An antigen produced by splicing of noncontiguous peptides in the reverse order
CD8+ T lymphocytes recognize peptides that are usually derived from the degradation of cellular proteins and are presented by class I molecules of the major histocompatibility complex. In an article published in Science, Warren et al.1 describe a human minor histocompatibility antigen created by a polymorphism in the SP110 nuclear phosphoprotein gene. The antigenic peptide comprises two noncontiguous SP110 peptide segments spliced together in reverse order of that in which they occur in the predicted SP110 protein. The antigenic peptide could be produced in vitro by incubation of precursor peptides with highly purified 20S proteasomes. Cutting and splicing probably occur within the proteasome by transpeptidation. The results of the authors' studies indicate that the reordered spliced peptide SLPRGTSTPK derived by proteasomal processing of the Arg299 SP110 protein is the naturally processed antigen recognized by CTL DRN-7 and that the splicing reaction occurs in the proteasome by transpeptidation involving an acyl-enzyme intermediate. In contrast to the two previous examples of spliced peptides, the peptide recognized by CTL DRN-7 is produced by ligation of two noncontiguous peptide fragments in the reverse order. The observation that this antigen is expressed in some normal cells indicates that peptide splicing is not restricted to tumor cells. The proteasome, by virtue of its proteolytic capacity, participates in the generation of active transcription factor domains from inactive precursors, controls the levels of numerous regulatory proteins, and serves as the major source of peptides recognized by CD8+ T cells. The ability of the proteasome to splice together peptide fragments from a protein in either the initial or the reverse order has profound implications for the diversity of peptides that can be presented on the cell surface for recognition by CD8+ T cells and could also have other unanticipated consequences.
A perspective by Nilabh Shastri in the same issue2 discusses an unexpected mechanism by which cells generate these presented antigenic peptides, increasing the breadth of proteins generated from the genetic code. Shastri states that, irrespective of the still obscure rules for making noncontiguous peptides, their existence shows that genetic information can be scrambled and yet be useful. Cells do not simply discard scrambled peptides and, like defective or cryptic translation products, use them to add to the peptide-bound major histocompatibility complex class I (pMHCI) repertoire. This view implies that such scramblings are not random accidents and would have to be generated in other cell types as well. In particular, cells constituting immune organs that are responsible for eliminating self-reactive T cells must generate the same scrambled peptides. This is to ensure that autoimmunity will not result from the sudden appearance of previously unseen pMHC I in some tissues. What fraction of the total pMHC I repertoire represents cryptic translation products or spliced and scrambled peptides, and whether and how they might be regulated, remain important unanswered questions. Nonetheless, it is clear, according to Shastri, that peptides from these sources are a functional aspect of the antigen presentation mechanism that keeps an eye on the genome, including its unexpected and unpredictable products. (Science 2006; 313: 1444–1447. Science 2006; 313: 1398–1399)
Marc De Broe
The antigen presentation pathway.
Circulating soluble endoglin as a potential test for preeclampsia
Recent work suggests that antiangiogenic proteins produced by the placenta are involved in the development of preeclampsia and eclampsia. Specifically, circulating soluble fms-like tyrosine kinase 1 (sFlt1) and endoglin, a soluble co-receptor for transforming growth factor-
1, are elevated in women with preeclampsia. Also, introduction of adenoviruses encoding both sFlt1 and soluble endoglin to rats produced severe hypertension, proteinuria, hepatitis, and hemolytic anemia, all manifestations of preeclampsia and eclampsia.
Levels of soluble endoglin according to weeks before the onset of preeclampsia.
