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R Timpl

Publications and source records attributed to R Timpl.

At least 73 records · Page 4Linked to original sources
PubMed1989

Structure of basement membranes in malignant melanoma and nevocytic nevi.

Basement membranes found around tumor cells in nevocytic nevi, Spitz's nevi, and malignant melanomas were analyzed by electron microscopy and antibody staining for several basement membrane proteins. Nevocytic nevi and Spitz's nevi showed a distinct, occasionally discontinuous lamina densa regardless of whether they were located in junctional zones of the epidermis or within the dermis. All basement membranes around nests of aggregated nevus cells, however, lacked anchoring fibrils. This correlated with the absence of type VII collagen. In contrast, type IV collagen, laminin, and nidogen were present at the periphery of the nevus cell clusters in agreement with the presence of an intact lamina densa. Aggregated tumor cells in malignant melanomas were bordered by a lamina densa when located in a junctional position and lacked this structure when they had migrated into the dermis. This process was accompanied by a drastically reduced staining for collagen type IV and nidogen, whereas laminin was still detectable. Anchoring fibrils and their molecular correlate, type VII collagen, were consistently absent. These observations demonstrate major alterations in the composition of basement membranes around malignant melanomas, which can be an important factor for the invasive growth and formation of metastases of these tumors.

Antibodies↗
PubMed1989

High antibody levels against mouse laminin with specificity for galactosyl-(alpha 1-3)galactose in patients with inner ear diseases.

Sera of 413 patients with inner ear diseases were examined for ELISA binding to mouse laminin and compared to a normal group. Strongly enhanced antilaminin antibody reactions were observed in a large number of patients with sensorineural hearing loss (68%), tinnitus (60%), and sudden deafness (46%), but not in those with Meniere's disease (14%) or normal individuals (8%). Absorption experiments demonstrated that these antibodies are also responsible for the antisarcolemmal antibody pattern in the immunofluorescence test and for the reaction with pig kidney microsomes in the ELISA. Immunochemical studies showed that laminin binding of high or low titer in sera from patients and normal individuals is due to the Gal alpha 1-3Gal epitope present in N-linked oligosaccharides of mouse laminin. No reactions were observed with human laminin, which lacks this epitope. The findings suggest that high levels of antibodies against carbohydrate structures are triggered by persistent infections, providing some new insights about the possible cause of these diseases.

Adult↗
PubMed1989

Human nidogen: cDNA cloning, cellular expression, and mapping of the gene to chromosome Iq43.

A human placental lambda gt11 expression cDNA library was screened for nidogen cDNAs by hybridizations with a heterologous mouse nidogen cDNA. A total of four positive overlapping clones were identified, and the sizes of the inserts were shown to vary from 0.8 to 2.8 kb. Nucleotide sequencing of the human cDNAs revealed that the largest clone, cHPN-16, contained both a 5' open reading frame encoding 582 amino acids and a 3' untranslated region of 1,063 nucleotides. Comparison of human cDNA sequences with mouse nidogen sequences revealed 84% identity on the nucleotide level and 88% identity with the deduced amino acid sequence. The deduced amino acid sequence of the human cDNAs revealed the presence of cysteine-rich epidermal growth factor-like repeats and the sequence Arg-Gly-Asp (RGD), a potential cell binding site, two features previously identified in mouse nidogen. The sequence Asn-Pro-Ser, a consensus sequence for N-linked glycosylation, was also noted. The newly isolated human cDNAs were utilized to analyze the expression of the nidogen gene by cultured human cells. Northern hybridizations revealed a single mRNA transcript of approximately 6.0 kb in human skin fibroblast and in HT 1080 fibrosarcoma cell cultures. However, the human choriocarcinoma cell line JEG-3, which expressed laminin genes, did not contain detectable levels of nidogen mRNAs. Quantitation of the relative nidogen mRNA abundance in HT 1080 fibrosarcoma cells indicated that nidogen mRNA levels were approximately the same as those for the laminin B2 chain. Finally, one of the nidogen cDNAs was used to map the nidogen gene onto locus q43 of chromosome 1.

Amino Acid Sequence↗
PubMed1989

Biochemistry of basement membranes.

