Peptides derived from a short stretch of diphtheria toxin bind to heparin-binding epidermal growth factor-like growth factor.

Membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) is the receptor for diphtheria toxin (DT). Mutated or truncated, non-toxic DT has been used earlier for HB-EGF-targeted drug delivery and to modulate HB-EGF signaling. In the present work, we have synthesized a peptide corresponding to a 26 amino acid long stretch of the receptor-binding domain of DT. This region of DT makes multiple contacts with HB-EGF and has residues critical for binding to HB-EGF. We show that this peptide and two of its mutants bind to HB-EGF. We have also created recombinant proteins by fusing Maltose-binding Protein (MBP) with these peptides. These recombinant MBP-tagged peptides bind to HB-EGF with affinities in the range of 10-7 to 10-8 M. We have observed that these MBP-tagged peptides can modulate molecular signaling of HB-EGF. Therefore, this 26 amino acid long stretch of DT can be considered as an independent functional segment for binding to HB-EGF. Peptides corresponding to this region may be used for HB-EGF targeted cellular delivery of molecular cargo or to modulate HB-EGF signaling.

Biological Stability of Plasma Rich in Growth Factors-Derived Autologous Topical Serum After Three-Months Storage.

In this study, the stability of a novel autologous topical serum (ATS) derived from plasma rich in growth factors technology (PRGF) has been evaluated. As skin ages, mechanical, protective and restorative properties decrease leading to multiple clinical conditions. In recent years, topical administration of growth factors has emerged as a promising therapeutic alternative to promote wound healing and skin regeneration. Determination of stability is a crucial step in the formulation process in order to develop an effective product. Blood from 8 healthy donors was harvested and the autologous topical serum was obtained. Resulting ATS samples were either kept fresh or stored for 1, 2, and 3 months at 4ºC. Physical properties and growth factor content were determined in ATS samples at each time of storage. The effect on human dermal fibroblast proliferation and the sterility of the samples was also studied. All the analyzed parameters remained stable along the storage time while pH values increased slightly with respect to fresh samples. No microbial contamination was detected in any of the samples. Preservation of the autologous topical serum up to 3 months under refrigeration does not affect either its physical or mechanical properties or neither alters the growth factors´ composition, thus preserving its biological potential. This achievement enables patients with chronic disorders to maintain their treatment with a lower frequency of blood extractions without affecting the efficacy of PRGF therapy. J Drugs Dermatol. 2018;17(10):1115-1121.

Selective Synergism Created by Interactive Nacre Framework-Associated Proteins Possessing EGF and vWA Motifs: Implications for Mollusk Shell Formation.

In the nacre layer of the Pinctada fucata oyster shell there exists a multimember proteome, known as the framework family, which regulates the formation of the aragonite mesoscale tablets and participates in the creation of an organic coating around each tablet. Several approaches have been developed to understand protein-associated mechanisms of nacre formation, yet we still lack insight into how protein ensembles or proteomes manage nucleation and crystal growth. To provide additional insights we have created a proportionally defined combinatorial model consisting of two recombinant framework proteins, r-Pif97 (containing a von Willebrand Factor Type A domain (vWA)) and r-n16.3 (containing an EGF-like domain), whose individual in vitro mineralization functionalities are distinct from one another. We find that at 1:1 molar ratios r-Pif97 and r-n16.3 exhibit little or no synergistic activity regarding modifying existing calcite crystals. However, during the early stages of nucleation in solution, we note synergistic effects on nucleation kinetics and ACC formation/stability (via dehydration) that are not observed for the individual proteins. This selective synergism is generated by Ca2+-mediated protein-protein interactions (∼4 molecules of r-n16.3 per 1 molecule of r-Pif97) which lead to the formation of nucleation-responsive hybrid hydrogel particles in solution. Interestingly, in the absence of Ca2+ there are no significant interactions occurring between the two proteins. This unique behavior of the framework-associated n16.3 and Pif97 proteins suggests that the Asp/Glu-containing regions of the vWA and EGF-like domains may play a role in both nacre matrix formation and mineralization.

Composite membranes of alginate and chitosan reinforced with cotton or linen fibers incorporating epidermal growth factor.

Suture threads of cotton or linen, in crossed and random orientation, were added to alginate-chitosan membranes intended to wound coatings application to improve the mechanical properties. The elongation at break increased to about 5 and 8 times for membranes with linen and cotton, respectively, both in the crossed orientation. The addition of the threads increased roughness and opacity of the membranes and reduced the liquid absorption capacity and water vapor transmission rate. The lowest toxicity to human fibroblasts was observed for extracts of membranes produced with linen, and incorporation in them of epidermal growth factor was able to slightly increase cell proliferation.

Epidermal growth factor loaded heparin-based hydrogel sheet for skin wound healing.

A heparin-based hydrogel sheet composed of thiolated heparin and diacrylated poly (ethylene glycol) was prepared via photo polymerization and human epidermal growth factor (hEGF) were loaded into it for the purpose of wound healing. It showed a sustained release profile of hEGF in vitro. In order to evaluate its function on wound healing in vivo, full thickness wounds were created on the dorsal surface of mice. Application of hEGF loaded heparin-based hydrogel sheet accelerated the wound closure compared to the non-treated control group, hEGF solution, and hEGF loaded PEG hydrogel sheet. Histological and immunohistological examinations also demonstrated an advanced granulation tissue formation, capillary formation, and epithelialization in wounds treated by hEGF loaded heparin-based hydrogel compared to other groups, and no biocompatibility issue was observed. In conclusion, the delivery of hEGF using the heparin-based hydrogel could accelerate the skin wound healing process.

