 enzyme in all cases as were all other epithelial as well as endothelial cells. mRNA for the Mr 92,000 type IV collagenase was present in all 9 squamous cell and in 3 of the 5 basal cell carcinomas. In all these cases, a subpopulation of tissue macrophages was found to be positive, while malignant cells showed a signal for Mr 92,000 type IV collagenase in 6 of the squamous cell carcinomas but in none of the basal cell carcinomas. In all cases, the signal for this mRNA was confined to cells located at the tumoral/stromal interface or in the close vicinity of tumor nodules. No mRNA for any of the 2 collagenases was detected in 3 biopsies of normal skin. In vitro studies have indicated that collagenases are involved in the degradation of the extracellular matrix during cancer invasion. The present findings are consistent with such a role of the Mr 72,000 and 92,000 type IV collagenases in squamous and basal cell carcinomas in situ. The findings also demonstrate that degradative enzymes are not necessarily produced by the malignant cells themselves but may be generated by induction or recruitment of nonmalignant stromal cells.

Basal Cell Carcinoma↗
PubMed1992

Association between the expression of murine 72 kDa type IV collagenase by odontoblasts and basement membrane degradation during mouse tooth development.

In situ hybridization was used to study the expression of the 72 kDa type IV collagenase gene and its association with morphogenesis and cell differentiation during advancing mouse tooth development. The epithelia were completely negative during all developmental stages. The dental mesenchyme was uniformly positive during the early stages of tooth morphogenesis, and no association of type IV collagenase with morphogenetic events was observed. However, at the bell stage the expression increased in differentiating preodontoblasts. Expression was intense in the odontoblasts during secretion of the first predentine matrix. The expression was, however, transient; it decreased around the time when mineralization of dentine started until it completely ceased. Transcripts for 72 kDa type IV collagenase also gradually disappeared from the dental pulp. The expression of 72 kDa type IV collagenase was also strong in the osteoblastic cell lineage. The preosteoblasts at the beginning of the formation of mandibular bone as well as the osteoblasts of the alveolar bone expressed more 72 kDa type IV collagenase than did other mesenchymal cells. The increased gene expression in the odontoblasts correlates with the disappearance of the dental basement membrane as shown by immunolabelling with antibodies against type IV collagen. The onset of increased expression in the odontoblasts preceded the disappearance of the basement membrane and at the time when type IV collagenase transcripts were lost from all odontoblasts the basement membrane was completely removed. It can be speculated that during early stages of tooth development the 72 kDa type IV collagenase acts as a gelatinase whereas during later stages, when odontoblasts and ameloblasts differentiate and the deposition of predentine and enamel matrix is initiated, the enzyme may act as a type IV collagenase and contribute to the degradation of the dental basement membrane.

Animals↗
PubMed1992

Proteolytic processing of the 72,000-Da type IV collagenase by urokinase plasminogen activator.

Regulation of the activity of proteolytic enzymes is of major importance in the turnover of connective tissues. The search for physiologically relevant activation mechanisms of principal tissue-degrading enzymes, e.g., metalloproteinases, has therefore been of wide interest. We have now studied whether the initiating factor of the fibrinolytic system, urokinase plasminogen activator (u-PA), may also function in the early steps of activation of one of the metalloproteinases, the M(r) 72,000 gelatinase/type IV collagenase produced by cultured fibroblasts. Treatment of the secreted M(r) 72,000 proteinase by u-PA yielded a cleavage product of M(r) 62,000 as revealed by fluorography of radioactively labeled proteins as well as by gelatin zymography SDS-PAGE gels. The u-PA-catalyzed cleavage of the M(r) 72,000 proteinase was blocked by anti-u-PA antibodies, but was unaffected by the plasmin inhibitor aprotinin, thus indicating a specific action for the activator. On the contrary, the tissue activator of plasminogen, t-PA, did not cleave the type IV collagenase in similar assays. u-PA-catalyzed cleavage of recombinant type IV collagenase, produced in a baculovirus expression system, yielded a similar M(r) 62,000 activity in gelatinolysis assay. Zymograms of the isolated pericellular matrices of cultured fibroblasts also revealed M(r) 72,000 gelatinolytic polypeptide that was converted to an M(r) 62,000 form by u-PA. Both polypeptides were recognized in immunoblotting by antibodies against the gelatinase/type IV collagenase, suggesting immunological identity with the secreted enzyme. Thus the M(r) 72,000 gelatinase/type IV collagenase is not only secreted, but also deposited into the pericellular fibroblast matrix, and both forms are substrates for u-PA. The results suggest a new potential role for u-PA as a direct regulator of metalloproteinase-mediated extracellular proteolysis via the cleavage of the M(r) 72,000 gelatinase/type IV collagenase to an M(r) 62,000 form.

