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M Kurkinen

Publications and source records attributed to M Kurkinen.

At least 55 records · Page 3Linked to original sources
PubMed1987

Basement membrane procollagen IV and its specialized carboxyl domain are conserved in Drosophila, mouse, and human.

Interaction with the extracellular matrix is important for the proliferation and differentiation of cells during development. A specialized extracellular matrix, basement membrane, is built around a scaffold of procollagen IV molecules. We report the sequence of a 2.5-kilobase cDNA which contains the carboxyl end of a Drosophila melanogaster procollagen IV. The amino acid sequence of the carboxyl-terminal domain, which forms an essential intermolecular linkage between procollagen IV molecules, is 59% identical in Drosophila and vertebrate procollagens IV, and an additional 17% of residues are conservatively substituted. This implies that the nature of the linkage is also conserved. We suggest that intermolecular junctions through procollagen IV carboxyl domains are fundamental elements of the molecular architecture of Metazoan basement membranes and have been conserved during evolution. The isolation and identification of this basement membrane collagen gene of Drosophila will help in deducing the function of procollagen IV in basement membranes.

Amino Acid Sequence↗
PubMed1987

Partial characterization of a low molecular weight human collagen that undergoes alternative splicing.

A cDNA library prepared from RNA isolated from a cultured human tumor cell line, HT-1080, was screened with a mouse cDNA clone coding for part of the -Gly-Xaa-Yaa- domain of the alpha 2(IV) collagen chain. Four overlapping cDNA clones were characterized that coded for a low molecular weight human collagen. The cDNA clones did not, however, code for the short-chain collagens, types IX and X. The amino acid sequences derived from the clones resembled type IV collagen in that there were short interruptions in the repeating -Gly-Xaa-Yaa- sequence. The noncollagenous, carboxyl-terminal domain was, however, much shorter and contained only 18 amino acid residues. Interestingly, one of the cDNA clones contained an additional 36 nucleotides not found in an overlapping clone. The 36 nucleotides encoded four -Gly-Xaa-Yaa- repeats without changing the reading frame. Nuclease S1 mapping demonstrated that the difference between the clones was due to existence of two different mRNAs. A synthetic 24-residue peptide corresponding to the last two -Gly-Xaa-Yaa- triplets and the entire carboxyl-terminal domain was used to generate polyclonal antibodies. Electrophoretic transfer blot analysis of HT-1080 cells and normal human skin fibroblasts identified two polypeptides, Mr 67,000 and Mr 62,000, that were sensitive to bacterial collagenase.

Amino Acid Sequence↗
PubMed1986

The gene for the alpha 1(IV) chain of human type IV procollagen: the exon structures do not coincide with the two structural subdomains in the globular carboxy-terminus of the protein.

Previous studies on the coding sequences of DNAs for the alpha 1(IV) chain of basement membrane collagen demonstrated a striking homology between the first 115 and the second 114 amino acids of the globular (NC1) domain of the protein. Also, alignment of the 12 cysteine residues indicated that the homology was particularly strong around three paired clusters of amino acids around cysteine residues. Here we have isolated a cosmid clone containing the 3'-end of the gene. Analysis of the clone and previously isolated lambda clones demonstrated that the intron--exon patterns of the gene does not reflect the homology in the protein. Therefore the homology cannot have arisen in any simple manner from gene duplications.

Amino Acid Sequence↗
PubMed1986

Large introns in the 3' end of the gene for the pro alpha 1 (IV) chain of human basement membrane collagen.

Using a recently characterized cDNA clone (HT-21) coding for the pro alpha 1 (IV) chain of human type IV procollagen, we have isolated three clones from a bacterio-phage lambda Charon 4A library of human genomic DNA. The intron/exon structure of the pro alpha 1 (IV) genomic clones was analyzed by heteroduplex electron microscopy and nucleotide sequencing. The analysis showed that the introns separating exons 2-9 are large and have a total length of over 12,000 base pairs (bp). Six of seven exons at the 3' end of the gene coded for -Gly-Xaa-Yaa-repeats of the collagenous part of the chain. Five of the -Gly-Xaa-Yaa- coding exons (numbers 5-9) varied in size between 72 bp and 134 bp, and none of them were 54 bp or multiples thereof. A sixth exon (exon 4) was a junction exon containing 71 bp coding for -Gly-Xaa-Yaa- sequences and 142 bp coding for the carboxyl-terminal noncollagenous domain (NC-1). The seventh exon (exon 3, 178 bp) coded for sequences of the NC-1 domain. Five of the six -Gly-Xaa-Yaa- coding exons began with the second base coding for glycine, and only one exon began with a complete glycine codon at the 5' end. The results (i) suggest that the gene for the pro alpha 1(IV) chain of human basement membrane collagen is significantly larger than the genes for fibrillar collagens and (ii) show that it lacks the 54-bp exon repeats characteristic of fibrillar collagen genes.

