Recombinant Human C-C motif chemokine 4-like protein (CCL4L1) (Active)

What is C-C motif chemokine 4-like protein (CCL4L1) and how does it differ from CCL4?

CCL4L1 is a human chemokine encoded by the CCL4L1 gene located on chromosome 17q21. It differs from the related non-allelic isoform CCL4 by only a single amino acid in the mature protein. Despite this minimal difference, CCL4L1 demonstrates unique binding properties to chemokine receptors including D6, CCR3, and CCR5 .

Both CCL4 and CCL4L1 show functional redundancy in competitive binding studies using HEK-293/CCR5 cells, producing comparable EC50 values. Chemotaxis assays with cells expressing CCR1, CCR3, or CCR5 also revealed no substantial differences between the two proteins .

What is known about CCL4L1 gene copy number variation?

Unlike many genes that maintain a consistent two copies per diploid genome, CCL4L1 demonstrates significant copy number variation:

• Can range from 1-6 copies per diploid genome in Caucasians

• Can occasionally be completely absent in some individuals

• Other isoforms (CCL3 and CCL4) typically remain at two copies per diploid genome

• Higher gene copy number correlates with increased ratio of CCL4L1 versus CCL3 mRNA

• Enhanced CCL4L1 production is associated with higher copy numbers

This variation may impact susceptibility to diseases in which this chemokine plays a role, including potential influences on inflammatory and autoimmune conditions.

How can researchers measure CCL4L1 in biological samples?

Multiple methodologies are available for CCL4L1 quantification:

For optimal results in protein quantification, researchers should reconstitute freeze-dried recombinant protein in 10mM PBS (pH7.4) to a concentration of 0.1-1.0 mg/mL, avoiding vortexing which may damage protein structure .

How does CCL4L1 gene copy number influence immune function?

In LPS-activated monocytes (a model system of pro-inflammatory chemokine production), higher CCL4L1 gene copy number directly correlates with:

• Increased ratio of CCL4L1 versus CCL3 mRNA

• Enhanced chemokine production

• Improved ability to chemoattract CCR5-expressing cells

• Effects that can be blocked with anti-CCL3/CCL3-L1 antibodies

These findings suggest that genetic variation in CCL4L1 copy number may affect susceptibility to diseases where inflammation plays a critical role. The relationship between gene dosage and functional impact provides insight into how quantitative genetic variation translates to phenotypic differences in immune response.

What is the role of CCL4L1 in HIV infection research?

CCL4L1 has particular significance in HIV research due to its interaction with CCR5, a co-receptor for HIV-1 entry:

• CCL4L1 demonstrates strong interaction with CCR5

• It is susceptible to cleavage by CD26, creating a truncated –2 form with enhanced affinity for CCR1 and CCR5

• Both CCL4L1 and particularly its –2 variant are among the most potent natural HIV entry inhibitors described

• CCL4L1 appears somewhat more effective at inhibiting HIV-1 replication in PBMCs than CCL4, though the difference was not statistically significant

These properties make CCL4L1 an important molecule for researchers investigating novel approaches to HIV prevention and treatment strategies.

How do environmental factors influence CCL4L1 expression?

Research has identified several environmental factors that modify CCL4L1 expression through various mechanisms:

These findings highlight the importance of considering environmental exposures when studying CCL4L1 expression in different experimental and clinical contexts.

What are the optimal conditions for working with recombinant CCL4L1?

For maximum stability and activity of recombinant CCL4L1:

• Store at 2-8°C for one month or aliquot and store at -80°C for 12 months

• Avoid repeated freeze/thaw cycles which can degrade protein

• The thermal stability shows less than 5% loss rate when incubated at 37°C for 48h

• Reconstitute in 10mM PBS (pH7.4) to a concentration of 0.1-1.0 mg/mL

• Do not vortex the reconstituted protein as this may disrupt structure

• Buffer formulation typically includes PBS, pH7.4, containing 0.01% SKL, 5% Trehalose

How should researchers design functional assays to evaluate CCL4L1 activity?

