muramidase and arginyl-glycyl-aspartic-acid

The assembly of protein-inorganic nanoparticles is an important yet challenging approach that is utilized to develop functional materials in numerous areas, such as bio-catalysis, drug delivery, and biosensing. In this study, we report on a facile, photo-inducible self-assembly method to generate protein-inorganic hybrid nanoplatforms. More specifically, photo-treated disulfide bond rich proteins of lysozyme (LYS) were able to be used as host materials in order to encapsulate nanoparticles (i.e., as-synthesized hydrophobic NIR quantum dots (QDs)) and anti-cancer small molecule drugs (i.e., paclitaxel (PTX)), constructing functional theranostic protein-inorganic hybrid nanoparticles. The modification of the functional polymer of cRGD-PEG contributes to the active tumour targeting characteristic of this protein-inorganic nanocarrier. This novel PTX loaded protein-inorganic hybrid nanoplatform showed high tumour homing accumulation as well as effective tumour inhibition. We believe that this general approach represents a new direction for the development of a photo-induced assembly of protein-inorganic nanoparticles towards versatile applications in both materials science and biomedical fields.

Topics: Animals; Cell Line, Tumor; Cell Survival; Drug Carriers; Humans; Male; Mice; Mice, Inbred BALB C; Microscopy, Atomic Force; Muramidase; Nanoparticles; Neoplasms; Oligopeptides; Paclitaxel; Polyethylene Glycols; Quantum Dots; Theranostic Nanomedicine; Tissue Distribution; Transplantation, Heterologous; Ultraviolet Rays

To increase the cell adhesion activity of 74RGD4, an RGDS-inserted mutant between Val74 and Asn75 of human lysozyme, one more site for the RGD introduction was investigated in the lysozyme molecule. We found that 47RGD4 with RGDS in place of AGDR (residues 47 to 50) in a beta-turn region possesses the same level of adhesion activity as that of 74RGD4. The acceptance of the RGD introduction in the beta-turn region of human lysozyme is in good agreement with recent studies on the functional conformation of RGD. We constructed (47,74)RGD4, a mutant containing RGD at two sites, by combining the N-terminal domain of 47RGD4 and the C-terminal domain of 74RGD4. The (47,74)RGD4 lysozyme, with two functional RGD sequences, exhibits even higher cell adhesion activity than that of 74RGD4 or 47RGD4.

Topics: Animals; Base Sequence; Cell Adhesion; Cell Adhesion Molecules; Cell Line; Cricetinae; Crystallography, X-Ray; DNA Primers; Humans; Molecular Sequence Data; Muramidase; Mutagenesis, Site-Directed; Oligopeptides; Protein Conformation

The cell-adhesive protein Cys-RGD4 has been constructed using a yeast expression system by inserting the sequence Cys-Arg-Gly-Asp-Ser-Cys (CRGDSC) between Val74 and Asn75 of human lysozyme [Yamada, T., Uyeda, A., Kidera, A. & Kikuchi, M. (1994b) Biochemistry 33, 11678-11683]. The Cys74a, Arg74b, Gly74c, Asp74d, Ser74e, Cys74f-lysozyme mutant, purified from the yeast culture supernatant contained glycosylated variants, in addition to the unglycosylated form. Peptide mapping analyses suggested that the glycosylation occurred at the Thr70 residue in the Cys-RGD4 molecule. Electrospray ionization mass spectrometric analysis demonstrated the presence of two hexose residues in the major variant, and one, three, four, or five hexose residues in the minor variants. All of these hexose residues were identified as mannose by analysis of the oligosaccharide mixture obtained by mild alkaline treatment of the variants. No other glycosylation was observed, although the Cys-RGD4 molecule possesses a total of 12 threonine and serine residues. In addition, the Thr70 residue is not glycosylated in either native lysozyme or the Arg-Gly-Asp-Ser (RGDS)-inserted mutant, RGD4 [Yamada, T., Matsushima, M., Inaka, K., Ohkubo, T., Uyeda, A., Maeda, T., Titani, K., Sekiguchi, K. & Kikuchi, M. (1993) J. Biol. Chem. 268, 10588-10592]. Thus, this O-glycosylation seems to be specific for both the mutant lysozyme molecule and the site of the threonine residue. Structural analyses of these lysozymes by X-ray crystallography suggest that the conformation of the serine-containing or threonine-containing region can affect the specificity of yeast O-glycosylation.

Topics: Amino Acid Sequence; Cell Adhesion Molecules; Glycosylation; Molecular Sequence Data; Muramidase; Mutation; Oligopeptides; Threonine; Yeasts

To examine the effect of a conformational constraint introduced into the Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we constructed a mutant protein by inserting an RGD-containing sequence flanked by two Cys residues between Val74 and Asn75 of human lysozyme. The CRGDSC-inserted lysozyme was expressed in yeast, purified, and designated as Cys-RGD4. Using baby hamster kidney cells, Cys-RGD4 was shown to possess even higher cell adhesion activity than that of the RGDS-inserted lysozyme, RGD4. The Cys-RGD4 protein was co-crystallized with a lysozyme inhibitor, tri-N-acetylchitotriose, and the three-dimensional structure was determined at 1.6-A resolution by x-ray crystallography. In contrast to RGD4, the inserted RGD-containing region of Cys-RGD4 was well defined. The structural analysis revealed that the two inserted Cys residues form a new disulfide bond in Cys-RGD4, as expected, and that the RGD region assumes a type II' beta-turn conformation of Gly-Asp with a hydrogen bond between the C = O of Arg and the H-N of Ser. In addition, it was confirmed that two more hydrogen bonds are present in the RGD region of the Cys-RGD4 lysozyme. These results suggest that the conformation of the RGD-containing region is rigid and stable in the Cys-RGD4 molecule and that the type II' beta-turn structure of RGD is essential for binding to integrins with high affinity.