Because soluble endoglin acts together with sFlt1 to induce a severe preeclampsia-like syndrome in pregnant rats, Levine et al. performed a study of healthy nulliparous women that included 72 women who had preterm preeclampsia (<37 weeks), as well as 480 randomly selected women — 120 women with preeclampsia at term (at 
37 weeks), 120 women with gestational hypertension, 120 normotensive women who delivered infants who were small for their gestational age, and 120 normotensive control subjects who delivered infants who were not small for their gestational age. They found that circulating soluble endoglin levels increased markedly beginning 2–3 months before the onset of preeclampsia (see Figure). After the onset of clinical disease, the mean serum level in women with preterm preeclampsia was 46.4 ng per ml, compared with 9.8 ng per ml in controls. The mean serum level in women with preeclampsia at term was 31.0 ng per ml, compared with 13.3 ng per ml in controls. At 17 through 20 weeks of gestation, soluble endoglin levels were significantly higher in women in whom preterm preeclampsia later developed than in controls (10.2 ng per ml versus 5.8 ng per ml), and at 25 through 28 weeks of gestation, the levels were significantly higher in women in whom term preeclampsia developed than in controls (8.5 ng per ml versus 5.9 ng per ml). A reliable test to predict preeclampsia has been sorely missing. The new study by Levine et al., while retrospective and cross-sectional, places the measurement of circulating proteins that control angiogenesis at the front of potentially useful tests to predict preeclampsia and eclampsia in clinical practice. (N Engl J Med 2006; 355: 992–1005)
Juan Oliver
Role of calcineurin in aquaporin 2 trafficking: a new model of nephrogenic diabetes insipidus
Long-term inhibition of calcineurin with cyclosporin A results in polyuria, decreased urine osmolality, and downregulation of several aquaporins, including aquaporin 2 (AQP2). It is unknown, however, whether cyclosporin A directly alters AQP2 action or whether it alters urine-concentrating capacity through other mechanisms.
AQP2 is a highly glycosylated protein, and it is processed through the endoplasmic reticulum and Golgi network, where it is folded and targeted to vesicles that are directed to the subapical region of the plasma membrane. Water permeability of the inner medullary collecting duct cells can then be rapidly regulated by the binding of the antidiuretic hormone arginine vasopressin to its V2 receptors, located mainly in the basolateral membrane of principal cells. Activation of the V2 receptors stimulates adenylyl cyclase, leading to elevation of cyclic adenosine monophosphate and subsequent activation of protein kinase A (PKA). In turn, PKA phosphorylates AQP2, resulting in enhanced accumulation of AQP2-bearing vesicles in the plasma membrane, where AQP2 is inserted by an exocytosis-like process.
In addition to PKA, several factors participate in the normal trafficking of AQP2, including the Golgi network casein kinase, and A kinase anchoring proteins (AKAPs), which are scaffolding proteins that facilitate interaction of AQP2 and PKA. Recent work suggests that the serine/threonine phosphatase calcineurin also regulates the trafficking of AQP2. Calcineurin binds to AKAPs and was identified in a complex with AQP2 and an AKAP protein. In addition, calcineurin dephosphorylated APQ2 in vitro, and the 
isoform of calcineurin A subunit (CnA) and AQP2 colocalize in collecting duct principal cells. Finally, calcineurin inhibition with cyclosporin A decreased the phosphorylation of AQP2 and redistributed it away from the apical membrane. To clarify the role of CnA in AQP2 trafficking in vivo, Gooch et al. analyzed CnA-/- mice. They found decreased phosphorylation of AQP2 in response to vasopressin and significantly less AQP2 protein in inner medullary collecting duct vesicles and, consequently, in the apical membrane. Instead, AQP2 appeared to be retained in an intracellular compartment, consistent with a defect in intracellular trafficking of the protein. Inhibition of calcineurin activity with cyclosporin A recapitulated the phenotype of CnA-/- mice, suggesting that phosphatase activity of calcineurin is required for normal AQP2 trafficking and function. As a result, CnA-/- mice are a new model of nephrogenic diabetes insipidus characterized by an impaired response to vasopressin as a result of altered trafficking and phosphorylation of AQP2. (J Cell Sci 2006; 119: 2468–2476)
Juan Oliver
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