Base Sequence↗
PubMed1986

Biosynthesis and supramolecular assembly of procollagen IV in neonatal lung.

The rate of biosynthesis of procollagen IV, the principal collagen of basement membranes, and the concentration of specific RNAs coding for procollagen IV were measured in neonatal rat lungs. Both decreased sharply at birth and then recovered again a few days later. The supramolecular assembly of procollagen IV was followed in neonatal rat, mouse, and chick lungs, which actively elaborate endothelial and alveolar basement membranes, and in chick embryo gizzard which is rich in smooth muscle. The tetramer of four procollagen IV molecules linked covalently through their amino ends was isolated as an assembly intermediate from all these tissues. While noncovalent association of the carboxyl ends of two procollagen IV molecules occurred readily, the subsequent establishment of covalent cross-links was substantially slower in the junctional complexes of the carboxyl ends than of the amino ends. Both disulfide bonds and other, unidentified covalent links formed. The six component carboxyl peptides of a junctional complex became progressively covalently linked into two kinds of carboxyl peptide pairs. We conclude that both amino-linked tetramers and carboxyl-linked dimers of procollagen IV molecules are intermediates in the biological assembly of the collagen networks of these basement membranes.

Age Factors↗
PubMed1985

cDNA clones coding for the pro-alpha1(IV) chain of human type IV procollagen reveal an unusual homology of amino acid sequences in two halves of the carboxyl-terminal domain.

We report the isolation and characterization of cDNA clones coding for part of the pro-alpha1(IV) chain of human type IV procollagen. A cDNA library was prepared from total RNA isolated from a cultured human tumor cell line, HT-1080, and screened with a cloned mouse cDNA coding for the pro-alpha1(IV) chain. The largest cDNA clone encoded for 185 amino acid residues of the -Gly-X-Y-sequence of the human pro-alpha1(IV) chain, all of the globular carboxyl-terminal domain, and the 3' noncoding region. The results provide the first complete sequence for the carboxyl-terminal globular portion of a type IV procollagen chain. A striking feature of the carboxyl-terminal globular domain was a homology between the first and second half of the structure. The homology involved all 12 cysteine residues, the spacing between the cysteine residues, and many adjacent amino acids. The results raised the possibility that evolution of the globular domain involved duplication of an ancestral sequence coding for about 100 amino acids, 6 of which were cysteine. The homology, however, was more apparent in the amino acid sequence than in the nucleotide sequence, and, therefore, the results suggested that the homology reflects selective pressure on the function of the protein more than conservation of the nucleotide sequences in the gene. The nucleotide sequences of the 3' noncoding region of the cDNAs contained four polyadenylation signals of AATAAA. Three or four of the polyadenylation signals were probably used in transcription, since one major and two minor smaller RNA species from human skin fibroblasts hydridized with the cDNAs. In further studies, sorted human chromosomes were used to locate the gene for the pro-alpha1(IV) chain on chromosome 13.

Amino Acid Sequence↗
PubMed1985

Chromosomal assignments of the genes coding for human types II, III, and IV collagen: a dispersed gene family.

The human type II collagen gene, COL2A1, has been assigned to chromosome 12, the type III gene, COL3A1, to chromosome 2, and one of the type IV genes, COL4A1, to chromosome 13. These assignments were made by using cloned genes as probes on Southern blots of DNA from a panel of mouse/human somatic cell hybrids. The two genes of type I collagen, COL1A1 and COL2A1, have been mapped previously to chromosomes 17 and 7, respectively. This family of conserved genes seems therefore to be dispersed throughout the genome.

Animals↗
PubMed1984

Cell surface-associated proteins which bind native type IV collagen or gelatin.

Gelatin coupled to Sepharose has been used to isolate [35S]methionine-labeled polypeptides of Mr = 47,000, 56,000, 62,000, and 65,000 from the 12,000 X g supernatant of detergent extracts of mouse embryo parietal endoderm cells. The polypeptides can also be recovered from various established cell lines which synthesize type IV procollagen, and in these cells the Mr = 47,000 polypeptide is the major gelatin-binding component. Several lines of evidence, including the results of continuous labeling and pulse-chase experiments, show that the polypeptides are not derived by proteolytic cleavage of larger precursors and are distinct from fragments of fibronectin. The Mr = 47,000, 62,000, and 65,000 polypeptides all contain N-linked oligosaccharide side chains, as judged by their labeling with [3H]mannose and their sensitivity to tunicamycin. The Mr = 62,000 and 65,000 polypeptides can be resolved by two-dimensional gel electrophoresis into species with different isoelectric points, and the Mr = 47,000 has a pI of 7.5-8.0. None of the gelatin-binding polypeptides appear to accumulate in the culture medium, and the Mr = 47,000, 62,000, and 65,000 species are labeled in a lactoperoxidase-catalyzed iodination of intact cells, suggesting that they are associated with the cell surface. Only the Mr = 47,000 glycoprotein binds to native type IV collagen. Possible functions for these surface components in vivo are discussed.

