Identification of three alternatively spliced variants of human CD28 mRNA.

CD28, expressed by T cells, plays a central role in providing costimulatory signals to T cells. The cd28 gene is organized into 4 exons. An alternatively spliced CD28 mRNA lacking most of the exon 2 has been previously evidenced. We report here that non stimulated human T cells express three additional alternatively spliced variants of CD28 mRNA (CD28a-c) in. The CD28a variant, expressed at similar levels to that of the full length CD28 mRNA encoding for the membrane form, lacks exon 3. This deletion introduces (i) a frame shift resulting in the addition of two extra amino acids and a premature stop codon and, (ii) induces the loss of the transmembrane region, suggesting that it could encodes for a soluble monomeric molecule which conserves the binding sites of CD28. The CD28b and CD28c variants, expressed at a low level compared with CD28a, are generated by deletion of most of the 3' end of exon 2 plus exon 3 and exon 2 plus exon 3, respectively. Activated T cells express only the membrane CD28 mRNA. These results suggest that resting human T cells may constitutively express both membrane and soluble CD28 which can differentially regulate the outcome of the T cell response.     

CD44 isoforms containing exon V3 are responsible for the presentation of heparin-binding growth factor

Glycosaminoglycan-modified isoforms of CD44 have been implicated in growth factor presentation at sites of inflammation. In the present study we show that COS cell transfectants expressing CD44 isoforms containing the alternatively spliced exon V3 are modified with heparan sulfate (HS). Binding studies with three HS-binding growth factors, basic-fibroblast growth factor (b-FGF), heparin binding-epidermal growth factor (HB-EGF), and amphiregulin, showed that the HS-modified CD44 isoforms are able to bind to b-FGF and HB-EGF, but not AR. b-FGF and HB-EGF binding to HS-modified CD44 was eliminated by pretreating the protein with heparitinase or by blocking with free heparin. HS- modified CD44 immunoprecipitated from keratinocytes, which express a CD44 isoform containing V3, also bound to b-FGF. We examined whether HS- modified CD44 isoforms were expressed by activated endothelial cells where they might present HS-binding growth factors to leukocytes during an inflammatory response. PCR and antibody-binding studies showed that activated cultured endothelial cells only express the CD44H isoform which does not contain any of the variably spliced exons including V3. Immunohistological studies with antibodies directed to CD44 extracellular domains encoded by the variably spliced exons showed that vascular endothelial cells in inflamed skin tissue sections do not express CD44 spliced variants. Keratinocytes, monocytes, and dendritic cells in the same specimens were found to express variably spliced CD44. 35SO4(-2)-labeling experiments demonstrated that activated cultured endothelial cells do not express detectable levels of chondroitin sulfate or HS-modified CD44. Our results suggest that one of the functions of CD44 isoforms expressing V3 is to bind and present a subset of HS-binding proteins. Furthermore, it is probable that HS- modified CD44 is involved in the presentation of HS-binding proteins by keratinocytes in inflamed skin. However, our data suggests that CD44 is not likely to be the proteoglycan principally involved in presenting HS- binding growth factors to leukocytes on the vascular cell wall.     

The effect of intracellular trafficking of CD1d on the formation of TCR repertoire of NKT cells

CD1 molecules belong to non-polymorphic MHC class I-like proteins and present lipid antigens to T cells. Five different CD1 genes (CD1a-e) have been identified and classified into two groups. Group 1 include CD1a-c and present pathogenic lipid antigens to αβ T cells reminiscence of peptide antigen presentation by MHC-I molecules. CD1d is the only member of Group 2 and presents foreign and self lipid antigens to a specialized subset of αβ T cells, NKT cells. NKT cells are involved in diverse immune responses through prompt and massive production of cytokines. CD1d-dependent NKT cells are categorized upon the usage of their T cell receptors. A major subtype of NKT cells (type I) is invariant NKT cells which utilize invariant Vα14-Jα18 TCR alpha chain in mouse. The remaining NKT cells (type II) utilize diverse TCR alpha chains. Engineered CD1d molecules with modified intracellular trafficking produce either type I or type II NKT cell-defects suggesting the lipid antigens for each subtypes of NKT cells are processed/generated in different intracellular compartments. Since the usage of TCR by a T cell is the result of antigen-driven selection, the intracellular metabolic pathways of lipid antigen are a key in forming the functional NKT cell repertoire. [BMB Reports 2014; 47(5): 241-248]     

Genomic structure, alternative splicing, and physical mapping of the killer cell lectin-like receptor G1 gene (KLRG1), the mouse homologue of MAFA

The mouse killer cell lectin-like receptor G1 (KLRG1), the mouse homologue of the mast cell function-associated antigen (MAFA), is an inhibitory C-type lectin expressed on natural killer (NK) cells and activated CD8 T cells. Here we report the complete nucleotide sequence, alternatively spliced variants, and the physical mapping of the KLRG1 gene in the mouse. The gene spans about 13 kb and consists of five exons. Short interspersed repeats of the B1 and B2 family, a LINE-1-like element, and a (CTT)170 triplet repeat were found in intron sequences. In contrast to human KLRG1 and to the murine KLR family members, mouse KLRG1 locates outside the NK complex on Chromosome 6 between the genes encoding CD9 and CD4.     

Regulation of CD44 binding to hyaluronan by glycosylation of variably spliced exons

The hyaluronan (HA)-binding function (lectin function) of the leukocyte homing receptor, CD44, is tightly regulated. Herein we address possible mechanisms that regulate CD44 isoform-specific HA binding. Binding studies with melanoma transfectants expressing CD44H, CD44E, or with soluble immunoglobulin fusions of CD44H and CD44E (CD44H-Rg, CD44E-Rg) showed that although both CD44 isoforms can bind HA, CD44H binds HA more efficiently than CD44E. Using CD44-Rg fusion proteins we show that the variably spliced exons in CD44E, V8-V10, specifically reduce the lectin function of CD44, while replacement of V8-V10 by an ICAM-1 immunoglobulin domain restores binding to a level comparable to that of CD44H. Conversely, CD44 bound HA very weakly when exons V8-V10 were replaced with a CD34 mucin domain, which is heavily modified by O- linked glycans. Production of CD44E-Rg or incubation of CD44E- expressing transfectants in the presence of an O-linked glycosylation inhibitor restored HA binding to CD44H-Rg and to cell surface CD44H levels, respectively. We conclude that differential splicing provides a regulatory mechanism for CD44 lectin function and that this effect is due in part to O-linked carbohydrate moieties which are added to the Ser/Thr rich regions encoded by the variably spliced CD44 exons. Alternative splicing resulting in changes in protein glycosylation provide a novel mechanism for the regulation of lectin activity.