Recombinant Human C-C motif chemokine 3-like 1 protein (CCL3L1) (Active)

What is CCL3L1 and how does it function within the immune system?

CCL3L1 (also known as LD78-beta) is a member of the CC chemokine family encoded by a gene located on chromosome 17q12. It functions as a chemoattractant for lymphocytes and monocytes, playing a critical role in inflammatory and immunoregulatory processes. The protein acts by binding to several chemokine receptors, most notably CCR1, CCR3, and CCR5 . The fully processed form of CCL3L1, LD78-beta(3-70), demonstrates significantly higher chemotactic activity (20-30 fold higher) compared to other related proteins and serves as a potent inhibitor of HIV-1 infection .

CCL3L1 is expressed at lower levels than CCL3 but has a substantially stronger binding affinity to the CCR5 receptor. This enhanced receptor affinity appears to be biologically significant, suggesting that even modest variations in CCL3L1 expression could have meaningful physiological consequences . Expression studies in LPS-stimulated monocytes from 55 individuals have demonstrated consistent gene dosage effects, confirming the relationship between copy number and protein function in immunologically relevant cell types .

How does CCL3L1 differ structurally and functionally from related chemokines?

CCL3L1 is part of a gene cluster that includes CCL3, CCL3L2, and CCL3L3. These genes share significant sequence homology, ranging from 50% to 99% in complete gene sequences and 70-100% in exonic regions . Most notably, CCL3L1 and CCL3L3 have identical exonic sequences despite showing some differences in their untranslated regions (UTRs) and introns. Specifically, there is at least one SNP in the UTR and two in the introns of CCL3L1, and one in the intron of CCL3L3 .

At the protein level, human CCL3L1/LD78-beta is generated from a 93 amino acid precursor with a 23 amino acid signal peptide that is cleaved to produce a 70 amino acid mature protein . When compared to CCL3/LD78-alpha, CCL3L1/LD78-beta demonstrates higher binding affinity to CCR5, which functionally translates to more potent chemotactic activity and stronger HIV-1 entry inhibition . This is evidenced by experimental data showing that CCL3L1/LD78-beta has an ED50 of 0.1-0.4 ng/mL for chemotaxis of CCR5-transfected cells, compared to higher required concentrations for related chemokines .

What is the significance of CCL3L1 copy number variation in human populations?

While most genes exist in two copies per diploid genome, CCL3L1 exhibits copy number variation (CNV) across human populations. Most individuals have between 1-6 copies of CCL3L1, though some may have zero copies or more than six . This variation appears to have population-specific distributions, with studies consistently showing higher median copy numbers in African populations (3-4 copies) compared to European populations (2 copies) .

The biological significance of this variation remains an active area of research. Copy number has been directly linked to CCL3L1 gene expression levels, with higher copy numbers correlating with increased mRNA production. In one study, copy number accounted for approximately 50% of the variation in CCL3L1:CCL3 mRNA ratio . Since CCL3L1 has higher affinity to CCR5 than CCL3, this variation may influence immune responses and disease susceptibility, particularly for conditions where CCR5 plays a role, such as HIV-1 infection .

What are the most reliable methods for accurately determining CCL3L1 copy number?

Accurate measurement of CCL3L1 copy number presents significant technical challenges due to its high sequence homology with other genes in the cluster. The current gold standard approach is the paralogue ratio test (PRT), particularly when implemented as a triplex assay that provides multiple independent estimates of copy number .

PRT is a comparative PCR method that amplifies both a test and reference locus using the same primer pair, followed by quantification through capillary electrophoresis. The triplex PRT assay provides three independent copy number estimates, increasing reliability through internal validation. In implementation studies, this approach showed clear separation of integer copy numbers with Gaussian distributions centered on each integer value, and demonstrated high consistency (95% of samples showed agreement across all three measurements) .

Alternative approaches include quantitative PCR-based methods and sequencing-based approaches. The latter has been implemented using read depth analysis of whole-genome sequencing data from the 1000 Genomes Project. This approach involves calculating sequence read depth across the genomic region in 500bp windows, followed by segmentation score clustering using Gaussian mixture models to infer integer copy numbers . While potentially more comprehensive, these methods require sophisticated bioinformatics pipelines and high-coverage sequencing data.

How can researchers address the challenges of distinguishing between CCL3L1 and highly homologous genes?

