Sialylation Of Glycans Research Articles

Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

The Spike of SARS-CoV-2 recognizes a transmembrane protease, angiotensin-converting enzyme 2 (ACE2), on host cells to initiate infection. Soluble derivatives of ACE2, in which Spike affinity is enhanced and the protein is fused to Fc of an immunoglobulin, are potent decoy receptors that reduce disease in animal models of COVID-19. Mutations were introduced into an ACE2 decoy receptor, including adding custom N-glycosylation sites and a cavity-filling substitution together with Fc modifications, which increased the decoy's catalytic activity and provided small to moderate enhancements of pharmacokinetics following intravenous and subcutaneous administration in humanized FcRn mice. Most prominently, sialylation of native glycans increases exposures by orders of magnitude, and the optimized decoy is therapeutically efficacious in a mouse COVID-19 model. Ultimately, an engineered and highly sialylated decoy receptor produced using methods suitable for manufacture of representative drug substance has high exposure with a 5- to 9-day half-life. Finally, peptide epitopes at mutated sites in the decoys generally have low binding to common HLA class II alleles and the predicted immunogenicity risk is low. Overall, glycosylation is a critical molecular attribute of ACE2 decoy receptors and modifications that combine tighter blocking of Spike with enhanced pharmacokinetics elevate this class of molecules as viable drug candidates.

Sialyltransferase ST3GAL4 confers osimertinib resistance and offers strategies to overcome resistance in non-small cell lung cancer

The third-generation EGFR-TKI osimertinib is widely used in EGFR-mutated positive non-small cell lung cancer (NSCLC) patients, but drug resistance is inevitable. The currently known mechanisms only explain resistance in a small proportion of patients. For most patients, the mechanism of osimertinib resistance is still unclear, especially for EGFR-independent resistance. Herein, we thoroughly investigated the novel mechanism of osimertinib resistance and treatment strategies. We identified that ST3GAL4, a sialyltransferase, catalyzes terminal glycan sialylation of receptor protein tyrosine kinases, which induces acquired resistance to osimertinib in vitro and in vivo. In addition, ST3GAL4 is generally overexpressed in osimertinib-resistant patients with unknown resistance mechanisms. ST3GAL4 modifies MET glycosylation on N785 with sialylation, which antagonizes K48-related ubiquitin-dependent MET degradation and subsequently activates MET and its downstream proliferation signaling pathways. Meanwhile, ST3GAL4 knockdown or inhibition by brigatinib resensitizes resistant non-small cell lung cancer cells to osimertinib in vitro and in vivo This study suggests that ST3GAL4 can induce acquired resistance to osimertinib, which may be an important EGFR-independent resistance mechanism Furthermore, targeting ST3GAL4 with brigatinib provides new strategies to overcome osimertinib resistance.

A14 ABLATION OF THE INTESTINAL EPITHELIAL SIALOME PREDISPOSES TO SPONTANEOUS COLITIS AND COLITIS-ASSOCIATED TUMORIGENESIS VIA MUCUS-DEPENDENT AND -INDEPENDENT MECHANISMS IN MICE

Abstract Background Recent work suggests epithelial sialylation (capping of glycoconjugates with the acidic sugar sialic acid) can protect the gut mucosa from inflammatory bowel disease by promoting mucus function. However, these studies are based on partial sialylation knockouts, potentially underestimating the role of glycoconjugate sialylation in mucosal homeostasis. Aims We sought to uncover how the complete intestinal epithelial sialome (the total glycan sialylation repetoire) regulates colitis susceptibility in vivo. Methods We generated a model of total gut epithelial sialylation deficiency by conditionally and inducibly (via tamoxifen, TM) targeting the CMP-Sia transporter, Slc35a1, in epithelial cells using Cre-Lox technology (Slc35a1f/f; VillinCre(ERT2) or (TM-)IEC Slc35a1-/- mice). We analyzed phenotypes clinically, histologicaly, and via multi-omic apporaches at baseline and in response to challenge. Results We confirmed the ablation of mucus sialylation via lectin staining, epithelial Slc35a1 gene expression, and O-glycomics of extracted mucins. In contrast to partial sialylation deficiencies, we found that IEC Slc35a1-/- mice developed mild spontaneous microbiota-dependent chronic colitis that was associated with tumorigenesis in the rectum in 30% of aged mice (>12 mo.). TM-IEC Slc35a1-/- mice developed less severe spontaneous colitis, but were susceptible to acute injury with 1% DSS. Ablation of the gut epithelial sialome led to reduced mucus thickness on fecal sections and within tissues, although the b1/b2 barrier layers were still intact. TM-IEC Slc35a1-/- mice showed altered microbiota composition with an increase in Clostridium spp. at the expense of a global reduction in the abundance of at least 20 unique taxa; however metabolomic analysis did not show any significant differences in short-chain fatty acid levels. IEC Slc35a1-/- Muc2 was more susceptible to fecal protease degradation vs.WT Muc2. While no spontaneous phenotype was observed in the small intestine (SI), treatment with 5-fluorouracil led to worsened SI mucositis and morbidity vs. WT littermates. This was traced to impaired stem cell activities via reduced Lgr5+ cell representation in small intestinal crypts in IEC Slc35a1-/-;Lgr5GFP mice. Conclusions Ablation of the intestinal epithelial sialome impacts the mucus-microbiota-stem cell axis with consequences on colonic mucosal homeostasis based on the type of injury. At baseline, loss of sialylation impairs mucus stability and the stem cell niche leading to microbiota-dependent spontaneous colitis and tumorigenesis. However, these same epithelial defects impair epithelial renewal and worsen colitis following acute injury likely due to the cytotoxic environment caused by the chemical agent. Funding Agencies CCC

