Identification of a Golgi GPI-N-acetylgalactosamine transferase with tandem transmembrane regions in the catalytic domain.

Many eukaryotic proteins are anchored to the cell surface via the glycolipid glycosylphosphatidylinositol (GPI). Mammalian GPIs have a conserved core but exhibit diverse N-acetylgalactosamine (GalNAc) modifications, which are added via a yet unresolved process. Here we identify the Golgi-resident GPI-GalNAc transferase PGAP4 and show by mass spectrometry that PGAP4 knockout cells lose GPI-GalNAc structures. Furthermore, we demonstrate that PGAP4, in contrast to known Golgi glycosyltransferases, is not a single-pass membrane protein but contains three transmembrane domains, including a tandem transmembrane domain insertion into its glycosyltransferase-A fold as indicated by comparative modeling. Mutational analysis reveals a catalytic site, a DXD-like motif for UDP-GalNAc donor binding, and several residues potentially involved in acceptor binding. We suggest that a juxtamembrane region of PGAP4 accommodates various GPI-anchored proteins, presenting their acceptor residue toward the catalytic center. In summary, we present insights into the structure of PGAP4 and elucidate the initial step of GPI-GalNAc biosynthesis.

A GPI processing phospholipase A2, PGAP6, modulates Nodal signaling in embryos by shedding CRIPTO.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) can be shed from the cell membrane by GPI cleavage. In this study, we report a novel GPI-processing enzyme, termed post-glycosylphosphatidylinositol attachment to proteins 6 (PGAP6), which is a GPI-specific phospholipase A2 mainly localized at the cell surface. CRIPTO, a GPI-AP, which plays critical roles in early embryonic development by acting as a Nodal coreceptor, is a highly sensitive substrate of PGAP6, whereas CRYPTIC, a close homologue of CRIPTO, is not sensitive. CRIPTO processed by PGAP6 was released as a lysophosphatidylinositol-bearing form, which is further cleaved by phospholipase D. CRIPTO shed by PGAP6 was active as a coreceptor in Nodal signaling, whereas cell-associated CRIPTO activity was reduced when PGAP6 was expressed. Homozygous Pgap6 knockout mice showed defects in early embryonic development, particularly in the formation of the anterior-posterior axis, which are common features with Cripto knockout embryos. These results suggest PGAP6 plays a critical role in Nodal signaling modulation through CRIPTO shedding.

Mutations in PIGY: expanding the phenotype of inherited glycosylphosphatidylinositol deficiencies.

Glycosylphosphatidylinositol (GPI)-anchored proteins are ubiquitously expressed in the human body and are important for various functions at the cell surface. Mutations in many GPI biosynthesis genes have been described to date in patients with multi-system disease and together these constitute a subtype of congenital disorders of glycosylation. We used whole exome sequencing in two families to investigate the genetic basis of disease and used RNA and cellular studies to investigate the functional consequences of sequence variants in the PIGY gene. Two families with different phenotypes had homozygous recessive sequence variants in the GPI biosynthesis gene PIGY. Two sisters with c.137T>C (p.Leu46Pro) PIGY variants had multi-system disease including dysmorphism, seizures, severe developmental delay, cataracts and early death. There were significantly reduced levels of GPI-anchored proteins (CD55 and CD59) on the surface of patient-derived skin fibroblasts (∼20-50% compared with controls). In a second, consanguineous family, two siblings had moderate development delay and microcephaly. A homozygous PIGY promoter variant (c.-540G>A) was detected within a 7.7 Mb region of autozygosity. This variant was predicted to disrupt a SP1 consensus binding site and was shown to be associated with reduced gene expression. Mutations in PIGY can occur in coding and non-coding regions of the gene and cause variable phenotypes. This article contributes to understanding of the range of disease phenotypes and disease genes associated with deficiencies of the GPI-anchor biosynthesis pathway and also serves to highlight the potential importance of analysing variants detected in 5'-UTR regions despite their typically low coverage in exome data.

IL-2 withdrawal induces HTLV-1 expression through p38 activation in ATL cell lines.

Expression of human T-cell leukemia virus type-1 (HTLV-1) in adult T-cell leukemia (ATL) cells is known to be marginal in vivo and inducible in short-term culture. In this study, we demonstrated that withdrawal of interleukin (IL)-2 from IL-2-dependent ATL cell lines resulted in induction of HTLV-1 mRNA and protein expression, and that viral induction was associated with phosphorylation of the stress kinase p38 and its downstream CREB. Pharmacological inhibitors of the p38 pathway suppressed viral expression induced by IL-2 depletion. These results indicate that the stress-induced p38 pathway might up-regulate HTLV-1 gene expression through at least CREB activation.

Augmentation of chemokine production by severe acute respiratory syndrome coronavirus 3a/X1 and 7a/X4 proteins through NF-kappaB activation.

Severe acute respiratory syndrome (SARS) is characterized by rapidly progressing respiratory failure resembling acute/adult respiratory distress syndrome (ARDS) associated with uncontrolled inflammatory responses. Here, we demonstrated that, among five accessory proteins of SARS coronavirus (SARS-CoV) tested, 3a/X1 and 7a/X4 were capable of activating nuclear factor kappa B (NF-kappaB) and c-Jun N-terminal kinase (JNK), and significantly enhanced interleukin 8 (IL-8) promoter activity. Furthermore, 3a/X1 and 7a/X4 expression in A549 cells enhanced production of inflammatory chemokines that were known to be up-regulated in SARS-CoV infection. Our results suggest potential involvement of 3a/X1 and 7a/X4 proteins in the pathological inflammatory responses in SARS.