The high sequence similarity between CCL3L1 and related genes (particularly CCL3L3) presents substantial challenges for specific quantification. Analysis of four different PCR-based assays used in the literature demonstrated significant inconsistencies in both population-level and individual-level measurements .

Through sequence alignment analysis, researchers have shown that primers and probes from previously published assays align with overlapping sequences in at least two of the four genes in the cluster, indicating that these assays lack specificity for CCL3L1 alone . The concordance between different assays ranges from 0.44-0.83, suggesting individual discordant calls and inconsistencies with assay performance .

To overcome these challenges, researchers should:

• Design assays targeting regions with unique sequence variations between CCL3L1 and related genes

• Include known controls with validated copy numbers in each experiment

• Consider using multiple independent methodologies to cross-validate results

• Implement more comprehensive approaches such as long-read sequencing or fiber-FISH that can distinguish between highly homologous regions

• Be explicit about the potential cross-reactivity of their assays with related genes

Researchers should also consider that while CCL3L1 and CCL3L3 have identical exonic sequences, they differ in their regulatory regions, which may affect expression patterns despite sequence similarity .

What is the relationship between CCL3L1 gene copy number and protein expression?

Studies have demonstrated a clear gene dosage effect of CCL3L1 copy number on mRNA expression levels. Analysis of RNAseq data from lymphoblastoid cell lines showed that CCL3L1 copy number significantly influences both absolute CCL3L1 expression and the CCL3L1:CCL3 expression ratio .

The relationship follows a linear pattern, with each additional copy of CCL3L1 producing a proportional increase in expression. This gene dosage effect appears robust, with copy number accounting for approximately 50% of the total variation in CCL3L1:CCL3 mRNA ratio . This is particularly significant given that CCL3L1 (LD78-beta) has stronger receptor affinity than CCL3 (LD78-alpha), suggesting functional consequences of this variation.

What controls and validation approaches should be included when working with recombinant CCL3L1?

When working with recombinant CCL3L1 protein, researchers should implement several critical controls and validation steps:

• Protein authenticity verification: Confirm the identity and purity of recombinant CCL3L1 using techniques such as mass spectrometry, N-terminal sequencing, or specific antibody recognition. This is particularly important given the high homology with related proteins.

• Functional validation: Assess the biological activity of the recombinant protein through:

  • Chemotaxis assays using CCR5-expressing cells (expected ED50: 0.1-0.4 ng/mL)

  • Receptor binding assays comparing affinity to CCR1, CCR3, and CCR5

  • HIV inhibition assays if relevant to the research question

• Dose-response characterization: Establish complete dose-response curves rather than single-point measurements, as CCL3L1 may show bell-shaped activity curves typical of chemokines.

• Specificity controls: Include related chemokines (particularly CCL3) as comparative controls to demonstrate the specific effects of CCL3L1 versus related family members.

• Endotoxin testing: Ensure recombinant preparations are endotoxin-free, as contamination can confound immunological experiments.

• Stability assessment: Verify protein stability under experimental conditions through multiple freeze-thaw cycles and at experimental temperatures.

The implementation of these controls will enhance reproducibility and reliability of experimental results involving recombinant CCL3L1.

How should researchers interpret conflicting data about CCL3L1 copy number and disease associations?

Conflicting reports regarding CCL3L1 copy number associations with disease susceptibility (particularly HIV-1) have appeared in the literature . These discrepancies likely stem from several methodological and biological considerations that researchers should address:

• Measurement methodology: Different assays for copy number determination show poor concordance. Without a universally accepted gold standard, researchers should clearly specify their methodology and its limitations .

• Population stratification: CCL3L1 copy number distributions vary between populations. Studies should either focus on homogeneous populations or properly account for population structure in their analyses .

• Composite genetic effects: CCL3L1 may interact with other genetic factors. For example, CCR5 variants like CCR5Δ32 may modify the effect of CCL3L1 copy number .

• Gene versus protein effects: Copy number influences mRNA levels, but post-transcriptional regulation may modify protein expression. Both genotype and phenotype measurements may be necessary .

• Gene specificity: Most assays cannot distinguish between CCL3L1 and CCL3L3. Results attributed to CCL3L1 may actually reflect the combined effect of multiple genes .

To address these challenges, researchers should:

• Employ multiple independent methods for copy number determination

• Include large, well-characterized cohorts with appropriate controls

• Account for potential confounding factors and gene-gene interactions

• Be cautious in interpreting marginal associations

• Consider functional validation of genetic associations

What experimental approaches can distinguish the specific effects of CCL3L1 from related chemokines?

