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H P Erickson

Publications and source records attributed to H P Erickson.

At least 55 records · Page 3Linked to original sources
PubMed1997

Backbone dynamics of homologous fibronectin type III cell adhesion domains from fibronectin and tenascin.

BACKGROUND: Fibronectin type III domains are found as autonomously-folded domains in a large variety of multidomain proteins, including extracellular matrix proteins. A subset of these domains employ an Arg-Gly-Asp (RGD) tripeptide motif to mediate contact with cell-surface receptors (integrins). This motif mediates protein-protein interactions in a diverse range of biological processes, such as in tissue development, would healing and metastasis. The molecular basis for affinity and specificity of cell adhesion via type III domains has not been clearly established. The tenth type III domain from fibronectin (FNfn10) and the third type III domain from tenascin-C (TNfn3) have 27% sequence identity and share the same overall protein fold, but present the RGD motifs in different structural contexts. The dynamical properties of the RGD motifs may affect the specificity and affinity of the FNfn10 and TNfn3 domains. Structure-dynamics correlations for these structurally homologous proteins may reveal common molecular features which are important to the dynamical properties of proteins. RESULTS: The intramolecular dynamics of the protein backbones of FNfn10 and TNfn3 have been studied by 15N nuclear spin relaxation. The FG loop in FNfn10, which contains the RGD motif, exhibits extensive flexibility on picosecond to nanosecond timescales, but motions on microsecond to millisecond timescales are not observed. The equivalent region in TNfn3 is as rigid as regular elements of secondary structure. The CC' loop also is more flexible on picosecond-nanosecond timescales in FNfn10 than in TNfn3. Conformational exchange, reflecting flexibility on microsecond-millisecond timescales, is observed in beta strands A and B of both FNfn10 and TNfn3. CONCLUSIONS: Comparison of the structures of the FNfn10 and TNfn3 reveals several features related to their different dynamical properties. The larger amplitude motions of loops in FNfn10 are consistent with the hypothesis that flexibility of these regions facilitates induced-fit recognition of fibronectin by multiple receptors. Similarly, the more rigid loops of TNfn3 may reflect greater specificity for particular integrins. The correlations observed between structural features and dynamical properties of the homologous type III domains indicate the influence of hydrogen bonding and hydrophobic packing on dynamical fluctuations in proteins.

Amino Acid Sequence↗
PubMed1997

Two oligomeric forms of plasma ficolin have differential lectin activity.

Ficolins are plasma proteins with binding activity for carbohydrates, elastin, and corticosteroids. The ficolin polypeptide has a collagen-like domain that presumably brings three subunits together in a triple helical rod, a C-terminal fibrinogen-like domain (fbg) similar to that of tenascin, which presumably has the binding activities, and a small N-terminal domain that we find to be the primary site for forming the ficolin oligomer. By sedimentation equilibrium we determined that the main plasma form, which we call big ficolin, had mass of 827,000 Da, consistent with 24 subunits. Little ficolin, about half this size, was obtained after binding to a GlcNAc affinity column. Electron microscopy of little ficolin showed a parachute-like structure, with a small globe at one end, corresponding to the 12 N-terminal domains, and the fbg domains clustered together at the ends of the collagen rods. Big ficolin was formed by the face to face fusion of the fbg domains of two little ficolins, leaving the rods and N-terminal domains projecting at opposite ends. Little ficolin maintained a high affinity for the GlcNAc column, and big ficolin had a low affinity or none. The binding sites for ligands may be obscured in this big ficolin oligomer, providing a regulation of their activity.

Animals↗
PubMed1997

Tenascin supports lymphocyte rolling.

