Genetic Removal of Terminal Sialic Acid Residues from the O-Linked Glycans Adjacent to the HPA-9b Polymorphism of Platelet Membrane Glycoprotein IIb Improves the Binding and Detection of HPA-9b Patient Alloantibodies

Sialic acids occupy the terminal position of glycan chains, and have the potential to influence the antigenicity of glycoproteins. Antibody binding sites on a glycoprotein can be solely protein in nature, or can include or be affected by nearby glycan chains, which may either mask the epitope, or conversely comprise part of the antibody binding site. The polymorphisms responsible for formation of the Human Platelet Alloantigens (HPA)-3 (Ile843Ser) and HPA-9 (Val837Met) are next to each other near the C-terminus of the extracellular domain of platelet membrane glycoprotein (GP)IIb, and are adjacent to sialyl-Core 1 O-glycans that emanate from serines 845 and 847. Previous studies have shown that these O-linked glycans are required to support the binding of some of HPA-3a alloantibodies. Loss of these glycans, especially terminal sialic acid residues, during platelet storage or preparation, can present major difficulties in detecting clinically important anti-HPA-3a alloantibodies in suspected cases of fetal/neonatal alloimmune thrombocytopenia (FNAIT). Similarly, detection and identification of anti-HPA-9b alloantibodies from FNAIT patient sera can also be extremely challenging, resulting in the inability to resolve clinical cases of this bleeding disorder. Whether the nearby O-glycans on serines 845 and 847 of GPIIb affect the antigenicity of HPA-9b, and/or influence the binding of anti-HPA-9b alloantibodies in clinically significant cases of FNAIT is unknown. We previously reported the generation of bioengineered, HLA class I-negative, HPA-9a or -9b allele-specific megakaryocytes (MKs) from human induced pluripotent stem cells (iPSCs) that are suitable for whole-cell flow cytometric detection of anti-HPA-9b alloantibodies (Blood 2019;134(22):e1-e8). Unexpectedly, treatment of these allele-specific MKs with neuraminidase actually enhanced the binding of anti-HPA-9b alloantibodies, suggesting that terminal sialic acids on GPIIb partially mask the HPA-9b epitope. To test the hypothesis that removal of terminal sialic acids on nearby O-glycans, or removal of the entire O-glycan chains emanating from Ser845/847 of GPIIb, might enhance the detection of anti-HPA-9b patient alloantibodies, we created a series of deletion mutants in two major sialidases, ST3GAL1 and ST3GAL2, known to be responsible for transferring terminal sialic acid residues to Core 1 O-glycans, in our HPA-9a and -9b allele-specific iPSCs. Immunoprecipitation/western blot analysis confirmed the complete removal of terminal sialic acids on the O-glycan chains of GPIIb in ST3GAL1/2 knockout (KO) iPSC-derived MKs, as reported by the binding of the lectin PNA to the exposed Core 1 structure on GPIIb. These sialylation-deficient ST3GAL1/2 KO HPA-9b MKs exhibited dramatically increased anti-HPA-9b alloantibody binding, further confirming the notion that HPA-9b epitopes are partially masked by terminal sialic acids on nearby GPIIb O-glycan chains. Finally, allele-specific iPSCs lacking the complete O-glycan chains attached to serines 845 and 847 of GPIIb were generated by mutating those residues to alanines using a similar CRISPR/Cas9 gene editing approach. Interestingly, O-glycan chain-deficient Ala845/847 mutant MKs carrying the HPA-9b polymorphism exhibited slightly to moderately reduced binding of anti-HPA-9b alloantibodies, indicating that the presence of the Core 1 O-glycan chains attached to GPIIb serine residues 845 and 847 contribute to the presentation of the HPA-9b epitope - perhaps by stabilizing the conformation of the glycoprotein in this region. Taken together, these data suggest that detection of anti-HPA-9b alloantibodies may be enhanced through the use of iPSC-derived HPA-9b-specific MKs that have been genetically altered to lack nearby terminal sialic acid residues, but retain the glycan chains to which they are attached. Disclosures Curtis: Rallybio: Consultancy. Newman: Rallybio: Consultancy, Research Funding.

