Structural study of AAVrh.10 Receptor and Antibody Interactions

Recombinant Adeno-associated virus (rAAV) vectors are one of the leading tools for the delivery of therapeutic genes in human gene therapy applications. For a successful transfer of their payload, the AAV vectors have to circumvent potential pre-existing neutralizing host antibodies and bind to the receptor of the target cells. Both these aspects have not been structurally analyzed for AAVrh.10. Here, cryo-electron microscopy (cryo-EM) and three-dimensional image reconstruction were used to map the binding site of sulfated N -Acetyllactosamine (LacNAc, previously shown to bind AAVrh.10) and a series of four monoclonal antibodies (MAbs). LacNAc was found to bind to a pocket located on the side of the 3-fold capsid protrusion, that is mostly conserved to AAV9 and equivalent to its galactose-binding site. As a result, AAVrh.10 was also shown to be able to bind to cell surface glycans with terminal galactose. For the antigenic characterization, it was observed that several anti-AAV8 MAbs cross-react with AAVrh.10. The binding sites of these antibodies were mapped to the 3-fold capsid protrusions. Based on these observations, the AAVrh.10 capsid surface was engineered to create variant capsids that escape these antibodies while maintaining infectivity. Importance Gene therapy vectors based on Adeno-associated virus rhesus isolate 10 (AAVrh.10) have been used in several clinical trials to treat monogenetic diseases. However, compared to other AAV serotypes little is known about receptor binding and antigenicity of the AAVrh.10 capsid. Particularly, pre-existing neutralizing antibodies against capsids are an important challenge that can hamper treatment efficiency. This study addresses both topics and identifies critical regions of the AAVrh.10 capsid for receptor and antibody binding. The insights gained were utilized to generate AAVrh.10 variants capable of evading known neutralizing antibodies. The findings of this study could further aid the utilization of AAVrh.10 vectors in clinical trials and help the approval of the subsequent biologics.

Neural-specific alterations in glycosphingolipid biosynthesis and cell signaling associated with two human ganglioside GM3 Synthase Deficiency variants

GM3 Synthase Deficiency (GM3SD) is a neurodevelopmental disorder resulting from pathogenic variants in the ST3GAL5 gene, which encodes GM3 synthase, a glycosphingolipid (GSL)-specific sialyltransferase. This enzyme adds a single alpha3-linked sialic acid to the terminal galactose of lactosylceramide (LacCer) to produce the monosialylated ganglioside GM3. In turn, GM3 is extended by other glycosyltransferases to generate nearly all the complex gangliosides enriched in neural tissue. Pathogenic mechanisms that account for neural phenotypes associated with GM3SD are not known. To explore how loss of GM3 impacts neural-specific glycolipid glycosylation and cell signaling, GM3SD patient fibroblasts bearing one of two different ST3GAL5 variants were reprogrammed to induced pluripotent stem cells (iPSCs) and then differentiated to neural crest cells (NCCs). GM3 and GM3-derived gangliosides were undetectable in iPSCs and NCCs from both variants, while LacCer precursor levels were elevated compared to wildtype (WT). NCCs of both variants synthesized elevated levels of neutral lacto- and globo-series, as well as minor alternatively sialylated, GSLs compared to WT. Shifts in ceramide profiles associated with iPSC and NCC GSLs were also detected in GM3SD variants. Altered GSL profiles in the GM3SD cells were accompanied by dynamic changes in the cell surface proteome, protein O-GlcNAcylation, and receptor tyrosine kinase abundance. GM3SD cells also exhibited increased apoptosis and sensitivity to erlotnib, an inhibitor of epidermal growth factor receptor signaling. Pharmacologic inhibition of O-GlcNAcase increased protein O-GlcNAcylation and significantly rescued baseline and erlotnib-induced apoptosis. Collectively, these findings indicate broad effects on cell signaling during differentiation of GM3SD patient-derived iPSCs to NCCs. Thus, human GM3SD cells provide a novel platform to investigate structure/function relationships that connect GSL diversity to cell signaling, cell survival, and neural differentiation.

Novel lamprey antibody recognizes terminal sulfated galactose epitopes on mammalian glycoproteins

The terminal galactose residues of N- and O-glycans in animal glycoproteins are often sialylated and/or fucosylated, but sulfation, such as 3-O-sulfated galactose (3-O-SGal), represents an additional, but poorly understood modification. To this end, we have developed a novel sea lamprey variable lymphocyte receptor (VLR) termed O6 to explore 3-O-SGal expression. O6 was engineered as a recombinant murine IgG chimera and its specificity and affinity to the 3-O-SGal epitope was defined using a variety of approaches, including glycan and glycoprotein microarray analyses, isothermal calorimetry, ligand-bound crystal structure, FACS, and immunohistochemistry of human tissue macroarrays. 3-O-SGal is expressed on N-glycans of many plasma and tissue glycoproteins, but recognition by O6 is often masked by sialic acid and thus exposed by treatment with neuraminidase. O6 recognizes many human tissues, consistent with expression of the cognate sulfotransferases (GAL3ST-2 and GAL3ST-3). The availability of O6 for exploring 3-O-SGal expression could lead to new biomarkers for disease and aid in understanding the functional roles of terminal modifications of glycans and relationships between terminal sulfation, sialylation and fucosylation.

