Leptin Salamander
Description
Introduction to Leptin Salamander
Leptin Salamander refers to a recombinant form of leptin protein derived from salamanders, specifically produced in Escherichia coli for research purposes. Leptin itself is a peptide hormone known for its role in regulating food intake, body mass, and energy balance in vertebrates, including mammals and non-mammals like amphibians . The salamander leptin is of particular interest due to its evolutionary conservation and potential applications in understanding leptin's role across different species.
Structure and Production
The recombinant Leptin Salamander protein is a single, non-glycosylated polypeptide chain containing 146 amino acids with an additional alanine at the N-terminus. It has a molecular mass of approximately 16 kDa . This protein is produced through recombinant DNA technology in E. coli and purified using proprietary chromatographic techniques .
Evolutionary Conservation
Studies have demonstrated that leptin is conserved across vertebrates, including salamanders. The leptin gene in the Chinese giant salamander (Andrias davidianus) shows a conserved structure with mammals, indicating its evolutionary significance . This conservation suggests that leptin's roles in energy balance and other physiological processes are fundamental across different species.
Role in Development
In amphibians like Xenopus laevis, leptin influences limb growth and differentiation. Experiments have shown that recombinant leptin injections can enhance hind-limb development in tadpoles, suggesting leptin acts as a growth factor during early vertebrate development .
Wound Healing
Leptin has been identified as a promoter of wound healing in amphibians. It enhances wound closure by stimulating cellular migration and immune responses, reducing bacterial infections . This role is similar to its function in mammals, where leptin promotes keratinocyte proliferation and wound repair.
Table 1: Characteristics of Recombinant Leptin Salamander
| Characteristic | Description |
|---|---|
| Molecular Mass | Approximately 16 kDa |
| Amino Acid Length | 146 amino acids + Ala at N-terminus |
| Production Method | Recombinant DNA technology in E. coli |
| Purification | Proprietary chromatographic techniques |
Table 2: Biological Functions of Leptin in Salamanders
| Function | Description |
|---|---|
| Energy Balance | Regulates food intake and body mass |
| Development | Influences limb growth and differentiation |
| Wound Healing | Promotes tissue repair and reduces infection |
Product Specs
Q&A
What is leptin and has it been identified in salamanders?
Leptin is a multifunctional cytokine initially discovered in mammals as the protein product of the obese (ob or Lep) gene. In mammals, leptin functions as a key hormone secreted by adipose tissue that regulates neuroendocrine function, energy homeostasis, and various physiological processes including reproduction, immune function, and bone formation .
Leptin has been successfully identified in salamander species, specifically in the tiger salamander (Ambystoma tigrinum) and the Chinese giant salamander (Andrias davidianus) . Before these discoveries, leptin had only been characterized in two amphibian species . The identification in salamanders, which represent early diverging amphibian lineages, provides important evolutionary evidence for the presence of leptin-like genes outside mammals .
In methodology, the tiger salamander leptin was identified through analysis of EST (Expressed Sequence Tag) data that shared 56% nucleotide sequence identity with human leptin cDNA, followed by experimental confirmation through RT-PCR of salamander testis and stomach mRNAs .
How does salamander leptin compare structurally to mammalian leptin?
Despite relatively low amino acid sequence identity (approximately 29%) with mammalian leptins, salamander leptin maintains critical structural characteristics that define the leptin family . Comparative analysis reveals:
| Feature | Salamander Leptin | Mammalian Leptin | Notes |
|---|---|---|---|
| Total amino acids | 169 residues | 167 residues (human) | Similar length |
| Mature peptide | 146 residues | 146 residues | Identical mature peptide length |
| Tertiary structure | Four-helix bundle | Four-helix bundle | Conserved structural motif |
| Conserved cysteines | Present | Present | Required for proper folding |
| Sequence identity | - | ~29% with mammals | Low primary sequence conservation |
Tertiary structure prediction using SWISS-MODEL with human leptin as a template confirms that salamander leptin maintains the characteristic four-helix bundle structure of mammalian leptins, suggesting that despite sequence divergence, the three-dimensional conformation has been evolutionarily conserved . This structural conservation indicates strong selective pressure to maintain functional aspects of the protein.
What is the tissue distribution pattern of leptin expression in salamanders?
Leptin expression in salamanders shows a tissue-specific distribution pattern that differs notably from mammals. While mammalian leptin is predominantly expressed in adipose tissue, salamander leptin demonstrates a broader expression profile .
In the tiger salamander, RT-PCR analysis revealed variable expression patterns between individuals, with the most widespread expression observed in smaller females. Northern hybridization of poly(A)+ RNA from salamander testis with a salamander leptin cDNA probe yielded a single band of RNA with a molecular size of 1.9kb, confirming expression in this tissue .
