A case of inherited glycosylphosphatidylinositol deficiency caused by PGAP3 variant with uniparental isodisomy on chromosome 17.

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. METHOD: 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. CONCLUSION: This is the first report of inherited GPI deficiency caused by UPiD. Inherited GPI deficiency must be considered in patients with unexplained hyperphosphatasemia.

Structure and Function of the Glycosylphosphatidylinositol Transamidase, a Transmembrane Complex Catalyzing GPI Anchoring of Proteins.

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.

Glycosylphosphatidylinositol anchor biosynthesis pathway-based biomarker identification with machine learning for prognosis and T cell exhaustion status prediction in breast cancer.

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.

Rare Genetic Developmental Disabilities: Mabry Syndrome (MIM 239300) Index Cases and Glycophosphatidylinositol (GPI) Disorders.

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.

Sensitive Method To Analyze Cell Surface GPI-Anchored Proteins Using DNA Hybridization Chain Reaction-Mediated Signal Amplification.

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 GPI-anchor biosynthesis pathway is critical for syncytiotrophoblast differentiation and placental development.

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.

Targeting the GPI transamidase subunit GPAA1 abrogates the CD24 immune checkpoint in ovarian cancer.

CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages, providing a "don't eat me" signal that prevents targeting and phagocytosis by macrophages. Factors promoting CD24 expression could represent novel immunotherapeutic targets for ovarian cancer. Here, using a genome-wide CRISPR knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment 1), a factor that catalyzes the attachment of a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins, as a positive regulator of CD24 cell surface expression. Genetic ablation of GPAA1 abolishes CD24 cell surface expression, enhances macrophage-mediated phagocytosis, and inhibits ovarian tumor growth in mice. GPAA1 shares structural similarities with aminopeptidases. Consequently, we show that bestatin, a clinically advanced aminopeptidase inhibitor, binds to GPAA1 and blocks GPI attachment, resulting in reduced CD24 cell surface expression, increased macrophage-mediated phagocytosis, and suppressed growth of ovarian tumors. Our study highlights the potential of targeting GPAA1 as an immunotherapeutic approach for CD24+ ovarian cancers.

The immunologic abnormalities in patients with paroxysmal nocturnal hemoglobinuria are associated with disease progression.

OBJECTIVES: 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). CONCLUSION: Our results demonstrated that immune abnormalities are associated with PNH development.

Intact cell lipidomics using the Bruker MBT lipid Xtract assay allows the rapid detection of glycosyl-inositol-phospho-ceramides from Aspergillus fumigatus.

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.

PIGA Mutations and Glycosylphosphatidylinositol Anchor Dysregulation in Polyposis-Associated Duodenal Tumorigenesis.

The pathogenesis of duodenal tumors in the inherited tumor syndromes familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP) is poorly understood. This study aimed to identify genes that are significantly mutated in these tumors and to explore the effects of these mutations. Whole exome and whole transcriptome sequencing identified recurrent somatic coding variants of phosphatidylinositol N-acetylglucosaminyltransferase subunit A (PIGA) in 19/70 (27%) FAP and MAP duodenal adenomas, and further confirmed the established driver roles for APC and KRAS. PIGA catalyzes the first step in glycosylphosphatidylinositol (GPI) anchor biosynthesis. Flow cytometry of PIGA-mutant adenoma-derived and CRISPR-edited duodenal organoids confirmed loss of GPI anchors in duodenal epithelial cells and transcriptional profiling of duodenal adenomas revealed transcriptional signatures associated with loss of PIGA. IMPLICATIONS: PIGA somatic mutation in duodenal tumors from patients with FAP and MAP and loss of membrane GPI-anchors may present new opportunities for understanding and intervention in duodenal tumorigenesis.

Protein sorting upon exit from the endoplasmic reticulum dominates Golgi biogenesis in budding yeast.

Cells sense and control the number and quality of their organelles, but the underlying mechanisms of this regulation are not understood. Our recent research in the yeast Saccharomyces cerevisiae has shown that long acyl chain ceramides in the endoplasmic reticulum (ER) membrane and the lipid moiety of glycosylphosphatidylinositol (GPI) anchor determine the sorting of GPI-anchored proteins in the ER. Here, we show that a mutant strain, which produces shorter ceramides than the wild-type strain, displays a different count of Golgi cisternae. Moreover, deletions of proteins that remodel the lipid portion of GPI anchors resulted in an abnormal number of Golgi cisternae. Thus, our study reveals that protein sorting in the ER plays a critical role in maintaining Golgi biogenesis.

Production of CA125 with Tn antigens using a glycosylphosphatidylinositol anchoring system.