Discovery of a haptoglobin glycopeptides biomarker panel for early diagnosis of hepatocellular carcinoma.

There is a need for new serum biomarkers for early detection of hepatocellular carcinoma (HCC). Haptoglobin (Hp) N-glycosylation is altered in HCC, but the diagnostic value of site-specific Hp glycobiomarkers is rarely reported. We aimed to determine the site-specific glycosylation profile of Hp for early-stage HCC diagnosis. Hp glycosylation was analyzed in the plasma of patients with liver diseases (n=57; controls), early-stage HCC (n=50) and late-stage HCC (n=32). Hp phenotype was determined by immunoblotting. Hp was immunoisolated and digested into peptides. N-glycopeptides were identified and quantified using liquid chromatography-mass spectrometry. Cohort samples were compared using Wilcoxon rank-sum (Mann-Whitney U) tests. Diagnostic performance was assessed using receiver operating characteristic (ROC) curves and area under curve (AUC). Significantly higher fucosylation, branching and sialylation of Hp glycans, and expression of high-mannose glycans, was observed as disease progressed from cirrhosis to early- and late-stage HCC. Several glycopeptides demonstrated high values for early diagnosis of HCC, with an AUC of 93% (n=1), >80% (n=3), >75% (n=13) and >70% (n=11), compared with alpha-fetoprotein (AFP; AUC of 79%). The diagnostic performance of the identified biomarkers was only slightly affected by Hp phenotype. We identified a panel of Hp glycopeptides that are significantly differentially regulated in early- and late-stage HCC. Some glycobiomarkers exceeded the diagnostic value of AFP (the most commonly used biomarker for HCC diagnosis). Our findings provide evidence that glycobiomarkers can be effective in the diagnosis of early HCC - individually, as a panel of glycopeptides or combined with conventional serological biomarkers.

Pathogenicity ofIgG-Fc desialylation andits association with Th17 cells inan animal model ofsystemic lupus erythematosus.

Decreased sialylation of IgG-Fc glycans has been reported in autoimmune diseases, but its role in systemic lupus erythematosus (SLE) is not fully understood. In this study, we examined the pathogenicity of IgG desialylation and its association with Th17 in SLE using an animal model. B6SKG mice, which develop lupus-like systemic autoimmunity due to the ZAP70 mutation, were used to investigate the pathogenicity of IgG desialylation. The proportion of sialylated IgG was compared between B6SKG and wild-type mice with or without β-glucan treatment-induced Th17 expansion. Anti-interleukin (IL)-23 and anti-IL-17 antibodies were used to examine the role of Th17 cells in IgG glycosylation. Activation-induced cytidine deaminase-specific St6gal1 conditionally knockout (cKO) mice were generated to examine the direct effect of IgG desialylation. The proportions of sialylated IgG were similar between B6SKG and wild-type mice in the steady state. However, IgG desialylation was observed after β-glucan-induced Th17 expansion, and nephropathy also worsened in B6SKG mice. Anti-IL-23/17 treatment suppressed IgG desialylation and nephropathy. Glomerular atrophy was observed in the cKO mice, suggesting that IgG desialylation is directly involved in disease exacerbation. IgG desialylation contributes to the progression of nephropathy, which is ameliorated by blocking IL-17A or IL-23 in an SLE mouse model.

