Heinz Furthmayr

Shapes, domain organizations and flexibility of laminin and fibronectin, two multifunctional proteins of the extracellular matrix

Abstract Laminin from a mouse tumor basement membrane and fibronectin from human blood plasma were examined by electron microscopy using rotary shadowing and negative staining and by transmission scanning electron microscopy of unstained samples. Laminin was visualized as a rigid, asymmetric cross consisting of a long (77 nm) and three apparently identical short (36 nm) arms. The rod-like arms (diameter about 2 nm) terminated in globular units (diameter 5 to 7 nm). Additional globules were found near the terminal units in the short arms. A large pepsin-resistant fragment of laminin appeared as a rigid structure with three arms (length 26 nm, preferred angle 90 °), which presumably represented parts of the three short arms of laminin. Fibronectin could be visualized as two identical strands (length 61 nm, diameter about 2 nm), which did not reveal distinct globular units. These strands very likely comprised single peptide chains connected to each other at one end, enclosing a fixed angle of about 70 °. Electron microscopy also indicated a limited flexibility of the arms of both laminin and fibronectin, comparable to the stiffness of tropomyosin or DNA. The electron microscopic images of the shapes and dimensions of laminin, of fragments of laminin, and of fibronectin are consistent with the specific molecular weights and with the hydrodynamic properties determined in solution. The arms of fibronectin showed three distinct regions at which preferential bending occurred. These sites apparently correspond to flexible segments connecting more compact domains previously identified in biochemical studies. No sites of preferential bending were visible in the arms of laminin. Although laminin and fibronectin have some similar biological activities (binding of cells, collagen, glycosaminoglycans), the corresponding functional domains are differently arranged in the two molecules.

Dynamic interaction of VCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelial docking structure for adherent leukocytes

Ezrin, radixin, and moesin (ERM) regulate cortical morphogenesis and cell adhesion by connecting membrane adhesion receptors to the actin-based cytoskeleton. We have studied the interaction of moesin and ezrin with the vascular cell adhesion molecule (VCAM)-1 during leukocyte adhesion and transendothelial migration (TEM). VCAM-1 interacted directly with moesin and ezrin in vitro, and all of these molecules colocalized at the apical surface of endothelium. Dynamic assessment of this interaction in living cells showed that both VCAM-1 and moesin were involved in lymphoblast adhesion and spreading on the endothelium, whereas only moesin participated in TEM, following the same distribution pattern as ICAM-1. During leukocyte adhesion in static or under flow conditions, VCAM-1, ICAM-1, and activated moesin and ezrin clustered in an endothelial actin-rich docking structure that anchored and partially embraced the leukocyte containing other cytoskeletal components such as α-actinin, vinculin, and VASP. Phosphoinositides and the Rho/p160 ROCK pathway, which participate in the activation of ERM proteins, were involved in the generation and maintenance of the anchoring structure. These results provide the first characterization of an endothelial docking structure that plays a key role in the firm adhesion of leukocytes to the endothelium during inflammation.

Collagen polymorphism in the lung: An immunochemical study of pulmonary fibrosis

The localization of type I, III, IV, and AB2 (V) collagens in normal and fibrotic human lungs was studied by indirect immunofluorescence techniques with affinity purified antibodies specific for these types of collagen. In the normal lung type I collagen appears to be localized to the interstitium of alveolar septa in irregular patterns. Type III appears to have a more prominent, irregular localization in septa and a perivascular localization. Types IV and V codistribute in linear patterns on alveolar and capillary basement membranes. Type V is also present in the interstitium. Dramatic changes in relative amounts and localization were noted in fibrotic lung samples by fluorescence microscopy. In fibrotic lung samples there is a marked increase in type I in thickened septae. Type III is markedly reduced and seen only in perivascular localization. Type V is markedly increased in the interstitium and located in areas of smooth muscle cell proliferation. No apparent change is noted in amount or localization of type IV collagen. These findings correlate well with biochemical data relating to the amounts of various collagens isolated by enzymatic extraction techniques. These results are discussed with respect to staging and evolution of the fibrotic process.