Animals↗
PubMed1984

Reichert's membrane as a model for studying the biosynthesis and assembly of basement membrane components.

The major components of Reichert's membrane (laminin, type IV procollagen, entactin and heparan sulphate proteoglycan) are all synthesized by the parietal endoderm cells of the mouse embryo. Fibronectin is found mainly on the trophoblast side of Reichert's membrane and does not appear to be a major structural component. Parietal endoderm cells are thought to differentiate and migrate as individual cells from the margins of the epithelial visceral endoderm layer. They may interact with the type IV collagen in Reichert's membrane via a surface-associated protein of Mr = 47000 known as 'colligin'. Parietal endoderm cells are a rich source of mRNAs for basement membrane components and have been used to prepare a cDNA library in the expression vectors pUC8 and pUC9 from which cDNAs for type IV collagen and laminin B chains have been isolated.

Animals↗
PubMed1984

Sequencing of laminin B chain cDNAs reveals C-terminal regions of coiled-coil alpha-helix.

cDNAs for laminin B chains have been isolated from a parietal endoderm cDNA library in pUC8 and pUC9. Identification is based on: ability to direct the synthesis in Escherichia coli of polypeptides carrying laminin antigen determinants, in vitro translation of hybrid selected mRNA, and hybridization to high mol. wt. RNA differentially expressed in cells synthesizing large amounts of laminin. The plasmid pPE9 hybrid selects mRNA for the B2 (mol. wt. 185 000) chain and provides 217 residues of C-terminal amino acid sequence. The plasmids pPE386 and 49 both hybrid select mRNAs for the B1a (mol. wt. 205 000) and B1b (mol. wt. 200 000) chains. These two cDNAs are identical over much of their sequence, but pPE386 includes 133 nucleotides of 3' non-coding sequence and a poly(A) tail. Together they provide 495 residues of C-terminal amino acid sequence. Analysis of the predicted sequences reveals a striking heptad repeat, with a high probability that residues a and d are hydrophobic. Such a repeat is typical of the coiled-coil alpha-helices found in proteins such as myosin, tropomyosin and desmin (2-stranded) and fibrinogen (3-stranded).

Amino Acid Sequence↗
PubMed1983

Isolation of cDNA clones for basal lamina components: type IV procollagen.

We have isolated cDNA clones for mouse type IV procollagen from a library constructed from total poly A+RNA of 13.5 day mouse embryo parietal endoderm (PE) cells. In Northern analysis these clones hybridise to a 6.8 kb RNA which is abundant in embryonic PE cells and in differentiated F9 teratocarcinoma cells. Hybrid selection and in vitro translation of the cDNA specific mRNA produced a single polypeptide of Mr = 165 000. This polypeptide was specifically immunoprecipitated with mouse type IV procollagen antisera and comigrated on SDS-gel electrophoresis with one of the two in vitro synthesised chains of type IV procollagen. Undifferentiated F9 teratocarcinoma cells can be induced by retinoic acid and dibutyryl cAMP to differentiate in vitro into endoderm-like cells which resemble mouse PE cells in synthesising large amounts of basement membrane proteins, including type IV procollagen. Here we show, using one of the cDNA clones as a probe for type IV procollagen, that an increase in cellular concentration of type IV procollagen mRNA occurs within 24 to 48 hours of induction, reaching a constant high level by 72 hours.

Animals↗
PubMed1983

In vitro synthesis of laminin and entactin polypeptides.

Total RNA and poly(A+) RNA, isolated from 13.5-day-old mouse embryo parietal endoderm cells and from differentiated F9 teratocarcinoma cells that synthesize laminin and entactin, were translated in the reticulocyte lysate. Antiserum raised against purified and denatured laminin B chains specifically immunoprecipitated from the translation reaction polypeptides of Mr = 205,000, 200,000, and 185,000. Antiserum against the native complex of laminin and entactin also immunoprecipitated these polypeptides, although less efficiently. In addition, this antiserum immunoprecipitated polypeptides of Mr = 300,000, 270,000, and 140,000. Antiserum against purified and denatured entactin immunoprecipitated only the Mr = 140,000 polypeptide. In contrast, no polypeptides were immunoprecipitated from translation reactions programmed with RNA from undifferentiated F9 cells that produce only small amounts of laminin and entactin. The in vitro synthesized polypeptides migrate on NaDodSO4-polyacrylamide gel electrophoresis slower than the respective unglycosylated laminin and entactin chains isolated from cells treated with tunicamycin. Supplementing the reticulocyte lysate with dog pancreas microsomal membranes yields in vitro translation products which co-migrate with the respective glycosylated laminin and entactin chains of control cells. Taken together, these results suggest that the polypeptides described represent in vitro synthesized laminin and entactin chains.