In the Chinese giant salamander, leptin (Adlep) is widely distributed across tissues but with varying expression levels:
| Tissue | Relative Expression Level in A. davidianus |
|---|---|
| Muscle | Highest |
| Other tissues | Variable (lower than muscle) |
This divergent tissue expression pattern suggests that leptin may serve additional or different physiological functions in salamanders compared to mammals, potentially reflecting adaptations to their unique physiological and environmental requirements .
What is the gene structure of salamander leptin?
Salamander leptin maintains a conserved gene structure comparable to that observed in other vertebrates. PCR amplification of genomic DNA from the tiger salamander using primers designed to amplify the leptin cDNA generated a 6 kb fragment, indicating the presence of substantial intronic sequence .
Partial sequencing of this genomic fragment confirmed:
| Feature | Observation in Salamander | Comparison to Mammals |
|---|---|---|
| Exon-intron organization | At least two coding exons separated by an intron | Conserved pattern |
| First exon | Identified 105 bp of exonic sequence | Homologous to mammalian first exon |
| Intronic sequence | Identified portions of intronic sequence (405 bp + 164 bp) | Introns present in similar positions |
The identification of an intron between coding exons provides definitive evidence that the leptin gene is present in the salamander genome and maintains a gene structure similar to that in other vertebrates . This conservation of gene structure further supports the orthology of salamander leptin with mammalian leptin and suggests that the basic genomic organization of leptin genes emerged early in vertebrate evolution.
How was leptin originally identified in salamander species?
The identification of leptin in salamanders followed a methodical research process:
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Initial discovery: A leptin-like EST (Expressed Sequence Tag) from the tiger salamander was identified, sharing 56% nucleotide sequence identity with human leptin cDNA .
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Experimental confirmation: To verify natural expression, researchers designed primers based on the EST sequence and amplified a 409 bp cDNA fragment by RT-PCR from salamander testis and stomach mRNAs .
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Sequence validation: The amplified cDNA sequence was 99.5% identical to the EST sequence, differing by only two nucleotide substitutions (G to A at position 409, and A to G at position 476). One substitution resulted in a conservative amino acid change from arginine to glutamine .
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Expression confirmation: Northern hybridization using a salamander leptin cDNA probe identified a single 1.9kb RNA transcript in testis tissue .
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Genomic verification: PCR amplification of genomic DNA generated a 6 kb fragment containing exonic and intronic sequences, confirming the presence of this gene in the salamander genome .
The comprehensive approach combining bioinformatic analysis, molecular cloning, and expression studies provided robust evidence for the identification of a genuine leptin ortholog in salamander species.
How does the tertiary structure of salamander leptin compare to mammalian leptin?
Comparative molecular modeling reveals that salamander leptin maintains the characteristic four-helix bundle structure that defines mammalian leptins, despite sharing only approximately 29% amino acid identity . This structural conservation was determined through sophisticated computational analysis:
The tertiary structure prediction was performed using the SWISS-MODEL server in Alignment Interface with a ClustalW alignment of mammalian protein sequences (mouse, rat, human, cow, pig, dog, fat-tailed dunnart) together with the salamander sequence. The salamander leptin sequence was modeled against the selected template human sequence (1AX8.pdb) .
This remarkable conservation of tertiary structure despite considerable sequence divergence suggests strong evolutionary pressure to maintain the three-dimensional conformation of leptin across distant vertebrate lineages. This structural conservation likely reflects functional constraints, as the specific folding pattern is presumably essential for receptor binding and biological activity .
What are the evolutionary implications of leptin conservation between salamanders and mammals?
The identification of leptin in salamanders, which represent early diverging amphibian lineages, has significant implications for understanding the evolutionary history of leptin:
| Evolutionary Aspect | Finding | Implication |
|---|---|---|
| Phylogenetic positioning | Salamander leptin clusters according to expected vertebrate phylogeny | Leptin evolution tracks species evolution |
| Gene structure | Conserved exon-intron organization | Ancient genomic organization maintained |
| Sequence divergence | ~29% amino acid identity with mammals | Functional constraints on specific residues |
| Structural conservation | Four-helix bundle maintained | Strong selection on tertiary structure |
| Gene duplication | Single gene in salamanders vs. duplicated in teleosts | Different evolutionary trajectories |
The presence of a single leptin gene in salamanders contrasts with teleost fishes, which generally possess duplicated leptins. These teleost duplications appear to have arisen through a Clupeocephala-specific gene duplication event rather than the whole genome duplication that occurred in teleosts . This finding suggests that the single leptin gene found in salamanders and mammals represents the ancestral condition, providing important context for understanding leptin gene evolution across vertebrates.
What experimental approaches are used to study leptin signaling pathways in salamanders?