Tumor cell-intrinsic and tumor microenvironmental conditions co-determine signaling by the glycoimmune checkpoint receptor Siglec-7

Tumors create an immunosuppressive tumor microenvironment by altering protein expression, but also by changing their glycosylation status, like altered expression of sialoglycans. Sialoglycans are capped with sialic acid sugar residues and are recognized by Siglec immune receptors. Siglec-7 is an inhibitory immune receptor similar to PD-1, and is emerging as glycoimmune checkpoint exploited by cancer cells to evade the immune system. However, the exact cellular and molecular conditions required for Siglec-7-mediated immune cell inhibition remain largely unknown. Here, we report on the development of a chimeric Siglec-7 cell system that enables dissection of Siglec-7 signaling, rather than Siglec-7 binding. Antibody-induced clustering, sialic acid-containing polymers, and highly sialylated erythrocytes effectively induced Siglec-7 signaling, thereby validating functionality of this reporter system. Moreover, the system reveals tumor cell-dependent Siglec-7 signaling. Tumor-associated conditions important for Siglec-7 signaling were defined, such as Siglec-7 ligand expression levels, presence of the known Siglec-7 ligand CD43, and sialic acid availability for sialylation of glycans. Importantly, therapeutic targeting of the Siglec-7/sialic acid axis using a sialyltransferase inhibitor resulted in strong reduction of Siglec-7 signaling. In conclusion, using a newly established cellular tool, we defined a set of tumor-associated conditions that influence Siglec-7 signaling. Moreover, the system allows to assess the efficacy of novel cancer drugs interfering with the Siglec-7/sialic acid axis as immunotherapy to treat cancer.

Mucus sialylation determines intestinal host-commensal homeostasis

Abstract Intestinal mucus forms the first line of defense against bacterial invasion while providing nutrition to support microbial symbiosis. How the host controls mucus barrier integrity and commensalism is unclear. We show that terminal sialylation of glycans on intestinal mucus by ST6GALNAC1 (ST6), the dominant sialyltransferase specifically expressed in goblet cells and induced by microbial pathogen-associated molecular patterns, is essential for mucus integrity and protecting against excessive bacterial proteolytic degradation. Glycoproteomic profiling and biochemical analysis of ST6 mutations identified in patients show that decreased sialylation causes defective mucus proteins and congenital inflammatory bowel disease (IBD). Mice harboring a patient ST6 mutation have compromised mucus barriers, dysbiosis, and susceptibility to intestinal inflammation. Based on our understanding of the ST6 regulatory network, we show that treatment with sialylated mucin or a Foxo3 inhibitor can ameliorate IBD. Supported by the Intramural Research Program of the NIAID, NIH, United States and by National Multiple Sclerosis Society Career Transition Award, United States (TA 3059-A-2 to C.W.).

Retrospective screening of serum IgG glycosylation biomarker for primary Sjögren's syndrome using lectin microarray.

Primary Sjögren's syndrome (PSS) is a systemic autoimmune disease resulting in significant loss of systemic gland secretory function. IgG glycosylation abnormalities had been found to play important roles in autoimmune diseases. Here, we aim to explore the specific changes of IgG glycosylation in PSS patient serum that could serve as potential biomarkers for disease diagnosis and differential diagnosis. From 2012 to 2018, patients diagnosed with PSS or primary biliary cholangitis (PBC) admitted consecutively to the department of Rheumatology at Peking Union Medical College Hospital were retrospectively included in this study. Glycan profiles of serum IgG from 40 PSS patients, 50 PBC patients, and 38 healthy controls were detected with lectin microarray containing 56 lectins. Lectins with significantly different signal intensity among groups were selected and validated by lectin blot assay. Lectin microarray analysis revealed that binding levels of Amaranthus Caudatus Lectin (ACL, prefers glycan Galβ3GalNAc, P = 0.011), Morniga M Lectin (MNA-M, prefers glycan mannose. P = 0.013), and Lens Culinaris Agglutinin (LCA, prefers glycan fucose) were significantly increased, while Salvia sclarea Agglutinin (SSA, prefers glycan sialylation, P = 0.001) was significantly decreased in PSS patients compared to PBC group. Compared to healthy controls, MNA-M (P = 0.001) and LCA (P = 0.028) were also significantly increased, while Phaseolus Vulgaris Erythroagglutinin and Phaseolus Vulgaris Leucoagglutinin (PHA-E and PHA-L, prefer glycan galactose, P = 0.004 and 0.006) were significantly decreased in PSS patients. The results of LCA and MNA-M were further confirmed using lectin blot assay. Changes in serum IgG glycosylation in PSS increased binding levels of LCA and MNA-M lectins using microarray techniques compared to PBC patients and healthy controls, which could provide potential diagnostic value. Increased core fucose and mannose alteration of IgG may play important roles in PSS disease.