Cell Line↗
PubMed1992

Different mutations in the COL4A5 collagen gene in two patients with different features of Alport syndrome.

Alport syndrome is a hereditary renal disease in which progressive renal failure is often accompanied by sensorineural deafness and ocular abnormalities. Recently, mutations were detected in the type IV collagen alpha 5 chain gene in Alport syndrome patients. We searched for mutations in this gene in 18 unrelated patients, and in two patients abnormalities were detected. In the gene of patient BB we identified a complex deletion, which included the exons encoding the non-collagenous domain and part of the collagenous region. This patient showed early onset nephritis (end-stage renal disease at 17 years) with deafness. Within a year after receiving a kidney from an unrelated donor, he developed an antiglomerular basement membrane nephritis. In patient WJ a point-mutation was detected, changing a tryptophane into a serine in the non-collagenous domain. His clinical features are milder (renal failure at 33 years, no hearing loss), and a recent renal allograft did not provoke antiglomerular basement membrane disease. These initial data suggest that differences in the extent of disruption of the non-collagenous domain may correlate with the severity and/or heterogeneity of Alport syndrome and with the development of nephritis in renal allografts.

Amino Acid Sequence↗
PubMed1992

Alport syndrome and diffuse leiomyomatosis: deletions in the 5' end of the COL4A5 collagen gene.

Alport syndrome (AS) is an hereditary glomerulonephritis that is mainly inherited as a dominant X-linked trait. Structural abnormalities in the type IV collagen alpha 5 chain gene (COL4A5), which maps to Xq22, have recently been detected in several patients with AS. The association of AS with diffuse esophageal leiomyomatosis (DL) has been reported in 24 patients, most of them also suffering from congenital cataract. The mode of transmission and the location of the gene(s) involved in this association have not been elucidated. Southern blotting using cDNA probes spanning the whole COL4A5 and a 5' end COL4A5 genomic probe showed that three out of three patients with the DL-AS association had a deletion in the 5' part of the COL4A5 gene extending beyond its 5' end. This indicates that the same gene, COL4A5, is involved in classical AS and in DL-AS and that the transmission of DL-AS is X-linked dominant. These results also suggest that leiomyomatosis might be due to the alteration of a second gene involved in smooth muscle cell proliferation, which is located upstream of the COL4A5 gene, and that there might be a contiguous gene deletion syndrome, involving at least the genes coding for congenital cataract, DL and AS.

Adolescent↗
PubMed1992

Deletions of the COL4A5 gene in patients with Alport syndrome.

Mutations in the COL4A5 gene encoding the alpha 5 chain of type IV collagen have been found in linkage with X-chromosomal Alport syndrome (AS). To identify COL4A5 mutations in patients from Germany with clinically defined AS, DNA from 20 unrelated patients was analyzed by conventional Southern blotting. By using full length alpha 5(IV) cDNA probes, large COL4A5 deletions could be detected in two patients. In one case, a 34 kb deletion affecting the 14 most 3' exons of the gene was observed. The second patient harbored a complete COL4A5 deletion. In both cases, functional alpha 5(IV) mRNA was unlikely to be present. Clinically, both patients developed end-stage renal failure before age 30. Furthermore, they had characteristic retinal flecks, and sensorineural hearing loss with typical changes on the audiogram. The patient with the complete deletion of COL4A5 lost the renal allograft due to an anti-GBM mediated glomerulonephritis.

Adolescent↗
PubMed1992

Human basement membrane heparan sulfate proteoglycan core protein: a 467-kD protein containing multiple domains resembling elements of the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor.