Cooperativity of catalytic and lectin-like domain of T. congolense trans-sialidase modulates its catalytic activity

Trans-sialidases (TS) represent a multi-gene family of unusual enzymes, which catalyse the transfer of terminal sialic acids from sialoglycoconjugates to terminal galactose or N-acetylgalactosamine residues of oligosaccharides without the requirement of CMP-Neu5Ac, the activated Sia used by typical sialyltransferases. Most work on trypanosomal TS has been done on enzymatic activities of TS from T. cruzi (causing Chagas disease in Latin America), subspecies of T. brucei, (causing human sleeping sickness in Africa) and T. congolense (causing African Animal Trypanosomosis in livestock). Previously, we demonstrated that T. congolense TS (TconTS) lectin domain (LD) binds to several carbohydrates, such as 1,4-β-mannotriose. To investigate the influence of TconTS-LD on enzyme activities, we firstly performed in silico analysis on structure models of TconTS enzymes. Findings strongly supports the potential of domain swaps between TconTS without structural disruptions of the enzymes overall topologies. Recombinant domain swapped TconTS1a/TS3 showed clear sialidase and sialic acid (Sia) transfer activities, when using fetuin and lactose as Sia donor and acceptor substrates, respectively. While Sia transfer activity remained unchanged from the level of TconTS1a, hydrolysis was drastically reduced. Presence of 1,4-β-mannotriose during TS reactions modulates enzyme activities favouring trans-sialylation over hydrolysis. In summary, this study provides strong evidence that TconTS-LDs play pivotal roles in modulating enzyme activity and biological functions of these and possibly other TS, revising our fundamental understanding of TS modulation and diversity.

A novel class of human milk oligosaccharides based on 6-galactosyllactose and containing N-acetylglucosamine branches extended by oligogalactoses

Human milk oligosaccharides (HMOs) have attracted much attention in recent years not only as a prebiotic factor, but in particular as an essential component in infant nutrition related to their impact in innate immunity. The backbone structures of complex HMOs generally contain single or repetitive lacto-N-biose (type 1) or lactosamine (type 2) units in either linear or branched chains extending from a lactose core. While all known branched structures originate from 3,6-substitution of the lactosyl core galactose, we here describe a new class of HMOs that tentatively branch at terminal galactose of 6-galactosyllactose. Another novel feature of this class of HMOs was found in linear oligo-galactosyl chains linked to one of the N-acetylglucosamine (GlcNAc) branches. The novel structures exhibit general formulas with hexose vs. hexosamine contents of 5/2 to 8/2 and can be designated as high-galactose (HG)-HMOs. In addition, up to three fucosyl residues are linked to the octa- to dodecasaccharides, which were detected in two human milk samples from Lewis blood group defined donors. Structural analyses of methylated glycans and their alditols comprised MALDI mass spectrometry, ESI-(CID)MS and linkage analyses by GC-MS of the derived partially methylated alditol acetates. Enzymatic degradation by application of β1-3,4-specific galactosidase supported the presence of terminal galactose linked [beta]1-6 to one of the two GlcNAc branches.

Knockout of the CMP–Sialic Acid Transporter SLC35A1 in Human Cell Lines Increases Transduction Efficiency of Adeno-Associated Virus 9: Implications for Gene Therapy Potency Assays

Recombinant adeno-associated viruses (AAV) have emerged as an important tool for gene therapy for human diseases. A prerequisite for clinical approval is an in vitro potency assay that can measure the transduction efficiency of each virus lot produced. The AAV serotypes are typical for gene therapy bind to different cell surface structures. The binding of AAV9 on the surface is mediated by terminal galactose residues present in the asparagine-linked carbohydrates in glycoproteins. However, such terminal galactose residues are rare in cultured cells. They are masked by sialic acid residues, which is an obstacle for the infection of many cell lines with AAV9 and the respective potency assays. The sialic acid residues can be removed by enzymatic digestion or chemical treatment. Still, such treatments are not practical for AAV9 potency assays since they may be difficult to standardize. In this study, we generated human cell lines (HEK293T and HeLa) that become permissive for AAV9 transduction after a knockout of the CMP–sialic acid transporter SLC35A1. Using the human aspartylglucosaminidase (AGA) gene, we show that these cell lines can be used as a model system for establishing potency assays for AAV9-based gene therapy approaches for human diseases.

