FGF 2 Human (147 a.a.)

What is Human FGF-2 (147 a.a.) and how does it differ from the full-length protein?

Human FGF-2 (147 a.a.) is a truncated form of the full-length Fibroblast Growth Factor 2 protein. This variant results from degradation of the full-length FGF-2 N-terminus during isolation from biological sources, yielding a protein with 147 amino acids rather than the complete 154 amino acids . The protein has a predicted molecular weight of 16.5 kDa and functions as a mediator of angiogenesis when expressed by endothelial cells . Importantly, while the N-terminal extensions influence the subcellular localization of FGF-2, they do not affect its biological activity, resulting in no detectable differences in activity between the 147 a.a. and 154 a.a. variants . FGF-2 is also known by several alternative names including HBGF-2, basic fibroblast growth factor, heparin-binding growth factor 2, FGFB, BFGF, and prostatropin .

What are the primary physiological functions of FGF-2?

FGF-2 serves multiple critical physiological functions, though interestingly, FGF-2 knockout mice are viable and fertile with relatively mild phenotypes compared to knockouts of other FGF family members . Key physiological roles include:

• Maintenance of vascular tone, as evidenced by administration of FGF-2 lowering blood pressure in rats and restoring nitric oxide synthase activity in hypertensive models

• Angiogenesis promotion through stimulation of endothelial cell migration and proliferation, though the absence of spontaneous vascular defects in FGF-2 overexpression models suggests compensatory mechanisms among growth factors mediating angiogenesis

• Cardioprotection during heart injury

• Critical component for embryonic stem cell culture systems, necessary for maintaining cells in an undifferentiated state

• Possible roles in inflammation through stress-induced activation of caspase 1

• Potential involvement in asthma by enabling airway smooth muscle cells to proliferate in response to triggers

• Possible regulation of human adipogenesis as a proliferative factor for human preadipocytes

How is the biological activity of FGF-2 (147 a.a.) determined in laboratory settings?

The biological activity of human FGF-2 (147 a.a.) is typically assessed through dose-dependent induced proliferation of NR6R 3T3 cells . In standard activity assays, the protein demonstrates full biological activity compared to reference standards. The activity is typically measured at concentrations less than 5 ng/ml, corresponding to an expected specific activity of approximately 2.0 × 10^5 units/mg . This standardized assay provides researchers with a reliable method to confirm protein functionality before experimental use. When evaluating newly produced or stored FGF-2 preparations, proliferation assays with these specific cell lines offer quantifiable metrics of growth factor potency.

What is the amino acid sequence of Human FGF-2 (147 a.a.)?

The complete amino acid sequence of Human FGF-2 (147 a.a.) is as follows:

MPALPEDGGS GAFPPGHFKD PKRLYCKNGG FFLRIHPDGR VDGVREKSDP HIKLQLQAEE RGVVSIKGVC ANRYLAMKED GRLLASKCVT DECFFFERLE SNNYNTYRSR KYTSWYVALK RTGQYKLGSK TGPGQKAILF LPMSAKS

This sequence encodes the complete 147 amino acid protein with a predicted molecular weight of 16.5 kDa. The sequence information is crucial for researchers designing experiments involving protein-protein interactions, structural analyses, or genetic manipulations of FGF-2. The protein maintains the critical β-trefoil core structure typical of FGF family members, consisting of 12 antiparallel β-strands that are essential for receptor binding and biological activity .

What molecular mechanisms govern FGF-2 interaction with its receptors?

The FGF-2-receptor interaction involves a complex molecular architecture with specific binding domains and cofactors. A functional FGF-FGFR unit consists of two 1:1:1 FGF-FGFR-HSGAG complexes arranged in a symmetrical dimer . The interaction involves several key components:

• Each FGF-2 ligand binds both receptors in the dimer, while the two receptors make direct contact through a patch at the base of the D2 domain

• Each ligand interacts with the D2 domain of a second receptor through a secondary receptor binding site

• Mutation of ligand residues within this secondary binding site reduces receptor dimerization and signaling without affecting primary ligand-receptor binding