Fractionation of the major sialoglycopeptides of the human red blood cell membrane

Summary The major sialoglycopeptides extracted from human red blood cell membranes by the LIS-phenol procedure can be separated into two distinct components by gel filtration in Ammonyx-LO. The main fraction, comprising ∼ 75% of the total, is a sialoglycopeptide containing 131 amino acids and 16 oligosaccharide chains and has been designated glycophorin A. A second glycopeptide, which can be distinguished from glycophorin A on the basis of its amino acid composition and tryptic peptide pattern, elutes in two fractions and has the capacity to form high molecular weight aggregates when analyzed by SDS gel electrophoresis.

The intramembranous segment of human erythrocyte glycophorin A

Abstract The intramembranous segment of glycophorin A has been localized to a 35-amino acid peptide. This has been isolated by a new procedure in which acid-insoluble peptides of a tryptic digest of detergent-purified glycophorin A are fractionated by countercurrent distribution. Amino acid sequence analyses, using both manual and automatic Edman degradation techniques, indicate that this peptide has a unique sequence in contrast to earlier work ( J. P. Segrest, I. Kahane, R. L. Jackson, and V. T. Marchesi, 1973 , Biochem. Biophys. Res. Commun. , 49 , 964–969). Ambiguities at three positions have been resolved, and sequencing errors at two additional positions have been corrected. One segment of this peptide has an uninterrupted stretch of 22 uncharged amino acids, and it is likely that this is the part which spans the lipid bilayer of the membrane. The complete 35-residue peptide has an apparent molecular weight in the 6000–8000 range, when analyzed on sodium dodecyl sulfate gels, suggesting that it forms dimers under these conditions. This result is consistent with our earlier proposal that intact glycophorin A molecules exist as dimers in sodium dodecyl sulfate which are stabilized by noncovalent associations between hydrophobic segments of their polypeptide chains.

Immunochemical evidence for the transmembrane orientation of glycophorin A. Localization of ferritin-antibody conjugates in intact cells.

Abstract Antibodies were raised in rabbits to a 51-amino acid cyanogen bromide-derived peptide of human erythrocyte glycophorin A which has been shown to represent the C-terminal end of the 131-residue polypeptide chain. Antibodies prepared by immunoadsorption were found to be directed against a chymotryptic-derived peptide (residues 102 to 118) of glycophorin A but were unreactive with either intact or proteolytically modified red blood cells. No cross-reactivity was observed with glycophorin B of human or sialoglycoproteins prepared from red blood cells of other mammalian species. Ferritin-antibody conjugates of such sera were applied to thin sections of intact red blood cells (frozen or protein embedded) and were found to localize exclusively to sites distributed uniformly along the inner surfaces of the membrane. No staining was seen on sections prepared from red blood cells from other species nor on sections of human red cells pretreated with unconjugated antisera. These results provide additional evidence in intact, fixed human erythrocytes that glycophorin A has a transmembrane orientation.

Immunoelectronmicroscopic localization of extracellular matrix components produced by bovine corneal endothelial cells in vitro