Investigating leptin signaling pathways in salamanders requires specialized methodological approaches that integrate molecular, biochemical, and bioinformatic techniques:
| Approach | Methodology | Application |
|---|---|---|
| Gene identification | RT-PCR, cloning, sequencing | Identifying leptin receptor and pathway components |
| Expression analysis | RT-PCR, qPCR, Northern blotting | Determining tissue-specific expression patterns |
| Pathway mapping | Bioinformatic analysis | Mapping genes to known leptin signaling pathways |
| Tertiary structure prediction | Comparative modeling | Predicting protein structure based on homology |
| Phylogenetic analysis | Multiple sequence alignment, tree construction | Determining evolutionary relationships |
In the Chinese giant salamander, researchers have successfully mapped the leptin receptor and other genes to three known leptin signaling pathways, providing evidence that these signaling mechanisms are conserved in salamanders . This approach involves identifying orthologous genes for each component of the signaling cascade and confirming their sequence similarity and predicted functional domains.
For expression analysis in the tiger salamander, researchers employed RT-PCR with leptin-specific primers on mRNA isolated from various tissues, with cyclophilin A used as a control to normalize expression levels . Northern hybridization using a salamander leptin riboprobe synthesized by in vitro transcription was also employed to confirm transcript size and expression .
These methodological approaches enable researchers to establish the presence and conservation of leptin signaling pathways in salamanders, providing a foundation for functional studies of leptin in these species.
How does tissue-specific leptin expression in salamanders differ from mammals?
The tissue distribution pattern of leptin expression in salamanders exhibits notable differences compared to mammals, suggesting potential functional divergence:
| Tissue | Expression in Salamanders | Expression in Mammals | Potential Implication |
|---|---|---|---|
| Adipose tissue | Variable | Primary expression site | Different energy storage signaling |
| Muscle | Highest in A. davidianus | Limited | Unique role in muscle metabolism |
| Testis | Confirmed expression | Limited | Potential reproductive function |
| Stomach | Confirmed expression | Present | Conserved role in feeding regulation |
| Other tissues | Variable expression | Limited | Broader physiological roles |
In mammals, leptin is predominantly expressed in adipose tissue, functioning as an adiposity signal that communicates information about energy stores to the brain . In contrast, salamander leptin shows a more diverse tissue distribution pattern, with notable expression in tissues not typically associated with high leptin production in mammals .
The tiger salamander demonstrates variable leptin expression across tissues, with expression patterns differing between individuals. The most widespread expression was observed in the smallest animal, a female . In the Chinese giant salamander, leptin (Adlep) shows highest expression in muscle tissue, a pattern distinctly different from the adipose-centric expression in mammals .
These differences in tissue expression patterns suggest that leptin may serve additional or different physiological functions in salamanders, potentially reflecting evolutionary adaptations to their unique physiological demands, including their ectothermic metabolism, aquatic or semi-aquatic lifestyle, and different energy utilization strategies.
What are the methodological challenges in studying leptin function in salamander models?
Investigating leptin function in salamander models presents several unique methodological challenges that researchers must address:
| Challenge | Description | Potential Solutions |
|---|---|---|
| Limited genetic tools | Fewer tools for gene manipulation compared to mammalian models | Development of CRISPR-Cas9 protocols specific for salamanders |
| Complex life cycles | Metamorphosis may affect leptin function across life stages | Stage-specific analysis of leptin expression and function |
| Large genome size | Salamanders often have large and complex genomes | Targeted sequencing approaches, transcriptomics |
| Longer development | Extended timeline for longitudinal studies | Focus on specific developmental windows or endpoints |
| Limited antibody availability | Few commercial antibodies for salamander leptin | Development of custom antibodies or epitope tagging |
| Physiological differences | Ectothermy vs. endothermy affects metabolism | Careful experimental design accounting for temperature effects |
| Housing requirements | Specialized aquatic or semi-aquatic housing needed | Development of standardized housing protocols |
These challenges necessitate creative experimental approaches, including the use of heterologous expression systems, in vitro functional assays, and comparative studies with other amphibian models. Researchers may need to develop custom molecular tools, such as species-specific antibodies or expression constructs, to effectively study leptin function in salamanders.
Additionally, the physiological differences between salamanders and mammals, particularly their ectothermic metabolism, must be carefully considered when designing experiments and interpreting results, as these differences may significantly impact leptin's functional roles and regulatory mechanisms.
How can phylogenetic analysis of salamander leptin inform our understanding of leptin evolution?