Animals↗
PubMed1983

Isolation of cDNA clones for the human transferrin receptor.

A cDNA clone bank containing 30 000 clones was constructed from sucrose gradient-fractionated mRNA from human placenta. mRNA coding for transferrin receptor (TR) was enriched by polysome immuno-adsorbed chromatography with monospecific rabbit IgG and protein-A Sepharose. The library was screened for hybridisation to 32P-labelled cDNA synthesised from immunoselected TR mRNA and from poly(A)+ RNA of the polysome fraction that failed to bind to protein-A Sepharose. Plasmids isolated from colonies showing hybridisation only to the probe made from immunoselected mRNA were then subjected to hybrid selection. Two clones, pTR-48 and pTR-67, were able to hybridise the mRNA coding for the TR.

Cell Line↗
PubMed1982

In vitro synthesis of type IV procollagen.

Total RNA was isolated from parietal endoderm cells of 131/2-day mouse embryos that synthesize large amounts of type IV procollagen. In vitro translation of this RNA in the reticulocyte lysate supplemented with a ribonuclease inhibitor yielded two equally prominent polypeptides of Mr = 165,000 and 168,000, immunoprecipitable with anti-mouse type IV collagen serum. The Mr = 165,000 polypeptide was shown by one-dimensional peptide mapping to represent an unmodified chain of type IV procollagen. The Mr = 168,000 polypeptide, the in vitro synthesis of which was barely detectable in the absence of a ribonuclease inhibitor, most likely represents the other genetically distinct chain of type IV procollagen. Similar results to those described were also obtained using poly(A) + RNA prepared from murine F9 embryonal carcinoma cells induced to differentiate in vitro into parietal endoderm.

Animals↗
PubMed1982

Deposition of basement membrane proteins in attachment and neurite formation of cultured murine C-1300 neuroblastoma cells.

The deposition of the basement membrane glycoproteins, laminin, fibronectin, and type IV procollagen was studied by indirect immunofluorescence microscopy during the attachment and differentiation of murine C-1300 neuroblastoma cells. A typical cytoplasmic perinuclear staining for the basement membrane antigens was seen both in undifferentiated and differentiated cells. Freshly seeded suspended cells lacked surface fluorescence but in two hours after plating, distinct punctate laminin deposits became discernible on the ventral surface of the cells. Notably, in sparsely seeded undifferentiated cultures, the cell-associated extracellular laminin deposits could only be detected under the primary attaching cells, whereas daughter cells in clonal cell colonies lacked such fluorescence. In cultures induced to neurite formation with dibutyryl cyclic AMP, laminin deposition was also detected in association with the growing cytoplasmic extensions. No distinct differences were found between the secreted proteins of cultures of differentiated and nondifferentiated neuroblastoma cells, but the patterns of fucosylation of high-molecular weight proteins in the two cultures were markedly different. We conclude that cultured neuroblastoma cells both synthesize, secrete and deposit laminin. The distribution of laminin during neuroblastoma cell attachment and neurite extension suggests that this glycoprotein may be involved in cell-to-substratum interactions in C-1300 cell cultures.

Animals↗
PubMed1982

Synthesis and localization of two sulphated glycoproteins associated with basement membranes and the extracellular matrix.

Two sulphated glycoproteins (sgps) of apparent molecular weight (Mr) 180,000 and 150,000, are synthesized by murine PYS and PF HR9 parietal endoderm and Swiss 3T3 cells. The Mr 150,000 sgp has a similar chemical structure to the sulphated glycoprotein, C, synthesized and laid down in Reichert's membrane by mouse embryo parietal endoderm cells (Hogan, B. L.M., A. Taylor, and A.R. Cooper, 1982, Dev. Biol., 90:210-214). Both the Mr 180,000 and 150,000 sgps are deposited in the detergent-insoluble matrix of cultured cells, but they do not apparently undergo any disulphide-dependent intermolecular interactions and are not precursors or products of each other. They contain asparagine-linked oligosaccharides, but these are not the exclusive sites of sulphate labeling. Antiserum raised against the Mr 150,000 sgp C of Reichert's membranes has been used in an immunohistochemical analysis of rat skin. In early foetal and adult skin the antigen is present only in basement membranes, but transiently before and after birth it is also found throughout the upper part of the dermis. This suggests that 150,000 sgp C is at times synthesized by nonepithelial cells and contributes to the extracellular matrix of mesenchymal tissues.

Animals↗