MCP 3 Rat
Description
Biological Functions
MCP-3/CCL7 binds to CCR1, CCR2, and CCR3 receptors, mediating monocyte, eosinophil, and lymphocyte recruitment .
Key Roles
-
Monocyte Mobilization: Critical for monocyte egress from bone marrow and recruitment to inflammatory sites .
-
Atherosclerosis: Promotes vascular smooth muscle cell (VSMC) proliferation and contributes to restenosis .
-
Intestinal Inflammation: Elevated in active IBD, suggesting a role in epithelial repair and immune modulation .
Monocyte Homeostasis
Studies in knockout mice reveal MCP-3’s necessity for maintaining blood monocyte counts. MCP-3–/– mice exhibit reduced inflammatory monocytes, indicating its role in CCR2-dependent monocyte mobilization .
| Model | Observation | Source |
|---|---|---|
| MCP-3–/– Mice | 65% reduction in blood monocytes; impaired recruitment to inflamed sites . | |
| CCR2–/– Mice | Defective monocyte egress from bone marrow, despite normal marrow stores . |
Vascular Pathophysiology
In rat VSMCs, MCP-3 is upregulated by TNF-α, driving proliferation and contributing to atherosclerosis. Adenoviral MCP-3-shRNA reduces TNF-α-induced VSMC growth .
Intestinal Inflammation
MCP-3 is highly expressed in inflamed intestinal epithelial cells, correlating with mucosal damage severity. Enhanced production in IBD suggests immunoregulatory roles .
Therapeutic and Research Implications
Product Specs
Q&A
What is MCP (CD46) in rats and how does it compare to human MCP?
Rat Membrane Cofactor Protein (MCP/CD46) is a regulatory complement protein encoded in the regulators of complement activation (RCA) gene cluster. Like human MCP, it functions as a cofactor for Factor I-mediated cleavage of C3b. The domain structure of rat MCP consists of four short consensus repeats (SCRs), followed by a serine-threonine-proline (STP) domain, a transmembrane segment, and a cytoplasmic tail. This structure is identical to mouse MCP but differs somewhat from human MCP. Rat and mouse MCP share 77% amino acid identity and 88% nucleotide identity, while showing weaker homology with human and pig MCPs .
What is the tissue expression pattern of MCP in rats?
Unlike human MCP, which is widely expressed, rat MCP exhibits a highly restricted expression pattern. Northern blot analysis reveals high-level expression almost exclusively in the testis, with a single product of approximately 1.6 kb. Using more sensitive reverse transcription-polymerase chain reaction (RT-PCR) methods, low-level expression can be detected in lung, small intestine, and kidney, with even weaker expression in brain, spleen, skeletal muscle, liver, and heart. No expression is detected in the ovary. This restricted expression pattern suggests that rat MCP may have evolved functions distinct from or additional to complement regulation, particularly in reproduction .
How does rat MCP relate to other complement regulators in rodents?
Rats possess multiple complement regulators, including MCP, decay-accelerating factor (DAF/CD55), and Crry. While MCP shows restricted expression primarily in testis, both DAF and Crry are widely distributed. Crry possesses both decay-accelerating and cofactor functions and is essential for complement homeostasis in vivo. Given these activities and distributions, Crry appears to be the major regulator of alternative pathway activation on most rat cell types, while classical pathway regulation likely involves both DAF and Crry. MCP may provide additional alternative pathway regulation specifically in testis, though its unique expression at this site strongly suggests additional roles in spermatogenesis or sperm survival .
What methods are most effective for cloning and expressing rat MCP?
The most effective approach for cloning rat MCP involves screening both cDNA and genomic libraries. Researchers have successfully identified rat MCP by:
-
Screening a rat testis cDNA library using a probe based on mouse MCP cDNA sequence
-
Isolating positive clones containing sequences compatible with MCP
-
Complementing missing portions through screening of a rat genomic library
-
Sequencing the genomic clone (such as pBSRG-1) with a reverse primer designed from the cDNA sequence
For expression studies, rat MCP can be effectively produced in recombinant form using adenoviral constructs in human fetal foreskin fibroblasts (HFFF). When expressed as a soluble protein, rat MCP appears as a single major protein species of approximately 25,000 Da molecular weight on SDS-PAGE analysis .
How can tissue expression of rat MCP be accurately determined?
A multi-technique approach is recommended for comprehensive assessment of rat MCP expression:
-
Northern blotting: Best for detecting high-level expression in tissues like testis, showing a single product of approximately 1.6 kb
-
RT-PCR: More sensitive for detecting low-level expression in other tissues
-
In situ hybridization: Optimal for cellular localization within tissues
For in situ hybridization specifically, anti-sense probes derived from rat MCP cDNA can localize MCP mRNA to specific cells within tissues. In testis, this technique reveals expression in areas containing Sertoli cells and spermatogonia within seminiferous tubules, while Leydig cells, connective tissue, and mature sperm in the tubule lumen remain negative .