• Heparan sulfate glycosaminoglycans (HSGAGs) bind to a basic canyon formed on the membrane-distal end of the symmetric dimer, strengthening protein-protein contacts

• HSGAGs facilitate FGF-FGFR dimerization by simultaneously binding both FGF and FGFR, promoting and stabilizing protein-protein contacts both within individual FGF-FGFR complexes and between the two complexes in the dimer

• Beyond facilitating binding, HSGAGs also stabilize FGFs against degradation, act as storage reservoirs for the ligand, and determine the radius of ligand diffusion

Understanding these molecular interactions is crucial for designing experiments that investigate signaling pathway activation or for developing therapeutic approaches targeting FGF-2 activity.

How do heparan sulfate glycosaminoglycans (HSGAGs) influence FGF-2 stability and function?

HSGAGs play multiple crucial roles in regulating FGF-2 activity and stability through several mechanisms:

• Structural stabilization: HSGAGs bind to a basic canyon formed in the FGF-FGFR dimer, strengthening protein-protein contacts and facilitating the dimerization necessary for signal transduction

• Protection from degradation: HSGAGs significantly extend the half-life of FGF-2 by protecting it from proteolytic degradation. When heparin is co-administered with FGF-2, the protein's half-life (normally about 7.6 hours) is substantially extended

• Storage function: The extracellular matrix serves as a reservoir for FGF-2 through HSGAG binding, allowing for controlled release of the growth factor over time

• Regulation of diffusion: HSGAGs determine the radius of FGF-2 diffusion through tissues, helping create concentration gradients essential for developmental processes and tissue homeostasis. The differing morphogenetic activities of FGF family members (such as FGF7 and FGF10) in branching organs correlate with differences in their HSGAG affinity and resultant diffusion properties

• Stabilizing mutations: Research has shown that specific amino acid mutations can increase the half-life of FGF-1 in the presence of heparin, and similar approaches may be applicable to FGF-2. Additionally, stabilizing mutations within the β-barrel structure can dramatically decrease protein unfolding, potentially enhancing stability independent of HSGAG binding

For optimal experimental outcomes when working with FGF-2, researchers should consider including appropriate HSGAGs or heparin in their buffers and storage solutions to maintain protein stability and biological activity.

What are the key considerations for using FGF-2 in stem cell culture systems?

FGF-2 (147 a.a.) is critical for maintaining embryonic stem cells in an undifferentiated state . When designing stem cell culture protocols involving FGF-2, researchers should consider the following methodological aspects:

• Reconstitution protocol: Centrifuge the vial before opening and suspend the lyophilized product by gently pipetting the recommended solution down the sides of the vial without vortexing. Allow several minutes for complete reconstitution. If a precipitate forms, centrifuge the solution thoroughly and use only the soluble fraction

• Storage conditions: For prolonged storage, dilute to working aliquots in a 0.1% BSA solution and store at -80°C. Avoid repeated freeze-thaw cycles that can degrade the protein and reduce activity

• Concentration optimization: Typical active concentrations are less than 5 ng/ml, but optimal dosage should be empirically determined for each stem cell line and culture system

• Half-life considerations: Given the relatively short half-life of FGF-2 (approximately 7.6 hours), culture media may require more frequent changes or supplementation compared to other growth factors with longer stability

• Formulation: The recommended formulation for reconstitution includes 10 mM sodium phosphate and 75 mM sodium chloride at pH 7.5, which helps maintain protein stability and activity

• Cofactor inclusion: Consider supplementing with heparin or heparan sulfate to extend stability and enhance activity in long-term culture applications

Careful attention to these technical details will ensure consistent performance of FGF-2 in stem cell maintenance protocols and reduce experimental variability.

What experimental approaches have been used to study FGF-2's role in cardiovascular disease and therapeutic angiogenesis?

