MIP 5 (68 a.a) Human

What is MIP-5 (68 a.a) Human and what are its alternative nomenclatures?

MIP-5 (68 a.a) Human refers to a specific form of the human CC chemokine CCL15, spanning amino acids Ser46 to Ile113 of the complete sequence . This chemokine is known by several alternative names including Leukotactin-1 (LKN-1), MIP-5, HCC-2, and NCC-3 .

For proper identification in research:

• Use HGNC (HUGO Gene Nomenclature Committee) approved name CCL15 as the primary identifier

• Include alternative nomenclature in methods sections

• Always specify the exact amino acid sequence or residue range when working with truncated forms

• Document the specific recombinant preparation used (e.g., E. coli-derived, with or without carrier protein)

What receptors does MIP-5 (68 a.a) Human interact with?

MIP-5/CCL15 primarily exerts its biological activities through interactions with CC chemokine receptors CCR1 and CCR3 . These G protein-coupled receptors typically signal through heterotrimeric Gi proteins, as demonstrated by inhibition studies with pertussis toxin .

Methodological approaches to study receptor interactions include:

• Radioligand binding assays using 125I-labeled chemokines

• Competitive binding assays with other known receptor ligands

• Chemotaxis assays with receptor antagonists or blocking antibodies

• Pertussis toxin inhibition studies to confirm Gi protein involvement

What are the reconstitution protocols for recombinant MIP-5 (68 a.a) Human?

Optimal reconstitution of recombinant MIP-5/CCL15 (68 a.a) protein depends on the preparation:

For preparations with carrier protein (BSA):

• Reconstitute at 10 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin

• The product is typically shipped at ambient temperature

• Upon receipt, store immediately at recommended temperatures

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles

For carrier-free preparations:

• Reconstitute at 100 μg/mL in sterile PBS

• Follow the same shipping and storage recommendations as above

The carrier-free version is recommended for applications where BSA might interfere with experimental outcomes .

What is the biological significance of MIP-5 (68 a.a) Human in normal physiology?

MIP-5/CCL15 plays several key roles in normal physiology:

• Immune cell chemotaxis: Attracts cells expressing CCR1 and CCR3 receptors

• Angiogenesis: Promotes endothelial cell migration and differentiation

• Vascular development: Stimulates sprouting of vessels in various model systems

These functions can be evaluated through:

• In vitro chemotaxis assays using Boyden chambers or Transwell systems

• Endothelial tube formation assays

• Aortic ring sprouting assays

• Chick chorioallantoic membrane (CAM) assays for in vivo angiogenesis

How does N-terminal truncation affect the biological activity of MIP-5 (68 a.a) Human?

N-terminal truncation dramatically enhances the biological activity of MIP-5/CCL15. Research demonstrates that the N-terminal truncated form CCL15(25-92) is at least 100-fold more potent than the full-length CCL15(1-92) in stimulating endothelial cell migration and differentiation . The 68 a.a form (Ser46-Ile113) represents another truncated version with potentially altered activity profile .

This effect is consistent with observations from other chemokines where N-terminal processing by proteases can significantly enhance receptor binding and activation. To investigate these effects:

• Generate multiple recombinant proteins with different N-terminal truncations

• Compare binding affinities to CCR1 and CCR3 using surface plasmon resonance

• Perform dose-response curves in chemotaxis and angiogenesis assays

• Use site-directed mutagenesis to identify critical residues in the N-terminus

What methodologies are appropriate for investigating MIP-5's angiogenic activity?

MIP-5/CCL15 demonstrates significant angiogenic properties, with the truncated form being particularly potent . Both CCL15(1-92) and the N-terminal truncated CCL15(25-92) stimulate chemotactic endothelial cell migration and differentiation, with the truncated form showing approximately 100-fold greater potency .

In vitro methods:

• Endothelial cell migration assays (Boyden chamber, wound healing)

• Tube formation assays on Matrigel

• Aortic ring sprouting assays

In vivo methods:

• Chick chorioallantoic membrane (CAM) assay

• Matrigel plug assay in mice

• Zebrafish vascular development models

Mechanism investigation:

• Receptor blocking with antibodies against CCR1 and CCR3

• Pertussis toxin treatment to inhibit Gi signaling

• Analysis of downstream signaling pathways

Research has shown that treatment with pertussis toxin (PTX), anti-CCR1, or anti-CCR3 antibodies inhibits the CCL15(25-92)-induced endothelial cell migration, confirming the involvement of these specific receptors and Gi-coupled signaling in the angiogenic response .

How do MIP-5 (68 a.a) Human interactions with receptors compare to other chemokines?

The structural basis for chemokine recognition and receptor activation provides important insights into how MIP-5/CCL15 might interact with its receptors. While specific structural data for MIP-5/CCR1 or CCR3 complexes is limited, research on related chemokine-receptor pairs offers valuable comparison points.

