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Biomedical subjects

H Furthmayr

Publications and source records attributed to H Furthmayr.

At least 73 records · Page 4Linked to original sources
PubMed1985

Structure and macromolecular organization of type VI collagen.

Collagen VI is a large, disulfide-bonded protein complex which is widely distributed in connective tissue. The constituent polypeptide chains (Mr = 110,000-140,000) consist of collagenous and noncollagenous segments, are degraded to chains of about half the size when collagen VI is solubilized by pepsin, and assemble to a unique pattern of oligomers. As revealed by electron microscopy, the triple-stranded protomer consists of a triple helix 105 nm in length flanked on each side by globular domains of similar size (diameter about 7 nm). Protomers are assembled to dimers by an antiparallel staggered alignment of triple-helical segments. This leads to inner regions, 75 nm in length, of two slightly supercoiled triple helices flanked by globular domains. At both sides 30-nm-long outer triple-helical segments emerge that are terminated by globules. Tetramers are formed from laterally aligned dimers that cross with their outer triple-helical segments in a scissors-like fashion. The same structures, except with much smaller globular domains, are found in pepsin-treated collagen VI. Disulfide-linked collagen VI produced by cultured fibroblasts has a size similar to that of genuine collagen VI found in tissue extracts. Larger forms of collagen VI are assembled from tetramers by end-to-end aggregation which because of an overlap of the outer segments brings all globular domains close together. This arrangement predicts microfibrillar structures in tissues with a periodicity of 100-110 nm and a diameter of 5-10 nm. Structures consistent with this proposal were indeed found by immunoelectron microscopy of placenta and aorta using the ferritin technique. Large, lateral aggregates of collagen VI microfibrils may in addition exist in cell cultures and tissues ("zebra collagen," "Luse bodies") and are presumably maintained by contacts between globular domains.

Animals↗
PubMed1985

Role of the carbohydrate domains of glycophorins as erythrocyte receptors for invasion by Plasmodium falciparum merozoites.

Solubilized preparations of purified glycophorins and specific domains of these molecules were assessed for their effects as inhibitors of Plasmodium falciparum invasion of human erythrocytes in vitro. The ability of newly invaded merozoites to continue developing and incorporating [3H]hypoxanthine during a 24-h period after their invasion was used as an assay for merozoite invasion. Glycophorins A, B, and C were found to be equally effective as inhibitors. Previous studies had shown N-acetylglucosamine, a sugar component of glycophorins A and C but not B, to be an effective inhibitor. Accordingly, molecular domains common to all of the glycophorins were further assessed. Sialic acid was shown to act almost as effectively as N-acetylglucosamine, presumably because of the structural similarities between these sugars. The inhibitory ability of sialic acid is considerably enhanced when presented to the parasite in a clustered form, as in an oligosaccharide. The acetyl group of these sugars does not appear to play an essential role in this inhibition. How the P. falciparum merozoite recognizes and interacts with the sugar domains of the glycophorin molecule remains to be determined.

Animals↗
PubMed1984

Self-assembly of basement membrane collagen.

The in vitro self-assembly of murine type IV collagen was examined by using biochemical and morphological techniques. Dimeric collagen undergoes a rapid and reversible thermal gelation at neutral pH without an appreciable lag period. The process is seen to be concentration dependent and inhibited by 2 M urea. The formed complex can be visualized by electron microscopy rotary shadowing as an irregular polygonal lattice network with extensive side by side associations within the collagenous triple-helical part of the molecules, two and three strands thick. Measurements on the matrix suggest a median stagger dimension of 170 nm, one-fifth the length of a dimer. The conversion of pepsin-generated monomers into N-terminally bound tetramers can also be demonstrated in vitro. This process is also concentration dependent and inhibited and reversed by 2 M urea but is thermally irreversible and occurs at a slow rate relative to the lateral associations. These tetramers can be seen by rotary shadowing as four-armed "spider" structures. It is proposed that lateral associations, by virtue of their faster rate of formation, precede 7S bond formation, and several models for the assembly of basement membrane collagen are discussed.