Deficiency in ST6GAL1, one of the two α2,6-sialyltransferases, has only a minor effect on the pathogenesis of prion disease.

Prion diseases are a group of fatal neurodegenerative diseases caused by misfolding of the normal cellular form of the prion protein or PrPC, into a disease-associated self-replicating state or PrPSc. PrPC and PrPSc are posttranslationally modified with N-linked glycans, in which the terminal positions occupied by sialic acids residues are attached to galactose predominantly via α2-6 linkages. The sialylation status of PrPSc is an important determinant of prion disease pathogenesis, as it dictates the rate of prion replication and controls the fate of prions in an organism. The current study tests whether a knockout of ST6Gal1, one of the two mammalian sialyltransferases that catalyze the sialylation of glycans via α2-6 linkages, reduces the sialylation status of PrPSc and alters prion disease pathogenesis. We found that a global knockout of ST6Gal1 in mice significantly reduces the α2-6 sialylation of the brain parenchyma, as determined by staining with Sambucus Nigra agglutinin. However, the sialylation of PrPSc remained stable and the incubation time to disease increased only modestly in ST6Gal1 knockout mice (ST6Gal1-KO). A lack of significant changes in the PrPSc sialylation status and prion pathogenesis is attributed to the redundancy in sialylation and, in particular, the plausible involvement of a second member of the sialyltransferase family that sialylate via α2-6 linkages, ST6Gal2.

Sialylated glycoproteins as biomarkers and drivers of progression in prostate cancer

Sialic acids have been implicated in cancer initiation, progression, and immune evasion in diverse human malignancies. Sialylation of terminal glycans on cell surface and secreted glycoproteins is a long-recognized feature of cancer cells. Recently, immune checkpoint inhibitor immunotherapy has tremendously improved the outcomes of patients with various cancers. However, available immunotherapy approaches have had limited efficacy in metastatic castration-resistant prostate cancer. Sialic acid modified glycoproteins in prostate cancers and their interaction with Siglec receptors on tumor infiltrating immune cells might underlie immunosuppressive signaling in prostate cancer. Here, we summarize the function of sialic acids and relevant glycosynthetic enzymes in cancer initiation and progression. We also discuss the possible uses of sialic acids as biomarkers in prostate cancer and the potential methods for targeting Siglec-sialic acid interactions for prostate cancer treatment.

Overlapping and unique substrate specificities of ST3GAL1 and 2 during hematopoietic and megakaryocytic differentiation

AbstractAlthough the sialyltransferases ST3GAL1 and ST3GAL2 are known to transfer sialic acid to the galactose residue of type III disaccharides (Galβ1,3GalNAc) in vitro, sialylation of O-linked glycosylated proteins in living cells has been largely attributed to ST3GAL1. To examine the role of ST3GAL2 in O-sialylation, we examined its expression during differentiation of human-induced pluripotent stem cells (iPSCs) into hematopoietic progenitor cells (HPCs) and megakaryocytes (MKs). ST3GAL1 and ST3GAL2 each became highly expressed during the differentiation of iPSCs to HPCs but decreased markedly in their expression upon differentiation into MKs, suggesting coordination of expression during megakaryopoiesis. To further delineate their role in these processes, we generated ST3GAL1-, ST3GAL2-, and doubly deficient human iPSC lines. Binding of the peanut agglutinin lectin, which reports the presence of unsialylated Galβ1,3GalNAc glycan chains, was strongly increased in HPCs and MKs derived from double-knockout iPSCs and remained moderately increased in cells lacking either one of these sialyltransferases, demonstrating that both can serve as functional cellular O-glycan sialyltransferases. Interestingly, the HPC markers CD34 and CD43, as well as MK membrane glycoprotein (GP) GPIbα, were identified as major GP substrates for ST3GAL1 and ST3GAL2. In contrast, O-sialylation of GPIIb relied predominantly on the expression of ST3GAL2. Finally, although disruption of ST3GAL1 and ST3GAL2 had little impact on MK production, their absence resulted in dramatically impaired MK proplatelet formation. Taken together, these data establish heretofore unknown physiological roles for ST3GAL1 and ST3GAL2 in O-linked glycan sialylation in hemato- and megakaryocytopoiesis.