Topics: Asparagine; Base Sequence; Computer Graphics; Crystallography, X-Ray; Humans; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Muramidase; Mutagenesis, Insertional; Oligodeoxyribonucleotides; Oligopeptides; Protein Conformation; Recombinant Proteins; Valine

To examine the effect of a conformational constraint introduced into the Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we have constructed mutant proteins by inserting RGD-containing sequences flanked by two Cys residues between Val74 and Asn75 of human lysozyme. CRGDC-, CRGDSC-, and CGRGDSC-inserted mutant lysozymes were expressed in yeast, purified, and designated as Cys-RGD3, Cys-RGD4, and Cys-RGD5, respectively. In baby hamster kidney cells, these mutants were shown to possess high cell adhesion activity by interaction with vitronectin receptor (integrin alpha v beta 3), and this activity is 2-3-fold higher than that of the RGDS-inserted mutant lysozyme, RGD4. The mutant proteins also inhibited the binding of human fibrinogen to its receptor (integrin alpha IIb beta 3) at a lower concentration than the RGD4 protein. Peptide mapping and mass spectrometric analyses showed that the two inserted Cys residues in these mutants are linked to each other without any effects on the mode of the four disulfide bonds present in native human lysozyme. These results suggest that the introduction of a conformational constraint into the RGD region significantly increases the cell adhesion activity. The conformation of the RGD region in Cys-RGD4 was modeled by a Monte Carlo simulation. Most of the sampled conformations were grouped into three classes; the first is characterized by an extended Gly conformation, the second assumes a type II' beta turn, and the third has a salt bridge between Arg and Asp.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Base Sequence; Cell Adhesion; Computer Simulation; Cysteine; Disulfides; Fibrinogen; Humans; Integrins; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; Monte Carlo Method; Muramidase; Mutagenesis, Insertional; Oligopeptides; Peptide Mapping; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Conformation; Tumor Cells, Cultured

The cell adhesive protein RGD8 has been constructed using a yeast expression system by inserting eight amino acid residues (TGRGDSPA) between Val74 and Asn75 of human lysozyme [Yamada et al. (1993) J. Biol. Chem. 268, 10588-10592]. Purified RGD8 from yeast culture supernatant was found to contain glycosylated variants, in addition to the unglycosylated form. Peptide mapping analyses suggested that the glycosylation occurred at the inserted Thr residue in the RGD8 molecule. Electrospray ionization mass spectrometric analysis demonstrated the presence of four or five hexose residues in the glycosylated variants. Only mannose was detected in the sugar analysis of the oligosaccharide mixture obtained by mild alkaline treatment of the variants, and the structures of these carbohydrate chains were identified as Man alpha 1-3Man alpha 1-2Man alpha 1-2Man alpha and Man alpha 1-3Man alpha 1-3Man alpha 1-2Man alpha 1-2Man alpha by 1H-NMR spectroscopy. No other glycosylation was found, although the RGD8 molecule possesses a total of 13 Thr and Ser residues. In addition, no O-glycosylation was observed when the RGD8 protein was expressed in mouse L-cells. Thus, this O-glycosylation looks specific for yeast and the site of the Thr residue. The O-glycosylated variants of RGD8 exhibited a high level of adhesion activity to baby hamster kidney cells, which was almost comparable to that of the unglycosylated form.

Topics: Amino Acid Sequence; Animals; Carbohydrate Sequence; Cell Adhesion Molecules; Crystallography, X-Ray; Glycosylation; Humans; L Cells; Mass Spectrometry; Mice; Molecular Sequence Data; Muramidase; Oligopeptides; Protein Processing, Post-Translational; Protein Structure, Tertiary; Saccharomyces cerevisiae

To determine the functional conformation of the Arg-Gly-Asp (RGD) sequence, we have constructed mutant proteins by inserting 4-12 amino acid residues from the RGD region of human fibronectin between Val74 and Asn75 of human lysozyme. RGDS-, GRGDSP-, TGRGDSPA-, VTGRGDSPAS-, and AVTGRGDS-PASS-introduced mutant lysozymes were expressed in yeast, purified, and designated as RGD4, -6, -8, -10, and -12, respectively. Using baby hamster kidney cells, RGD8, RGD10, and RGD12 were shown to possess high cell adhesion activity nearly equal to 10% of human vitronectin activity. RGD4 and RGD6 exhibited somewhat lower cell adhesion activity. The activities of these mutant proteins were inhibited by the addition of either GRGDSP peptide or polyclonal antibody against vitronectin receptor, as was the case for the vitronectin activity. The results suggest that the cell adhesion signals are transduced to cells through the interaction with the vitronectin receptor. The three-dimensional structures of RGD4 and RGD8 were determined at 1.8-A resolution by x-ray crystallography. A model of the inserted region in RGD4 could be built in the electron density map, but the positions of the preceding residues, Ala73-Val74, were uncertain. The inserted region in RGD8 did not demonstrate continuous electron densities. The results suggest that these RGD sequence-containing regions are highly flexible and that such flexibility could allow the conformation of the RGD regions to be induced to fit into the binding pocket of the integrin receptor.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Adhesion; Cell Line; Codon; Cricetinae; Fibronectins; Humans; Kidney; Kinetics; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Muramidase; Mutagenesis, Insertional; Oligodeoxyribonucleotides; Oligopeptides; Protein Conformation; Restriction Mapping; Transfection; X-Ray Diffraction