Animals↗
PubMed1984

The ultrastructural organization and architecture of basement membranes.

Basement membranes are ubiquitous complex, multicomponent structures having diverse functions. They are morphologically distinct and exhibit specific structural details including the lamina rara and lamina densa. In addition, the interstitial stroma abutting the lamina densa has a unique organization. While the composition of basement membranes is still incompletely known, several components have been identified, including collagen types IV and V, laminin and heparan sulphate proteoglycan. High resolution immunoelectron microscopic studies have allowed the development of various models of the organization and architecture of the basement membrane, suggesting specific localizations of the various collagen types and specific domains of the collagen molecules, laminin and other components. In addition, high resolution metal shadow casting techniques have allowed the development of molecular models of specific components of the basement membrane and methods of studying the domain structure and interactions of these components.

Animals↗
PubMed1984

Studies on the interaction of human plasma-fibronectin with native type I calf skin collagen molecules using the rotary shadowing technique.

Fibronectin is a ubiquitous glycoprotein found in plasma, on the surface of a number of cell types and in the extracellular matrix. It is believed to function as an adhesive protein for cells by mediating their interaction with connective tissue macromolecules. This study uses the rotary shadowing technique to investigate the interaction between human plasma fibronectin and native calf skin type I collagen molecules. Purified human plasma fibronectin appears fibrillar with a total length of 152 +/- 48 nm (n = 127). Individual molecules of fibronectin interact with one another in an apparent concentration dependent process to form linear polymeric structures up to 10 molecules by end-to-end association. Incubation of various concentrations of fibronectin with collagen results in the interaction of fibronectin with specific sites on the native collagen molecules. In addition, polymeric forms of fibronectin interact with collagen molecules and occasionally bridging structures between collagen molecules are formed. This study provides direct visual demonstration of an interaction between fibronectin and native collagen molecules. Possible physiologic implications of these observations are discussed.

Animals↗
PubMed1983

A monoclonal antibody specific for the amino terminal cleavage site of procollagen type I.

A monoclonal mouse IgG1 antibody was produced against the aminopropeptide of dermatosparactic sheep procollagen type I by using the hybridoma technique. Radioimmunoassays demonstrated an apparent affinity constant of 10(8) l X mol-1. The antibody reacted with a 19-amino-acid-long sequence spanning the procollagen N-proteinase cleavage site with stronger binding to structures contributed by the aminopropeptide. The antibody showed strong cross-reactions with similar antigens of bovine, human or chick origin but failed to react with the aminopropeptide of procollagen type III. Incubation of chick or sheep procollagen type I with stoichiometric amounts of antibody blocked the release from procollagen molecules of the aminopropeptide by procollagen N-proteinase. Thus, this antibody seems useful for studying various biological problems encountered in the conversion of procollagen.

Animals↗
PubMed1983

Ultrastructural morphology and domain structure of a unique collagenous component of basement membranes.

A disulfide cross-linked collagenous fragment (7 S) has been isolated by pepsin solubilization from several tissues rich in basement membranes including bovine lung, human placenta, and the murine EHS tumor. Examination of this material by the rotary shadowing technique indicates that these fragments are similar to but not identical with the 7S collagen described recently [Risteli, J., Bächinger, H.P., Engel, J., Furthmayr, H., & Timpl, R. (1980) Eur. J. Biochem. 108, 239-250]. The central rodlike portion of the particles was found to be similar in length; however, the peripheral four arms of 7S particles from bovine and murine sources are 10 nm longer in comparison to those from human sources. In addition, about 5-7% of all the particles contain a fifth arm. Specific antibodies to bovine 7 S cross-react with murine 7 S but only to a rather limited extent with human 7 S. These antibodies react with antigenic sites located at the ends of the peripheral arms of the fragment as visualized directly with rotary shadowing techniques. The data are consistent with a structural difference in type IV collagens from bovine, human, and mouse which leads to pepsin cleavage at different sites in a particular noncollagenous region adjacent to 7 S. However, since bovine 7S antibodies cross-react with human and murine tissues by immunofluorescence despite the lack of complete serological cross-reactivity, it is suggested that type IV collagens from all three species have some degree of homology in this region.

