LGMN Mouse
Description
Cancer Biology
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Tumor Metastasis: Overexpression of LGMN promotes tumor invasion by activating pro-MMP-2 and integrin αvβ3 signaling .
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Biomarker Potential: Elevated LGMN correlates with advanced tumor grades in cervical, breast, and ovarian cancers .
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Therapeutic Target: DNA vaccines targeting LGMN reduced tumor burden in murine models by suppressing oncogenic cytokines .
Cardiovascular Pathology
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Aortic Dissection: LGMN deficiency in mice (Lgmn −/−) reduced aortic rupture risk by 40% and mitigated extracellular matrix degradation .
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Mechanism: Macrophage-derived LGMN binds integrin αvβ3 in vascular smooth muscle cells (VSMCs), disrupting Rho GTPase signaling and promoting VSMC dedifferentiation .
In Vitro and In Vivo Models
Enzymatic Regulation
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Activation: Auto-cleavage at acidic pH (≤5.5) generates mature 46 kDa and 36 kDa forms .
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Inhibition: Cystatin E/M and RGD motif-targeted nanoparticles suppress LGMN activity .
Therapeutic Implications
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Small-Molecule Inhibitors: Aza-Asn epoxides block LGMN activity, reducing breast cancer cell proliferation .
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Nanoparticle Vaccines: Mutant DNA vaccines with RGD modifications enhanced immunogenicity and tumor suppression in mice .
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Cardiovascular Protection: Pharmacologic LGMN inhibitors attenuated aortic dissection progression in murine models .
Product Specs
Produced in Sf9 Baculovirus cells, LGMN is a single, glycosylated polypeptide chain consisting of 426 amino acids (18-435a.a.) with a molecular weight of 48.6kDa. This protein is expressed with an 8 amino acid His tag at the C-terminus and purified using proprietary chromatographic methods.
Q&A
What is the biological role of LGMN in mouse models, and how is it experimentally validated?
LGMN, an asparaginyl endopeptidase, mediates proteolytic processing in lysosomes and extracellular environments. In mouse cancer models, its overexpression accelerates tumor cell invasion by activating matrix metalloproteinases (MMP-2/MMP-9) and PI3K/AKT pathways . Validation methods include:
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Knockout (KO) models: Lgmn<sup>-/-</sup> mice exhibit reduced tumor growth in breast cancer xenografts .
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Activity assays: Fluorogenic substrates (e.g., Z-Ala-Ala-Asn-AMC) quantify enzymatic activity in tissue lysates .
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Immunohistochemistry (IHC): Spatial expression patterns in tumor-associated macrophages (TAMs) and neovascular endothelia are mapped using anti-LGMN antibodies .
How do researchers detect LGMN expression and activity in murine tissues?
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qRT-PCR: Primers targeting Lgmn exons 3–5 (e.g., Forward: 5′-CTG GAC CCT GGA GAA GAT GA-3′; Reverse: 5′-TCC TTG GTG CTC TTG TTC TG-3′) quantify transcriptional levels .
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Western blot: Distinguishes pro-LGMN (56 kDa), intermediate (46 kDa), and mature (36 kDa) forms under pH-controlled conditions .
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In vivo imaging: Legumain-activated fluorescent probes (e.g., Cy5-AAN) visualize tumor margins in orthotopic models .
What experimental designs resolve contradictions in LGMN’s dual roles in tumor promotion vs. suppression?
Discrepancies arise from tissue-specific contexts:
Methodological resolution:
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Conditional knockout systems: Myeloid- (Lyz2-Cre) or VSMC-specific (Myh11-Cre) deletions isolate cell-type contributions .
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Pathway inhibition: Co-administer PI3K inhibitors (LY294002) or ROCK inhibitors (Y-27632) to dissect signaling crosstalk .
How does LGMN regulate integrin signaling in vascular smooth muscle cells (VSMCs)?
LGMN binds integrin αvβ3 via its RGD motif, blocking downstream Rho GTPase activation and suppressing contractile markers (e.g., α-SMA, SM22α) . Experimental approaches:
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Co-culture assays: Macrophage-VSMC co-cultures + LGMN-neutralizing antibodies restore α-SMA expression by 2.5-fold .
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Surface plasmon resonance: KD ≈ 12 nM for LGMN-integrin αvβ3 binding .
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Phenotypic rescue: ROCK inhibitor Y-27632 reverses LGMN-induced VSMC dedifferentiation .
