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M Paulsson

Publications and source records attributed to M Paulsson.

At least 127 records · Page 7Linked to original sources
PubMed1989

Matrix-associated heparan sulfate proteoglycan: core protein-specific monoclonal antibodies decorate the pericellular matrix of connective tissue cells and the stromal side of basement membranes.

Cultured human lung fibroblasts produce a large, nonhydrophobic heparan sulfate proteoglycan that accumulates in the extracellular matrix of the monolayer (Heremans, A., J. J. Cassiman, H. Van den Berghe, and G. David. 1988. J. Biol. Chem. 263: 4731-4739). A panel of four monoclonal antibodies, specific for four distinct epitopes on the 400-kD core protein of this extracellular matrix heparan sulfate proteoglycan, detects similar proteoglycans in human epithelial cell cultures. Immunohistochemistry of human tissues with the monoclonal antibodies reveals that these proteoglycans are concentrated at cell-matrix interfaces. Immunogold labeling of ultracryosections of human skin indicates that the proteoglycan epitopes are nonhomogeneously distributed over the width of the basement membrane. Immunochemical investigations and amino acid sequence analysis indicate that the proteoglycan from the fibroblast matrix shares several structural features with the large, low density heparan sulfate proteoglycan isolated from the Engelbreth-Holm-Swarm sarcoma. Thus, both epithelial cell sheets and individual mesenchymal cells accumulate a large heparan sulfate proteoglycan(s) at the interface with the interstitial matrix, where the proteoglycan may adopt a specific topological orientation with respect to this matrix.

Amino Acid Sequence↗
PubMed1988

Binding of Ca2+ influences susceptibility of laminin to proteolytic digestion and interactions between domain-specific laminin fragments.

Ca2+ was found to influence the patterns of limit digests of laminin obtained with various neutral proteases. In the presence of Ca2+, larger fragments were obtained from the central part of laminin than in its absence. This was interpreted as being due to a stabilization of the central short-arm domains of laminin by bound Ca2+. When proteolytic fragments were tested for their ability to aggregate, only large fragments containing intact short arms were active, indicating an important role for these domains in laminin self-aggregation.

Calcium↗
PubMed1988

Cartilage proteoglycans. Assembly with hyaluronate and link protein as studied by electron microscopy.

Aggregates formed by the interaction of cartilage proteoglycan monomers and fragments thereof with hyaluronate were studied by electron microscopy by use of rotary shadowing [Wiedemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333]. The differences in shape and packing of the proteins bound along the hyaluronate strand in aggregates formed in the presence and in the absence of link protein were examined in detail. The high resolution of the method allowed examination of the involvement in hyaluronate binding of the globular core-protein domains G1, G2 and G3 [Wiedemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333; Paulsson, Mörgelin, Wiedemann, Beardmore-Gray, Dunham, Hardingham, Heinegård, Timpl & Engel (1987) Biochem. J. 245, 763-772]. Fragments comprising the globular hyaluronate-binding region G1 form complexes with hyaluronate with an appearance of necklace-like structures, statistically interspaced by free hyaluronate strands. The closest centre-to-centre distance found between adjacent G1 domains was 12 nm. Another fragment comprising the binding region G1 and the adjacent second globular domain G2 attaches to hyaluronate only by one globule. Also, the core protein obtained by chondroitinase digestion of proteoglycan monomer binds only by domain G1, with domain G3 furthest removed from the hyaluronate. Globule G1 shows a statistical distribution along the hyaluronate strands. In contrast, when link protein is added, binding is no longer random, but instead uninterrupted densely packed aggregates are formed.

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

The role of Ca2+ binding in the self-aggregation of laminin-nidogen complexes.