What are the appropriate experimental controls when studying rat MCP function?
When studying rat MCP function, particularly its cofactor activity, the following controls are essential:
-
Negative control: C3ma (methylamine-inactivated human C3) incubated with factor I alone
-
Positive control: C3ma incubated with factor I and soluble human CR1 (sCR1)
-
Background control: Supernatant from cells infected with control virus containing no insert
These controls allow for proper assessment of cofactor activity as demonstrated by the appearance of C3 cleavage products of molecular weights 43,000 and 46,000 Da. For in situ hybridization studies, sense probes should be used as controls to assess nonspecific binding .
How can rat models be integrated into comprehensive studies of MCP function?
For researchers conducting comprehensive studies of MCP function using rat models, an integrated experimental design is recommended that follows these principles:
This integrated approach allows for comprehensive assessment of MCP function across different physiological contexts while optimizing animal use .
What are the functional implications of the restricted expression pattern of rat MCP?
The highly restricted expression pattern of rat MCP, primarily in testis, raises important research questions about its specialized functions:
-
Reproductive role: High expression in Sertoli cells and spermatogonia but absence in mature sperm suggests a role in spermatogenesis rather than sperm function
-
Complement regulation specificity: Despite showing factor I cofactor activity, the restricted expression indicates MCP is unlikely to be a major systemic complement regulator in rats
-
Evolutionary significance: The distinct expression pattern compared to human MCP suggests evolutionary divergence of function, with rodents relying more on Crry for systemic complement regulation
Researchers should design experiments that specifically address these potential specialized functions, particularly investigating roles in spermatogenesis that may be independent of complement regulation .
How do contradictory findings about rat MCP's role in complement regulation reconcile with its expression pattern?
The apparent contradiction between rat MCP's demonstrated cofactor activity and its highly restricted expression pattern can be addressed through several hypotheses that researchers may investigate:
-
MCP may provide localized complement protection specifically in the testis microenvironment
-
The primary role of rat MCP may have evolved away from complement regulation toward reproduction-specific functions
-
MCP may serve as a backup or specialized complement regulator in specific physiological contexts
To resolve these contradictions, researchers should design experiments that:
-
Compare the relative efficiency of rat MCP versus Crry in complement regulation
-
Investigate potential non-complement regulatory functions in testicular cells
-
Examine MCP expression under various physiological and pathological conditions
How does rat MCP compare with MCP in other species in experimental models?
| Species | Tissue Distribution | Domain Structure | Amino Acid Identity with Rat MCP | Primary Functions |
|---|---|---|---|---|
| Rat | Primarily testis, low expression in other tissues | 4 SCRs, STP-C domain, transmembrane, cytoplasmic tail | 100% | Factor I cofactor activity, possible reproductive role |
| Mouse | Primarily testis | 4 SCRs, STP domain, transmembrane, cytoplasmic tail | 88% | Factor I cofactor activity, possible reproductive role |
| Human | Widely expressed | 4 SCRs, STP domain (variable), transmembrane, cytoplasmic tail | Lower homology | Complement regulation, reproductive roles, pathogen receptor |
| Pig | Various tissues | Similar to human | Lower homology | Complement regulation |
This comparative analysis highlights the evolutionary divergence of MCP across species, with rodents showing a more specialized expression pattern compared to humans .
What are the recommended experimental designs for studying rat MCP in vivo?
For researchers investigating rat MCP in vivo, the following integrated experimental design is recommended:
-
Animal selection: Use Sprague-Dawley rats that meet OECD guidelines requirements
-
Exposure window: Include prenatal, lactational, and neonatal exposures when studying developmental effects
-
Multiple endpoints: Integrate protocols that assess developmental, reproductive, immunological, and long-term outcomes
-
Cohort design: Organize experimental animals into cohorts for specific endpoint assessment:
-
Cohort 1: Reproductive and developmental toxicity testing
-
Cohort 2: Developmental neurotoxicity testing
-
Cohort 3: Developmental immunotoxicity testing
-
-
Ethical considerations: Avoid culling when possible and utilize all pups generated during experiments to minimize unnecessary sacrifice of animals
This approach maximizes data collection while adhering to the 3Rs principles (replacement, reduction, refinement) .
What methods can be used to assess the cofactor activity of rat MCP?
The cofactor activity of rat MCP can be assessed using the following methodology:
-
Express soluble rat MCP (sMCP) using adenoviral constructs in human fetal foreskin fibroblasts
-
Collect supernatant containing sMCP (approximately 20 μg/ml total protein, with MCP accounting for about 50%)
-
Incubate 45 μl of supernatant (approximately 0.45 μg sMCP) with 0.5 μg purified methylamine-inactivated human C3 (C3ma) and 0.75 μg human factor I in a total volume of 50 μl
-
Incubate at 37°C for 16 hours
-
Dilute the mixture in an equal volume of SDS-PAGE sample buffer
-
Subject to SDS-PAGE on 10% acrylamide gels under reducing conditions
-
Blot onto nitrocellulose and probe with polyclonal antibody against human C3c
-
Develop using appropriate detection systems (e.g., ECL system)
-
Assess cleavage by identifying C3 cleavage products of 43,000 and 46,000 Da
This protocol allows for quantitative assessment of the cofactor activity of rat MCP compared to appropriate controls .