Basement membranes are thin (20 to 300 nm) extracellular matrices with a ubiquitous occurrence in the body. They consist mainly of collagenous and noncollagenous glycoproteins, are formed early during embryonic development, and in mature tissues they compartmentalize various types of cells and tissue structures. The close apposition to cells is the most remarkable feature of basement membranes, which can either surround cells completely (muscle, fat, nerve axons) or separate them from underlying stroma in a polarized fashion (epithelium, endothelium). These cell contacts are mediated by cellular receptors and specific matrix components and have profound effects on polarization, differentiation, and proliferation of cells and on the control of their migratory behavior. In addition, basement membranes represent barriers regulating filtration of macromolecules and penetration by cells. The supramolecular organization of basement membranes is still insufficiently known. In transmission electron microscopy cross-sections show a two-layered morphology that includes an electron dense (lamina densa) and an electron lucent (lamina rara) zone, the latter being closer to the cells. Other specialized basement membranes such as those in renal glomeruli appear more complex and contain two laminae rarae. Here, presumably epithelial and invading endothelial cells each produce their own basement membrane, which then fuse together to form the filtration units of the glomerulus. Further structural and functional study of authentic basement membranes has been limited because these membranes comprise only a small fraction of tissues and most of their components are notoriously insoluble. Rodent tumors that produce large amounts of basement membrane material and the use of recombinant DNA technology in the past decade have paved the way for a more precise biochemical characterization of several basement membrane proteins. These components include collagen type IV, the cell-binding protein laminin, several proteoglycans, and other proteins. In the following pages we will briefly review the biochemical properties of the basement membranes and discuss their possible functions and contributions to supramolecular structures. For more extensive discussions, including certain biologic and pathologic aspects of basement membrane function, we refer the reader to several recent reviews.

Basement Membrane↗
PubMed1988

Amino acid sequence of the triple-helical domain of human collagen type VI.

The complete amino acid sequence of the triple-helical domain of human collagen VI was deduced from sequences of appropriate cDNA clones and confirmed to about 50% by Edman degradation of tryptic peptides. This domain consists of three different peptide segments containing some 335-336 amino acid residues originating from central portions of the alpha 1 (VI), alpha 2(VI), and alpha 3(VI) chains, respectively. Sequence identity in the X/Y positions of the Gly-X-Y repeats is rather low (10-15%) between the chains. Peculiar features of these sequences include 3 cysteine residues about 50 (alpha 3(VI)) and 89 (alpha 1(VI), alpha 2(VI)) residues away from the N-terminus and several Gly-X-Y interruptions clustered in the C-terminal two-thirds of the triple helix. These structures are presumably required for cross-linking collagen VI oligomers and for super-coiling of triple helices in the dimers. Other features include 11 Arg-Gly-Asp sequences, some of which are likely to be used as cell-binding sites, and four Asn-X-Thr sequences, allowing N-linked glycosylation along the triple helix. Junctional areas close to the helix contain short, cysteine-rich segments which may seal the triple-helical domain through disulfide bond formation, endowing it with high stability. These features, together with a low sequence homology to fiber-forming and basement-membrane collagens, document the unique character of collagen VI, whose triple helix is specifically adjusted for forming microfibrils in tissues.

Amino Acid Sequence↗
PubMed1988

Characterization of proteolytic fragments of the laminin-nidogen complex and their activity in ligand-binding assays.