Cancer antigen 125 (CA125) is a serum marker associated with ovarian cancer. Despite its widespread use, CA125 levels can also be elevated in benign conditions. Recent reports suggest that detecting serum CA125 that carries the Tn antigen, a truncated O-glycan containing only N-acetylgalactosamine on serine or threonine residues, can improve the specificity of ovarian cancer diagnosis. In this study, we engineered cells to express CA125 with a Tn antigen. To achieve this, we knocked out C1GALT1 and SLC35A1, genes encoding Core1 synthase and a transporter for cytidine-5'-monophospho-sialic acid respectively, in human embryonic kidney 293 (HEK293) cells. In ClGALT1-SLC35A1-knockout (KO) cells, the expression of the Tn antigen showed a significant increase, whereas the expression of the T antigen (galactose-ß1,3-N-acetylgalactosamine on serine or threonine residues) was decreased. Due to the inefficient secretion of soluble CA125, we employed a glycosylphosphatidylinositol (GPI) anchoring system. This allowed for the expression of GPI-anchored CA125 on the cell surface of ClGALT1-SLC35A1-KO cells. Cells expressing high levels of GPI-anchored CA125 were then enriched through cell sorting. By knocking out the PGAP2 gene, the GPI-anchored form of CA125 was converted to a secretory form. Through the engineering of O-glycans and the use of a GPI-anchoring system, we successfully produced CA125 with Tn antigen modification.

Glycosylation in malaria parasites: what do we know?

In malaria parasites, although post-translational modification of proteins with N-. O-, and C-glycosidic bond-linked glycans is limited, it is confined to relatively fewer proteins in which the glycans are present at significant levels and may have important functions. Furthermore, several proteins are modified with glycosylphosphatidylinositols (GPIs) which represent the predominant glycan synthesized by parasites. Modification of proteins with GPIs is obligatory for parasite survival as GPI-anchored proteins (GPI-APs) play essential roles in all life cycle stages of the parasites, including development, egress, gametogenesis, motility, and host cell adhesion and invasion. Here, we discuss the current knowledge on the structures and potential functions of the glycan moieties of parasite proteins. The knowledge has important implications for the development of drugs and vaccines for malaria.

Molecular basis of the inositol deacylase PGAP1 involved in quality control of GPI-AP biogenesis.

The secretion and quality control of glycosylphosphatidylinositol-anchored proteins (GPI-APs) necessitates post-attachment remodeling initiated by the evolutionarily conserved PGAP1, which deacylates the inositol in nascent GPI-APs. Impairment of PGAP1 activity leads to developmental diseases in humans and fatality and infertility in animals. Here, we present three PGAP1 structures (2.66-2.84 Å), revealing its 10-transmembrane architecture and product-enzyme interaction details. PGAP1 holds GPI-AP acyl chains in an optimally organized, guitar-shaped cavity with apparent energetic penalties from hydrophobic-hydrophilic mismatches. However, abundant glycan-mediated interactions in the lumen counterbalance these repulsions, likely conferring substrate fidelity and preventing off-target hydrolysis of bulk membrane lipids. Structural and biochemical analyses uncover a serine hydrolase-type catalysis with atypical features and imply mechanisms for substrate entrance and product release involving a drawing compass movement of GPI-APs. Our findings advance the mechanistic understanding of GPI-AP remodeling.

Recent research progress in glycosylphosphatidylinositol-anchored protein biosynthesis, chemical/chemoenzymatic synthesis, and interaction with the cell membrane.

Glycosylphosphatidylinositol (GPI) attachment to the C-terminus of proteins is a prevalent posttranslational modification in eukaryotic species, and GPIs help anchor proteins to the cell surface. GPI-anchored proteins (GPI-APs) play a key role in various biological events. However, GPI-APs are difficult to access and investigate. To tackle the problem, chemical and chemoenzymatic methods have been explored for the preparation of GPI-APs, as well as GPI probes that facilitate the study of GPIs on live cells. Substantial progress has also been made regarding GPI-AP biosynthesis, which is helpful for developing new synthetic methods for GPI-APs. This article reviews the recent advancements in the study of GPI-AP biosynthesis, GPI-AP synthesis, and GPI interaction with the cell membrane utilizing synthetic probes.

GPI-anchored Gas1 protein regulates cytosolic proteostasis in budding yeast.

The decline in protein homeostasis (proteostasis) is a hallmark of cellular aging and aging-related diseases. Maintaining a balanced proteostasis requires a complex network of molecular machineries that govern protein synthesis, folding, localization, and degradation. Under proteotoxic stress, misfolded proteins that accumulate in cytosol can be imported into mitochondria for degradation through the "mitochondrial as guardian in cytosol" (MAGIC) pathway. Here, we report an unexpected role of Gas1, a cell wall-bound glycosylphosphatidylinositol (GPI)-anchored ß-1,3-glucanosyltransferase in the budding yeast, in differentially regulating MAGIC and ubiquitin-proteasome system (UPS). Deletion of GAS1 inhibits MAGIC but elevates protein ubiquitination and UPS-mediated protein degradation. Interestingly, we found that the Gas1 protein exhibits mitochondrial localization attributed to its C-terminal GPI anchor signal. But this mitochondria-associated GPI anchor signal is not required for mitochondrial import and degradation of misfolded proteins through MAGIC. By contrast, catalytic inactivation of Gas1 via the gas1-E161Q mutation inhibits MAGIC but not its mitochondrial localization. These data suggest that the glucanosyltransferase activity of Gas1 is important for regulating cytosolic proteostasis.