Sugar Matters: Improving In Vivo Clearance Rate of Highly Glycosylated Recombinant Plasma Proteins for Therapeutic Use

Correct glycosylation of proteins is essential for production of therapeutic proteins as glycosylation is important for protein solubility, stability, half-life and immunogenicity. The heavily glycosylated plasma protein C1-inhibitor (C1-INH) is used in treatment of hereditary angioedema attacks. In this study, we used C1-INH as a model protein to propose an approach to develop recombinant glycoproteins with the desired glycosylation. We produced fully functional recombinant C1-INH in Chinese hamster ovary (CHO) cells. In vivo we observed a biphasic clearance, indicating different glycosylation forms. N-glycan analysis with mass spectrometry indeed demonstrated heterogeneous glycosylation for recombinant C1-INH containing terminal galactose and terminal sialic acid. Using a Ricinus Communis Agglutinin I (RCA120) column, we could reduce the relative abundance of terminal galactose and increase the relative abundance of terminal sialic acid. This resulted in a fully active protein with a similar in vivo clearance rate to plasmaderived C1-INH. In summary, we describe the development of a recombinant human glycoprotein using simple screening tools to obtain a product that is similar in function and in vivo clearance rate to its plasma-derived counterpart. The approach used here is of potential use in the development of other therapeutic recombinant human glycoproteins.

The terminal sialic acid of stage-specific embryonic antigen-4 has a crucial role in binding to a cancer-targeting antibody

Cancer remains a leading cause of morbidity and mortality worldwide, requiring ongoing development of targeted therapeutics such as monoclonal antibodies. Carbohydrates on embryonic cells are often highly expressed in cancer and are therefore attractive targets for antibodies. Stage-specific embryonic antigen-4 (SSEA-4) is one such glycolipid target expressed in many cancers, including breast and ovarian carcinomas. Here, we defined the structural basis for recognition of SSEA-4 by a novel monospecific chimeric antibody (ch28/11). Five X-ray structures of ch28/11 Fab complexes with the SSEA-4 glycan headgroup, determined at 1.5–2.7 Å resolutions, displayed highly similar three-dimensional structures indicating a stable binding mode. The structures also revealed that by adopting a horseshoe-shaped conformation in a deep groove, the glycan headgroup likely sits flat against the membrane to allow the antibody to interact with SSEA-4 on cancer cells. Moreover, we found that the terminal sialic acid of SSEA-4 plays a dominant role in dictating the exquisite specificity of the ch28/11 antibody. This observation was further supported by molecular dynamics simulations of the ch28/11-glycan complex, which show that SSEA-4 is stabilized by its terminal sialic acid, unlike SSEA-3, which lacks this sialic acid modification. These high-resolution views of how a glycolipid interacts with an antibody may help to advance a new class of cancer-targeting immunotherapy.

Implication of Sialidases in Salmonella Infection: Genome Release of Sialidase Knockout Strains from Salmonella enterica Serovar Typhimurium LT2

ABSTRACT Sialidases, which are widely distributed in nature, cleave the α-ketosidic bond of terminal sialic acid residue. These emerging virulence factors degrade the host glycan. We report here the release of seven sialidase and one sialic acid transporter deletion in Salmonella enterica serovar Typhimurium strain LT2, which are important in cellular invasion during infection.

Polyoxometalates as sialidase mimics: selective and non-destructive removal of sialic acid from a glycoprotein promoted by phosphotungstic acid

The selective hydrolysis of the glycosidic bond between the terminal sialic acid and the penultimate sugar has been achieved in the alpha-2-HS-glycoprotein (Fetuin-A) in the presence of H3PW12O40, a Keggin type polyoxometalate.

Streptococcus oralis Neuraminidase Modulates Adherence to Multiple Carbohydrates on Platelets

ABSTRACT Adherence to host surfaces is often mediated by bacterial binding to surface carbohydrates. Although it is widely appreciated that some bacterial species express glycosidases, previous studies have not considered whether bacteria bind to multiple carbohydrates within host glycans as they are modified by bacterial glycosidases. Streptococcus oralis is a leading cause of subacute infective endocarditis. Binding to platelets is a critical step in disease; however, the mechanisms utilized by S. oralis remain largely undefined. Studies revealed that S. oralis, like Streptococcus gordonii and Streptococcus sanguinis, binds platelets via terminal sialic acid. However, unlike those organisms, S. oralis produces a neuraminidase, NanA, which cleaves terminal sialic acid. Further studies revealed that following NanA-dependent removal of terminal sialic acid, S. oralis bound exposed β-1,4-linked galactose. Adherence to both these carbohydrates required Fap1, the S. oralis member of the serine-rich repeat protein (SRRP) family of adhesins. Mutation of a conserved residue required for sialic acid binding by other SRRPs significantly reduced platelet binding, supporting the hypothesis that Fap1 binds this carbohydrate. The mechanism by which Fap1 contributes to β-1,4-linked galactose binding remains to be defined; however, binding may occur via additional domains of unknown function within the nonrepeat region, one of which shares some similarity with a carbohydrate binding module. This study is the first demonstration that an SRRP is required to bind β-1,4-linked galactose and the first time that one of these adhesins has been shown to be required for binding of multiple glycan receptors.