Various experimental approaches have been employed to investigate FGF-2's potential in cardiovascular therapeutics, with mixed results. Key methodological considerations include:

• Delivery methods: Several administration routes have been tested with varying efficacy:

  • Intracoronary injection retains only 3-5% of the dose in the myocardium after 150 minutes and virtually none after 24 hours

  • Intravenous delivery is even less effective due to first-pass pulmonary metabolism

  • Intramyocardial delivery provides the best tissue retention (up to tenfold higher than intracoronary injection) and allows targeting of specific ischemic regions

  • Implantation of heparin beads containing adsorbed FGF-2 over ischemic myocardium has shown more sustainable positive effects in clinical trials, with benefits lasting up to 3 years in follow-up studies

• Clinical trial designs: Several approaches have been tested:

  • Single bolus administration showed initial promise in reducing ischemic regions and improving performance but failed to show sustained benefits in the FGF Initiating RevaScularization Trial (FIRST)

  • Intra-arterial administration for peripheral circulation conditions showed improved calf blood flow compared to placebo, but the TRAFFIC study found that immediate improvements were not ultimately statistically significant

• Protein engineering approaches: Research has explored ways to overcome FGF-2's short half-life:

  • Single amino acid mutations have been shown to increase FGF-1's half-life in the presence of heparin

  • Stabilizing mutations within the β-barrel structure can significantly decrease protein unfolding

  • Co-administration with heparin extends FGF-2's half-life

These experimental approaches highlight the challenges and potential solutions for harnessing FGF-2's angiogenic properties in therapeutic applications, providing valuable methodological insights for researchers in this field.

How do FGF-2 knockout models inform our understanding of its physiological roles?

FGF-2 knockout mouse models have provided surprising insights into the physiological importance of this growth factor. Studies of these models reveal:

• Viability and fertility: Unlike knockouts of many other FGF family members, FGF-2 knockout mice (Fgf2^-/-) are viable and fertile, suggesting redundancy or compensatory mechanisms for its developmental functions

• Vascular phenotypes: Fgf2^-/- mice display loss of vascular tone and exhibit a reduced response to vasoconstrictors, demonstrating FGF-2's role in maintaining vascular function . Despite experiencing some hypotension due to decreased smooth muscle contractility, these mice can still regulate their blood pressure, indicating partial compensation by other factors

• Neurological effects: Knockout mice show a slight loss of cortex neurons, suggesting a role in neuronal maintenance or development

• Compensatory mechanisms: Normal vascularization is retained even in double knockout Fgf2^-/-; Fgf1^-/- mice, highlighting substantial compensation among growth factors mediating angiogenesis

• Comparison with other FGF knockouts: The table below contrasts FGF-2 knockout phenotypes with those of other FGF family members:

This comparative analysis demonstrates that while FGF-2 is not essential for embryonic development (unlike FGF4, FGF8, or FGF10), it does play specific roles in vascular and neuronal function that cannot be fully compensated by other factors .

What methods can researchers use to enhance FGF-2 stability for experimental applications?

Maintaining FGF-2 stability is crucial for reliable experimental results. Researchers can employ several methodological approaches to enhance stability:

• Proper reconstitution: Centrifuge the vial before opening and gently pipette the recommended solution down the sides without vortexing. Allow several minutes for complete reconstitution to maintain structural integrity

• Formulation optimization: Use buffers containing 10 mM sodium phosphate and 75 mM sodium chloride at pH 7.5 for initial reconstitution

• Storage with carrier proteins: For prolonged storage, dilute FGF-2 in a 0.1% BSA solution to prevent protein adsorption to container surfaces and protect against degradation

• Temperature management: Store at -80°C for long-term preservation and avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Heparin co-administration: Include heparin in storage and experimental buffers to extend FGF-2's half-life, which is normally about 7.6 hours but significantly increased in the presence of heparin

• Protein engineering approaches: Consider using stabilized FGF-2 variants:

  • Single amino acid mutations similar to those that increase FGF-1's half-life in the presence of heparin may be applicable to FGF-2

  • Stabilizing mutations within the β-barrel structure can dramatically decrease protein unfolding

• Controlled release systems: For in vivo applications, heparin beads or other controlled release systems can provide sustained delivery of active FGF-2, as demonstrated in clinical trials where implanting heparin beads with adsorbed FGF-2 over ischemic myocardium provided benefits lasting up to 3 years