Some 12 new nidogen and laminin fragments were purified from elastase, thrombin and trypsin digests and characterized by their sizes (22 kDa to greater than 300 kDa), subunit patterns on electrophoresis, partial amino acid sequences, content of specific epitopes and their binding to laminin or nidogen structures in radioligand assays. This permitted the various fragments to be ordered along the dumbbell-shaped structure of nidogen and to compare them with previously described nidogen fragments arising by endogenous proteolysis. Two nidogen fragments (E-50, E-90; 50 kDa and 90 kDa) remain associated with a large laminin fragment in elastase digests of the complex and could be dissociated with 2 M guanidine.HCl. Recombination studies demonstrated Kd = 10-20 nM for this interaction. Nidogen fragments devoid of binding activity included the tryptic peptide T-40 (40 kDa) corresponding to the rod-like domain and several larger fragments extending more to the N-terminus of nidogen. An N-terminal thrombin fragment of about 50 kDa was also inactive. Together the data show a lack of laminin binding to the N-terminal globule and rod of nidogen and provide indirect evidence that this activity is located within or close to its C-terminal globular domain. Nidogen-binding structures of laminin were obtained as two large fragments (greater than 300 kDa), P1X and E1X. They correspond to the short arm structure of laminin with one (E1X) or two (P1X) arms decreased in size to the inner rod-like segment. Shortening in E1X is mainly due to the B1 chain segment including the central globular domain which was identified as a new laminin fragment E10. Binding of E1X and P1X to nidogen was comparable to that of laminin while much lower activity was found for other laminin fragments. A 10-fold lower binding potential was also observed for the laminin-nidogen complex whose structure can now be defined in more precise molecular terms.

Amino Acid Sequence↗
PubMed1988

Role of laminin A chain in the development of epithelial cell polarity.

Kidney organ culture was used to study the conversion of embryonic mesenchymal cells into a polarized, differentiated kidney epithelium. We examined the expression of laminin, a basement membrane glycoprotein, during this conversion. The B chains of laminin were constitutively expressed, whereas the appearance of the A chain of laminin was dependent on embryonic induction and coincided with the onset of cell polarization. Antisera against the carboxy-terminal end of laminin inhibited polarization but did not affect the developmental events that precede polarization. Antisera against N-terminal parts of laminin failed to inhibit morphogenesis. Since the fragments at the carboxy-terminal end contain parts of the A chain, we suggest that the appearance of this chain is fundamental for initiation of cell polarity.

Animals↗
PubMed1988

Cloning and complete amino acid sequences of human and murine basement membrane protein BM-40 (SPARC, osteonectin).

Amino acid sequences of 285 and 286 residues, respectively, were deduced for mouse and human BM-40 from cDNA clones isolated from expression libraries. The sequences showed 92% identity and were also essentially identical to those of bone osteonectin and of the parietal endoderm protein SPARC. About 60% of the mouse BM-40 sequence was confirmed by Edman degradation. Two of the seven disulfide bonds were localized which apparently separate two distinct domains of mouse BM-40.

Amino Acid Sequence↗
PubMed1988

Reductive cleavage and reformation of the interchain and intrachain disulfide bonds in the globular hexameric domain NC1 involved in network assembly of basement membrane collagen (type IV).

The formation of collagen IV dimers in the extracellular space requires the association of two C-terminal globular domains giving rise to a large hexameric structure NC1 (Mr = 170,000). NC1 hexamer was purified from collagenase digests of a mouse tumor and several human tissues. It was shown by electrophoresis to consist of two kinds of cross-linked, dimeric segments, Da and Db (Mr about 50,000), and monomeric segments in a molar ratio of about 3:1. In the native hexamers free SH groups were detectable by N-[14C]ethylmaleimide and other sulfhydryl reagents. They account for 4-11% of the total number of cysteine residues with some variations between preparations from different sources and in the distribution between monomers and dimers. Reduction with 10 mM dithioerythritol under non-denaturing condition completely converted dimers into monomers and allowed the alkylation of all twelve cysteine residues present in each monomeric NC1 segment. A monomeric intermediate with four to six free SH groups and a higher electrophoretic mobility than the final product was observed. Generation of this intermediate from dimers Da and Db follows apparently different routes proceeding either directly or through a dimeric intermediate respectively. The time course of conversion is best described by a mechanism consisting of two (Db) or three (Da) consecutive steps with pseudo-first-order rate constants ranging from 0.14 ms-1 to 0.5 ms-1. Glutathione-catalyzed reoxidation of completely reduced NC1 in the presence of 2 M urea results in a product indistinguishable from native material by ultracentrifugation and electrophoresis pattern. The data suggest that in situ formation of NC1 structures is catalyzed by a small fraction (5-10%) of intrinsic SH groups leading to the formation and stabilization of dimers by rearrangement of disulfide bonds.

