CD40L/CD154/TRAP Protein, Human (HEK293, hFc-Flag)

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 human, cleaved into 2 chains of membrane form (1-261 a.a.) and soluble form (113-261 a.a.) which serves as a ligand for integrins (ITGA5:ITGB1 and ITGAV:ITGB3). CD40L/CD154/TRAP Protein, Human (HEK293, hFc-Flag) has a total length of 248 amino acids (M13-L261), is expressed in HEK392 cells with N-terminal hFc- and Flag-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 Human is highly similar to Rhesus macaque (98.08%), but very different from Rat and Mouse with similarities of 77.31% and 77.69%, respectively. CD40L in Human is cleaved into 2 chains of membrane form (1-261 a.a.) and soluble form (113-261 a.a.), while the soluble form 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].

CD40 Ligand (2.86 ng/mL or 2.09 ng/mL) has been demonstrated to be a mark of cardiovascular risk, while the level (2.86 ng/mL) in who subsequently developed myocardial infarction, stroke, or cardiovascular death (cases) is higher than control group (2.09 ng/mL)^[8].

CD40 Ligand (1 ng/mL; 24 hr) induces endothelial cell vascular cell adhesion molecule-1 (VCAM-1) expression. CD40 Ligand shows costimulation effect with IL-4 against VCAM-1^[7].
CD40 Ligand (1 ng/mL; 24 hr) combined with IL-4 enhances IL-6 and inhibits IL-8 production by HUVEC^[7].

1.Measured by its binding ability in a functional ELISA. Immobilized CD40L at 2 μg/mL can bind Anti-CD40L Rabbit Monoclonal Antibody, the EC₅₀ is ≤3.3 ng/mL.
2.Measured by its binding ability in a functional ELISA. Immobilized CD40 at 2 μg/mL can bind CD40L, the EC₅₀ is 3.112-3.858 ng/mL.
3.Human CD40 protein hFc tag captured on COOH chip can bind Human CD40L protein hFc and Flag tag with an affinity constant < 2.06 nM as detected by LSPR Assay.
4. Immobilized Human CD40 Ligand Trimer, His Tag at 1 μg/mL (100 μl/Well) on the plate. Dose response curve for Human CD40, hFc Tag with the EC₅₀ of 1.29 μg/mL determined by ELISA.
5.Measured in a cell proliferation assay using human B cells (Ramos) in the presence of 10 ng/mL Human IL-4 (HY-P70445). The ED₅₀ for this effect is 2.718 ng/mL, corresponding to a specific activity is 3.679×10⁵ units/mg.

• 
  Measured in a cell proliferation assay using human B cells (Ramos) in the presence of 10ng/mL Human IL-4 (HY-P70445). The ED₅₀ for this effect is 2.718 ng/mL, corresponding to a specific activity is 3.679×10⁵ units/mg.

Human

HEK293

N-hFc;N-Flag

P29965 (M113-L261)

959  [NCBI]

MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

Approximately 44-50 kDa due to the glycosylation.

• 
  Greater than 95% as determined by reducing SDS-PAGE.

Lyophilized powder

Lyophilized from a 0.2 μm solution of PBS, 6% Trehalose, pH 7.4 or PBS, pH 7.4, 8% trehalose.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O.

Stored at -20°C for 2 years. 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]. 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]

  [2]. 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]

  [3]. 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]

  [4]. 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]

  [5]. 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]

  [6]. 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]

  [7]. Kotowicz K, et al. Biological function of CD40 on human endothelial cells: costimulation with CD40 ligand and interleukin-4 selectively induces expression of vascular cell adhesion molecule-1 and P-selectin resulting in preferential adhesion of lymphocytes. Immunology. 2000 Aug;100(4):441-8.  [Content Brief]

  [8]. Schönbeck U, et al. Soluble CD40L and cardiovascular risk in women. Circulation. 2001 Nov 6;104(19):2266-8.  [Content Brief]

  [9]. Lee N, et al. Comparison of Flow-cytometric Antibody Secreting Cell Assay and Mabtech Immunoglobulin ELISpot Assay. Transplant Proc. 2017 Jun;49(5):963-966.  [Content Brief]