Mucin 21 confers resistance to apoptosis in an O-glycosylation-dependent manner

Highly glycosylated mucins protect epithelial surfaces from external insults and are related to malignant behaviors of carcinoma cells. However, the importance of carbohydrate chains on mucins in the process of cellular protection is not fully understood. Here, we investigated the effect of human mucin-21 (MUC21) expression on the susceptibility to apoptosis. MUC21 transfection into HEK293 cells decreased the number of apoptotic cells in culture media containing etoposide or after ultraviolet light irradiation. We used Chinese hamster ovary (CHO) cell variants to investigate the importance of MUC21 glycosylation in the resistance to apoptosis. When MUC21 was expressed in CHO-K1 cells, it was glycosylated with sialyl T-antigen and the cells showed resistance to etoposide-induced apoptosis. MUC21 transfection into Lec2 cells, a variant of CHO cells lacking sialylation of glycans, revealed that the presence of nonsialylated T-antigen also renders cells resistant to etoposide-induced apoptosis. MUC21 was transfected into ldlD cells and the glycosylation was manipulated by supplementation to the medium. Nonsupplemented cells and cells supplemented with N-acetylgalactosamine showed no resistance to etoposide-induced apoptosis. In contrast, these cells supplemented with N-acetylgalactosamine plus galactose expressed sialyl T-antigen and exhibited resistance to etoposide-induced apoptosis. Finally, galectin-3 knockdown in MUC21 transfectants of HEK293 cells did not significantly affect MUC21-dependent induction of apoptosis resistance. The results suggest that T-antigen with or without sialic acid is essential to the antiapoptotic effect of MUC21.

Mucus sialylation determines intestinal host-commensal homeostasis

Intestinal mucus forms the first line of defense against bacterial invasion while providing nutrition to support microbial symbiosis. How the host controls mucus barrier integrity and commensalism is unclear. We show that terminal sialylation of glycans on intestinal mucus by ST6GALNAC1 (ST6), the dominant sialyltransferase specifically expressed in goblet cells and induced by microbial pathogen-associated molecular patterns, is essential for mucus integrity and protecting against excessive bacterial proteolytic degradation. Glycoproteomic profiling and biochemical analysis of ST6 mutations identified in patients show that decreased sialylation causes defective mucus proteins and congenital inflammatory bowel disease (IBD). Mice harboring a patient ST6 mutation have compromised mucus barriers, dysbiosis, and susceptibility to intestinal inflammation. Based on our understanding of the ST6 regulatory network, we show that treatment with sialylated mucin or a Foxo3 inhibitor can ameliorate IBD.

Role of sialylation of N-linked glycans in prion pathogenesis.

Mammalian prion or PrPSc is a proteinaceous infectious agent that consists of a misfolded, self-replicating state of the prion protein or PrPC. PrPC and PrPSc are posttranslationally modified with N-linked glycans, which are sialylated at the terminal positions. More than 30years have passed since the first characterization of the composition and structural diversity of N-linked glycans associated with the prion protein, yet the role of carbohydrate groups that constitute N-glycans and, in particular, their terminal sialic acid residues in prion disease pathogenesis remains poorly understood. A number of recent studies shed a light on the role of sialylation in the biology of prion diseases. This review article discusses several mechanisms by which terminal sialylation dictates the spread of PrPSc across brain regions and the outcomes of prion infection in an organism. In particular, relationships between the sialylation status of PrPSc and important strain-specific features including lymphotropism, neurotropism, and neuroinflammation are discussed. Moreover, emerging evidence pointing out the roles of sialic acid residues in prion replication, cross-species transmission, strain competition, and strain adaptation are reviewed. A hypothesis according to which selective, strain-specified recruitment of PrPC sialoglycoforms dictates unique strain-specific disease phenotypes is examined. Finally, the current article proposes that prion strains evolve as a result of a delicate balance between recruiting highly sialylated glycoforms to avoid an "eat-me" response by glia and limiting heavily sialylated glycoforms for enabling rapid prion replication.