Amino Acids↗
PubMed1983

Electron-microscopical approach to a structural model of intima collagen.

Intima collagen was studied by electron microscopy (rotary shadowing and negative staining) and by analytical ultracentrifugation. It was found that the monomeric unit (Mr 170 000) consists of a 105 nm-long triple helix terminated by a small globular domain (Mr about 30 000) at one end and a large globular domain (Mr about 40 000) at the other end. The monomer was produced by selective reduction of interchain disulphide bridges. Before reduction, dimers, tetramers and larger filamentous structures were found. Dimers are lateral staggered aggregates of two monomers aligned in an anti-parallel fashion. This gives rise to an inner 75 nm-long region of two slightly intertwisted triple helices flanked by the large globular domains. The outer triple-helical segments (length 30 nm) with the small globular domains at their ends emerge at both sides of this structure. Interchain disulphide bridges are probably located in the vicinity of the large domains. Only the outer segments could be degraded by bacterial collagenase. In tetramers the outer segments of two dimers are covalently linked, forming a scissors-like structure. In the fibrous forms several tetramers are assembled end-to-end with an overlap between the outer segments. The molecular masses and sedimentation coefficients were calculated for these various forms from the electron-microscopically observed dimensions and agreed with results obtained by ultracentrifugation. The unique structure of intima collagen suggests that it originates from a microfibrillar component and that it can be considered a unique collagenous protein, for which we propose the designation type VI collagen.

Chemical Phenomena↗
PubMed1983

Human glioma-mesenchymal extracellular matrix antigen defined by monoclonal antibody.

The distribution and localization of a glioma-associated antigen defined by monoclonal antibody 81C6 has been examined using human cultured cell lines and tissues. Monoclonal antibody 81C6 was selected from a hybridoma fusion of spleen cells of mice immunized with the glial fibrillary acidic protein-positive human glioma cell line U-251 MG. Results of cell surface radioimmunoassay and absorption analysis demonstrated that 81C6 defined a glioma-mesenchymal extracellular matrix (GMEM) antigen expressed by 14 of 16 gliomas, 1 of 3 neuroblastomas, 1 of 7 melanomas, 2 of 6 sarcoma cell lines, and 8 of 9 cultured fibroblast lines. GMEM was not expressed by carcinoma or by the myeloid-lymphoid cell lines examined. Within the central nervous system, GMEM was expressed in 10 of 11 glioblastomas but was undetected in 5 of 6 astrocytomas and in normal adult and fetal brain by peroxidase-antiperoxidase immunohistology. In glioblastomas, the GMEM antigen was localized to basement membranes of the distinctive glomeruloid endothelial proliferations and hyperplastic blood vessels. The GMEM antigen was also expressed in 3 of 3 glioblastoma cell lines and 6 of 8 glioblastoma biopsy xenografts in athymic nude mice. Among non-central nervous system tissues and tumors, GMEM was found by peroxidase-antiperoxidase immunohistology in normal liver sinusoids, spleen red pulp sinusoids, kidney medullary tubule interstitium, and glomerular mesangium and in association with vascular and stromal elements of several undifferentiated tumors. The GMEM antigen is distinct from previously described forms of fibronectin, laminin, collagen types I to V, hyaluronic acid, chondroitin sulfate, and heparin, as determined by absorption analysis and immunohistological localization in tissues. The expression of GMEM in glioblastoma but not normal brain, association with glioblastoma-proliferative endothelium basement membranes, and expression in glioblastoma cell lines and nude mouse xenografts suggest that GMEM may be a useful marker of gliomas in vivo and in vitro.