Folate Receptor Alpha-A Novel Approach to Cancer Therapy.

Folate receptor α (FR) was discovered many decades ago, along with drugs that target intracellular folate metabolism, such as pemetrexed and methotrexate. Folate is taken up by the cell via this receptor, which also targeted by many cancer agents due to the over-expression of the receptor by cancer cells. FR is a membrane-bound glycosyl-phosphatidylinositol (GPI) anchor glycoprotein encoded by the folate receptor 1 (FOLR1) gene. FR plays a significant role in DNA synthesis, cell proliferation, DNA repair, and intracellular signaling, all of which are essential for tumorigenesis. FR is more prevalent in cancer cells compared to normal tissues, which makes it an excellent target for oncologic therapeutics. FRα is found in many cancer types, including ovarian cancer, non-small-cell lung cancer (NSCLC), and colon cancer. FR is widely used in antibody drug conjugates, small-molecule-drug conjugates, and chimeric antigen-receptor T cells. Current oncolytic therapeutics include mirvetuximab soravtansine, and ongoing clinical trials are underway to investigate chimeric antigen receptor T cells (CAR-T cells) and vaccines. Additionally, FRα has been used in a myriad of other applications, including as a tool in the identification of tumor types, and as a prognostic marker, as a surrogate of chemotherapy resistance. As such, FRα identification has become an essential part of precision medicine.

PIGB maintains nuclear lamina organization in skeletal muscle of Drosophila.

The nuclear lamina (NL) plays various roles and participates in nuclear integrity, chromatin organization, and transcriptional regulation. Lamin proteins, the main components of the NL, form a homogeneous meshwork structure under the nuclear envelope. Lamins are essential, but it is unknown whether their homogeneous distribution is important for nuclear function. Here, we found that PIGB, an enzyme involved in glycosylphosphatidylinositol (GPI) synthesis, is responsible for the homogeneous lamin meshwork in Drosophila. Loss of PIGB resulted in heterogeneous distributions of B-type lamin and lamin-binding proteins in larval muscles. These phenotypes were rescued by expression of PIGB lacking GPI synthesis activity. The PIGB mutant exhibited changes in lamina-associated domains that are large heterochromatic genomic regions in the NL, reduction of nuclear stiffness, and deformation of muscle fibers. These results suggest that PIGB maintains the homogeneous meshwork of the NL, which may be essential for chromatin distribution and nuclear mechanical properties.

Proteomic screens of SEL1L-HRD1 ER-associated degradation substrates reveal its role in glycosylphosphatidylinositol-anchored protein biogenesis.

Endoplasmic reticulum-associated degradation (ERAD) plays indispensable roles in many physiological processes; however, the nature of endogenous substrates remains largely elusive. Here we report a proteomics strategy based on the intrinsic property of the SEL1L-HRD1 ERAD complex to identify endogenous ERAD substrates both in vitro and in vivo. Following stringent filtering using a machine learning algorithm, over 100 high-confidence potential substrates are identified in human HEK293T and mouse brown adipose tissue, among which ~88% are cell type-specific. One of the top shared hits is the catalytic subunit of the glycosylphosphatidylinositol (GPI)-transamidase complex, PIGK. Indeed, SEL1L-HRD1 ERAD attenuates the biogenesis of GPI-anchored proteins by specifically targeting PIGK for proteasomal degradation. Lastly, several PIGK disease variants in inherited GPI deficiency disorders are also SEL1L-HRD1 ERAD substrates. This study provides a platform and resources for future effort to identify proteome-wide endogenous substrates in vivo, and implicates SEL1L-HRD1 ERAD in many cellular processes including the biogenesis of GPI-anchored proteins.

Neuronal Wiring Receptors Dprs and DIPs Are GPI Anchored and This Modification Contributes to Their Cell Surface Organization.

The Drosophila Dpr and DIP proteins belong to the immunoglobulin superfamily of cell surface proteins (CSPs). Their hetero- and homophilic interactions have been implicated in a variety of neuronal functions, including synaptic connectivity, cell survival, and axon fasciculation. However, the signaling pathways underlying these diverse functions are unknown. To gain insight into Dpr-DIP signaling, we sought to examine how these CSPs are associated with the membrane. Specifically, we asked whether Dprs and DIPs are integral membrane proteins or membrane anchored through the addition of glycosylphosphatidylinositol (GPI) linkage. We demonstrate that most Dprs and DIPs are GPI anchored to the membrane of insect cells and validate these findings for some family members in vivo using Drosophila larvae, where GPI anchor cleavage results in loss of surface labeling. Additionally, we show that GPI cleavage abrogates aggregation of insect cells expressing cognate Dpr-DIP partners. To test if the GPI anchor affects Dpr and DIP localization, we replaced it with a transmembrane domain and observed perturbation of subcellular localization on motor neurons and muscles. These data suggest that membrane anchoring of Dprs and DIPs through GPI linkage is required for localization and that Dpr-DIP intracellular signaling likely requires transmembrane coreceptors.