Recombinant Rhesus Macaque Pro-interleukin-16 [Cleaved into: Interleukin-16 protein (IL16) (Active)

What is the molecular structure and basic characteristics of Rhesus Macaque IL-16?

Rhesus Macaque Interleukin-16 (IL-16) is a cytokine with a molecular weight of approximately 12.5 kDa, consisting of 121 amino acid residues in its mature form. The expression region spans amino acids 510-630 of the precursor protein . The complete sequence is: SAASASAASD VSVESSAEAT VYTVTLEKMS AGLGFSLEGG KGSLHGDKPL TINRIFKGAA SEQSETIQPG DEILQLAGTA MQGLTRFEAW NIIKALPDGP VTIVIRRKSL QPKETTAAAD S . This cytokine shares 95% amino acid sequence identity with human IL-16 and 85% with murine IL-16, making it valuable for comparative immunological studies across species .

How does Pro-IL-16 differ from mature IL-16 in structure and function?

Pro-interleukin-16 serves as the precursor form that undergoes proteolytic cleavage to generate the mature, bioactive Interleukin-16. The pro-form is involved in T-cell cycle progression and appears to participate in transcriptional regulation, potentially acting as part of a transcriptional repression complex on the core promoter of the SKP2 gene . After cleavage, the mature IL-16 functions primarily as a chemoattractant and immunomodulatory cytokine. The processing of Pro-IL-16 represents an important regulatory step that controls the release and activity of the mature form in immunological responses.

What conservation patterns exist between Rhesus Macaque IL-16 and other species' IL-16?

Recombinant Rhesus Macaque IL-16 demonstrates high sequence conservation with human IL-16 (95% amino acid identity) and moderate conservation with murine IL-16 (85% identity) . This high degree of conservation, particularly with human IL-16, makes the Rhesus Macaque model valuable for translational research. The conserved regions likely correspond to functional domains essential for receptor binding and signaling, while variations may reflect species-specific adaptations in immune regulation.

What are the primary immunological functions of Rhesus Macaque IL-16?

Rhesus Macaque IL-16, also known as lymphocyte chemoattractant factor (LCF), performs several crucial immunological functions. It stimulates migratory responses in CD4+ lymphocytes, monocytes, and eosinophils . Additionally, it primes CD4+ T-cells for responsiveness to IL-2 and IL-15, induces T-lymphocyte expression of the IL-2 receptor, and functions as a ligand for CD4 . The cytokine is also involved in suppressing human immunodeficiency virus (HIV) replication and inhibiting T-cell antigen receptor/CD3-mediated T-cell stimulation in mixed lymphocyte reactions . These functions collectively position IL-16 as a significant regulator of immune cell trafficking and activation.

Through which receptor pathways does IL-16 mediate its biological effects?

IL-16 primarily signals through the CD4 receptor, which serves as its main cognate receptor . This interaction is critical for initiating downstream signaling cascades that result in chemotaxis and immune cell activation. The CD4-dependent signaling explains IL-16's selective effects on CD4+ immune cells, including T-helper lymphocytes and monocytes. The binding of IL-16 to CD4 triggers intracellular pathways that influence cell migration, activation, and responsiveness to other cytokines.

How does IL-16 contribute to immune cell trafficking and inflammation in rhesus macaque models?

IL-16 functions as a potent chemoattractant for CD4+ cells, orchestrating their migration to sites of inflammation. In rhesus macaque models, IL-16 mediates the recruitment of CD4+ T lymphocytes, monocytes, and eosinophils, thereby regulating the composition of inflammatory infiltrates . This directed migration of immune cells plays a crucial role in both normal immune surveillance and pathological inflammatory responses. IL-16's ability to prime these cells for responses to other cytokines, particularly IL-2 and IL-15, further amplifies inflammatory cascades and shapes adaptive immune responses.

What are the optimal conditions for reconstituting lyophilized Rhesus Macaque IL-16?