Preparation of bioactive and antimicrobial PLGA membranes by magainin II/EGF functionalization.

Development of dual functional materials that are capable of both reducing bacterial interaction and encouraging host tissue integration has gained importance in design of biomaterials. In this study, we prepared a bilayer poly (lactide co-glycolide) fibrous membrane with antibacterial and bioactive properties by electrospinning. The antibacterial layer was produced by covalent immobilization of antimicrobial peptide, Magainin II. The bioactive layer incorporating epidermal growth factor (EGF) molecules was subsequently electrospun on the antibacterial layer. The membranes were characterized by X-ray photoelectron spectroscopy, scanning electron microscopy and fluorescence microscopy. EGF release was detected by enzyme-linked immunosorbent assay. The antibacterial activity was tested against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The ability to support tissue cell integration was detected by using L-929 mouse fibroblasts. The dual functional membranes established enhanced antibacterial properties and increased tissue cell compatibility. This combined approach suggests a promising strategy for wound dressings, vascular grafts and dental membranes as well as catheters and fixation devices.

Stability analysis of an inline peptide-based conjugate for metal delivery: nickel(II)-claMP Tag epidermal growth factor as a model system.

Metals are a key component of many diagnostic imaging and biotechnology applications, and the majority of cancer patients receive a platinum-based drug as part of their treatment. Significant effort has been devoted to developing tight binding synthetic chelators to enable effective targeted delivery of metal-based conjugates, with most successes involving lanthanides rather than transition metals for diagnostic imaging. Chemical conjugation modifies the protein's properties and generates a heterogeneous mixture of products. Chelator attachment is typically carried out by converting the amino group on lysines to an amide, which can impact the stability and solubility of the targeting protein and these properties vary among the set of individual conjugate species. Site-specific attachment is sought to reduce complexity and control stability. Here, the metal abstraction peptide technology was applied to create the claMP Tag, an inline platform for generating site-specific conjugates involving transition metals. The claMP Tag was genetically encoded into epidermal growth factor (EGF) and loaded with nickel(II) as a model system to demonstrate that the tag within the homogeneous inline conjugate presents sufficient solution stability to enable biotechnology applications. The structure and disulfide network of the protein and chemical stability of the claMP Tag and EGF components were characterized.

The length of the linker between the epidermal growth factor-like domains in factor VIIa is critical for a productive interaction with tissue factor.

Formation of the factor VIIa (FVIIa)-tissue factor (TF) complex triggers the blood coagulation cascade. Using a structure-based rationale, we investigated how the length of the linker region between the two epidermal growth factor (EGF)-like domains in FVIIa influences TF binding and the allosteric activity enhancement, as well as the interplay between the γ-carboxyglutamic acid (Gla)-containing and protease domains. Removal of two residues from the native linker was compatible with normal cofactor binding and accompanying stimulation of the enzymatic activity, as was extension by two (Gly-Ser) residues. In sharp contrast, truncation by three or four residues abolished the TF-mediated stabilization of the active conformation of FVIIa and abrogated TF-induced activity enhancement. In addition, FVIIa variants with short linkers associated 80-fold slower with soluble TF (sTF) as compared with wild-type FVIIa, resulting in a corresponding increase in the equilibrium dissociation constant. Molecular modeling suggested that the shortest FVIIa variants would have to be forced into a tense and energetically unfavorable conformation in order to be able to interact productively with TF, explaining our experimental observations. We also found a correlation between linker length and the residual intrinsic enzymatic activity of Ca(2+)-free FVIIa; stepwise truncation resulting in gradually higher activity with des(83-86)-FVIIa reaching the level of Gla-domainless FVIIa. The linker appears to determine the average distance between the negatively charged Gla domain and a structural element in the protease domain, presumably of opposite charge, and proximity has a negative impact on apo-FVIIa activity.

claMP Tag: a versatile inline metal-binding platform based on the metal abstraction peptide.

Molecularly targeted research and diagnostic tools are essential to advancing understanding and detection of many diseases. Metals often impart the desired functionality to these tools, and conjugation of high-affinity chelators to proteins is carried out to enable targeted delivery of the metal. This approach has been much more effective with large lanthanide series metals than smaller transition metals. Because chemical conjugation requires additional processing and purification steps and yields a heterogeneous mixture of products, inline incorporation of a peptide tag capable of metal binding is a highly preferable alternative. Development of a transition metal binding tag would provide opportunity to greatly expand metal-based analyses. The metal abstraction peptide (MAP) sequence was genetically engineered into recombinant protein to generate the claMP Tag. The effects of this tag on recombinant epidermal growth factor (EGF) protein expression, disulfide bond formation, tertiary structural integrity, and transition metal incorporation using nickel were examined to confirm the viability of utilizing the MAP sequence to generate linker-less metal conjugates.