Nomenclature of prominin-1 (CD133) splice variants - an update.

Prominin-1 (CD133), a pentaspan membrane glycoprotein that constitutes an important cell surface marker of various, either normal or cancerous, stem cell populations is widely used to isolate or characterize such cells in different systems. Occurring throughout the metazoan evolution with a remarkably conserved genomic organization, it may be expressed as different splice variants with distinctive characteristics. A rational nomenclature has been proposed earlier for their consistent designation across species. Although generally accepted, it seems to be misunderstood in view of the recent report of novel prominin-1 complementary DNAs in rhesus monkey and humans with improper naming. As this may lead to confusion, we have reexamined the genomic organization of prominin-1 in various primates to provide an update that should further clarify the rationale of the nomenclature for prominin-1 gene products. This report comprises (i) the determination of the genomic organization of prominin-1 gene in two non-human primates, i.e. Macaca mulatta and Pan troglodytes, commonly used in research, (ii) the mapping of a new exon that creates an alternative cytoplasmic C-terminal end of prominin-1, (iii) the identification of various potential PDZ-binding domains generated by alternative cytoplasmic C-terminal tails, suggesting that different prominin-1 splice variants might interact with distinct protein partners, and (iv) a summing up of the different prominin-1 splice variants.

Rat prominin, like its mouse and human orthologues, is a pentaspan membrane glycoprotein.

Mouse prominin is the first characterized member of a novel family of membrane glycoproteins. It displays a characteristic membrane topology with five transmembrane segments and two large glycosylated extracellular loops. Prominin orthologues and paralogues have been identified in human, fish, fly, and worm. Recently, a cDNA sequence encoding the rat homologue of mouse prominin has been reported [Zhu et al. (2001) Biochem. Biophys. Res. Commun. 281, 951-956]. Surprisingly, due to a single nucleotide deletion that shifts the reading frame and introduces a premature stop codon, the protein predicted from this cDNA would correspond to a C-terminally truncated form of prominin with only four transmembrane segments. Here we report evidence that is in contrast to the report of Zhu et al. (2001). We isolated a rat prominin cDNA devoid of any frameshift mutation, demonstrate that rat prominin, like the other mammalian prominins, is a full-length 120-kDa pentaspan membrane glycoprotein, and have not been able to detect any C-terminally truncated form of rat prominin.

Conformational alterations during biosynthesis of HLA-DR3 molecules controlled by invariant chain and HLA-DM.

HLA-DM is known to catalyze the exchange of class II-associated invariant chain (Ii) peptide (CLIP) for cognate peptide during biosynthesis. In DM-negative cells HLA-DR3 molecules have been shown to predominantly present CLIP and to lack the DR3-specific mAb epitope 16.23, which has led to the assumption that CLIP prevents binding of mAb 16.23. In the present study we show that CLIP does not prohibit 16.23 epitope expression, but that the formation of this epitope is directly influenced by interactions of the DR molecule with Ii and DM. Detergent solubilized DR3 from wild-type as well as DM(-) cells bound CLIP in a 16.23(+) mode. On cells, however, neither CLIP nor antigenic peptide bound to DR3 in a 16.23(+) conformation, unless HLA-DM was expressed. Thus, HLA-DM appears to alter the conformation of DR3 in a peptide-independent fashion. Since in DM-deficient cells that also lack Ii, DR3 molecules assembled in a 16.23(+) conformation, we conclude that during biosynthesis Ii and DM exert opposing conformational constraints, characterized by suppressing or releasing 16.23 epitope expression. These results imply that DR3/peptide complexes, including DR3/ CLIP, can exist in two conformations depending on previous interaction with DM, but independent of the nature of the peptide bound. We show that these naturally occurring class II conformers can be selectively recognized by T cells.

Prominin: a story of cholesterol, plasma membrane protrusions and human pathology.

Prominin is the first identified member of a novel family of polytopic membrane proteins conserved throughout the animal kingdom. It has an unusual membrane topology, containing five transmembrane domains and two large glycosylated extracellular loops. In mammals, prominin is expressed in various embryonic and adult epithelial cells, as well as in nonepithelial cells, such as hematopoietic stem cells. At the subcellular level, prominin is selectively localized in microvilli and other plasma membrane protrusions, irrespective of cell type. At the molecular level, prominin specifically interacts with membrane cholesterol and is a marker of a novel type of cholesterol-based lipid 'raft'. A frameshift mutation in the human prominin gene, which results in a truncated protein that is no longer transported to the cell surface, is associated with retinal degeneration. Given that prominin is concentrated in the plasma membrane evaginations at the base of the outer segment of rod photoreceptor cells, which are essential precursor structures in the biogenesis of photoreceptive disks, it is proposed that prominin has a role in the generation of plasma membrane protrusions, their lipid composition and organization and their membrane-to-membrane interactions.

Identification of residues in the V domain of CD80 (B7-1) implicated in functional interactions with CD28 and CTLA4.

The CD80 (B7-1) molecule is a 45-60-kD member of the immunoglobulin superfamily that is expressed on a variety of cell types of haematopoietic origin. CD80 can provide a critical costimulatory signal to T cells by interacting with the T cell surface molecule CD28. CD80 also binds to the CD28-related molecule CTLA4, which is expressed on activated T cells, Recently, additional ligands of CD28 and CTLA4 have been described in mice and humans. One of them, CD86 (B-70 or B7-2) was characterized at the molecular level. Although similar in predicted structure to CD80, it is distantly related in amino acid sequence. In this study, human CD80 mutants were generated and tested for their ability to maintain the interaction with CD28 leading to adhesion and enhanced IL-2 production. Two hydrophobic residues in the V-like domain of CD80 were identified as critical for binding to CD28 and are also important for the interaction with CTLA4. These residues are adjacent to the epitope of the BB1 antibody, which inhibits CD28-CD80 interactions. One of these residues, Y87, is conserved in all CD80 and CD86 cloned from various species. These results being to unravel the structural requirements for binding to CD28 and CTLA4.

Purification and partial characterization of rat macrophage Fc receptor and binding factor for IgA.

By using a biotinylated ligand and Western blotting techniques, a receptor (RFc alpha) and a binding factor (BF) for IgA were detected, respectively, on membrane and in the cell-free culture supernatant of rat peritoneal macrophages. Extraction of the RFc alpha was obtained by solubilization of macrophages with Nonidet P-40, and purification was performed by HPLC affinity chromatography on a column derivatized with IgA. RFc alpha is formed of two subunits, with molecular masses of 56 and 70 kDa, which are both involved in the IgA binding ability of rat peritoneal macrophages. IgABF was recovered from the cell-free supernatant of a short-term culture of rat macrophages and was affinity-purified in the same manner as RFc alpha. Like RFc alpha, IgABF retained its IgA binding activity in its native, as well as denatured form. Since the molecular masses of RFc alpha and IgABF are similar, and IgABF competes with RFc alpha for IgA binding, one can assume that IgABF probably represents a shed RFc alpha.