Phylogenetic analysis incorporating salamander leptin provides critical insights into the evolutionary history of this important hormone:
| Evolutionary Feature | Finding | Significance |
|---|---|---|
| Phylogenetic topology | Matches accepted vertebrate relationships | Confirms orthologous relationship of leptin genes |
| Sequence conservation | Specific residues conserved across vertebrates | Identifies functionally critical amino acids |
| Gene duplication patterns | Single gene in salamanders vs. duplicated in teleosts | Clarifies timing of duplication events |
| Structural conservation | Four-helix bundle maintained despite sequence divergence | Reveals selective pressure on protein folding |
The phylogenetic tree constructed using non-synonymous substitutions for salamander leptin and leptin from other vertebrates (human, sheep, fat-tailed dunnart, mouse, dog, pig, Xenopus laevis, salmon, and cow) shows a topology consistent with the accepted evolutionary relationships of these species . This consistency suggests that leptin genes have evolved without major convergent or highly divergent evolutionary events in most vertebrate lineages.
By including leptin sequences from salamanders in phylogenetic analyses, researchers can better identify conserved versus derived features. The salamander represents an important evolutionary position as an early diverging tetrapod, helping to bridge the gap between fish and amniote vertebrates in our understanding of leptin evolution.
The analysis of selection pressures on different regions of leptin sequences across vertebrates, including salamanders, can identify functionally important domains that have been conserved throughout evolution and regions that have undergone adaptive evolution in specific lineages, providing insights into the functional evolution of this hormone.
What functional implications arise from salamander leptin's structural differences?
Despite maintaining the characteristic four-helix bundle structure, salamander leptin exhibits significant sequence divergence from mammalian leptins, which may have important functional implications:
The structural differences observed in salamander leptin may reflect adaptations to the unique physiological needs of salamanders as ectotherms. These adaptations could include:
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Modified receptor binding properties that optimize signaling at variable body temperatures experienced by ectotherms
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Altered signaling pathway activation kinetics appropriate for the slower metabolism of salamanders
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Adapted functionality for the unique energy utilization strategies of amphibians, including extended periods of low metabolic activity
Understanding these functional implications requires further experimental studies, including receptor binding assays, signaling pathway analyses, and comparative physiological studies between salamander and mammalian leptins.
How can salamander leptin research contribute to understanding evolution of energy homeostasis?
Salamander leptin research provides a valuable evolutionary perspective on energy homeostasis mechanisms by representing an early diverging tetrapod lineage:
| Research Focus | Potential Insight | Significance |
|---|---|---|
| Ancestral functions | Identify core functions shared across vertebrates | Distinguishes fundamental vs. derived leptin roles |
| Ectotherm adaptation | How leptin functions in variable temperature conditions | Reveals temperature-independent regulatory mechanisms |
| Fasting tolerance | Leptin regulation during extended periods without food | Illuminates ancient starvation response mechanisms |
| Tissue-specific expression | Evolutionary shifts in primary sites of leptin action | Identifies functional diversification across lineages |
| Signaling pathway conservation | Core pathways maintained across vertebrates | Reveals essential regulatory networks |
By studying leptin in salamanders, researchers can gain insights into which aspects of energy homeostasis regulation are ancestral (shared with other vertebrates) versus derived (specific to certain lineages). The presence of leptin signaling pathways in salamanders similar to those in mammals suggests fundamental conservation of energy regulation mechanisms across vertebrates, despite hundreds of millions of years of separate evolution .
The distinct tissue expression pattern of leptin in salamanders, particularly the high expression in muscle in the Chinese giant salamander , may indicate evolutionary shifts in the primary sites of leptin production and action across vertebrate evolution. Understanding these differences can help reconstruct how energy homeostasis mechanisms have evolved and adapted to different physiological demands across vertebrate lineages.
Many salamander species demonstrate remarkable metabolic flexibility, including the ability to survive extended periods without food. Investigating how leptin regulates metabolism during these periods could provide valuable insights into ancient mechanisms of metabolic adaptation that may have been modified or lost in mammalian lineages.
Product Science Overview
Leptin Salamander Recombinant is a recombinant form of leptin derived from salamanders. It is produced in Escherichia coli (E. coli) and is a single, non-glycosylated polypeptide chain containing 146 amino acids, with an additional alanine at the N-terminus. The molecular mass of this recombinant protein is approximately 16 kDa .
- Formulation: The lyophilized leptin is typically reconstituted in a sterile solution of 0.4% NaHCO3 at pH 9, at a concentration of not less than 100 µg/ml. This solution can then be further diluted for various applications .
- Stability: The lyophilized form is stable at room temperature for up to three weeks but should be stored desiccated below -18°C for long-term storage. Once reconstituted, it should be stored at 4°C for short-term use (2-7 days) and below -18°C for long-term use. It is recommended to add a carrier protein (0.1% HSA or BSA) to prevent freeze-thaw cycles .
- Purity: The purity of Leptin Salamander Recombinant is greater than 99.0%, as determined by gel-filtration and SDS-PAGE analysis .
