scholarly journals Delivery of bFGF for Tissue Engineering by Tethering to the ECM

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Chawapun Suttinont ◽  
Yasumasa Mashimo ◽  
Masayasu Mie ◽  
Eiry Kobatake
Keyword(s):  
Tissue Engineering ◽  
Extracellular Matrix ◽  
Amino Acid ◽  
Growth Factor ◽  
Functional Unit ◽  
Basic Amino Acid ◽  
Target Cells ◽  
C Terminus ◽  
Acid Domain ◽  
Amino Acid Domain

Delivery of growth factors to target cells is an important subject in tissue engineering. Towards that end, we have developed a growth factor-tethered extracellular matrix (ECM). Here, basic fibroblast growth factor (bFGF) was tethered to extracellular matrix noncovalently. The designed ECM was comprised of 12 repeats of the APGVGV peptide motif derived from elastin as a stable structural unit and included the well-known cell adhesive RGD peptide as an active functional unit. To bind bFGF to the ECM, an acidic amino acid-rich sequence was introduced at the C-terminus of the ECM protein. It consisted of 5 repeats of 4 aspartic acids and a serine, DDDDS. bFGF has a highly basic amino acid domain. Therefore, bFGF was tethered to the ECM protein by electrostatic interaction. Cells cultured on bFGF-tethered ECM were well attached to the ECM and induced proliferation without addition of soluble bFGF.

scholarly journals Primary- and secondary-structural analysis of a unique prokaryotic metallothionein from a Synechococcus sp. cyanobacterium

1988 ◽  
Vol 251 (3) ◽  
pp. 691-699 ◽  
Author(s):  
R W Olafson ◽  
W D McCubbin ◽  
C M Kay
Keyword(s):  
Amino Acid ◽  
Metal Binding ◽  
Helical Structure ◽  
Basic Amino Acid ◽  
Cysteine Residues ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Empirical Relations ◽  
Heavy Metal Binding ◽  
Synechococcus Sp

Biochemical and physiological studies of Synechococcus cyanobacteria have indicated the presence of a low-Mr heavy-metal-binding protein with marked similarity to eukaryotic metallothioneins (MTs). We report here the characterization of a Synechococcus prokaryotic MT isolated by gel-permeation and reverse-phase chromatography. The large number of variants of this molecule found during chromatographic separation could not be attributed to the presence of major isoproteins as assessed by amino acid analysis and amino acid sequencing of isoforms. Two of the latter were shown to have identical primary structures that differed substantially from the well-described eukaryotic MTs. In addition to six long-chain aliphatic residues, two aromatic residues were found adjacent to one another near the centre of the molecule, making this the most hydrophobic MT to be described. Other unusual features included a pair of histidine residues located in repeating Gly-His-Thr-Gly sequences near the C-terminus and a complete lack of association of hydroxylated residues with cysteine residues, as is commonly found in eukaryotes. Similarly, aside from a single lysine residue, no basic amino acid residues were found adjacent to cysteine residues in the sequence. Most importantly, sequence alignment analyses with mammalian, invertebrate and fungal MT sequences showed no statistically significant homology aside from the presence of Cys-Xaa-Cys structures common to all MTs. On the other hand, like other MTs, the prokaryotic molecule appears to be free of alpha-helical structure but has a considerable amount of beta-structure, as predicted by both c.d. measurements and the Chou & Fasman empirical relations. Considered together, these data suggested that some similarity between the metal-thiolate clusters of the prokaryote and eukaryote MTs may exist.

scholarly journals C-Terminal Deletions Can Suppress Temperature-Sensitive Mutations and Change Dominance in the Phage Mu Repressor

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 661-672 ◽  
Author(s):  
Jodi L Vogel ◽  
Vincent Geuskens ◽  
Lucie Desmet ◽  
N Patrick Higgins ◽  
Ariane Toussaint
Keyword(s):  
Binding Activity ◽  
Temperature Sensitive ◽  
Dna Binding Activity ◽  
Heat Stable ◽  
C Terminus ◽  
Acid Domain ◽  
Mutant Forms ◽  
Amino Acid Domain

