CD40L/CD154/TRAP Protein, Mouse (HEK293, Fc)

CD40L (CD154; TRAP) is a ligand to CD40/TNFRSF5, acts function by generating a costimulatory signal that up-regulates IL-4 synthesis^[1]. CD40L is specifically expressed on activated CD4+ T-lymphocytes and involves in activation of NF-κB/MAPK pathway^[2][3]. CD40L also involves in B cell differentiation, maturation, and apoptosis^[4]. CD40L in mouse, cleaved into 2 chains of membrane form (1-260 a.a.) and soluble form (112-260 a.a.) which serves as a ligand for integrins (ITGA5:ITGB1 and ITGAV:ITGB3). CD40L/CD154/TRAP Protein, Mouse (HEK293, Fc) is expressed in HEK392 cells with N-terminal hFc-tag.

CD40 Ligand (CD40L; CD154; TRAP) belongs to the tumor necrosis factor (TNF) family, is the ligand for CD40/TNFRSF5, specifically expressed on activated CD4+ T-lymphocytes^[1].
CD40L is a type II transmembrane protein on B cells triggers important signals for B cell differentiation, maturation, and apoptosis^[4].
CD40L acts function by cross-linking on T-cells to generate a costimulatory signal and thus enhances the production of IL4 and IL10 in conjunction with the TCR/CD3 ligation and CD28 costimulation, as well as promoting the production of interferon-γ, and TNF-α^[1][4].
CD40L, binding with CD40 on antigen-presenting cells (APC), activates TNFR-associated factor 2- and IKK2-dependent pathways with stimulating I-κB kinase (IKK), increasing NF-κB DNA binding, and p65 nuclear translocation. The activation of I-κB kinase leads to strongly c-Jun N-terminal kinase activation as well as GST-I-κB and GST-p65 phosphorylation^[2].
CD40L involves in MAPK pathways that strongly repress Bcl-6 with inducing the phosphorylation of Erk1/2, p38 and Jnk1/2 and activating IRF4 mediated by NF-κB^[3].
CD40L also binds to and signals through several integrins, including αvβ3 and α5β1, which bind to the trimeric interface of CD40L. CD40L plays a major role in immune response and is a major target for inflammation^[5].
CD40L is widely found in different animals, while the sequence in Mouse is highly similar to Rat (93.85%), but very different from Human and Rhesus macaque with similarities of 77.69% and 77.31%, respectively. CD40L in Mouse is cleaved into 2 chains of membrane form (1-260 a.a.) and soluble form (112-260 a.a.), while the soluble form in human derives from the membrane form by proteolytic processing. Release of soluble CD40L from platelets is partially regulated by GP IIb/IIIa, actin polymerization, and a matrix metalloproteinases (MMP) inhibitor-sensitive pathway^[6].

CD4 Ligand lacking in mice (CD4L-deficient) results CLP-induced edema and myeloperoxidase activity in the lung as well as neutrophil infiltration in the broncoalveolar space markedly reduced^[7].

CD4 Ligand (1 ng/mL; 1 min) doesn’t stimulate Mac-1 expression in murine blood neutrophils indicating a MIP-2-dependent manner^[7].
CD4 Ligand (1 ng/mL; 1 min) regulates plasma levels of MIP-2 in plasma of cecal ligation and puncture (CLP) mice^[7].

1.Measured by its binding ability in a functional ELISA. Immobilized Mouse CD40 His at 2 μg/mL (100 μl/well) can bind Mouse CD40 Ligand hFc, the EC₅₀ of Mouse CD40 Ligand hFc is 60-300 ng/mL.
2.Measured in a cell proliferation assay using human B cells (Ramos) in the presence of 10 ng/mL Human IL-4. The ED₅₀ this effect is 0.2651 ng/mL, corresponding to a specific activity is 3.7722×10⁶ units/mg.
3. Measured in a cell proliferation assay using mouse splenic B cells in the presence of 10 ng/mL IL-4. The ED₅₀ this effect is 0.39 ng/mL, corresponding to a specific activity is 2.56×10⁶ units/mg.

• 
  Measured in a cell proliferation assay using human B cells (Ramos) in the presence of 10 ng/mL Human IL-4. The ED₅₀ this effect is 0.2651 ng/mL, corresponding to a specific activity is 3.7722×10⁶ units/mg.

Mouse

HEK293

N-hFc

P27548 (G115-L260)

21947  [NCBI]

Full Length of CD40L soluble form

GDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKL

Approximately 46.23 kDa

• 
  ≥ 90%, as determined by reducing SDS-PAGE.

Lyophilized powder

1.Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4, 10% glycerol, 5% trehalose, 5% mannitol, 0.01% tween 80.
2.Lyophilized from a 0.22 μm filtered solution of 20 mM PB, 150 mM NaCl, pH 7.4.
Please refer to the lot-specific COA for specific buffer information.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O. For long term storage it is recommended to add a carrier protein (0.1% BSA, 5% HSA, 10% FBS or 5% Trehalose).

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.

• [1]. /biology-dictionary/hs27-cell.html  [Content Brief]

  [2]. Blotta MH, et al. Cross-linking of the CD40 ligand on human CD4+ T lymphocytes generates a costimulatory signal that up-regulates IL-4 synthesis. J Immunol. 1996 May 1;156(9):3133-40.  [Content Brief]

  [3]. Schwabe RF, et al. CD40 activates NF-kappa B and c-Jun N-terminal kinase and enhances chemokine secretion on activated human hepatic stellate cells. J Immunol. 2001 Jun 1;166(11):6812-9.  [Content Brief]

  [4]. Batlle A, et al. CD40 and B-cell receptor signalling induce MAPK family members that can either induce or repress Bcl-6 expression. Mol Immunol. 2009 May;46(8-9):1727-35.  [Content Brief]

  [5]. Mikolajczak SA, et al. The modulation of CD40 ligand signaling by transmembrane CD28 splice variant in human T cells. J Exp Med. 2004 Apr 5;199(7):1025-31.  [Content Brief]

  [6]. Takada YK, et al. Soluble CD40L activates soluble and cell-surface integrin αvβ3, α5β1, and α4β1 by binding to the allosteric ligand-binding site (site 2). J Biol Chem. 2021 Jan-Jun;296:100399.  [Content Brief]

  [7]. Pietravalle F, et al. Human native soluble CD40L is a biologically active trimer, processed inside microsomes. J Biol Chem. 1996 Mar 15;271(11):5965-7.  [Content Brief]

  [8]. Rahman M, et al. Platelet-derived CD40L (CD154) mediates neutrophil upregulation of Mac-1 and recruitment in septic lung injury. Ann Surg. 2009 Nov;250(5):783-90.  [Content Brief]