30 research outputs found
Determination of the primary structure and carboxyl pKAs of heparin-derived oligosaccharides by band-selective homonuclear-decoupled two-dimensional 1H NMR
Determination of the structure of heparin-derived oligosaccharides by 1H NMR is challenging because resonances for all but the anomeric protons cover less than 2 ppm. By taking advantage of increased dispersion of resonances for the anomeric H1 protons at low pD and the superior resolution of band-selective, homonuclear-decoupled (BASHD) two-dimensional 1H NMR, the primary structure of the heparin-derived octasaccharide ∆UA(2S)-[(1 → 4)-GlcNS(6S)-(1 → 4)-IdoA(2S)-]3-(1 → 4)-GlcNS(6S) has been determined, where ∆UA(2S) is 2-O-sulfated ∆4,5-unsaturated uronic acid, GlcNS(6S) is 6-O-sulfated, N-sulfated β-d-glucosamine and IdoA(2S) is 2-O-sulfated α-l-iduronic acid. The spectrum was assigned, and the sites of N- and O-sulfation and the conformation of each uronic acid residue were established, with chemical shift data obtained from BASHD-TOCSY spectra, while the sequence of the monosaccharide residues in the octasaccharide was determined from inter-residue NOEs in BASHD-NOESY spectra. Acid dissociation constants were determined for each carboxylic acid group of the octasaccharide, as well as for related tetra- and hexasaccharides, from chemical shift–pD titration curves. Chemical shift–pD titration curves were obtained for each carboxylic acid group from sub-spectra taken from BASHD-TOCSY spectra that were measured as a function of pD. The pKAs of the carboxylic acid groups of the ∆UA(2S) residues are less than those of the IdoA(2S) residues, and the pKAs of the carboxylic acid groups of the IdoA(2S) residues for a given oligosaccharide are similar in magnitude. Relative acidities of the carboxylic acid groups of each oligosaccharide were calculated from chemical shift data by a pH-independent method
Human Apolipoprotein A-I-Derived Amyloid: Its Association with Atherosclerosis
Amyloidoses constitute a group of diseases in which soluble proteins aggregate and deposit extracellularly in tissues. Nonhereditary apolipoprotein A-I (apoA-I) amyloid is characterized by deposits of nonvariant protein in atherosclerotic arteries. Despite being common, little is known about the pathogenesis and significance of apoA-I deposition. In this work we investigated by fluorescence and biochemical approaches the impact of a cellular microenvironment associated with chronic inflammation on the folding and pro-amyloidogenic processing of apoA-I. Results showed that mildly acidic pH promotes misfolding, aggregation, and increased binding of apoA-I to extracellular matrix elements, thus favoring protein deposition as amyloid like-complexes. In addition, activated neutrophils and oxidative/proteolytic cleavage of the protein give rise to pro amyloidogenic products. We conclude that, even though apoA-I is not inherently amyloidogenic, it may produce non hereditary amyloidosis as a consequence of the pro-inflammatory microenvironment associated to atherogenesis
Heparin-binding Sites In Granulocyte-macrophage Colony-stimulating Factor: Localization And Regulation By Histidine Ionization
The biological activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) is modulated by the sulfated glycosaminoglycans (GAGs) heparan sulfate and heparin. However, the molecular mechanisms involved in such interactions are still not completely understood. We have proposed previously that helix C, one of the four α-helices of human GM-CSF (hGM-CSF), contains a GAG-binding site in which positively charged residues are spatially positioned for interaction with the sulfate moieties of the GAGs (Wettreich, A., Sebollela, A., Carvalho, M. A., Azevedo, S. P., Borojevic, R., Ferreira, S. T., and Coelho-Sampaio, T. (1999) J. Biol. Chem. 274, 31468-31475). Protonation of two histidine residues (His83 and His87) in helix C of hGM-CSF appears to act as a pH-dependent molecular switch to control the interaction with GAGs. Based on these findings, we have now generated a triple mutant form of murine GM-CSF (mGM-CSF) in which three noncharged residues in helix C of the murine factor (Tyr83, Gln85, and