How can cellular localization of rat MCP be determined with precision?
Precise cellular localization of rat MCP requires a combination of techniques:
-
In situ hybridization for mRNA localization:
-
Prepare tissue sections (e.g., 5 μm thick)
-
Hybridize with antisense RNA probes derived from rat MCP cDNA
-
Include sense probes as negative controls
-
Process using standard in situ hybridization protocols
-
Counterstain to visualize tissue architecture
-
-
Immunohistochemistry for protein localization:
-
Generate specific antibodies against rat MCP
-
Process tissue sections using standard immunohistochemical techniques
-
Include appropriate negative controls (e.g., isotype antibodies)
-
Use confocal microscopy for precise subcellular localization
-
-
Flow cytometry for quantitative cellular expression:
-
Prepare single-cell suspensions from tissues of interest
-
Label with fluorescently-tagged anti-rat MCP antibodies
-
Analyze using flow cytometry to quantify expression levels
-
These complementary approaches provide comprehensive information about both the cellular and subcellular localization of rat MCP and its relative expression levels in different cell populations .
What are the most promising research directions for rat MCP studies?
Based on current knowledge, the most promising research directions include:
-
Reproductive biology: Investigating the specific roles of MCP in spermatogenesis, given its high expression in Sertoli cells and spermatogonia
-
Functional evolution: Comparing the activities and expression patterns of MCP across species to understand evolutionary divergence
-
Complement regulation specificity: Determining the relative contributions of MCP, DAF, and Crry in different tissues and physiological contexts
-
Antibody development: Generating specific antibodies against rat MCP to facilitate further studies of tissue distribution and function
-
Genetic manipulation: Creating conditional knockout models to assess the functional consequences of MCP deficiency in specific tissues or developmental stages
Progress in these areas will enhance understanding of the specialized functions of MCP in rats and its relevance to human biology and disease .
How might findings from rat MCP research translate to human applications?
The distinct expression patterns of MCP between rats and humans highlight both opportunities and limitations for translational research:
-
Reproductive medicine: Given the high expression in rat testis, findings may inform understanding of complement regulation in human reproductive tissues
-
Evolutionary insights: Comparative studies can illuminate how complement regulatory proteins have evolved different functions across species
-
Functional conservation: Despite expression differences, the demonstrated cofactor activity suggests functional conservation that may be relevant to human complement regulation
-
Model limitations: The restricted expression pattern in rats means they may not be optimal models for studying systemic functions of human MCP
-
Specialized niches: Rat models may be particularly valuable for studying tissue-specific roles of complement regulators in specialized microenvironments
Researchers should carefully consider these factors when designing studies intended to have translational relevance to human biology and disease .
Product Science Overview
Monocyte Chemotactic Protein-3 (MCP-3), also known as Chemokine (C-C motif) ligand 7 (CCL7), is a chemokine that plays a crucial role in the immune system by attracting monocytes, neutrophils, and other immune cells to sites of inflammation. It is part of the CC chemokine family, which is characterized by two adjacent cysteine residues near the amino terminus. MCP-3 is produced by various cell types, including macrophages and certain tumor cell lines .
Recombinant MCP-3 (CCL7) can be prepared using several methods. One common approach involves the expression of the protein in Escherichia coli (E. coli) cells. The gene encoding MCP-3 is cloned into an expression vector, which is then introduced into E. coli cells. The bacteria are cultured, and the protein is expressed as a fusion protein. The fusion protein is then purified using affinity chromatography techniques. The purified protein is often lyophilized for storage and reconstituted in sterile water or other aqueous solutions before use .
MCP-3 (CCL7) interacts with several G protein-coupled receptors, including CCR1, CCR2, and CCR3. These interactions trigger a cascade of intracellular signaling events that lead to the chemotaxis of immune cells. The biological activity of MCP-3 can be assessed using various assays, such as chemotaxis assays, which measure the ability of the protein to attract immune cells. Additionally, the protein’s purity and structural integrity can be analyzed using techniques like SDS-PAGE and RP-HPLC .
MCP-3 (CCL7) is involved in various physiological and pathological processes. It plays a significant role in the immune response by recruiting monocytes and other immune cells to sites of infection or injury. This chemokine is also implicated in the progression of chronic inflammatory diseases, such as cardiovascular disease, diabetes mellitus, and kidney disease. By attracting macrophages and monocytes, MCP-3 amplifies inflammatory processes and contributes to disease progression .
