Insights into the hyperglycosylation of human chorionic gonadotropin revealed by glycomics analysis.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone that is essential for the maintenance of pregnancy. Glycosylation of hCG is known to be essential for its biological activity. "Hyperglycosylated" variants secreted during early pregnancy have been proposed to be involved in initial implantation of the embryo and as a potential diagnostic marker for gestational diseases. However, what constitutes "hyperglycosylation" is not yet fully understood. In this study, we perform comparative N-glycomic analysis of hCG expressed in the same individuals during early and late pregnancy to help provide new insights into hCG function, reveal new targets for diagnostics and clarify the identity of hyperglycosylated hCG. hCG was isolated in urine collected from women at 7 weeks and 20 weeks' gestation. hCG was also isolated in urine from women diagnosed with gestational trophoblastic disease (GTD). We used glycomics methodologies including matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) and MS/MS methods to characterise the N-glycans associated with hCG purified from the individual samples. The structures identified on the early pregnancy (EP-hCG) and late pregnancy (LP-hCG) samples corresponded to mono-, bi-, tri-, and tetra-antennary N-glycans. A novel finding was the presence of substantial amounts of bisected type N-glycans in pregnancy hCG samples, which were present at much lower levels in GTD samples. A second novel observation was the presence of abundant LewisX antigens on the bisected N-glycans. GTD-hCG had fewer glycoforms which constituted a subset of those found in normal pregnancy. When compared to EP-hCG, GTD-hCG samples had decreased signals for tri- and tetra-antennary N-glycans. In terms of terminal epitopes, GTD-hCG had increased signals for sialylated structures, while LewisX antigens were of very minor abundance. hCG carries the same N-glycans throughout pregnancy but in different proportions. The N-glycan repertoire is more diverse than previously reported. Bisected and LewisX structures are potential targets for diagnostics. hCG isolated from pregnancy urine inhibits NK cell cytotoxicity in vitro at nanomolar levels and bisected type glycans have previously been implicated in the suppression of NK cell cytotoxicity, suggesting that hCG-related bisected type N-glycans may directly suppress NK cell cytotoxicity.

N-glycosylation profile analysis of Trastuzumab biosimilar candidates by Normal Phase Liquid Chromatography and MALDI-TOF MS approaches.

The pharmaceutical market has entered an era in which the production of new therapeutics is being often replaced by "biosimilars", copies of already commercialized products waiting for the patents to expire in order to be distributed in a more competitive and affordable manners. Due to its relevance, the ErbB2-targeted monoclonal antibody Trastuzumab (Herceptin) used as breast cancer therapy is one of the main targets in the production of biosimilars. A major challenge is to produce antibodies with the same or the closest N-glycosylation pattern seen in the commercialized drug. Several factors, such as growing conditions or cell types employed, can determine the final composition and structure of the glycans, significantly affecting the properties of the generated antibodies. Therefore, an appropriate characterization is essential. In the present study, we describe two different but complementary strategies to characterize the N-glycosylation of two biosimilar candidates of Trastuzumab. In the first case, N-glycans are fluorescently labeled and separated by Normal Phase HPLC. Different sugars will elute at different times and can be identified using specific oligosaccharide standards. In the second approach, released glycans are permethylated and analyzed by MALDI-TOF MS, being able to determine the structure because of the differential sugar masses. BIOLOGICAL SIGNIFICANCE: The characterization of the N-glycosylation sites of therapeutic recombinant monoclonal antibodies (mAbs) is usually one of the most critical and time consuming steps in the developing process of biosimilars or any other glycosylated drug. Herein we describe two different but complementary approaches to characterize mAbs glycosylation patterns, the use of glycan fluorescence labeling coupled to HPLC and MALDI-TOF MS profile analysis. This article is part of a Special Issue entitled: HUPO 2014.

Methylated glycans as conserved targets of animal and fungal innate defense.