Animals↗
PubMed1983

Lineage infidelity in acute leukemia.

Blast cells from 20 patients with acute leukemia (13 diagnosed myeloblastic and 7 as lymphoblastic, using the FAB classification) were studied using antibodies to lineage-specific differentiation markers. The phenotypic findings were usually consistent with the clinical diagnosis. However, examples were encountered where individual blast cells had a cytoplasmic marker of one lineage and a surface marker of a different lineage (lineage infidelity). Six examples of intramyeloid (two different myeloid lineages in the same cell) and three examples of interlineage infidelity (myeloid and lymphoid markers in the same blast cell) were encountered. No doubly marked cells were found in control material consisting of normal marrow cells, marrow regenerating after transplantation, or multilineage colonies derived from marrow in culture. A significant trend was observed relating the presence of lineage infidelity and failure of remission-induction. The data are interpreted as support for abnormal gene expression in leukemia.

Acute Disease↗
PubMed1983

Methods in laboratory investigation. Monoclonal antibodies to type IV collagen: probes for the study of structure and function of basement membranes.

Type IV collagen is one of the main constituents of basement membranes, yet it is unknown whether the structural framework at different sites is assembled from one unique type of molecule or whether different type IV collagen molecules exist. To study the composition, chemical identity, and organization of this protein in different organs we have prepared monoclonal antibodies to a type IV collagen preparation from human placenta. Swiss Webster mice were hyperimmunized, and splenic cells were fused with the three different myeloma cell lines SP2/0, NS1, and U1. Type IV collagen-specific hybrids were selected and cloned by limiting dilution and on hard agar. Monoclonal antibodies secreted by two clones were extensively characterized by ELISA-inhibition assay, immunoprecipitation, rotary shadowing, and immunofluorescence techniques. Unlike conventionally raised antibodies in rabbits, both monoclonal antibody reagents show species-specific binding exclusively to native type IV collagen from human placenta but not to a similar preparation from calf lung or to other types of collagen. After heat denaturation of the antigen binding was no longer observed. The M3F7 antibody-binding site is located within the triple helical domain of the type IV molecule, approximately 900 A removed from the amino terminal end as visualized by a metal shadow casting technique. The monoclonal antibody M3F7 precipitates material from pepsin-derived and radiolabeled type IV collagen, and analysis of the polypeptide chains in the immunoprecipitate by sodium dodecyl sulfate polyacrylamide gel electrophoresis suggests that two major fragments are contained in the precipitate, which yield polypeptides of about 100 and 50 kilodaltons. After rotary shadowing of antigen-antibody mixtures native collagen fragments of two different size classes that bind antibody are visualized. One fragment is approximately 1500 A in length, and the other measures about 2700 to 3000 A. The localization of the antigenic site on these fragments suggests that both are generated by pepsin cleavage at a site about 900 A removed from the amino terminal end. In immunofluorescence experiments the monoclonal antibodies stained all basement membranes in kidney, lung, placenta, or skin, suggesting that at least the type IV collagen molecule recognized by these monoclonal antibodies is shared by a variety of vascular and epithelial basement membranes.

Antibodies, Monoclonal↗
PubMed1983

Diverse specificities of five monoclonal antibodies reactive with glycophorin A of human erythrocytes.

Glycophorin A (GPA), the major sialoglycoprotein of human red cells, bears blood group MN determinants, and is a useful marker of the erythroid lineage in differentiating cells. Five monoclonal antibodies that react with GPA and possess a spectrum of serologic properties and fine specificities were obtained by immunization of mice with umbilical cord erythrocytes. Three antibodies, B22A, D22 and E11B, did not agglutinate En(a-) erythrocytes, genetic variants that lack GPA, and F11 and J11A agglutinated these cells very weakly. Antibodies B22A, E11B, and F11 agglutinated protease-treated cells more strongly than untreated erythrocytes, and they appeared to react with a peptide determinant located on the C-terminal side of the site at which trypsin cleaves GPA in the intact erythrocyte. In contrast to B22A and E11B, the hemagglutinating activity of F11 was not inhibited by purified GPA, nor did it bind to GPA in a solid phase immunoassay, but it immunoprecipitated GPA. Antibodies D22 and J11A appeared to be directed against carbohydrate determinants, or conformational determinants created by hydrogen bonding or electrostatic interactions between carbohydrate and protein. A preferential reaction of antibody J11A with MM over NN GPA was demonstrated by its reactions with enzyme-treated erythrocytes, its inhibition by purified GPA or its tryptic fragments, and by an ELISA assay.