For optimal reconstitution of lyophilized Rhesus Macaque IL-16, it is recommended to first briefly centrifuge the vial to bring the contents to the bottom. The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, it is advisable to add glycerol to a final concentration of 5-50% (with 50% being commonly recommended) and aliquot the solution before storing at -20°C or -80°C . This approach minimizes protein degradation that can occur with repeated freeze-thaw cycles and maintains the biological activity of the reconstituted protein.

What bioassays can effectively measure the biological activity of Rhesus Macaque IL-16?

The biological activity of Rhesus Macaque IL-16 is typically assessed using a chemotaxis bioassay with human peripheral T lymphocytes . In this functional assay, the protein demonstrates activity in a concentration range of 1.0-100 ng/ml . The chemotactic response of target cells provides a direct measurement of IL-16's ability to induce cell migration, which is one of its primary biological functions. Alternative assays may include measuring IL-2 receptor expression on T-cells or evaluating the inhibition of HIV replication in appropriate cellular models.

How can researchers validate the purity and integrity of recombinant Rhesus Macaque IL-16?

Researchers can validate the purity and integrity of recombinant Rhesus Macaque IL-16 through multiple analytical methods. SDS-PAGE and HPLC analyses are standard techniques used to confirm purity levels, which typically exceed 98% for research-grade preparations . Endotoxin contamination should be assessed using the Limulus Amebocyte Lysate (LAL) method, with levels below 1.0 EU/μg considered acceptable for most research applications . Additional validation may include mass spectrometry to confirm the exact molecular weight and protein sequence verification through techniques such as peptide mapping or amino acid analysis.

What storage conditions are critical for maintaining the stability of Rhesus Macaque IL-16?

For optimal stability, lyophilized Rhesus Macaque IL-16 should be stored desiccated at -20°C . Once reconstituted, the protein solution should be supplemented with 5-50% glycerol and stored in small aliquots at -20°C or -80°C to prevent degradation from repeated freeze-thaw cycles . Working aliquots can be stored at 4°C for up to one week, but longer periods risk loss of biological activity . The maintenance of these precise storage conditions is essential for preserving the structural integrity and functional properties of the protein over time.

How can researchers troubleshoot decreased activity in Rhesus Macaque IL-16 experiments?

When troubleshooting decreased activity in Rhesus Macaque IL-16 experiments, researchers should first verify proper reconstitution and storage procedures. Common issues include protein degradation from repeated freeze-thaw cycles, inadequate buffer conditions, or extended storage at suboptimal temperatures. The biological activity should be confirmed using a chemotaxis bioassay with the recommended concentration range (1.0-100 ng/ml) . Researchers should also verify the absence of interfering substances in the experimental system and consider the possibility of receptor desensitization or downregulation in target cells after prolonged exposure to IL-16.

What are the most effective experimental controls when using recombinant Rhesus Macaque IL-16?

Effective experimental controls when using recombinant Rhesus Macaque IL-16 include both positive and negative controls. A positive control might involve using IL-16 from another species with known activity (such as human IL-16) or another cytokine with established chemotactic properties. Negative controls should include buffer-only treatments and, when evaluating specificity, the use of appropriate blocking antibodies against IL-16 or the CD4 receptor. For migration assays, random migration controls (without chemotactic gradient) are essential for distinguishing directed chemotaxis from general cell motility. These controls help establish the specificity and magnitude of IL-16-mediated effects in experimental systems.

How can Rhesus Macaque IL-16 be utilized in HIV/SIV research models?

Rhesus Macaque IL-16 offers valuable applications in HIV/SIV research due to its documented ability to suppress HIV replication and its interactions with the CD4 receptor, which serves as the primary entry receptor for these viruses . In SIV-infected rhesus macaque models, researchers can investigate how IL-16 modulates viral entry, replication, and immune cell dynamics. The high homology between rhesus and human IL-16 (95%) makes findings potentially translatable to human HIV pathogenesis. Studies may examine how IL-16 levels correlate with disease progression, viral load, and CD4+ T-cell counts, or investigate therapeutic approaches targeting IL-16 pathways to modulate immune responses during infection.

