As the primary component of anti-tumor immunity, T cells are prone to exhaustion and dysfunction in the tumor microenvironment (TME). A thorough understanding of T cell exhaustion (TEX) in the TME is crucial for effectively addressing TEX in clinical settings and promoting the efficacy of immune checkpoint blockade therapies. In eukaryotes, numerous cell surface proteins are tethered to the plasma membrane via Glycosylphosphatidylinositol (GPI) anchors, which play a crucial role in facilitating the proper translocation of membrane proteins. However, the available evidence is insufficient to support any additional functional involvement of GPI anchors. Here, we investigate the signature of GPI-anchor biosynthesis in the TME of breast cancer (BC)patients, particularly its correlation with TEX. GPI-anchor biosynthesis should be considered as a prognostic risk factor for BC. Patients with high GPI-anchor biosynthesis showed more severe TEX. And the levels of GPI-anchor biosynthesis in exhausted CD8 T cells was higher than normal CD8 T cells, which was not observed between malignant epithelial cells and normal mammary epithelial cells. In addition, we also found that GPI -anchor biosynthesis related genes can be used to diagnose TEX status and predict prognosis in BC patients, both the TEX diagnostic model and the prognostic model showed good AUC values. Finally, we confirmed our findings in cells and clinical samples. Knockdown of PIGU gene expression significantly reduced the proliferation rate of MDA-MB-231 and MCF-7 cell lines. Immunofluorescence results from clinical samples showed reduced aggregation of CD8 T cells in tissues with high expression of GPAA1 and PIGU.

BACKGROUND: Inherited glycosylphosphatidylinositol (GPI) deficiency is an autosomal recessive disease and a set of syndromes caused by different genes involved in the biosynthesis of phosphatidylinositol characterized by severe cognitive disability, elevated serum alkaline phosphatase (ALP) levels, and distinct facial features. This report presents a patient with inherited GPI deficiency caused by a homozygous frameshift variant of PGAP3 due to uniparental isodisomy (UPiD) on chromosome 17.
METHODS: Clinical characteristics of the patient were collected. Microarray analysis followed by adaptive sampling sequencing targeting chromosome 17 was used for the identification of variants. Sanger sequencing was used to confirm the variant in the target region.
RESULTS: The patient was born at 38 weeks of gestation with a birthweight of 3893 g. He had a distinctive facial appearance with hypertelorism, wide nasal bridge, and cleft soft palate. Postnatal head magnetic resonance imaging revealed a Blake\'s pouch cyst. The serum ALP level was 940 IU/L at birth and increased to 1781 IU/L at 28 days of age. Microarray analysis revealed region of homozygosity in nearly the entire region of chromosome 17, leading to the diagnosis of UPiD. Adaptive sampling sequencing targeting chromosome 17 confirmed the homozygous variant NM_033419:c.778dupG (p.Val260Glyfs*14) in the PGAP3 gene, resulting in a diagnosis of inherited GPI deficiency.
CONCLUSIONS: This is the first report of inherited GPI deficiency caused by UPiD. Inherited GPI deficiency must be considered in patients with unexplained hyperphosphatasemia.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a ubiquitous posttranslational modification in eukaryotic cells. GPI-anchored proteins (GPI-APs) play critical roles in enzymatic, signaling, regulatory, and adhesion processes. Over 20 enzymes are involved in GPI synthesis, attachment to client proteins, and remodeling after attachment. The GPI transamidase (GPI-T), a large complex located in the endoplasmic reticulum membrane, catalyzes the attachment step by replacing a C-terminal signal peptide of proproteins with GPI. In the last three decades, extensive research has been conducted on the mechanism of the transamidation reaction, the components of the GPI-T complex, the role of each subunit, and the substrate specificity. Two recent studies have reported the three-dimensional architecture of GPI-T, which represent the first structures of the pathway. The structures provide detailed mechanisms for assembly that rationalizes previous biochemical results and subunit-dependent stability data. While the structural data confirm the catalytic role of PIGK, which likely uses a caspase-like mechanism to cleave the proproteins, they suggest that unlike previously proposed, GPAA1 is not a catalytic subunit. The structures also reveal a shared cavity for GPI binding. Somewhat unexpectedly, PIGT, a single-pass membrane protein, plays a crucial role in GPI recognition. Consistent with the assembly mechanisms and the active site architecture, most of the disease mutations occur near the active site or the subunit interfaces. Finally, the catalytic dyad is located ~22 Å away from the membrane interface of the GPI-binding site, and this architecture may confer substrate specificity through topological matching between the substrates and the elongated active site. The research conducted thus far sheds light on the intricate processes involved in GPI anchoring and paves the way for further mechanistic studies of GPI-T.