Abstract Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause temperature-sensitive DNA-binding activity. Surprisingly, amber mutations can conditionally correct the heat-sensitive defect in three mutant forms of the repressor gene, cts25 (D43-G), cts62 (R47-Q and cts71 (M28-I), and in the appropriate bacterial host produce a heat-stable Sts phenotype (for survival of temperature shifts). Sts repressor mutants are heat sensitive when in supE or supF hosts and heat resistant when in Sup° hosts. Mutants with an Sts phenotype have amber mutations at one of three codons, Q179, Q187, or Q190. The Sts phenotype relates to the repressor size: in Sup° hosts sts repressors are shorter by seven, 10, or 18 amino acids compared to repressors in supE or supF hosts. The truncated form of the sts62-1 repressor, which lacks 18 residues (Q179–V196), binds Mu operator DNA more stably at 42° in vitro compared to its full-length counterpart (cts62 repressor). In addition to influencing temperature sensitivity, the C-terminus appears to control the susceptibility to in vivo Clp proteolysis by influencing the multimeric structure of repressor.

scholarly journals Insulin-Like Growth Factor Binding Protein-5 Binds to Plasminogen Activator Inhibitor-I*

Endocrinology ◽  
1997 ◽  
Vol 138 (7) ◽  
pp. 2972-2978 ◽  
Author(s):  
Taek Jeong Nam ◽  
Walker Busby ◽  
David R. Clemmons
Keyword(s):  
Amino Acids ◽  
Extracellular Matrix ◽  
Amino Acid ◽  
Growth Factor ◽  
Heparan Sulfate ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

Abstract Insulin-like growth factor binding protein-5 (IGFBP-5) has been shown to bind to the extracellular matrix (ECM) of both fibroblasts and smooth muscle cells. The ECM-IGFBP-5 interaction is mediated in part by binding to heparan sulfate containing proteoglycans. Because proteoglycans may not be the only components of ECM that bind to IGFBP-5, we have determined its ability to bind to other ECM proteins. When a partially purified mixture of the proteins that were present in fibroblast conditioned medium was purified by IGFBP-5 affinity chromatography, a 55-kDa protein was eluted. Amino acid sequencing of the amino terminal 28 amino acids showed that it was human plasminogen activator inhibitor-1 (PAI-1). To determine if this interaction was specific, purified human PAI-1 was incubated with IGFBP-5 and the IGFBP-5/PAI-1 complex immunoprecipitated with anti-PAI-1 antiserum. When the precipitate was analyzed by immunoblotting using anti-IGFBP-5 antiserum, the intensity of the IGFBP-5 band was substantially increased compared with controls that did not contain human PAI-1. A synthetic IGFBP-5 peptide that contained the amino acid sequence between positions 201 and 218 inhibited IGFBP-5/PAI-1 interaction. Coincubation of IGFBP-5 mutants that contained substitutions for specific basic residues located between positions 201 and 218 with PAI-1 indicated that some of these amino acids were important for binding. Two mutants that contained neutral substitutions for specific basic amino acids within the glycosaminoglycan binding domain had reduced binding to PAI-1. In contrast, three other mutants that also had substitutions for charged residues in the same region had no reduction in binding. Heparin and heparan sulfate inhibited the IGFBP-5/PAI-1 interaction; however, several other glycosaminoglycans had no effect. PAI-1 was determined to be an important ECM component for binding because approximately 27% of total ECM binding could be inhibited with anti-PAI-1 antiserum. Competitive binding studies with unlabeled IGFBP-5 showed that the dissociation constant of PAI-1 for IGFBP-5 was 9.1 × 10−8m. In summary, IGFBP-5 binds specifically to plasminogen activator inhibitor-1. Because this is present in the extracellular matrix of several cell types, it may be one of the important binding components of ECM. PAI-1 binding partially protects IGFBP-5 from proteolysis, suggesting that it is one of the ECM components that is involved in mediating this effect.

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