Recombinant Notophthalmus viridescens Fibroblast growth factor 1 (fgf1)

What is the structural relationship between newt FGF1 and mammalian FGF1?

Newt FGF1 shares significant structural homology with mammalian counterparts while maintaining distinct characteristics. Amino acid sequence comparisons indicate that Notophthalmus viridescens FGF1 exhibits between 79-83% identity with FGF1 from mammalian and avian species . Despite this high primary sequence conservation, the newt protein demonstrates antigenic divergence, as evidenced by its failure to be detected in Western blot analysis using polyclonal antibodies directed against mammalian FGF1 . The three-dimensional structure of newt FGF1 consists of 12 beta-strands arranged antiparallely into a beta-barrel structure, similar to the human FGF1 structural organization .

What are the expression patterns of FGF receptors during newt limb regeneration?

The expression patterns of FGF receptors during limb regeneration in Notophthalmus viridescens follow distinct temporal and spatial dynamics:

Preblastema stages:

• FGFR2 expression is observed in the basal layer of the wound epithelium and in periosteal cells

• KGFR variant (of FGFR2) specifically appears in the basal layer of wound epithelium

• The bek variant (of FGFR2) is expressed in the periosteum cells

Blastema stages:

• FGFR2 expression continues in the basal wound epithelium layer with additional expression in blastema mesenchyme near bisected bones

• FGFR1 expression appears throughout the blastema mesenchyme but is notably absent from the wound epithelium

• KGFR expression continues in the wound epithelium with additional presence in blastema mesenchyme associated with bisected bones

Differentiation stages:

• Mesenchymal FGFR2 expression becomes restricted to condensing cartilage cells and later to the perichondrium

• Wound epithelium hybridization to FGFR2 is no longer observed in later regeneration stages

• A dorsoventral gradient of expression for both KGFR and bek variants of FGFR2 emerges, with opposing patterns

How does newt FGF1 function compare to human FGF1 despite sequence differences?

Despite the antigenic divergence between newt and human FGF1, functional studies demonstrate remarkable conservation of biological activity. Newt FGF1 exhibits the following functional properties:

• It binds to NIH/3T3 and Chinese hamster ovary cells overexpressing mammalian and amphibian FGF receptors with dissociation constants comparable to those reported for mammalian FGF1

• The recombinant protein successfully cross-links to receptors on NIH/3T3 cell surfaces

• It elicits a mitogenic response in NIH/3T3 cells that is indistinguishable from the response to human recombinant FGF1

• It maintains its role in regulating cell survival, division, angiogenesis, differentiation, and migration, functioning as a potent mitogen in vitro

What structural features might explain the differential stability between newt and human FGF1?

The conformational stabilities of newt FGF1 (nFGF-1) and human FGF1 (hFGF-1) differ significantly despite their structural homology. According to structural analyses, these stability differences can be attributed to:

These structural differences provide important insights for researchers designing experiments to investigate stability-function relationships or engineering recombinant FGF1 variants with enhanced properties.

What is the role of FGF1 in the context of other FGFs during amphibian limb regeneration?

The coordination of multiple FGF family members appears essential for successful limb regeneration in amphibians. Research indicates differential expression patterns and potential functional complementarity:

Expression patterns during regeneration:

• FGF8 and FGF10 show increased expression during limb regeneration

• FGF4 expression is completely absent during regeneration despite high expression in differentiated limbs

• FGF8 expression pattern in regenerating limbs differs from developmental patterns in other vertebrates, with highest expression found in blastema mesenchyme rather than distal epithelium

Tissue-specific expression:

• FGF signaling appears instrumental during limb field specification

• Developing flank tissue shows clear FGF expression that becomes severely downregulated in mature flank tissue

• Differential FGF expression between limb/shoulder (limb field) versus flank (non-limb field) suggests a role in regional identity determination

How do experimental treatments with FGF1 affect amphibian limb regeneration?

Studies examining the effects of growth factors on limb regeneration in adult newts have revealed that various FGF family members can stimulate regeneration with distinct effects. While the search results do not specifically address FGF1 administration, they provide context on related growth factors:

• Fibroblast growth factor-2 (FGF-2) stimulates bud emergence, reducing time to 8.3 ± 0.6 days compared to 11.4 ± 1.1 days for controls

• FGF-2 enhances progression to the cone stage (14.6 ± 0.5 days vs 16.5 ± 0.5 days in controls)

• Insulin-like growth factor I (IGF-I) similarly stimulates bud emergence (8.3 ± 0.7 days)

• Transforming growth factor beta 5 (TGF-β5) enhances progression to cone stage (15.4 ± 0.4 days)

These findings suggest that FGF1, with its functional similarity to FGF2, might also accelerate certain stages of limb regeneration, though direct experimental evidence would be necessary to confirm this hypothesis.