One-pot multienzyme (OPME) chemoenzymatic synthesis of brain ganglioside glycans with human ST3GAL II expressed in E. coli.

A human sialyltransferase ST3GAL II (hST3GAL II) was successfully expressed in Escherichia coli as an active soluble fusion protein with an N-terminal maltose-binding protein (MBP) and a C-terminal hexa-histidine tag. It was used as an efficient catalyst in a one-pot multienzyme (OPME) sialylation system for high-yield production of the glycans of ganglioside GM1b and highly sialylated brain gangliosides GD1a and GT1b. Further sialylation of GM1b and GD1a glycans using a bacterial α2-8-sialyltransferase in another OPME sialylation reaction led to the formation of the glycans of GD1c and brain ganglioside GT1a, respectively. The lower reverse glycosylation activity of the recombinant hST3GAL II compared to its bacterial sialyltransferase counterpart simplifies the handling of enzymatic synthetic reactions and has an advantage for future use in automated chemoenzymatic synthetic processes.

Fc glycan sialylation of biotherapeutic monoclonal antibodies has limited impact on antibody-dependent cellular cytotoxicity.

It has been well documented that the terminal sugars of Fc glycans can play a critical role in the safety and efficacy of therapeutic mAbs. However, many of the effects of highly heterogeneous Fc glycan structures have yet to be fully characterized. Different glycosylation patterns can affect Fc‐dependent activities, such as the ability of mAbs to bind Fcγ receptors on the effector cell surface, which is critical to immune effector functions, such as antibody‐dependent cellular cytotoxicity (ADCC). Previous studies on the impact of sialic acid in the Fc glycan on ADCC have not resulted in consistent conclusions. In our study, we tested sialic acid‐enriched species from a chimeric murine/human kappa light chain IgG1 (mAb1) with known Fcγ receptor IIIa binding and ADCC activities. These enriched species contained up to a fourfold increase in sialic acid‐containing glycans relative to the typical levels present in therapeutic mAbs, along with other attributes such as oxidized and deamidated species. The ADCC analysis of sialylated and asialo mAb1 provided herein shows evidence that sialic acids have little or no impact on ADCC activity. Altogether, our results highlight the value of novel glycan engineering strategies in designing therapeutic mAbs with high‐quality attributes and in improving production process controls.

Cellular and Molecular Engineering of Glycan Sialylation in Heterologous Systems.

Glycans have been shown to play a key role in many biological processes, such as signal transduction, immunogenicity, and disease progression. Among the various glycosylation modifications found on cell surfaces and in biomolecules, sialylation is especially important, because sialic acids are typically found at the terminus of glycans and have unique negatively charged moieties associated with cellular and molecular interactions. Sialic acids are also crucial for glycosylated biopharmaceutics, where they promote stability and activity. In this regard, heterogenous sialylation may produce variability in efficacy and limit therapeutic applications. Homogenous sialylation may be achieved through cellular and molecular engineering, both of which have gained traction in recent years. In this paper, we describe the engineering of intracellular glycosylation pathways through targeted disruption and the introduction of carbohydrate active enzyme genes. The focus of this review is on sialic acid-related genes and efforts to achieve homogenous, humanlike sialylation in model hosts. We also discuss the molecular engineering of sialyltransferases and their application in chemoenzymatic sialylation and sialic acid visualization on cell surfaces. The integration of these complementary engineering strategies will be useful for glycoscience to explore the biological significance of sialic acids on cell surfaces as well as the future development of advanced biopharmaceuticals.

GLYCOSYLATION FEATURES OF FOLLITROPIN DELTA (REKOVELLE®) MORE RESEMBLE THOSE OF NATURAL HUMAN FSH COMPARED TO FOLLITROPIN ALFA (GONAL F®).