Tenascin is a large extracellular matrix molecule expressed at specific sites in the adult, including immune system tissues such as the bone marrow, thymus, spleen, and T cell areas of lymph nodes. Tenascin has been reported to have both adhesive and anti-adhesive effects in static assays. We report here that tenascin supports the tethering and rolling of lymphocytes and lymphoblastic cell lines under flow conditions. Binding was calcium dependent and was not inhibited by treatment of lymphocytes with O-glycoprotease or a panel of glycosidases including neuraminidase and heparitinase but was inhibited by treatment of cells with proteinase K. Binding was to the fibrinogen-like terminal domain of tenascin as determined by antibody blocking studies and binding to recombinant tenascin proteins. When compared to rolling of the same cell type on E-selectin, rolling on tenascin was found to be smoother at all shear stresses tested, suggesting that cells formed a larger number of bonds on the tenascin substrate than on the E-selectin substrate. When protein plating densities were adjusted to give similar profiles of cell detachment under increasing shears, the density of tenascin was 8.5-fold greater than that of E-selectin. Binding to tenascin was not dependent on any molecules previously identified as tenascin receptors and is likely to involve a novel tenascin receptor on lymphocytes. We postulate that the ability of tenascin to support lymphocyte rolling may reflect its ability to support cell migration and that this interaction may be used by lymphocytes migrating through secondary lymphoid organs.

Binding Sites↗
PubMed1997

Tenascin-C knockout mouse has no detectable tenascin-C protein.

A recent study by Mitrovic and Schachner (J Neurosci Res 42:710-717, 1995) reported the detection of a small amount of truncated tensacin-C (TN-C) in the nervous system of the TN-C knockout mice created by Saga et al. (Genes Dev 6:1821-1831, 1992). The authors suggested that the truncated protein might be responsible for the failure to detect any phenotypic abnormalities in the knockout mice. We have reexamined the knockout mice in our laboratories by Western blot and immunocytochemistry, and have not detected any full-length or truncated TN-C protein. In addition, we note that the construction of the knockout gene deleted the signal sequence, so if any residual truncated protein were produced it would be trapped in the cytoplasm, and therefore inaccessible to extracellular ligands or receptors. We therefore conclude that the TN-C knockout created by Saga et al. is a valid TN-C null.

Animals↗
PubMed1997

How calcium causes microtubule depolymerization.

The effects of calcium (Ca) were assessed using video-enhanced differential interference contrast light microscopy on individual microtubules in vitro. Phosphocellulose-purified (PC) and microtubule associated protein (MAP)-containing preparations of porcine brain tubulin were assembled in a flow chamber onto sperm axoneme fragments and the pattern of growth and shortening of the microtubules was observed. Tubulin plus Ca was then added to the chamber and observation continued. Ca promoted the disassembly of microtubules by specifically promoting the catastrophe reaction in both PC- and MAP-containing microtubules, without an appreciable change in elongation rate. The effect on catastrophe frequency increased very rapidly above 0.5 mM free Ca, implying a possible cooperative effect. The rescue rate remained very high after Ca addition in MAP-containing microtubules, and the shortening rate was unchanged, while in phosphocellulose-purified microtubules, rescue appeared to be decreased by Ca addition and shortening rates increased 4 to 6-fold. These results illustrate that Ca can directly destabilize growing microtubule ends without changing the effective concentration of free tubulin, and that this effect can be seen even against the background of the profound differences in dynamics conferred by the microtubule-associated proteins. Considered within models of the GTP cap, the results imply that high Ca may act to increase the rate of GTP hydrolysis within the cap.

Animals↗
PubMed1997

Expression in Escherichia coli of the thermostable DNA polymerase from Pyrococcus furiosus.

Pfu, the DNA polymerase from Pyrococcus furiosus, has the lowest error rate of any known polymerase in polymerase chain reaction (PCR) amplification. Previously the protein has been purified from P. furiosus bacterial cultures, and a recombinant form has been produced in a baculovirus system. We have produced a pET plasmid for expression of Pfu in Escherichia coli (the expression plasmid pETpfu is available from ATCC, Accession No. 87496) and found that this plasmid is toxic or unstable in the expressing strain BL21(DE3), even in the absence of induction. However, the plasmid was stable in BL21(DE3) containing the pLysS plasmid, which suppresses expression prior to induction, and a 90-kDa protein was expressed upon addition of isopropyl beta-D-thiogalactopyranoside. The protein was purified by heating (to denature E. coli proteins), followed by chromatography on P11 phosphocellulose and mono Q columns. The purified protein had the same activity as the commercially obtained baculovirus-expressed Pfu in both DNA polymerase and PCR reactions. This bacterial expression system appears to be the method of choice for production of Pfu.