When assessing CCL4L1 functional activity, researchers should consider:

• Receptor binding assays:

  • Use HEK-293/CCR5 cells for competitive binding studies

  • Compare EC50 values with CCL4 as a reference standard

  • Include appropriate controls for non-specific binding

• Chemotaxis assays:

  • Test cells expressing relevant receptors (CCR1, CCR3, or CCR5)

  • Establish dose-response relationships

  • Include antibody blocking experiments (anti-CCL3/CCL3-L1) to confirm specificity

• HIV-1 inhibition assays:

  • Use peripheral blood mononuclear cells (PBMCs)

  • Compare activity with other chemokines including CCL4

  • Measure viral replication through appropriate markers

What are the key considerations for accurate gene copy number determination?

Determining CCL4L1 gene copy number requires careful experimental design:

• Use real-time PCR with appropriate primers that distinguish CCL4L1 from the highly similar CCL4

• Include reference genes that maintain consistent copy numbers (2 copies per diploid genome)

• Establish standard curves with samples of known copy number

• Consider technical limitations that might affect accuracy

• Validate unusual findings (complete absence or high copy numbers) using alternative methodologies

• Account for population differences in copy number distribution

How should researchers interpret differences between CCL4L1 and CCL4 in experimental settings?

When analyzing comparative data between CCL4L1 and CCL4:

• Consider the single amino acid difference in context of:

  • Receptor binding profiles

  • Functional redundancy in most assays

  • Subtle differences that may become significant in specific contexts

• Examine gene copy number influence:

  • Higher CCL4L1 copy number correlates with increased expression

  • Potential dosage effects on immune responses

• Evaluate disease relevance:

  • CCL4L1 variation may affect disease susceptibility or progression

  • Different effects may be observed in different disease models

• Statistical considerations:

  • Small differences may not reach statistical significance despite biological relevance

  • Consider power calculations when designing experiments to detect subtle effects

What control samples should be included in CCL4L1 research?

For robust experimental design, include these controls:

• For protein studies:

  • Recombinant CCL4 as a closely related protein control

  • Neutralizing antibodies (anti-CCL3/CCL3-L1) for specificity confirmation

  • Vehicle controls with identical buffer composition but no active protein

• For genetic studies:

  • Samples with known CCL4L1 copy numbers (1-6 copies)

  • Samples lacking CCL4L1 when available

  • Reference genes with stable copy numbers

• For functional assays:

  • Dose-response curves to establish concentration-dependent effects

  • Receptor antagonists to confirm receptor specificity

  • Positive and negative controls for each experimental system

What are the potential therapeutic applications of CCL4L1 research?

Based on current understanding, CCL4L1 research may contribute to therapeutic developments in:

• HIV infection:

  • CCL4L1's natural ability to inhibit HIV-1 entry through CCR5 interaction

  • Potential development of CCL4L1-based or CCL4L1-inspired entry inhibitors

  • Understanding how genetic variation in CCL4L1 copy number affects HIV susceptibility

• Inflammatory conditions:

  • Targeting CCL4L1 in diseases where chemokine-mediated inflammation plays a role

  • Understanding how copy number variation influences disease susceptibility or progression

  • Development of modulators of CCL4L1 function for therapeutic purposes

• Diagnostic applications:

  • CCL4L1 gene copy number as a potential biomarker for disease susceptibility

  • CCL4L1 protein levels as indicators of specific immune activation states

How can researchers study CCL4L1 in relation to specific disease models?

For disease-focused CCL4L1 research:

• Select appropriate models:

  • In vitro systems using primary cells from individuals with known CCL4L1 copy numbers

  • Animal models that recapitulate relevant aspects of human disease

  • Clinical samples from well-characterized patient cohorts

• Measure relevant parameters:

  • CCL4L1 gene copy number in study populations

  • CCL4L1 expression levels (mRNA and protein)

  • Functional outcomes (chemotaxis, receptor activation, disease progression)

• Consider disease-specific contexts:

  • HIV infection: focus on viral entry and replication

  • Autoimmune conditions: leukocyte recruitment and inflammatory responses

  • Cancer: potential roles in tumor microenvironment and metastasis