Animals↗
PubMed1988

Structure and distribution of N-linked oligosaccharide chains on various domains of mouse tumour laminin.

Asparagine-linked oligosaccharides were liberated from laminin and some of its fragments by hydrazinolysis, and after purification characterized by exoglycosidase digestions. This demonstrated the presence of nine forms of complex oligosaccharide chains, which differed in antennary and oligolactosamine structure, and of small amounts of high-mannose-type oligosaccharides. Additional variations were found with regard to substitutions by terminal alpha-galactose and sialic acid residues. Each of the various laminin fragments showed a unique but less complex repertoire of carbohydrate structures. These fragments also differed in mass, carbohydrate content, localization within the laminin molecule and functional activities such as cell-binding (fragments 1 and 6) and heparin- and collagen-binding (fragments 3 and 4). Fragment 7 with a particularly high carbohydrate content (72%) also showed the highest complexity of tri- and tetra-antennary structures. Further differences between the fragments were detected with human antibodies against the Gal alpha 1-3Gal epitope, which was expressed in either a high-affinity or a low-affinity form. Such differences in carbohydrate structure of topologically distinct laminin domains may have implications for their functions and in the regulation of post-translational modification events.

Amino Acid Sequence↗
PubMed1988

Assessment of disease activity in essential cryoglobulinemia by serum levels of a basement membrane antigen, laminin.

When compared with 40 normal controls, 46 patients with essential cryoglobulinemia had higher serum concentrations of a basement membrane antigen, laminin fragment P1 (LP1). Serum LP1 values were more pronounced in patients with visceral involvement, compared with those with skin vasculitis only. Asymptomatic patients or those with arthralgia usually had normal values. Increased disease activity was associated with a significant increase in LP1 (P less than 0.01). For evaluation of disease activity, determination of serum LP1 was a more sensitive measure than any complement factor assay.

Adolescent↗
PubMed1988

cDNA cloning, expression and mapping of human laminin B2 gene to chromosome 1q31.

A laminin B2 chain cDNA clone was isolated from a human lung cDNA library by screening with antibody against mouse laminin. The authenticity of the human cDNA clone was established by comparison of the nucleotide and deduced amino acid sequences of the cDNA insert with those of the previously reported mouse laminin cDNA clones. The human clone (LC7) contained an insert of 0.75 kb (kilobase pair) that corresponded to the last 232 amino acid residues in the carboxyl terminus of the B2 chain. Northern blot analyses with the LC7 probe detected two mRNA transcripts of 8.2 and 5.6 kb in both normal human skin fibroblasts and three human tumor cell lines. The cDNA probe was also used in Southern blot analysis of DNA from human rodent somatic cell hybrids to localize the gene to human chromosome 1. In situ hybridization of the cDNA with metaphase chromosome spreads confirmed the assignment and further mapped the human laminin B2 chain gene to the long arm of chromosome 1 in the band q31.

Amino Acid Sequence↗
PubMed1988

Structural requirements for the stimulation of neurite outgrowth by two variants of laminin and their inhibition by antibodies.

Laminin derived from the Engelbreth-Holm-Swarm (EHS) tumor and a lamininlike molecule synthesized by RN22 Schwannoma cells both stimulate rapid neurite outgrowth, consistent with a common neurite-promoting site. However, antilaminin antisera can only inhibit the activity of the EHS laminin. The blocking antibodies in such sera are directed against the terminal heparin-binding domain of the laminin long arm (Edgar, D., R. Timpl, and H. Thoenen. 1984. EMBO [Eur. Mol. Biol. Organ.] J. 3: 1463-1468). These epitopes are demonstrated by immunoblotting to be part of the A chain and to be absent in RN22 laminin, showing (through metabolic labeling) that the cells synthesized little if any 440-kD A chain. This indicates that the antibody inhibition was probably due to steric hindrance, a common neurite-promoting site, apparently not being antigenic in native molecules. Antibodies raised against a 25-kD proteolytic fragment derived from the long arm of laminin were then used as probes to identify other potential neurite-promoting structures. Although these antibodies do not cross-react with native laminin, they recognized the B chains of denatured EHS and RN22 molecules on immunoblots. The antibodies also bound to the large proteolytic fragment, derived from the long arm of laminin that contains the neurite-promoting site, thus inhibiting its activity. Taken together, these results point to the localization of normally nonantigenic, defined, B chain sequences within or close to the neurite-promoting site of laminin.