The aim of this study was to explore potential novel plasma protein biomarkers for lung adenocarcinoma (LUAD). A plasma proteomics analysis was carried out and candidate protein biomarkers were validated in 102 LUAD cases and 102 matched healthy controls. The same LUAD tumor tissues were detected to explore the correlation between the expression of candidate proteins in tissues and plasma and vascular normalization. A LUAD active metastasis mice model was constructed to explore the role of candidate proteins for lung metastasis. GPI and PGD were verified to be upregulated in plasma from LUAD patients, and the expression of GPI in tumor tissue was positively correlated with the expression of GPI in plasma and negatively correlated with the normalization of tumor blood vessels. Meanwhile, a negative correlation between the expression of GPI and PGD in plasma and tumor vascular normalization was discovered. In the LUAD active metastasis model, the lowest levels of vascular normalization and the highest expression of GPI and PGD were found in mice with lung metastases. This study found that GPI and PGD may be potential plasma biomarkers for LUAD, and monitoring those may infer the risk of metastasis and malignancy of the tumor. SIGNIFICANT: We identified GPI and PGD as potential novel diagnostic and prognostic biomarkers for LUAD. PGD and GPI can be used as diagnostic biomarkers in combination with other available strategies to assist in the screening and diagnosis of LUAD, and as prognostic biomarkers aid in predict the risk of tumor metastasis and malignancy in patients with LUAD.

Candida albicans is an opportunistic fungal pathogen that can switch between yeast and hyphal morphologies depending on the environmental cues it receives. The switch to hyphal form is crucial for the establishment of invasive infections. The hyphal form is also characterized by the cell surface expression of hyphae-specific proteins, many of which are GPI-anchored and important determinants of its virulence. The coordination between hyphal morphogenesis and the expression of GPI-anchored proteins is made possible by an interesting cross-talk between GPI biosynthesis and the cAMP-PKA signaling cascade in the fungus; a parallel interaction is not found in its human host. On the other hand, in the nonpathogenic yeast, Saccharomyces cerevisiae, GPI biosynthesis is shut down when filamentation is activated and vice versa. This too is achieved by a cross-talk between GPI biosynthesis and cAMP-PKA signaling. How are diametrically opposite effects obtained from the cross-talk between two reasonably well-conserved pathways present ubiquitously across eukarya? This Review attempts to provide a model to explain these differences. In order to do so, it first provides an overview of the two pathways for the interested reader, highlighting the similarities and differences that are observed in C. albicans versus the well-studied S. cerevisiae model, before going on to explain how the different mechanisms of regulation are effected. While commonalities enable the development of generalized theories, it is hoped that a more nuanced approach, that takes into consideration species-specific differences, will enable organism-specific understanding of these processes and contribute to the development of targeted therapies.

The glycosylphosphatidylinositol (GPI) biosynthetic pathway in the endoplasmic reticulum (ER) is crucial for generating GPI-anchored proteins (GPI-APs), which are translocated to the cell surface and play a vital role in cell signaling and adhesion. This study focuses on two integral components of the GPI pathway, the PIGL and PIGF proteins, and their significance in trophoblast biology. We show that GPI pathway mutations impact on placental development impairing the differentiation of the syncytiotrophoblast (SynT), and especially the SynT-II layer, which is essential for the establishment of the definitive nutrient exchange area within the placental labyrinth. CRISPR/Cas9 knockout of Pigl and Pigf in mouse trophoblast stem cells (mTSCs) confirms the role of these GPI enzymes in syncytiotrophoblast differentiation. Mechanistically, impaired GPI-AP generation induces an excessive unfolded protein response (UPR) in the ER in mTSCs growing in stem cell conditions, akin to what is observed in human preeclampsia. Upon differentiation, the impairment of the GPI pathway hinders the induction of WNT signaling for early SynT-II development. Remarkably, the transcriptomic profile of Pigl- and Pigf-deficient cells separates human patient placental samples into preeclampsia and control groups, suggesting an involvement of Pigl and Pigf in establishing a preeclamptic gene signature. Our study unveils the pivotal role of GPI biosynthesis in early placentation and uncovers a new preeclampsia gene expression profile associated with mutations in the GPI biosynthesis pathway, providing novel molecular insights into placental development with implications for enhanced patient stratification and timely interventions.

GPI-anchored proteins (GPI-APs) are ubiquitous and essential but exist in low abundances on the cell surface, making their analysis and investigation especially challenging. To tackle the problem, a new method to detect and study GPI-APs based upon GPI metabolic engineering and DNA-facilitated fluorescence signal amplification was developed. In this context, cell surface GPI-APs were metabolically engineered using azido-inositol derivatives to introduce an azido group. This allowed GPI-AP coupling with alkyne-functionalized multifluorophore DNA assemblies generated by hybridization chain reaction (HCR). It was demonstrated that this approach could significantly improve the detection limit and sensitivity of GPI-APs, thereby enabling various biological studies, including the investigation of live cells. This new, enhanced GPI-AP detection method has been utilized to successfully explore GPI-AP engineering, analyze GPI-APs, and profile GPI-AP expression in different cells.