What are the technical challenges in modeling LGMN’s role in tumor microenvironment (TME) crosstalk?
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Stromal heterogeneity: Single-cell RNA sequencing (scRNA-seq) of Lgmn<sup>GFP</sup> mice reveals TAM subtypes with divergent LGMN expression .
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Exosome trafficking: Ultracentrifugation (100,000 × g) isolates LGMN<sup>+</sup> exosomes from ovarian cancer ascites, which transfer functional protease activity to mesothelial cells .
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Dynamic imaging: Intravital microscopy captures real-time LGMN activity at tumor-stroma interfaces .
How to optimize LGMN inhibition studies in vivo?
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Pharmacologic inhibitors: Aza-Asn epoxides (AEPi) reduce tumor growth by 50% in MDA-MB-231 xenografts .
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Dosing regimens: 10 mg/kg AEPi i.p., twice weekly, balances efficacy and toxicity .
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Validation metrics: Plasma LGMN ELISAs and MMP-9 zymography confirm target engagement .
What controls are critical for genetic perturbation studies?
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Cre-negative littermates: Control for off-target effects in Lgmn<sup>fl/fl</sup>; Cre<sup>+</sup> models .
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Sham surgeries in TAD models: Differentiate BAPN-induced pathology from procedural artifacts .
Why do some studies report LGMN as a tumor suppressor?
Context-dependent roles emerge from:
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Isoform specificity: Nuclear LGMN (13–17% in colon cancer) may antagonize cytoplasmic forms .
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Substrate bias: Overexpression screens identify thrombospondin-1 cleavage → anti-angiogenic fragment generation .
Resolution strategy:
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Isoform-selective KO: CRISPR-Cas9 targeting nuclear localization signals (NLS).
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Substrate profiling: TAILS N-terminomics identifies context-dependent cleavage targets .
Future Directions Table
| Priority Area | Tools | Hypothesis |
|---|---|---|
| LGMN-immune crosstalk | Single-cell ATAC-seq | LGMN<sup>+</sup> TAMs exhibit distinct chromatin accessibility |
| Metabolic regulation | Stable isotope tracing | LGMN degrades LDHA → Warburg shift |
| Therapeutic targeting | PROTAC degraders | LGMN-directed degradation outperforms catalytic inhibition |
Product Science Overview
Legumain is synthesized as an inactive zymogen and undergoes autocatalytic cleavage to become active. The active form of legumain has a molecular weight of approximately 49 kDa . It is characterized by its ability to cleave peptide bonds on the C-terminal side of asparagine residues, which is a unique feature among proteases.
- Protein Degradation: Legumain is involved in the degradation of intracellular proteins within lysosomes. It contributes to the turnover of proteins and the maintenance of cellular homeostasis.
- Antigen Presentation: In the immune system, legumain plays a role in the processing of antigens for presentation by major histocompatibility complex (MHC) class II molecules. This is essential for the activation of T cells and the initiation of immune responses.
- Extracellular Matrix Remodeling: Legumain is implicated in the remodeling of the extracellular matrix (ECM), which is important for tissue repair and regeneration. It has been shown to degrade ECM components, such as collagen, and is involved in processes like wound healing and fibrosis .
Recombinant mouse legumain is produced using genetic engineering techniques. The gene encoding mouse legumain is cloned and expressed in a suitable host system, such as a mouse myeloma cell line (NS0). The recombinant protein is then purified to high levels of purity, typically greater than 95% as determined by SDS-PAGE .
The recombinant form of legumain retains its enzymatic activity and is used in various research applications, including studies on protein degradation, antigen processing, and ECM remodeling. It is also utilized in drug discovery and development, particularly in the context of diseases where legumain activity is dysregulated, such as cancer and cardiovascular diseases .
- Cancer Research: Legumain is overexpressed in several types of cancer, and its activity is associated with tumor progression and metastasis. Inhibitors of legumain are being explored as potential therapeutic agents for cancer treatment.
- Cardiovascular Diseases: Elevated levels of legumain have been linked to adverse outcomes in cardiovascular diseases, such as acute myocardial infarction (AMI). Research has shown that legumain contributes to ECM degradation and cardiac remodeling post-AMI, making it a potential target for therapeutic intervention .
- Immunology: The role of legumain in antigen processing makes it a valuable tool for studying immune responses and developing vaccines. Recombinant legumain can be used to investigate the mechanisms of antigen presentation and T cell activation.