What methodologies can differentiate between the effects of Pro-IL-16 and mature IL-16 in experimental systems?

Differentiating between Pro-IL-16 and mature IL-16 effects requires specialized methodologies that target their distinct functions. Researchers can employ antibodies specifically recognizing either the pro-domain or the mature form in immunodetection techniques. Experimentally, selective expression of truncated constructs containing only the pro-domain or the mature domain can help delineate their specific activities. For studying Pro-IL-16's nuclear functions in transcriptional regulation, chromatin immunoprecipitation (ChIP) assays examining interactions with the SKP2 gene promoter would be appropriate . Conversely, extracellular chemotaxis assays would primarily detect mature IL-16 activity. Cell fractionation techniques can also help distinguish between nuclear-localized Pro-IL-16 and secreted mature IL-16.

How do post-translational modifications affect the biological activity of Rhesus Macaque IL-16?

The biological activity of Rhesus Macaque IL-16 can be significantly influenced by post-translational modifications, although this area remains less comprehensively studied than other aspects of IL-16 biology. When producing recombinant IL-16 in E. coli expression systems , the protein lacks eukaryotic post-translational modifications, which may affect certain aspects of its activity compared to the naturally occurring cytokine. Researchers should consider that modifications such as glycosylation, phosphorylation, or specific proteolytic processing might alter protein stability, receptor binding affinity, or signaling potency. Comparative studies between E. coli-derived and eukaryotic cell-derived IL-16 could help elucidate the functional significance of these modifications.

How can differences between Rhesus Macaque IL-16 and human IL-16 impact translational research?

Despite the high sequence homology (95%) between Rhesus Macaque and human IL-16, the 5% difference in amino acid composition may influence certain aspects of protein function relevant to translational research. These differences might affect receptor binding affinity, signaling potency, or interactions with species-specific regulatory proteins. Researchers conducting translational studies should systematically evaluate these potential variations through comparative functional assays before extrapolating findings to human systems. The strategic use of both rhesus and human IL-16 in parallel experiments can help identify any species-specific effects and strengthen the translational value of the research.

What experimental design considerations are crucial when comparing IL-16 responses across different cell types?

When comparing IL-16 responses across different cell types, researchers must consider several critical experimental design factors. First, expression levels of the CD4 receptor vary significantly between cell types, directly impacting sensitivity to IL-16 . Standardized cell counting and viability assessments are essential for accurate interpretation of differential responses. Researchers should employ multiple readouts (chemotaxis, calcium flux, receptor expression, etc.) to comprehensively characterize cell type-specific responses. Time-course experiments are recommended, as response kinetics may differ substantially between cell populations. Finally, cell culture conditions should be optimized for each cell type while maintaining as many variables constant as possible to enable valid comparisons.

How can researchers design experiments to evaluate IL-16's role in complex immunological networks?

Designing experiments to evaluate IL-16's role in complex immunological networks requires a systems biology approach. Researchers should combine in vitro studies with ex vivo analyses and in vivo models to capture the multifaceted effects of IL-16. Techniques such as multiplexed cytokine profiling can help identify how IL-16 interacts with other immune mediators. Co-culture systems incorporating multiple cell types (T cells, monocytes, epithelial cells) better recapitulate the cellular complexity of immune responses compared to single-cell-type experiments. Selective neutralization or knockout approaches targeting IL-16 or its receptor in these systems can reveal its specific contributions to immune network dynamics. For in vivo studies in rhesus macaques, local or systemic administration of recombinant IL-16, coupled with comprehensive immune monitoring, can elucidate its effects on immune cell trafficking and activation in a physiologically relevant context.