Animals↗
PubMed1988

High resolution immunoelectron microscopic localization of functional domains of laminin, nidogen, and heparan sulfate proteoglycan in epithelial basement membrane of mouse cornea reveals different topological orientations.

Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain.

Animals↗
PubMed1988

Procollagen intermediates during tendon fibrillogenesis.

The purpose of this study was to correlate ultrastructural features of tendon collagen fibrils at various stages of development with the presence of procollagen, pN-collagen, pC-collagen, and the free amino propeptides and carboxyl propeptide of type I procollagen. Tendons from 10-, 14-, and 18-day chicken embryos reveal small, well-defined intercellular compartments containing collagen fibrils with diameters showing a unimodal distribution. At 21 days (hatching) and 9 days (post hatching) and at 5 weeks (post hatching), the compartments are larger, less well-defined, and there is multimodal distribution of tendon fibril diameters. Procollagen and the intermediates pN-collagen and pC-collagen are present in tendons up to 18 days. Thereafter there is a marked reduction in procollagen, whereas the intermediates persist throughout all stages of development. Similarly, free amino propeptides and carboxyl propeptides of type I procollagen were found at all stages. The amino propeptide of type III procollagen was restricted to the peritendineum until 7 weeks post hatching. At that time, a network of fibrils containing the amino propeptide of type III procollagen was seen delineating well-circumscribed compartments of collagen fibrils throughout the entire tendon. This study supports the notion that pN- and pC-collagen have an extracellular role and participate in collagen fibrillogenesis.

Animals↗
PubMed1988

Cloning and chromosomal localization of human genes encoding the three chains of type VI collagen.

Type VI collagen is a heterotrimer composed of three polypeptide chains, alpha 1(VI), alpha 2(VI), and alpha 3(VI). By immunological screening of an expression cDNA library, human cDNAs specific for each chain were isolated and characterized. Major mRNA species encoding these chains have a size of 4.2 kb (alpha 1), 3.5 kb (alpha 2), and 8.5 kb (alpha 3). The cDNA clones were also used to map the genes on human chromosomes by somatic cell hybrid analysis and in situ hybridization. The alpha 1 (VI) and alpha 2(VI) collagen genes were both located on chromosome 21, in band q223. This represents a third example of a possible physical proximity of two collagen loci. The alpha 3(VI) collagen gene was localized to chromosome 2, in the region 2q37. The alpha 3(VI) collagen gene is the fifth extracellular matrix gene to be localized to 2q, as four other extracellular matrix genes--i.e., the alpha 1(III) and alpha 2(V) collagen genes, the elastin gene, and the fibronectin gene--have been previously mapped to the distal region of the long arm of chromosome 2.

Amino Acid Sequence↗
PubMed1987

Characterization of three constituent chains of collagen type VI by peptide sequences and cDNA clones.

Pepsin-solubilized collagen VI was prepared from human placenta and used to separate three constituent chains for determining partial amino acid sequences. Antibodies raised against the chains assisted in the identification and purification of several cDNA clones from three expression lambda gt11 libraries. Most of the clones hybridized to either a 3.5-kb or 4.2-kb mRNA species which by matching peptide and nucleotide sequences could be identified as coding for the alpha 2(VI) or alpha 1(VI) chain, respectively. Other clones hybridized to either an 8.5-kb mRNA which very likely encoded the alpha 3(VI) chain or to an unknown 2.0-kb mRNA. Northern blots revealed a considerable variation in the mRNA levels for each collagen VI chain in both skin and cornea fibroblasts and in several tumor cell lines. Limited sequence data generated from peptides and cDNA clones demonstrated a characteristic cysteine pattern at the junction between N-terminal globular domain and triple helix in all three chains. In addition, the data showed occasional interruptions of triplet sequences within the triple-helical domain and the presence of two Arg-Gly-Asp sequences which are potential cell-binding structures.

Amino Acid Sequence↗