CD154 Variant Lacking Tumor Necrosis Factor Homologous Domain Inhibits Cell Surface Expression of Wild-type Protein

Abstract

X-linked hyper-IgM (XHIM) syndrome is an immunological disorder resulting from mutations in the CD154 gene. Some mutations occur in splicing sites and result in transcripts encoding wild-type and mutant proteins. These mutants lack the tumor necrosis factor homologous (TNFH) domain and consequently fail to trimerize. Given that the TNFH domain is responsible for trimerization, one may predict that the TNFH mutant can not participate in the assembly of wild-type CD154. Thus, it was puzzling why these patients exhibit XHIM phenotype, presumably resulting from a lack of functional CD154. One possibility is that the TNFH mutant exhibits a dominant negative effect over the wild-type protein.To investigate this, we coexpressed the wild-type protein and a TNFH mutant and examined the biochemical and functional properties of the resulting CD154 products. We demonstrate that despite the lack of the TNFH domain, the TNFH mutant can associate with the wildtype protein. Furthermore, such an association compromises the ability of the wild-type protein to mature onto the cell surface. These results provide a mechanism for the defect of CD154 in XHIM patients producing both wild-type and TNFH variants and suggest that besides the TNFH domain, the stalk region participates in the assembly of CD154 trimers.CD40 ligand (CD154) is a type II membrane protein expressed primarily on activated T cells. The interaction of CD154 with its receptor, CD40, is critical for the functions of T helper cells to induce differentiation, proliferation, and Ig isotype switching in B cells (for review see Refs. 1 and 2). The CD154 gene, located at Xq2.6 -2.7, spans over 12 kilobase pairs and consists of five exons (3). The first exon encodes the cytoplasmic region, the transmembrane domain, and 6 amino acids of the extracellular domain. The second and third exons encode the extracellular stalk region. The fourth and fifth exons encode the C-terminal 147 amino acids (3), which share a limited homology with other members of the TNF 1 family and is called the TNF homologous (TNFH) domain. The x-ray structure of the CD154 TNFH domain reveals that it contains a sandwichlike fold of two ␤ sheets with jellyroll or Greek key topology and forms a trimer similar to that seen in TNF and lymphotoxin-␣ (4). Given the facts that for CD154, the TNFH domain alone is capable of forming trimers (4, 5) and that deletion mutants missing a major portion of this domain did not trimerize (6), it appeared that the TNFH domain is necessary and sufficient for the assembly of trimeric CD154 protein.Mutations in the CD154 gene can prevent the expression of functional CD154 protein, which lead to an immunodeficiency characterized by an elevated IgM and low IgG and IgA levels in serum called X-linked hyper-IgM (XHIM) syndrome (7-11). Over 70 unique mutations in the CD154 gene have been identified in more than 100 patients (12). These mutations are very heterogeneous and include insertions, deletions, and point mutations. Thus, it is conceivable that the underlying mechanisms res...

Three novel mutations reflect the variety of defects causing phenotypically diverse X-linked hyper-IgM syndrome

“…The correctly spliced mRNA would be translated to generate wild‐type molecules that would associate in functional homotrimers [31–33]. Recently, Su and coworkers [34] demonstrated by a co‐transfection experiment in COS cells that CD40L mutants lacking the TNFH domain are still able to form a complex with the wild‐type molecule. However, this association compromises the ability of the complex to mature and be expressed on the cell surface do, however, and therefore the probabilities of assembling a functional homotrimer are greatly decreased.…”

Immunological and genetic analysis of 65 patients with a clinical suspicion of X linked hyper-IgM

“…7 In addition, patients may express a non-functional form of CD40L and thus, although normal by protein expression analysis, have XHIM syndrome. 10 Like most primary immunodeficiencies, no genotype-phenotype correlation has been described. 11 12 We have reviewed results obtained using a whole blood CD40L expression assay and describe the correlation between protein expression and mutation detection.…”