Distinguishing the specific biological effects of CCL3L1 from highly related chemokines requires sophisticated experimental designs:

• Receptor-specific functional assays: Utilize cells expressing individual receptors (CCR1, CCR3, or CCR5) to compare binding and signaling properties of CCL3L1 versus related chemokines. The ED50 for CCL3L1-induced chemotaxis in CCR5-expressing cells (0.1-0.4 ng/mL) can serve as a benchmark for comparison .

• Competitive binding assays: Perform displacement studies with labeled ligands to determine relative receptor affinities and binding kinetics of CCL3L1 compared to related chemokines.

• Genetic manipulation approaches:

  • Use CRISPR-Cas9 to specifically modify CCL3L1 without affecting related genes

  • Employ siRNA designed to target unique regions (likely in non-coding sequences)

  • Create transgenic models with controlled expression of human CCL3L1

• Structure-function studies: Identify amino acid residues unique to CCL3L1 that confer its distinct functional properties, then create point mutants to verify their role.

• Domain swapping: Create chimeric proteins between CCL3L1 and related chemokines to map functional domains.

• Specific neutralizing antibodies: Develop and validate antibodies that specifically recognize CCL3L1 but not related proteins for neutralization studies.

By employing these approaches, researchers can more definitively attribute biological effects to CCL3L1 rather than related chemokines, despite their high sequence similarity.

How might recombinant CCL3L1 be utilized in HIV research and potential therapeutic applications?

Recombinant CCL3L1 has several promising applications in HIV research and potential therapeutics, leveraging its high-affinity binding to CCR5 and potent HIV-1 inhibitory properties:

• HIV entry inhibition studies: Recombinant CCL3L1 can be used to investigate mechanisms of viral entry blockade. The processed form LD78-beta(3-70) shows 20-30 fold higher chemotactic activity than related chemokines and is a very potent inhibitor of HIV-1 infection .

• Structure-based drug design: Detailed understanding of CCL3L1-CCR5 interactions could inform the development of small molecule CCR5 antagonists with improved pharmacological properties over existing drugs.

• Combination therapy approaches: Studies examining synergistic effects between CCL3L1 and other entry inhibitors or antiretroviral drugs could identify optimal combination strategies.

• Genetic therapy concepts: The protective effect of higher CCL3L1 copy numbers against HIV-1 infection suggests gene therapy approaches that increase CCL3L1 expression might confer protection.

• Biomarker development: CCL3L1 copy number or expression levels might serve as biomarkers for HIV progression or treatment response.

• Population-specific intervention strategies: Given the variation in CCL3L1 copy number across populations, tailored approaches based on genetic background could be developed.

Future research should focus on optimizing recombinant CCL3L1 production, improving stability for therapeutic applications, and developing targeted delivery systems to maximize antiviral effects while minimizing potential inflammatory consequences.

What are the emerging research areas related to CCL3L1 beyond HIV/AIDS?

While much CCL3L1 research has focused on HIV-1, several emerging areas warrant investigation:

• Broader infectious disease susceptibility: Since CCL3L1 influences chemotaxis and immune cell recruitment, its role in other viral, bacterial, and parasitic infections should be explored.

• Inflammatory and autoimmune disorders: The potent pro-inflammatory properties of CCL3L1 suggest potential involvement in conditions like rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis. Copy number variation might contribute to disease risk or severity.

• Cancer immunology: Chemokines significantly impact tumor microenvironments. CCL3L1 might influence tumor-associated macrophage recruitment and function, potentially affecting tumor progression or response to immunotherapy.

• Neurodegenerative diseases: Neuroinflammation plays a role in conditions like Alzheimer's and Parkinson's diseases. CCL3L1's influence on microglial activation and neuroinflammatory processes deserves investigation.

• Transplantation biology: CCL3L1 might affect graft survival and rejection processes through its influence on immune cell trafficking.

• Developmental immunology: The role of CCL3L1 in immune system development and maturation remains poorly understood.

• Evolution and population genetics: The variation in CCL3L1 copy number across populations suggests potential selective pressures that might illuminate human evolutionary history and adaptation to infectious disease.

Methodological advances in accurately measuring CCL3L1 separate from related genes will be crucial to progress in these emerging research areas.