The case report by Mabry et al. (1970) of a family with four children with elevated tissue non-specific alkaline phosphatase, seizures and profound developmental disability, became the basis for phenotyping children with the features that became known as Mabry syndrome. Aside from improvements in the services available to patients and families, however, the diagnosis and treatment of this, and many other developmental disabilities, did not change significantly until the advent of massively parallel sequencing. As more patients with features of the Mabry syndrome were identified, exome and genome sequencing were used to identify the glycophosphatidylinositol (GPI) biosynthesis disorders (GPIBDs) as a group of congenital disorders of glycosylation (CDG). Biallelic variants of the phosphatidylinositol glycan (PIG) biosynthesis, type V (PIGV) gene identified in Mabry syndrome became evidence of the first in a phenotypic series that is numbered HPMRS1-6 in the order of discovery. HPMRS1 [MIM: 239300] is the phenotype resulting from inheritance of biallelic PIGV variants. Similarly, HPMRS2 (MIM 614749), HPMRS5 (MIM 616025) and HPMRS6 (MIM 616809) result from disruption of the PIGO, PIGW and PIGY genes expressed in the endoplasmic reticulum. By contrast, HPMRS3 (MIM 614207) and HPMRS4 (MIM 615716) result from disruption of post attachment to proteins PGAP2 (HPMRS3) and PGAP3 (HPMRS4). The GPI biosynthesis disorders (GPIBDs) are currently numbered GPIBD1-21. Working with Dr. Mabry, in 2020, we were able to use improved laboratory diagnostics to complete the molecular diagnosis of patients he had originally described in 1970. We identified biallelic variants of the PGAP2 gene in the first reported HPMRS patients. We discuss the longevity of the Mabry syndrome index patients in the context of the utility of pyridoxine treatment of seizures and evidence for putative glycolipid storage in patients with HPMRS3. From the perspective of the laboratory innovations made that enabled the identification of the HPMRS phenotype in Dr. Mabry\'s patients, the need for treatment innovations that will benefit patients and families affected by developmental disabilities is clear.

OBJECTIVE: To suggest the presence of a hyperimmune state in patients, and indicate that immune system attack on glycosylphosphatidylinositol (+) (GPI+) cells while escaping GPI- cell immunity.
METHODS: We retrospective the immune cell subtypes in peripheral blood from 25 patients visiting Tianjin Medical University General Hospital, Tianjin, China, with classical paroxysmal nocturnal hemoglobinuria (PNH) and 50 healthy controls.
RESULTS: The total CD3+ and CD3+CD8+ cell levels were higher in patients with PNH. The CD3+ cells are positively, correlated with lactate dehydrogenase (LDH; r=0.5453, p=0.0040), indirect bilirubin (r=0.4260, p=0.0379) and Flear- cells in monocytes (r=0.4099, p=0.0303). However, a negative correlation was observed between CD3+ cells and hemoglobin (r= -0.4530, p=0.0105). The total CD19+ cells decreased in patients, and CD19+ cells were negatively correlated with LDH (r= -0.5640, p=0.0077) and Flear- cells in monocytes (r= -0.4432, p=0.0341). Patients showed an increased proportion of total dendritic cells (DCs), with a higher proportion of myeloid DCs (mDCs) within the DC population. Moreover, the proportion of mDC/DC was positively correlated with CD59- cells (II + III types) in red cells (r=0.7941, p=0.0004), Flear- cells in granulocytes (r=0.5357, p=0.0396), and monocytes (r=0.6445, p=0.0095).
CONCLUSIONS: Our results demonstrated that immune abnormalities are associated with PNH development.

Glycosyl-inositol-phospho-ceramides (GIPCs) or glycosylphosphatidylinositol-anchored fungal polysaccharides are major lipids in plant and fungal plasma membranes and play an important role in stress adaption. However, their analysis remains challenging due to the multiple steps involved in their extraction and purification prior to mass spectrometry analysis. To address this challenge, we report here a novel simplified method to identify GIPCs from Aspergillus fumigatus using the new Bruker MBT lipid Xtract assay. A. fumigatus reference strains and clinical isolates were cultured, harvested, heat-inactivated and suspended in double-distilled water. A fraction of this fungal preparation was then dried in a microtube, mixed with an MBT lipid Xtract matrix (Bruker Daltonik, Germany) and loaded onto a MALDI target plate. Analysis was performed using a Bruker MALDI Biotyper Sirius system in the linear negative ion mode. Mass spectra were scanned from m/z 700 to m/z 2 000. MALDI-TOF MS analysis of cultured fungi showed a clear signature of GIPCs in Aspergillus fumigatus reference strains and clinical isolates. Here, we have demonstrated that routine MALDI-TOF in the linear negative ion mode combined with the MBT lipid Xtract is able to detect Aspergillus fumigatus GIPCs.