Tyr87) were replaced by the corresponding basic residues present in hGM-CSF (His 83, Lys85, and His87). Binding assays on heparin-Sepharose showed that, at acidic pH, the triple mutant mGM-CSF binds to immobilized heparin with significantly higher affinity than wild type (WT) mGM-CSF and that neither protein binds to the column at neutral pH. The fact that even WT mGM-CSF binds to heparin at acidic pH indicates the existence of a distinct, lower affinity heparin-binding site in the protein. Chemical modification of the single histidine residue (His15) located in helix A of WT mGM-CSF with diethyl pyrocarbonate totally abolished binding to immobilized heparin. Moreover, replacement of His15 for an alanine residue significantly reduced the affinity of mGM-CSF for heparin at pH 5.0 and completely blocked heparin binding to a synthetic peptide corresponding to helix A of GM-CSF. These results indicate a major role of histidine residues in the regulation of the binding of GM-CSF to GAGs, supporting the notion that an acidic microenvironment is required for GM-CSF-dependent regulation of target cells. In addition, our results provide insight into the molecular basis of the strict species specificity of the biological activity of GM-CSF. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.280363194931956Metcalf, D., (1991) Science, 254, pp. 529-533Modrowski, D., Lomri, A., Marie, P.J., (1998) J. Cell Physiol., 177, pp. 187-195Walter, M.R., Cook, W.J., Ealick, S.E., Nagabhushan, T.L., Trotta, P.P., Bugg, C.E., (1992) J. Mol. Biol., 224, pp. 1075-1085Bazan, J.F., (1990) Proc. Natl. Acad. Sci. U. S. A., 87, pp. 6934-6938Hayashida, K., Kitamura, T., Gorman, D.M., Arai, K.I., Yokota, T., Miyajima, A., (1990) Proc. Natl. Acad. Sci. U. S. A., 87, pp. 9655-9659D'Andrea, R.J., Gonda, T.J., (2000) Exp. Hematol., 28, pp. 231-243Harmer, N.J., Chirgadze, D., Kim, K.H., Pellegrini, L., Blundell, T.L., (2003) Biophys. Chem., 100, pp. 545-553Alvarez-Silva, M., Da Silva, L.C.F., Borojevic, R., (1993) J. Cell Sci., 104, pp. 477-484Alvarez-Silva, M., Borojevic, R., (1996) J. Leukocyte Biol., 59, pp. 435-441Modrowski, D., Basle, M., Lomri, A., Marie, P.J., (2000) J. Biol. Chem., 275, pp. 9178-9185Tumova, S., Woods, A., Couchman, J.R., (2000) Int. J. Biochem. Cell Biol., 32, pp. 269-288Schlessinger, J., Plotnikov, A.N., Ibrahimi, O.A., Eliseenkova, A.V., Yeh, B.K., Yayon, A., Linhardt, R.J., Mohammadi, M., (2000) Mol. Cell, 6, pp. 743-750Harmer, N.J., Ilag, L.L., Mulloy, B., Pellegrini, L., Robinson, C.V., Blundell, T.L., (2004) J. Mol. Biol., 339, pp. 821-834Monfardini, C., Canziani, G., Plugariu, C., Kieber-Emmons, T., Godillot, A.P., Kwah, J., Bajgier, J., Williams, W.V., (2002) Curr. Pharm. Des., 8, pp. 2185-2199Cardin, A.D., Weintraub, H.J., (1989) Arteriosclerosis, 9, pp. 21-32Mulloy, B., Linhardt, R.J., (2001) Curr. Opin. Struct. Biol., 11, pp. 623-628Wettreich, A., Sebollela, A., Carvalho, M.A., Azevedo, S.P., Borojevic, R., Ferreira, S.T., Coelho-Sampaio, T., (1999) J. Biol. Chem., 274, pp. 31468-31475Landt, O., Grunert, H.P., Hahn, U., (1990) Gene (Amst.), 96, pp. 125-128Mosmann, T., (1983) J. Immunol. Methods, 65, pp. 55-63Miles, E.W., (1977) Methods Enzymol., 47, pp. 431-442Baker, N.A., Sept, D., Joseph, S., Holst, M.J., McCammon, J.A., (2001) Proc. Natl. Acad. Sci. U. S. A., 98, pp. 10037-10041Delano, W.L., (2002) The PyMOL Molecular Graphics System, , DeLano Scientific, San Carlos, CADelamarter, J.F., Mermod, J.J., Liang, C.M., Eliason, J.F., Thatcher, D.R., (1985) EMBO J., 4, pp. 2575-2581Burgess, A.W., Begley, C.G., Johnson, G.R., Lopez, A.F., Williamson, D.J., Mermod, J.J., Simpson, R.J., Delamarter, J.F., (1987) Blood, 69, pp. 43-51Wingfield, P., Graber, P., Moonen, P., Craig, S., Pain, R.H., (1988) Eur. J. Biochem., 173, pp. 65-72Schrimsher, J.L., Rose, K., Simona, M.G., Wingfield, P., (1987) Biochem. J., 247, pp. 195-199Dexter, T.M., Garland, J., Scott, D., Scolnick, E., Metcalf, D., (1980) J. Exp. Med., 152, pp. 1036-1047Thompson, L.D., Pantoliano, M.W., Springer, B.A., (1994) Biochemistry, 33, pp. 3831-3840Matsumoto, R., Sali, A., Ghildyal, N., Karplus, M., Stevens, R.L., (1995) J. Biol. Chem., 270, pp. 