What expression systems are most effective for producing recombinant newt FGF1?

Based on the available research, prokaryotic expression systems have been successfully employed for newt FGF1 production:

• E. coli expression system: The full-length cDNA of newt FGF1 has been successfully cloned into prokaryotic expression vectors and purified from E. coli . This system offers:

  • High yield production

  • Established purification protocols

  • Cost-effective scale-up potential

  • Compatibility with structural and functional studies

For researchers seeking to produce recombinant newt FGF1, the following methodological considerations should be addressed:

• Codon optimization for E. coli expression may improve yields

• Inclusion of appropriate affinity tags (His-tag, GST-tag) facilitates purification

• Expression conditions (temperature, IPTG concentration, induction time) should be optimized

• Proper refolding protocols may be necessary if the protein forms inclusion bodies

What methods are appropriate for analyzing FGF receptor-ligand interactions in the context of limb regeneration?

Several complementary approaches have been employed to study FGF-receptor interactions in newt limb regeneration:

In situ hybridization:

• This technique has been extensively used to map the expression patterns of FGFR1 and FGFR2 during different stages of limb regeneration

• Allows visualization of spatial and temporal expression patterns in tissue sections

• Can distinguish between different splice variants (KGFR and bek) of FGFR2

Binding studies:

• Recombinant newt FGF1 binding to cells expressing FGF receptors can be assessed using:

  • Dissociation constant measurements

  • Cross-linking experiments with cell surface receptors

Functional assays:

• Mitogenic response assessment in NIH/3T3 cells

• In vivo administration of growth factors to regenerating limbs

• Semiquantitative reverse transcriptase-polymerase chain reaction to detect differential FGF expression

How can researchers investigate the structural properties of newt FGF1?

The three-dimensional solution structure of newt FGF1 has been determined using multidimensional NMR techniques, providing a framework for additional structural investigations . Researchers can employ the following methodological approaches:

NMR spectroscopy:

• Complete assignment of all atoms (¹H, ¹⁵N, and ¹³C) using triple resonance experiments

• Structure calculation using hybrid distance geometry-dynamical simulated annealing with constraints

Stability assessment:

• Comparison of conformational stabilities between newt and human FGF1

• Analysis of hydrogen bonding networks and solvent inaccessible cavities

Ligand binding studies:

• Investigation of binding to sucrose octasulfate (SOS) in a 1:1 stoichiometric ratio

• Identification of the binding site consisting of positively charged residues at the C-terminal end

How might comparative studies between newt and human FGF1 inform regenerative medicine approaches?

The evolutionary conservation of FGF1 structure and function, combined with the remarkable regenerative capacity of newts, offers valuable insights for translational research:

• The high degree of sequence similarity (79-83%) between newt and human FGF1 suggests conserved core functions despite millions of years of evolutionary divergence

• Identification of structural differences contributing to stability variations may inform protein engineering approaches to enhance human FGF1 properties

• Understanding the spatiotemporal expression patterns of FGF receptors during successful regeneration in newts provides templates for therapeutic intervention strategies in mammals

• The distinct roles of FGFR1 and FGFR2 in limb regeneration, despite sharing many FGF ligands, highlights the importance of receptor-specific targeting in potential therapies

What experimental approaches can assess the therapeutic potential of FGF1 in non-regenerative models?

Based on the documented effects of FGFs on amphibian limb regeneration, researchers might consider the following experimental approaches:

• Comparative administration studies:

  • Intraperitoneal or local administration of recombinant FGF1 following the protocols established for related growth factors

  • Assessment of tissue remodeling, cellular proliferation, and differentiation responses

• Combined growth factor treatments:

  • Since FGF2, IGF-I, and TGF-β5 show overlapping but distinct effects on regeneration stages , combination therapies with FGF1 might reveal synergistic potential

• Cross-species receptor activation:

  • Given that newt FGF1 can activate mammalian FGF receptors , investigation of its effects in mammalian wound healing models could identify unique regenerative properties

What are the critical knowledge gaps regarding newt FGF1 that require further investigation?

Despite significant progress in understanding newt FGF1, several important questions remain unanswered:

• The specific ligand-receptor relationships between FGF1 and the KGFR/bek variants of FGFR2 during different regeneration stages

• The upstream regulatory mechanisms controlling FGF1 expression during limb regeneration

• The potential role of FGF1 in reprogramming cellular identities during the dedifferentiation phase of regeneration

• The complete signaling cascade downstream of FGF1 activation specifically in the regeneration context

• The precise mechanism by which antigenic divergence occurs despite functional conservation between newt and mammalian FGF1