Cryo-electron microscopy (cryo-EM) and X-ray crystallography studies of chemokine-receptor complexes reveal important details about these interactions:

• Chemokines like MIP-1α and RANTES bind to CCR5 with distinct binding modes

• G protein coupling (particularly to Gi proteins) follows chemokine binding

• Toggle switch mechanisms and rearrangements in the receptor extracellular region are critical for activation

• Conserved residues, such as a tryptophan in helix II, can act as triggers for receptor activation

These structural insights can guide experimental designs for investigating MIP-5/CCL15 interactions with its receptors using similar methodologies.

What experimental models are most appropriate for studying MIP-5's role in disease?

As a chemokine involved in immune cell recruitment and angiogenesis, MIP-5/CCL15 may play roles in various pathological conditions including inflammation, cancer, and immune disorders. Appropriate experimental models include:

For inflammation studies:

• Acute inflammation models (air pouch, peritonitis)

• Chronic inflammation models (colitis, arthritis)

• Humanized mouse models expressing human CCR1/CCR3

For angiogenesis-related diseases:

• Tumor xenograft models to study cancer angiogenesis

• Ischemia models to study therapeutic angiogenesis

• Retinal neovascularization models

For mechanistic investigations:

• Transgenic models with cell-specific CCR1/CCR3 expression

• Receptor knockout models

• Models with fluorescent reporter tags for tracking cellular responses

These models can be complemented with analysis of human patient samples to establish clinical relevance.

How can researchers optimize functional assays for measuring MIP-5 (68 a.a) Human activity?

Optimizing functional assays for MIP-5/CCL15 requires careful consideration of several factors:

Chemotaxis assays:

• Cell type selection: Use cells naturally expressing CCR1/CCR3 or stable transfectants

• Medium composition: Low serum (0.1-1%) reduces background migration

• Incubation time: Typically 2-4 hours for maximal response

• Positive controls: Include established chemokines for CCR1/CCR3 (e.g., MIP-1α, RANTES)

Endothelial cell assays:

• Cell source: Primary human endothelial cells vs. cell lines

• Passage number: Low passage cells (P2-P5) maintain better receptor expression

• Readout methods: Direct cell counting vs. fluorescent labeling

• Assay duration: 4-24 hours depending on the specific assay

Receptor activation assays:

• Calcium flux: Rapid (seconds to minutes) with appropriate calcium indicators

• cAMP inhibition: Measure decreases in forskolin-stimulated cAMP levels

• ERK phosphorylation: Peak activation typically at 5-15 minutes

The ED50 for MIP-5/CCL15 (68 a.a) in chemotaxis assays ranges from 0.6-3 ng/mL for cells expressing CCR1 , providing a reference point for assay development.

What are potential applications of MIP-5 (68 a.a) Human in therapeutic development?

The biological properties of MIP-5/CCL15, particularly its angiogenic activity, suggest several potential therapeutic applications:

Pro-angiogenic applications:

• Therapeutic angiogenesis for ischemic diseases

• Wound healing enhancement

• Tissue engineering and regenerative medicine

Anti-inflammatory applications:

• Antagonism of MIP-5/CCL15 or its receptors in inflammatory disorders

• Modulation of specific immune cell recruitment

• Targeted delivery of anti-inflammatory agents

Development of these applications requires:

• Structure-activity relationship studies to identify key functional domains

• Development of stable analogs with improved pharmacokinetics

• Targeted delivery systems for tissue-specific effects

• Comprehensive safety profiling in relevant models

How can structural studies enhance our understanding of MIP-5 (68 a.a) Human function?

Advanced structural biology approaches provide critical insights into MIP-5/CCL15 function:

Structural techniques applicable to MIP-5/CCL15 research:

• X-ray crystallography of the chemokine alone or in complex with receptors

• Cryo-EM studies of receptor-chemokine-G protein complexes

• NMR spectroscopy for dynamic studies of chemokine-receptor interactions

• Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

Recent advances in cryo-EM have enabled visualization of chemokine-receptor-G protein complexes, revealing how chemokines like MIP-1α and RANTES bind to CCR5 and trigger G protein coupling . Similar approaches could illuminate the structural basis of MIP-5/CCL15 interactions with CCR1 and CCR3.

These structural insights can guide:

• Rational design of agonists or antagonists

• Understanding of receptor selectivity determinants

• Identification of allosteric binding sites for drug development

• Elucidation of activation mechanisms

What are current technical challenges in studying MIP-5 (68 a.a) Human?

Researchers face several technical challenges when working with MIP-5/CCL15:

Production and handling:

• Ensuring proper folding and disulfide bond formation in recombinant preparations

• Maintaining stability during storage and experimental procedures

• Controlling for batch-to-batch variations in activity

Specificity and selectivity:

• Distinguishing CCR1 vs. CCR3-mediated effects

• Accounting for potential binding to other receptors or proteoglycans

• Controlling for effects of endogenous chemokines in complex systems

Translational considerations:

• Species differences in receptor binding and signaling

• Context-dependent activity in different tissues

• Complex interactions with other chemokines and inflammatory mediators

Addressing these challenges requires rigorous experimental design, appropriate controls, and complementary approaches to validate findings.

Comparative Potency of MIP-5/CCL15 Forms

Receptor Specificity and Inhibition

Reconstitution Methods for Research Applications

Functional Assay Systems for MIP-5/CCL15 Research