Laminin-nidogen complexes were found to aggregate in the presence of divalent cations in a manner dependent on ion concentration. This effect shows a selectivity for Ca2+, as half-maximal aggregation is achieved already at about 10 microM Ca2+, while Mg2+ induces aggregation at 10-fold higher ion concentrations and always to a lesser extent. When binding of Ca2+ to laminin-nidogen complexes was measured by equilibrium dialysis, a total of about 16 binding sites with dissociation constants in the range of 5-300 microM could be identified. At 50 microM Ca2+, where the aggregation is maximal, only two to three Ca2+ ions are bound to laminin-nidogen complexes, indicating that the aggregation reaction is induced by the binding of Ca2+ to a small number of sites and possibly to a single distinct site. Analysis of Ca2+ binding to various proteolytic fragments of laminin allowed the tentative localization of a high affinity binding site to a large fragment comprising two of the short arms connected by the central part of the laminin molecule.

Animals↗
PubMed1988

Particle agglutination assays for rapid detection of fibronectin, fibrinogen, and collagen receptors on Staphylococcus aureus.

Latex beads (0.8-micron diameter; Difco Laboratories) were coated with fibronectin, fibrinogen, collagen type I, or denatured collagen (gelatin) and evaluated in a particle agglutination assay (PAA) for the rapid detection of fibronectin, fibrinogen, or collagen binding to Staphylococcus aureus. These assays were compared with a commercial test for detecting the binding of fibrinogen and immunoglobulin G (Staphaurex). Bacterial cells (approximately 10(10) cells per ml) suspended in 0.02 M potassium phosphate buffer (pH 6.8) caused the clumping of standard fibronectin, collagen, gelatin, and fibrinogen latex suspensions within 2 min on glass slides. The test results were scored semiquantitatively from strongly positive ( ) to weakly positive (+) and negative (-) reactions. The negative PAA reactions corresponded to a median value of 11.5% relative to the binding of 125I-labeled protein to strain Cowan 1, indicating the high sensitivity of the test. The reactions with fibronectin and fibrinogen latex suspensions and with Staphaurex were optimal for cells grown on tryptic soy and brain heart infusion broth media. Blood agar was optimal for reactions with collagen and gelatin latex suspensions. Media containing high salts (mannitol salt agar and staphylococcus medium 110) enhanced the tendency of cells to autoaggregate. These assays were also clinically evaluated on 187 S. aureus isolates. The PAA reagents were stable, and the assays were highly specific, sensitive, and reproducible, thus making PAA suitable for the rapid screening of the binding of various bacterial pathogens to serum and connective-tissue proteins.

Animals↗
PubMed1988

Structural and immunological relationship between laminin and the cysteine proteinase inhibitor stefin.

A sequence analogy was found between the C-terminal region of the B1 chain of laminin (residues 1538-1639) and cysteine proteinase inhibitors, in particular the stefin group. The sequence analogous with cysteine proteinase inhibitors occurs in fragment E8 obtained by elastase digestion of laminin. Antibodies raised against E 8 reacted with stefin but not with kininogen or cystatin.

Amino Acid Sequence↗
PubMed1987

Calcium binding domains and calcium-induced conformational transition of SPARC/BM-40/osteonectin, an extracellular glycoprotein expressed in mineralized and nonmineralized tissues.

SPARC, BM-40, and osteonectin are identical or very closely related extracellular proteins of apparent Mr 43,000 (Mr 33,000 predicted from sequence). They were originally isolated from parietal endoderm cells, basement membrane producing tumors, and bone, respectively, but are rather widely distributed in various tissues. In view of the calcium binding activity reported for osteonectin, we analyzed the SPARC sequence and found two putative calcium binding domains. One is an N-terminal acidic region with clusters of glutamic acid residues. This region, although neither gamma-carboxylated nor homologous, resembles the gamma-carboxyglutamic acid (Gla) domain of vitamin K dependent proteins of the blood clotting system in charge density, size of negatively charged clusters, and linkage to the rest of the molecule by a cysteine-rich domain. The other region is an EF-hand calcium binding domain located near the C-terminus. A disulfide bond between the E and F helix is predicted from modeling the EF-hand structure with the known coordinates of intestinal calcium binding protein. The disulfide bridge apparently serves to stabilize the isolated calcium loop in the extracellular protein. As observed for cytoplasmic EF-hand-containing proteins and for Gla domain containing proteins, a major conformational transition is induced in BM-40 upon binding of several Ca2+ ions. This is accompanied by a 35% increase in alpha-helicity. A pronounced sigmoidicity of the dependence of the circular dichroism signal at 220 nm on calcium concentration indicates that the process is cooperative. In view of its properties, abundance, and wide distribution, it is proposed that SPARC/BM-40/osteonectin has a rather general regulatory function in calcium-dependent processes of the extracellular matrix.