Archaeal Proteins↗
PubMed1997

Extracellular annexin II.

Annexin II belongs to a family of calcium-dependent, phospholipid binding proteins. Annexin II was first identified as an intracellular protein and attributed intracellular functions. Although it lacks a signal peptide and its mechanism of secretion is unknown, extracellular annexin II has recently been found in several tissues as both soluble and membrane-bound protein. Cell-surface annexin II has been identified as a receptor for a number of polypeptide ligands. Extracellular annexin II may be important in several biological processes, such as fibrinolysis, cell adhesion, ligand-mediated cell signaling and virus infection. These processes provide several possibilities for therapeutic approaches targeting extracellular annexin II, and future research should further illuminate the biology of this molecule.

Annexin A2↗
PubMed1997

Glycosaminoglycans modulate fibronectin matrix assembly and are essential for matrix incorporation of tenascin-C.

We have investigated the role of glycosaminoglycans in fibronectin matrix assembly and the incorporation of tenascin-C into matrix fibrils. Chinese hamster ovary cell mutants with a total block in heparan and chondroitin sulfate production failed to assemble a fibronectin matrix, and incorporated no tenascin-C. Another mutant with reduced heparan sulfate produced a normal fibronectin matrix but failed to incorporate tenascin-C. Excess soluble glycosaminoglycans inhibited the binding of tenascin-C to purified fibronectin in ELISA, and completely blocked incorporation into matrix fibrils. Treating cultured cells with xyloside, which interferes with glycosaminoglycan attachment to proteoglycans, also completely blocked their ability to incorporate tenascin-C into matrix fibrils. We conclude that proteoglycans bound to fibronectin fibrils play a major role in binding tenascin-C to these fibrils. We examined more closely the large heparan sulfate proteoglycan, perlecan, and found that it co-localizes with tenascin-C and fibronectin in the matrix. The perlecan binding site in tenascin-C was mapped to the fibronectin type III domains 3-5, but this binding was strongly enhanced for the small splice variant, which is the major form incorporated into the matrix. Apparently when the alternative splice segment is inserted after domain 5 it inhibits perlecan binding. Thus heparan sulfate glycosaminoglycans, and perlecan in particular, may play a role in incorporation of the small splice variant of tenascin-C into fibronectin matrix fibrils.

Animals↗
PubMed1996

Structural analysis of a human glial variant laminin.

Astrocytes secrete laminin-like molecules in culture and may represent a major source of laminin in the developing central nervous system, yet these laminins have not been extensively characterized. We previously reported the presence of an astrocyte-derived variant laminin in media conditioned by human U251 MG astrocytoma cells. This laminin was partially purified in a highly anionic Mono Q fraction with strong adhesion activity for fibroblasts and glial cells (Aukhil et al. (1990) Matrix 10: 98-111). We now show that glial laminin could be dissociated from an anionic species, perhaps an approximately 400-kDa keratan sulfate proteoglycan present in the preparation, by a second round of Mono Q anion exchange chromatography in the presence of 6 M urea. Cell adhesion activity remained tightly associated with laminin-containing fractions, suggesting that glial laminin was responsible for the adhesion activity in the original preparation. Immunochemical and SDS-PAGE gel analyses of laminin heterotrimers demonstrated that glial laminin contained the beta 2 and gamma 1 chains in disulfide-bonded heterotrimeric complexes with a 360-kDa chain, a 320-kDa chain, or a postulated approximately 200-kDa chain. While these chains were not recognized by antibodies directed against the alpha 1-, alpha 2-, or alpha 3-related laminin chains, rotary shadowed glial laminin molecules appeared to contain alpha chains, as judged by the presence of an apparent G-domain terminating the long arm of each laminin molecule. These findings suggest that glial laminin contains one or more variant alpha chains, perhaps related to one of the more recently described alpha chains, alpha 3B, alpha 4, or alpha 5. Together our results implicate human U251 MG glial laminin as a previously uncharacterized laminin isoform with strong adhesive activity for fibroblasts and glial cells.