Compare the glycosylation properties of follitropin delta, from a human cell line, with those of natural human derived FSH and with follitropin alfa from a hamster cell line. Follitropin delta (Rekovelle®) produced in PER.C6® human derived cell line, follitropin alfa (Gonal f®) produced in CHO derived cell line and natural human urinary derived FSH (Bravelle®) were used in the study. Isoelectric Focusing (IEF), was used to resolve and profile the isoforms present in FSH preparations. IEF was carried out using a gel with a pH range between pH 3-7. Glycans were released from FSH preparations by enzymatic digestion with PNGase and derivatized with 2-aminobenzamide (2AB). Glycans were sequenced with specific exoglycosidases and analysed using normal phase HPLC and weak anion exchange chromatography. A more acidic isoform profile was obtained by IEF for follitropin delta and human derived FSH in comparison to follitropin alfa. Compared to follitropin alfa, the released glycans from follitropin delta and natural human FSH both show higher abundance of tri- and tetra-antennary forms and a lower abundance of the di-antennary forms. Sialylation of glycans on follitropin delta and natural human FSH comprises both α2,3 linked and α2,6 linked sialic acid, whereas follitropin alfa exhibits only the α2,3 linked form. The resemblance of follitropin delta glycans to the human derived FSH was further supported by the presence of N-acetylgalactosamine (GalNAc) and bisecting N-acetylglucosamine (GlcNAc) which are absent in CHO-derived FSH (Table). Follitropin delta glycosylation is richer and more complex compared to follitropin alfa with a higher proportion of tri- and tetra-antennary and charged glycans, as well as by the presence of α2,6-linked sialic acid, GalNAc residues and bisecting GlcNAc. In these ways follitropin delta shares numerous glycosylation features and more resembles natural human derived FSH.

Thyrotropin N-glycosylation and Glycan Composition in Severe Primary Hypothyroidism.

ContextIn severe primary hypothyroidism (sPH), the serum thyrotropin (TSH) levels are elevated with an increased degree of sialylation. The circulating TSH comprises 2 different TSH glycoforms: TSHdi with 2 and TSHtri with 3 N-glycans and methods have developed to determine their contents of anionic monosaccharides (AMS), that is, sialic acid (SA) and sulfonated N-acetylglactosamine (SU) residues.ObjectiveCharacterize N-glycosylation and glycan composition of circulating TSH molecules and determine the effects during levothyroxine treatment in patients with sPH.MethodsSerum samples were obtained from 25 patients with sPH, from 159 euthyroid individuals, and from 12 women during treatment with levothyroxine for sPH. Degrees of N-glycosylation and concentrations of TSHdi and TSHtri as well as their contents of AMS, SA, and SU residues were determined.ResultsThe circulating TSH molecules in sPH patients had lower degrees of N-glycosylation, higher degrees of sialylation, and lower degrees of sulfonation than in euthyroid individuals. Levothyroxin restored sialylation and sulfonation of the glycans already at low free thyroxine (FT4) levels, while degree of N-glycosylation was not restored until the FT4 levels were normal.ConclusionsThe majority of TSH molecules in severe primary hypothyroidism were less N- glycosylated, more sialylated, and less sulfonated compared with euthyroid individuals. This glycan pattern favors a prolonged half-life in the circulation combined with lower in vitro biopotency at the target cells. During levothyroxine treatment of sPH patients, the sialylation and sulfonation of glycans were restored already at low FT4 levels, while N-glycosylation of TSH was not restored until the FT4 levels were normal.

VALIDATION OF HPTM-001, A HUMANIZED CANDIDATE THERAPEUTIC ANTIBODY FOR PROMOTING MUCOSAL WOUND HEALING IN IBD

Abstract A hallmark of the clinical course of patients with Inflammatory Bowel Disease (IBD) is poorly healing erosions and ulcers in the intestinal mucosa. Despite the adverse clinical consequences of non-healing mucosal wounds in IBD, current front-line therapies that selectively target mucosal wound healing are not available. Recent studies revealed an association between colonic inflammation and aberrant glycosylation of epithelial CD44v6. Under conditions of inflammation, epithelial CD44v6 was shown to be decorated with the terminal glycan sialyl Lewis A. Importantly, targeting sialylated Lewis glycans on CD44v6 with a murine antibody GM35 was shown to promote mucosal wound healing in cell lines and in biopsy based wounding assays as well as dextran sodium sulfate (DSS) induced murine colitis models. We sequenced CDRs from GM35 and produced a humanized antibody. Eight candidate human IgG1 clones were produced and screened. hPTM-001.4772 was selected from the eight candidates based on glycan affinity and selectivity compared to GM35. In vitro wound healing studies revealed that PTM-001.4772 was as effective as GM35 in promoting repair of scratch wounds with human intestinal epithelial cells. Furthermore, intraperitoneal injection of mice with hPTM-001.4772 during induction of DSS colitis resulted in reduced weight loss compared to control IgG. These results suggest that hPTM-001.4772 is well-positioned as a unique potential candidate therapeutic for IBD that can be used to selectively promote healing of mucosal wounds and ulcers.