Amino Acid Sequence↗
PubMed1987

Structure of low density heparan sulfate proteoglycan isolated from a mouse tumor basement membrane.

A large heparan sulfate proteoglycan of low buoyant density (p = 1.32 to 1.40 g/cm3 in 6 M-guanidine.HCl) was extracted from a tumor basement membrane with denaturing solvents and purified by chromatography and CsCl gradient centrifugation. Chemical, immunological, physical and electron microscopical analyses have demonstrated a high degree of purity and have allowed us to propose a structural model for this proteoglycan. It is composed of an 80 nm long protein core formed from a single polypeptide chain (Mr about 500,000) with intrachain disulfide bonds. This core is folded into a row of six globular domains of variable size as shown by electron microscopy after rotary shadowing and negative staining. A multidomain structure was confirmed by protease digestion experiments that allowed the isolation of a single heparan sulfate-containing peptide segment representing less than 5% of the total mass of the protein core. Electron microscopy has visualized generally three heparan sulfate chains in each molecule close to each other at one pole of the protein core. The molecular mass and length (100 to 170 nm) of the heparan sulfate chains were found to vary consistently between different preparations. The mass per length ratio (350 nm-1) indicated an extended conformation for the heparan sulfate side-chains. These structural features are distinctly different from those of the high density proteoglycan, suggesting that both forms of basement membrane heparan sulfate proteoglycan are genetically distinct and not derived from a common precursor.

Amino Acids↗
PubMed1987

The cellular interactions of laminin fragments. Cell adhesion correlates with two fragment-specific high affinity binding sites.

The molecular interactions of laminin with several tumor cell lines and skin fibroblasts were investigated by radioligand binding studies and cell attachment assays using laminin, the laminin-nidogen complex, and laminin fragments as substrates and also domain-specific antibodies as inhibitors of cell attachment. The majority of cells showed a dual binding pattern for fragments 1 and 8 which originate from short-arm or long-arm structures of laminin, respectively. Both of these fragments in solution bind to suspended cells with high affinity (KD = 1-10 nM), with the receptor numbers for each fragment depending on the cell type. Competition studies and independent variation of receptor numbers demonstrated that the cell-binding structures on each fragment are different, implicating the existence of two distinct cellular receptors for laminin. The ability of these fragments to act as substrates for cell adhesion correlated with the presence of high affinity binding sites on the cells. However, only antibodies to fragment 8 were able to block cell adhesion to laminin, despite the presence of binding sites for fragment 1. A few cells had very low numbers of high affinity receptors for either fragment 1 or 8. The latter cell type was used to demonstrate that complex formation between laminin and nidogen, which binds to fragment 1 structures, reduces the potential of laminin for cell binding.

Animals↗
PubMed1987

Extended and globular protein domains in cartilage proteoglycans.