Anion Exchange Resins↗
PubMed1996

Visualization of P-selectin glycoprotein ligand-1 as a highly extended molecule and mapping of protein epitopes for monoclonal antibodies.

P-selectin glycoprotein ligand-1 (PSGL-1), a sialomucin on human leukocytes, mediates rolling of leukocytes on P-selectin expressed by activated platelets or endothelial cells under shear forces. PSGL-1 requires both tyrosine sulfate and O-linked glycans to bind P-selectin. Electron microscopy of rotary-shadowed PSGL-1 purified from human neutrophils indicated that it is a highly extended molecule with an extracellular domain that is -50 nm long. Both individual PSGL-1 molecules and rosettes composed of several molecules presumably attached at their transmembrane segments were observed. The extracellular domain of PSGL-1 has 318 residues, including a signal peptide from residues 1-18 and a propeptide from residues 19-41. Using bacterially expressed fusion proteins and synthetic peptides derived from the extracellular domain, we mapped the epitopes for two IgG anti-PSGL-1 monoclonal antibodies, PL1 and PL2. PL2 bound to a region within residues 188-235 that is located in a series of decameric consensus repeats. PL1, which blocks binding of PSGL-1 to P-selectin, recognized an epitope spanning residues 49-62. This sequence overlaps the tyrosine sulfation sites at residues 46, 48, and 51 that have been implicated in binding of PSGL-1 to P-selectin. Our results demonstrate that PSGL-1 is a long, extended molecule and suggest that the P-selectin binding site is located near the N terminus, well above the membrane. This location may facilitate interactions of PSGL-1 with P-selectin under shear stress.

Amino Acid Sequence↗
PubMed1996

2.0 A crystal structure of a four-domain segment of human fibronectin encompassing the RGD loop and synergy region.

We have determined the 2.0 A crystal structure of a fragment of human fibronectin encompassing the seventh through the RGD-containing tenth type III repeats (FN7-10). The structure reveals an extended rod-like molecule with a long axis of approximately 140 A and highly variable relationships between adjacent domains. An unusually small rotation between domains 9 and 10 creates a distinctive binding site, in which the RGD loop from domain 10 and the "synergy" region from domain 9 are on the same face of FN7-10 and thus easily accessible to a single integrin molecule. The cell-binding RGD loop is well-ordered in this structure and extends approximately 10 A away from the FN7-10 core.

Amino Acid Sequence↗
PubMed1996

Bacterial cell division protein FtsZ assembles into protofilament sheets and minirings, structural homologs of tubulin polymers.

The bacterial cell division protein FtsZ is a homolog of tubulin, but it has not been determined whether FtsZ polymers are structurally related to the microtubule lattice. In the present study, we have obtained high-resolution electron micrographs of two FtsZ polymers that show remarkable similarity to tubulin polymers. The first is a two-dimensional sheet of protofilaments with a lattice very similar to that of the microtubule wall. The second is a miniring, consisting of a single protofilament in a sharply curved, planar conformation. FtsZ minirings are very similar to tubulin rings that are formed upon disassembly of microtubules but are about half the diameter. This suggests that the curved conformation occurs at every FtsZ subunit, but in tubulin rings the conformation occurs at either beta- or alpha-tubulin subunits but not both. We conclude that the functional polymer of FtsZ in bacterial cell division is a long thin sheet of protofilaments. There is sufficient FtsZ in Escherichia coli to form a protofilament that encircles the cell 20 times. The similarity of polymers formed by FtsZ and tubulin implies that the protofilament sheet is an ancient cytoskeletal system, originally functioning in bacterial cell division and later modified to make microtubules.