The primary structure of the large human basement membrane heparan sulfate proteoglycan (HSPG) core protein was determined from cDNA clones. The cDNA sequence codes for a 467-kD protein with a 21-residue signal peptide. Analysis of the amino acid sequence showed that the protein consists of five domains. The amino-terminal domain I contains three putative heparan sulfate attachment sites; domain II has four LDL receptor-like repeats; domain III contains repeats similar to those in the short arms of laminin; domain IV has lg-like repeats resembling those in neural cell adhesion molecules; and domain V contains sequences resembling repeats in the G domain of the laminin A chain and repeats in the EGF. The domain structure of the human basement membrane HSPG core protein suggests that this mosaic protein has evolved through shuffling of at least four different functional elements previously identified in other proteins and through duplication of these elements to form the functional domains. Comparison of the human amino acid sequence with a partial amino acid sequence from the corresponding mouse protein (Noonan, D. M., E. A. Horigan, S. R. Ledbetter, G. Vogeli, M. Sasaki, Y. Yamada, and J. R. Hassell. 1988. J. Biol. Chem. 263:16379-16387) shows a major difference between the species in domain IV, which contains the Ig repeats: seven additional repeats are found in the human protein inserted in the middle of the second repeat in the mouse sequence. This suggests either alternative splicing or a very recent duplication event in evolution. The multidomain structure of the basement membrane HSPG implies a versatile role for this protein. The heparan sulfate chains presumably participate in the selective permeability of basement membranes and, additionally, the core protein may be involved in a number of biological functions such as cell binding, LDL-metabolism, basement membrane assembly, calcium binding, and growth- and neurite-promoting activities.

Amino Acid Sequence↗
PubMed1992

A truncated laminin chain homologous to the B2 chain: structure, spatial expression, and chromosomal assignment.

We describe the identification of a novel laminin chain. Overlapping clones were isolated from a human fibrosarcoma HT1080 cell cDNA library spanning a total of 5,200 bp. A second set of clones contained an alternative 3' end sequence giving a total of 4,316 bp. The longer sequence contained an open reading frame for a 1,193-residue-long polypeptide. The alternative sequence was shortened at the carboxyl-terminal end coding for a 1,111-residue-long polypeptide. The amino acid sequence contained 21 amino acids of a putative signal peptide and 1,172 residues or alternatively 1,090 residues of a sequence with five distinct domains homologous to domains I-V in laminin chains. Comparison of the amino acid sequences showed that the novel laminin chain is homologous to the laminin B2 chain. However, the structure of the novel laminin chain isolated here differs significantly from that of the B2 chain in that it has no domain VI and domains V, IV, and III are shorter, resulting in a truncated laminin chain. The alternative sequence had a shortened domain I/II. In accordance with the current nomenclature, the chain characterized here is termed B2t. Calculation of possible chain interactions of laminin chains with the B2t chain domain I/II indicated that the B2t chain can replace the B2 chain in some laminin molecules. The gene for the laminin B2t chain (LAMB2T) was localized to chromosome 1q25-q31 in close proximity to the laminin B2 chain gene. Northern analysis showed that the B2t chain is expressed in several human fetal tissues but differently from the laminin B1 and B2 chains. By in situ hybridization expression of the B2t chain was localized to specific epithelial cells in skin, lung, and kidney as opposed to a general epithelial and endothelial cell expression of the laminin B2 chain in the same tissues.

Amino Acid Sequence↗
PubMed1992

Simultaneous expression of 70 kilodalton type IV collagenase and type IV collagen alpha 1 (IV) chain genes by cells of early human placenta and gestational endometrium.

BACKGROUND: In this study we used in situ hybridization to investigate the expression of the genes 70 kilodalton (kd) collagenase and the alpha 1(IV) collagen chain of type IV collagen in cells of early human placenta and gestational endometrium. EXPERIMENTAL DESIGN: The aim was to study the spatial distribution of these gene expressions within a developing tissue which possesses physiologic invasive potential. The results obtained for the 70 kd type IV collagenase mRNA expression were also compared with the immunohistochemical distribution of the corresponding antigen. RESULTS: Expression of mRNAs for these proteins was found in cells of trophoblastic columns, stromal cells of villi and in cells of decidua and endometrial stroma. The only differences between the expressions was the lower level of signals for 70 kd type IV collagenase in fibroblastic stromal cells and endothelial cells of villi and in the pericytic cells of spiral arteries. Otherwise the results for both types of mRNA were comparable. We also studied the immunohistochemical distribution of the 70 kd type IV collagenase using specific monoclonal antibodies against the enzyme. Immunohistochemistry supported well the findings obtained by in situ hybridization. CONCLUSIONS: The results indicate that the genes for the 70 kd type IV collagenase and for the alpha 1(IV) collagen chain are simultaneously active in cells of placenta and gestational endometrium and the same cells which produce type IV collagen also can produce the cleaving enzyme, the 70 kd type IV collagenase. The results also show that the cytotrophoblastic cells, which during early pregnancy invade the extracellular matrix and spiral arteries of uterine wall contain significant amount of mRNA for the 70 kd type IV collagenase. This finding supports the concept that the 70 kd type IV collagenase would be important for invasion, and in the case of this study, also for the physiologic invasion of placental cytotrophoblasts.

Amino Acid Sequence↗