Electron microscopy after rotary shadowing and negative staining of the large chondroitin sulphate proteoglycan from rat chondrosarcoma, bovine nasal cartilage and pig laryngeal cartilage demonstrated a unique multidomain structure for the protein core. A main characteristic is a pair of globular domains (diameter 6-8 nm), one of which forms the N-terminal hyaluronate-binding region. They are connected by a 25 nm-long rod-like domain of limited flexibility. This segment is continued by a 280 nm-long polypeptide strand containing most chondroitin sulphate chains (average length 40 nm) in a brush-like array and is terminated by a small C-terminal globular domain. The core protein showed a variable extent of degradation, including the loss of the C-terminal globular domain and sections of variable length of the chondroitin sulphate-bearing strand. The high abundance (30-50%) of the C-terminal domain in some extracted proteoglycan preparations indicated that this structure is present in the cartilage matrix rather than being a precursor-specific segment. It may contain the hepatolectin-like segment deduced from cDNA sequences corresponding to the 3'-end of protein core mRNA [Doege, Fernandez, Hassell, Sasaki & Yamada (1986) J. Biol. Chem. 261, 8108-8111; Sai, Tanaka, Kosher & Tanzer (1986) Proc. Natl. Acad. Sci. 83, 5081-5085; Oldberg, Antonsson & Heinegård (1987) Biochem. J. 243, 255-259].

Animals↗
PubMed1987

Laminin-nidogen complex. Extraction with chelating agents and structural characterization.

Large quantities of intact laminin-nidogen complex could be extracted from a mouse tumor basement membrane with a physiological buffer containing EDTA. Analysis of the purified complex demonstrated that the two proteins occur in an equimolar ratio and that anchoring of these complexes to the extracellular matrix requires divalent cations. Reversible dissociation of the complex was achieved with 2 M guanidine X HCl and has been used for purification of the individual components. Electron microscopy and binding studies using laminin fragments demonstrated that nidogen interacts specifically with the center of the cross-shaped laminin molecule as represented by the short-arm structure fragment 1. The complex was also useful to confirm and refine a previously proposed dumb-bell structure of nidogen and to prepare and characterize the cell-binding fragment 8 from the long arm of laminin.

Animals↗
PubMed1987

Structure and affinity for antithrombin of heparan sulfate chains derived from basement membrane proteoglycans.

Metabolically 35S- or 3H-labeled heparan sulfate was isolated from murine Reichert's membrane, an extraembryonic basement membrane produced by parietal endoderm cells, and from the basement membrane-producing Engelbreth-Holm-Swarm mouse tumor. The polysaccharides were subjected to structural analysis involving identification of products formed on deamination of the polysaccharides with nitrous acid. The polysaccharide from Reichert's membrane contained N- and O-sulfate groups in approximately equal proportions. It bound almost quantitatively and with high affinity to antithrombin. A high proportion of antithrombin-binding sequence was also indicated by the finding that 3-O-sulfated glucosamine residues accounted for about 10% of the total O-sulfate groups. In contrast, at least 80% of the sulfate residues in the heparan sulfate isolated from the mouse tumor were N-substituents. Only a minor proportion of this polysaccharide bound with high affinity to antithrombin, and no 3-O-sulfated glucosamine residues were detected. These results are discussed in relation to the possible functional role of heparan sulfate in basement membranes.

Animals↗
PubMed1987

Distinct antigenic characteristics of murine parietal yolk sac laminin.

Two monoclonal antibodies (LAM-A and LAM-B) specific for laminin from normal and neoplastic parietal yolk sac (PYS) cells were produced in rats immunized with a mouse yolk sac carcinoma cell line. Both antibodies immunoprecipitated the 400,000- and 200,000-Da chains of laminin and reacted with purified PYS laminin in ELISA. LAM-A reacted with mouse and rat PYS laminin, whereas LAM-B reacted only with mouse PYS laminin. Formaldehyde- and methanol-fixed adult and fetal somatic tissues were immunohistochemically unreactive with either of the two antibodies. In acetone-fixed tissue sections, both antibodies reacted weakly with some medullary tubules of the kidney, the follicular basement membrane of the ovaries, and the seminiferous tubules. The antibodies appear to react with the polypeptide determinants residing on the terminal portion of the long arm of laminin. It is concluded that laminin derived from normal or malignant PYS cells has distinct antigenic sites that are immunochemically not apparent in most other basement membranes.

Animals↗
PubMed1987

Cartilage.

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Animals↗