19524-19531Sasaki, T., Larsson, H., Kreuger, J., Salmivirta, M., Claesson-Welsh, L., Lindahl, U., Hohenester, E., Timpl, R., (1999) EMBO J., 18, pp. 6240-6248Lundblad, R.L., Noyes, C.M., (1984) Chemical Reagents for Protein Modification, 1, pp. 105-125. , CRC Press, Inc., Boca Raton, FLMeot-Ner, M., Sieck, L.W., (1991) J. Am. Chem. Soc., 113, pp. 4448-4460Carvalho, M.A., Arcanjo, K., Silva, L.C.F., Borojevic, R., (2000) Biol. Cell, 92, pp. 605-614Borojevic, R., Carvalho, M.A., Correa-Junior, J.D., Arcanjo, K., Gomes, L., Joazeiro, P.P., Balduino, A., Coelho-Sampaio, T., (2003) Cell Tissue Res., 313, pp. 55-62Carneiro, F.A., Stauffer, F., Lima, C.S., Juliano, M.A., Juliano, L., Da Poian, A.T., (2003) J. Biol. Chem., 278, pp. 13789-13794Wang, H.M., Loganathan, D., Linhardt, R.J., (1991) Biochem. J., 278, pp. 689-695Hileman, R.E., Fromm, J.R., Weiler, J.M., Linhardt, R.J., (1998) BioEssays, 20, pp. 156-167Margalit, H., Fischer, N., Bensasson, S.A., (1993) J. Biol. Chem., 268, pp. 19228-19231Hallgren, J., Backstrom, S., Estrada, S., Thuveson, M., Pejler, G., (2004) J. Immunol., 173, pp. 1868-1875Gallagher, J.T., (2001) J. Clin. Investig., 108, pp. 357-361Schnaar, R.L., (2004) Arch. Biochem. Biophys., 426, pp. 163-172De Cristan, G., Morbidelli, L., Alessandri, G., Ziche, M., Cappa, A.P.M., Gullino, P.M., (1990) J. Cell Physiol., 144, pp. 505-510Vyas, A.A., Patel, H.V., Fromholt, S.E., Heffer-Lauc, M., Vyas, K.A., Dang, J.Y., Schachner, M., Schnaar, R.L., (2002) Proc. Natl. Acad. Sci. U. S. A., 99, pp. 8412-8417Freire, E., Gomes, F.C.A., Linden, R., Neto, V.M., Coelho-Sampaio, T., (2002) J. Cell Sci., 115, pp. 4867-4876Freire, E., Gomes, F.C., Jhota-Mattos, T., Neto, V.M., Silva Filho, F.C., Coelho-Sampaio, T., (2004) J. Cell Sci., 117, pp. 4067-4076Arcanjo, K., Belo, G., Folco, C., Werneck, C.C., Borojevic, R., Silva, L.C.F., (2002) J. Cell Biochem., 87, pp. 160-172Kaushansky, K., Lin, N., Adamson, J.W., (1988) J. Clin. Investig., 81, pp. 92-9
Amyloidogenicity and cytotoxicity of recombinant mature human islet amyloid polypeptide (rhAPP).
Made available in DSpace on 2018-05-30T00:50:38Z (GMT). No. of bitstreams: 1
ID25095.pdf: 488162 bytes, checksum: 33a928ffb26cdec72c5327c47879449d (MD5)
Previous issue date: 2005-06-08bitstream/item/177879/1/ID-25095.pd
Synapse-Binding Subpopulations of Aβ Oligomers Sensitive to Peptide Assembly Blockers and scFv Antibodies
Amyloid β42 self-assembly is complex, with multiple
pathways
leading to large insoluble fibrils or soluble oligomers. Oligomers
are now regarded as most germane to Alzheimer’s pathogenesis.
We have investigated the hypothesis that oligomer formation itself
occurs through alternative pathways, with some leading to synapse-binding
toxins. Immediately after adding synthetic peptide to buffer, solutions
of Aβ42 were separated by a 50 kDa filter and fractions assessed
by SDS-PAGE silver stain, Western blot, immunoprecipitation, and capacity
for synaptic binding. Aβ42 rapidly assembled into aqueous-stable
oligomers, with similar protein abundance in small (<50 kDa) and
large (>50 kDa) oligomer fractions. Initially, both fractions were
SDS-labile and resolved into tetramers, trimers, and monomers by SDS-PAGE.
Upon continued incubation, the larger oligomers developed a small
population of SDS-stable 10–16mers, and the smaller oligomers
generated gel-impermeant complexes. The two fractions associated differently
with neurons, with prominent synaptic binding limited to larger oligomers.
Even within the family of larger oligomers, synaptic binding was associated
with only a subset of these species, as a new scFv antibody (NUsc1)
immunoprecipitated only a small portion of the oligomers while eliminating
synaptic binding. Interestingly, low doses of the peptide KLVFFA blocked
assembly of the 10–16mers, and this result was associated with
loss of the smaller clusters of oligomers observed at synaptic sites.
What distinguishes these smaller clusters from the unaffected larger
clusters is not yet known. Results indicate that distinct species
of Aβ oligomers are generated by alternative assembly pathways
and that synapse-binding subpopulations of Aβ oligomers could
be specifically targeted for Alzheimer’s therapeutics