Amino Acid Sequence↗
PubMed1982

Type V collagens of the human placenta: trimer alpha-chain composition, ultrastructural morphology and peptide analysis.

The alpha-chain trimer composition of type V collagen preparations from human placental membrane and villi was determined by two-dimensional electrophoresis on a non-denaturing polyacrylamide gel system followed by electrophoresis in the presence of sodium dodecylsulfate. In preparations isolated from placental membranes pure type V collagen was found with the alpha-chain composition [alpha 1(V)]2 alpha 2(V). In preparations from placental villi two different collagen trimers could be identified with alpha-chain compositions [alpha 1(V)]2 alpha 2(V) and [alpha 3(V)]3. Two-dimensional peptide maps after chymotryptic digestion of the various alpha-chain revealed distinct patterns for alpha 1(V), alpha 2(V) and alpha 3(V) suggesting unique structures for all three alpha-chains. Shadowing of the two collagen preparations with carbon-platinum by the rotary shadowing technique allowed the visualization of the individual molecules. In the placental membrane preparations, a uniform species of molecules was present while in placental villi preparations the same elongated form of collagen was found together with larger aggregates presumably containing molecules with the alpha 3-chain component. These data are interpreted to indicate that so-called type V collagen, at least in preparations from placental villi, contain two distinct collagen molecules.

Chymotrypsin↗
PubMed1981

Chemical and carbon-13 nuclear magnetic resonance studies of the blood group M and N active sialoglycopeptides from human glycophorin A.

The NH2-terminal sialoglycopeptides from human erythrocyte glycophorin A have been obtained by specific proteolytic cleavage and gel filtration chromatography. By cyanogen bromide cleavage, a glycosylated octapeptide was obtained from blood group M donors having an amino acid composition and 13C NMR spectrum consistent with the structure (formula: see text) was demonstrated. By Staphylococcus aureus protease cleavage, a glycosylated pentapeptide was obtained from N donors having the same structure as II, without the carboxyl-terminal sequence Val . Ala . Hse. Methanolysis/gas chromatographic analysis and 13C NMR spectroscopy of I and II and their asialo derivatives reveal that the M- and N-active sialoglyco-octapeptides both have identical oligosaccharide structures, each containing three O-linked tetrasaccharides with the structure NeuNAc alpha 2-3Gal beta 1-3(NeuNAc alpha 2-6)GalNAc alpha 1-O-Ser(Thr). The demonstration of the anomeric form of GalNAc-peptide linkages revealed by 13C NMR has previously been inaccessible by chemical analysis. Conformationally, I and II appear identical and both manifest several unusual resonance shifts suggestive of a glycopeptide secondary structure involving four specific hydrogen bonds. Calcium ion titration was also found to induce shifts in the NeuNAc 13C resonances that may be of functional significance. Serological studies reveal that both the M and N glyco-octapeptides and the N glyco-pentapeptide retain all of the M and N activity of the parent structure. Deamination and/or desialylation completely destroys this activity. These data are consistent with a model in which the M or N determinant is the NH2-terminal amino acid and a NeuNAc residue(s). From these data it is concluded that there is no chemical basis for assertions in the literature that M and N antigens differ in their oligosaccharide structure or that the N antigen is biosynthetically transformed to the M antigen by sialylation.

Amino Acid Sequence↗