Identification of Residues on CD40 and Its Ligand Which Are Critical for the Receptor-Ligand Interaction

Abstract

Interactions between gp39 (CD40L, TRAP, T-BAM) on activated T cells and CD40 on antigen-presenting cells play an important role in regulating antibody production by B cells, cytokine production by monocytes, and other immune responses which require T cell "help". Using structure-based sequence alignments, a molecular model of gp39, site-directed mutagenesis, and receptor-ligand binding assays, we have identified CD40 and gp39 surface residues which are important for receptor-ligand binding. Binding studies with CD40 or gp39 proteins containing single and double amino acid substitutions showed that CD40 residues Y82, D84, and N86 are involved in gp39 binding, while gp39 residues K143 and Y145 are important for CD40 binding. Analysis of the location of amino acid substitutions in the naturally occurring gp39 mutants expressed by the X-linked hyper-IgM (X-HIM) patients studied to date indicated the E129/G substitution found in the S128/R-E129/G double mutant affects a solvent-accessible residue which might participate in CD40/gp39 binding. Binding studies with E129/G and E129/A gp39 point mutants showed that this residue does not contribute directly to CD40/gp39 binding but that its substitution with a glycine disrupts the gp39 structure. Comparison of the gp39 and CD40 residues involved in receptor-ligand contacts with those previously identified as playing an important role in TNF-beta/TNFR binding suggests that some of the identified residues from contacts similar to those found in the TNF-beta/TNFR while others are unique to the CD40-gp39 interaction.

Incorporation of an Isoleucine Zipper Motif Enhances the Biological Activity of Soluble CD40L (CD154)

“…Such structural restraint of the N terminus may also affect the way in which the ligand interacts with the receptor and explain the enhanced bioactivity. Site-directed mutagenesis has shown that Lys 143 and Tyr 145 are critical for CD40L interaction with CD40 (27). These residues are in close proximity to the N terminus.…”

Early Diversification of the TNF Superfamily in Teleosts: Genomic Characterization and Expression Analysis

“…Similar trees were obtained using maximum parsimony analyses and by systematically adding or removing sequences from the phylogenetic analyses. (22). A potential glycosylation site (Asn 240 ) described in human CD40L is indicated ( ‡) (22).…”

“…(22). A potential glycosylation site (Asn 240 ) described in human CD40L is indicated ( ‡) (22). Abbreviations: Hs, Homo sapiens; Mm, Mus musculus; Xt, X. tropicalis; Om, O. mykiss; Tn, T. nigroviridis.…”

“…Through single and double amino acid substitutions, five amino acids (Gln 220 , Arg 203 , Lys 143 , Tyr 145 , and Tyr 146 ) have been found to be important for CD40L-CD40 binding in mammals (22). None of these residues were found to be conserved in alignment analysis of teleost O. mykiss, S. salar, Tetraodon nigroviridis, Fugu rubripes, D. rerio, P. olivaceus, and G. aculeatus CD40L sequences (Fig.…”

Functional Interaction of CD154 Protein with α5β1 Integrin Is Totally Independent from Its Binding to αIIbβ3 Integrin and CD40 Molecules

“…With the TNF-␤/TNF receptor interaction in mind, Bajorath et al (29) have identified the CD40 and CD154 residues that are ؉ BJAB cells. BJAB (A) or U937 (B) cell lines were stimulated at 37°C with 250 ng of sCD154WT or each of its mutants for the indicated time points.…”