Animals↗
PubMed1996

Tenascin-C expression in dystrophin-related muscular dystrophy.

The mdx mouse has a mutated dystrophin gene and is used as a model for the study of Duchenne muscular dystrophy (DMD). We investigated whether regenerating mdx skeletal muscle contains the extracellular matrix protein tenascin-C (TN-C), which is expressed in wound healing and nerve regeneration. Prior to the initiation of muscle degeneration, both normal and mdx mice displayed similar weak staining for TN-C in skeletal muscle, but by 3 weeks of age the mice differed substantially. TN-C was undetectable in normal muscle except at the myotendinous junction, while in dystrophic muscle, TN-C was prominent in degenerating/regenerating areas, but absent from undegenerated muscle. With increasing age, TN-C staining declined around stable regenerated mdx myofibers. TN-C was also observed in muscle from dogs with muscular dystrophy and in human boys with DMD. Therefore, in dystrophic muscle, TN-C expression may be stimulated by the degenerative process and remain upregulated unless the tissue undergoes successful regeneration.

Animals↗
PubMed1996

Mitogenesis, cell migration, and loss of focal adhesions induced by tenascin-C interacting with its cell surface receptor, annexin II.

In a previous study we demonstrated that the alternatively spliced region of tenascin-C, TNfnA-D, bound with high affinity to a cell surface receptor, annexin II. In the present study we demonstrate three changes in cellular activity that are produced by adding intact tenascin-C or TNfnA-D to cells, and we show that all three activities are blocked by antibodies against annexin II. 1) TNfnA-D added to confluent endothelial cells induced loss of focal adhesions. 2) TNfnA-D produced a mitogenic response of confluent, growth-arrested endothelial cells in 1% serum. TNfnA-D stimulated mitogenesis only when it was added to cells before or during exposure to other mitogens, such as basic fibroblast growth factor or serum. Thus the effect of TNfnA-D seems to be to facilitate the subsequent response to growth factors. 3) TNfnA-D enhanced cell migration in a cell culture wound assay. Antibodies to annexin II blocked all three cellular responses to TNfnA-D. These data show that annexin II receptors on endothelial cells mediate several cell regulatory functions attributed to tenascin-C, potentially through modulation of intracellular signalling pathways.

Animals↗
PubMed1995

Binding of tenascin-C to soluble fibronectin and matrix fibrils.

The small splice variant of tenascin-C (TN) has eight fibronectin type III (FN3) domains. The major large splice variant has three (in chicken) or seven (in human) additional FN3 domains inserted between domains five and six. Chiquet-Ehrismann et al. (Chiquet-Ehrismann, R., Matsuoka, Y., Hofer, U., Spring, J., Bernasconi, C., and Chiquet, M. (1991, Cell Regul. 2, 927-938) demonstrated that the small variant bound preferentially to fibronectin in enzyme-linked immunosorbent assay, and only the small variant was incorporated into the matrix by cultures of chicken fibroblasts. Here we have studied human TN, and confirmed that the small variant binds preferentially to purified fibronectin and to fibronectin-containing extracellular matrix. Thus this differential binding appears to be conserved across vertebrate species. Using bacterial expression proteins, we mapped the major binding site to the third FN3 domain of TN. Consistent with this mapping, a monoclonal antibody against an epitope in this domain did not stain TN segments bound to cell culture matrix fibrils. The enhanced binding of the small TN variant suggests the existence of another, weak binding site probably in FN3 domains 6-8, which is only positioned to bind fibronectin in the small splice variant. This binding of domains 6-8 may involve a third molecule present in matrix fibrils, as the enhanced binding of small TN was much more prominent to matrix fibrils than to purified fibronectin.

3T3 Cells↗