Bovine corneal endothelial cells deposit an extracellular matrix in short-term cultures, which contains various morphologically distinct structures when analysed by electron microscopy after negative staining. Amongst these were long-spacing fibers with a 150 nm periodicity, which appeared also to be assembled into more complex hexagonal lattices. Another structure was fine filaments, 10-40 nm in diameter, which occasionally exhibited 67 nm periodic cross-striation. Non-striated 10-20 nm filaments sometimes formed radially oriented bundles arranged in networks and fuzzy granular material was associated with the filaments in the bundles. Often, these bundles extended into solitary filaments, 10-20 nm in diameter, with a smooth surface. In addition, amorphous patches were seen, which contained dense aggregates of fibrillar and granular material. In longer-term cultures, some of the structures coalesced to form large fibrillar bundles. By using specific antibodies to various extracellular matrix components and immunolabeling with gold some of these structures could be identified as to their protein composition. Whereas fibronectin antibodies labeled a variety of structures--fine filaments with granular materials, radially oriented bundles, patchy amorphous aggregates and small granular material scattered throughout the background--type III collagen antibody predominantly labeled filaments with periodic banding (10-40 nm in diameter). A small amount of type III specific labeling was also observed over the networks of radially oriented fibrils and fine filaments associated with granular material. Type IV collagen and laminin antibodies localized in areas of the patchy amorphous aggregates. Type VI collagen antibodies, on the other hand, labeled fine filaments and the gold particles showed a pattern of 100 nm periodicity. Many of the fine 10-20 nm filaments exhibited a tubular appearance on cross-section, but they were not reactive with any of the antibodies used. Also negative were the long-spacing fibers and assemblies--including hexagonal lattices--containing this structural element.

Type V Collagens of the Human Placenta: Trimer α-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.

[21] Glycophorins: Isolation, orientation, and localization of specific domains

Publisher Summary Glycophorins are relatively small and heavily glycosylated proteins that are found in plasma membranes of many, possibly all, cell types. This chapter describes the isolation procedures and properties of intact glycophorins, and simplified procedures to obtain preparative amounts of the glycosylated domains of the three glycophorin molecules. The isolation procedures involve preparation of the crude glycophorin fraction, preparation of reagents, LIS/Phenol extraction, isolation of Glycophorin A, isolation of Glycophorin B and Glycophorin C, and preparation of the glycosylated (cell surface) domains of Glycophorins. Glycophorins may be associated to form dimeric structures in the membrane. Glycophorin A is a transmembrane protein, and the linear arrangement of the three domains—extracellular, intramembranous, and cytoplasmic—is evident not only from studies on the primary structure, but also from experiments performed with cells. However, there is no information available on the mode of insertion or transmembrane nature of glycophorins B and C.

Type IV Collagen “7S” Tetramer Formation: Aspects of Kinetics and Thermodynamics

Type IV collagen, a major structural element of basement membranes, is assembled into large polymers apparently held together by and carboxy-terminal2 as well as by lateral association^.^ This collagen, following extraction from the mouse EHS tumor6 (mostly as carboxy-terminal bound dimers), will self-assemble in vitro into a polygonal network containing side-by-side collagenous complexes which can be visualized in rotary shadow platinum replicas? Amino-terminal (“7s”) bond formation can be seen in platinum replicas after longer incubation periods? When type IV collagen is treated with pepsin in order to remove selectively the carboxy-terminal globular domain, amino-terminal tetramer formation, in the absence of other associations (at the concentration range studied), can be quantitated by velocity sediment a t i ~ n . ~ In this communication we report a study on rate and equilibrium characteristics of amino-terminal assembly. The rate of conversion of monomers to tetramers was examined at 28°C at different total concentrations of protein, and equilibrium was approached after about 24 hours. A plot of the reciprocal of the remaining monomeric concentration against time (at early time points when little product had formed) produced a straight line with nearly the same slope at various total protein concentrations, suggestive of a second-order reaction in the rate-limiting step in the forward direction. In addition small amounts of dimeric and trimeric intermediates could be detected at early as well as late time points as small shoulders on the main monomeric and tetrameric peaks. The presence of these forms could be confirmed by direct visualization in rotary shadow platinum replicas (FIGURE 1). A simple model for assembly which is consistent with the above data is one in which two monomers reversibly associate into a dimer (step l), one dimer binds to a monomer to produce a trimer (step 2), and then one trimer binds to a monomer to produce a tetramer (step 3). In such a model each foward rate step would be second order and each reverse step would be first order. Ignoring other possible interactions, it is possible to derive equations to describe the concentration dependence for all species and to compare this to observed data (FIGURE 2). The concentration of tetramer (C,) can be described with respect to monomer (C,) by: C, = C,‘/K,K,K,, where Ka, K,, and K, are the dissociation equilibrium constants for the first, second, and