Asialoglycoprotein receptor 1 is a novel PCSK9-independent ligand of liver LDLR that is shed by Furin

ABSTRACTThe hepatic carbohydrate-recognizing asialoglycoprotein receptor (ASGR1) mediates the endocytosis/lysosomal degradation of desialylated glycoproteins following binding to terminal galactose/N-acetylgalactosamine. Human heterozygote-carriers of ASGR1-deletions exhibited ~34% lower risk of coronary artery disease and ~10-14% non-HDL-cholesterol reduction. Since PCSK9 is a major degrader of LDLR, we examined the regulation of LDLR and/or PCSK9 by ASGR1. We investigated the role of endogenous/overexpressed ASGR1 on LDLR degradation and functionality in naïve HepG2 and HepG2-PCSK9-knockout cells by Western-blot and immunofluorescence.ASGR1, like PCSK9, targets LDLR and both interact with/enhance the degradation of the receptor independently. The lack of cooperativity between PCSK9 and ASGR1 on LDLR expression was confirmed in livers of wild-type (WT) versus Pcsk9-/- mice. ASGR1-knockdown in naïve HepG2 cells significantly increased total (~1.2-fold) and cell-surface (~4-fold) LDLR protein. In HepG2-PCSK9-knockout cells ASGR1-silencing led to ~2-fold higher levels of LDLR protein and DiI-LDL uptake associated with ~4-fold increased cell-surface LDLR. Overexpression of WT-ASGR1 reduced primarily the immature non-O-glycosylated LDLR (~110 kDa), whereas the triple Gln240/Trp244/Glu253 Ala-mutant (loss of carbohydrate-binding) reduced the mature form of the LDLR (~150 kDa), suggesting that ASGR1 binds the LDLR in sugar-dependent and -independent fashion. Furin sheds ASGR1 at RKMK103↓ into a secreted form, likely resulting in a loss-of-function on LDLR. LDLR is the first example of a liver-receptor ligand of ASGR1. Additionally, we demonstrate that lack of ASGR1 enhances LDLR levels and DiI-LDL incorporation, independently of PCSK9. Overall, silencing of ASGR1 and PCSK9 may lead to higher LDL-uptake by hepatocytes, thereby providing a novel approach to further reduce LDL-cholesterol.

Nutrient Responsive O-GlcNAcylation Dynamically Modulates Galectin 3 Secretion

Endomembrane glycosylation and cytoplasmic O-GlcNAcylation each play essential roles in nutrient sensing, and in fact, characteristic changes in glycan patterns have been described in disease states such as diabetes and cancer. These changes in glycosylation have important functional roles and can drive disease progression. However, little is known about the molecular mechanisms underlying how these signals are integrated and transduced into biological effects. Galectins are proteins that bind glycans that are secreted by a poorly characterized non-classical secretory mechanism. Once outside the cell, galectins bind to terminal galactose residues of cell surface glycans and modulate numerous extracellular functions like clathrin independent endocytosis (CIE). Originating in the cytoplasm, galectins are predicted substrates for O-GlcNAc addition and removal. This study shows that galectin 3 is O-GlcNAcylated, and that changes in O-GlcNAc cycling alters its secretion. Moreover, we determined that there is a significant difference in O-GlcNAcylation status between cytoplasmic and secreted galectin 3. We observed dramatic alterations in galectin 3 secretion in response to nutrient conditions and that these changes were dependent on dynamic O-GlcNAcylation. Finally, we showed that alterations in galectin 3 secretion via disrupted O-GlcNAcylation drove changes in CIE. These results indicate that dynamic O-GlcNAcylation of galectin 3 plays a role in modulating its secretion and can tune its function of transducing nutrient sensing information coded in cell surface glycosylation into biological effects.

High expression of tissue O-linked glycans is associated with a malignant phenotype of cholangiocarcinoma

Objective This study aimed to investigate the expression of O-linked glycoprotein glycans in tissue of patients with cholangiocarcinoma compared with adjacent normal tissue. Methods Sixty patients with cholangiocarcinoma were included in the study. Permethylated O-linked glycans from intrahepatic cholangiocarcinoma tissue and adjacent normal tissue were analyzed using nano-spray ionization-linear ion trap mass spectrometry. Histochemistry of peanut agglutinin lectin was used for detection and localization of galactose (Gal) 1, N-acetyl-galactosamine (GalNAc) 1. Results O-linked glycans from patients with cholangiocarcinoma were composed of di- to hexa-saccharides with a terminal galactose and sialic acids (N-acetylneuraminic acid [NeuAc]). A total of eight O-linked glycan structures were detected. Gal1GalNAc1 and Gal2 N-acetyl-glucosamine 1 GalNAc1 expression was significantly higher in tissue from patients with cholangiocarcinoma compared with adjacent normal tissue, while NeuAc1Gal1GalNAc1 expression was significantly lower. High Gal1GalNAc1 expression was significantly associated with the late stage of cholangiocarcinoma (stages II–IV), lymphatic invasion, and vascular invasion. Conclusion Our study shows expression of O-linked glycans in progression of cholangiocarcinoma and highlights the association of Gal1GalNAc1 with lymphatic and vascular invasion of cholangiocarcinoma.