Effector proteins of innate immune systems recognize specific non-self epitopes. Tectonins are a family of ß-propeller lectins conserved from bacteria to mammals that have been shown to bind bacterial lipopolysaccharide (LPS). We present experimental evidence that two Tectonins of fungal and animal origin have a specificity for O-methylated glycans. We show that Tectonin 2 of the mushroom Laccaria bicolor (Lb-Tec2) agglutinates Gram-negative bacteria and exerts toxicity toward the model nematode Caenorhabditis elegans, suggesting a role in fungal defense against bacteria and nematodes. Biochemical and genetic analysis of these interactions revealed that both bacterial agglutination and nematotoxicity of Lb-Tec2 depend on the recognition of methylated glycans, namely O-methylated mannose and fucose residues, as part of bacterial LPS and nematode cell-surface glycans. In addition, a C. elegans gene, termed samt-1, coding for a candidate membrane transport protein for the presumptive donor substrate of glycan methylation, S-adenosyl-methionine, from the cytoplasm to the Golgi was identified. Intriguingly, limulus lectin L6, a structurally related antibacterial protein of the Japanese horseshoe crab Tachypleus tridentatus, showed properties identical to the mushroom lectin. These results suggest that O-methylated glycans constitute a conserved target of the fungal and animal innate immune system. The broad phylogenetic distribution of O-methylated glycans increases the spectrum of potential antagonists recognized by Tectonins, rendering this conserved protein family a universal defense armor.

Chemoenzymatic synthesis of O-mannosylpeptides in solution and on solid phase.

O-mannosyl glycans are known to play an important role in regulating the function of α-dystroglycan (α-DG), as defective glycosylation is associated with various phenotypes of congenital muscular dystrophy. Despite the well-established biological significance of these glycans, questions regarding their precise molecular function remain unanswered. Further biological investigation will require synthetic methods for the generation of pure samples of homogeneous glycopeptides with diverse sequences. Here we describe the first total syntheses of glycopeptides containing the tetrasaccharide NeuNAcα2-3Galß1-4GlcNAcß1-2Manα, which is reported to be the most abundant O-mannosyl glycan on α-DG. Our approach is based on biomimetic stepwise assembly from the reducing end and also gives access to the naturally occurring mono-, di-, and trisaccharide substructures. In addition to the total synthesis, we have developed a "one-pot" enzymatic cascade leading to the rapid synthesis of the target tetrasaccharide. Finally, solid-phase synthesis of the desired glycopeptides directly on a gold microarray platform is described.

Tumor biomarker glycoproteins in the seminal plasma of healthy human males are endogenous ligands for DC-SIGN.

DC-SIGN is an immune C-type lectin that is expressed on both immature and mature dendritic cells associated with peripheral and lymphoid tissues in humans. It is a pattern recognition receptor that binds to several pathogens including HIV-1, Ebola virus, Mycobacterium tuberculosis, Candida albicans, Helicobacter pylori, and Schistosoma mansoni. Evidence is now mounting that DC-SIGN also recognizes endogenous glycoproteins, and that such interactions play a major role in maintaining immune homeostasis in humans and mice. Autoantigens (neoantigens) are produced for the first time in the human testes and other organs of the male urogenital tract under androgenic stimulus during puberty. Such antigens trigger autoimmune orchitis if the immune response is not tightly regulated within this system. Endogenous ligands for DC-SIGN could play a role in modulating such responses. Human seminal plasma glycoproteins express a high level of terminal Lewis(x) and Lewis(y) carbohydrate antigens. These epitopes react specifically with the lectin domains of DC-SIGN. However, because the expression of these sequences is necessary but not sufficient for interaction with DC-SIGN, this study was undertaken to determine if any seminal plasma glycoproteins are also endogenous ligands for DC-SIGN. Glycoproteins bearing terminal Lewis(x) and Lewis(y) sequences were initially isolated by lectin affinity chromatography. Protein sequencing established that three tumor biomarker glycoproteins (clusterin, galectin-3 binding glycoprotein, prostatic acid phosphatase) and protein C inhibitor were purified by using this affinity method. The binding of DC-SIGN to these seminal plasma glycoproteins was demonstrated in both Western blot and immunoprecipitation studies. These findings have confirmed that human seminal plasma contains endogenous glycoprotein ligands for DC-SIGN that could play a role in maintaining immune homeostasis both in the male urogenital tract and the vagina after coitus.

N-glycans of human protein C inhibitor: tissue-specific expression and function.

Protein C inhibitor (PCI) is a serpin type of serine protease inhibitor that is found in many tissues and fluids in human, including blood plasma, seminal plasma and urine. This inhibitor displays an unusually broad protease specificity compared with other serpins. Previous studies have shown that the N-glycan(s) and the NH2-terminus affect some blood-related functions of PCI. In this study, we have for the first time determined the N-glycan profile of seminal plasma PCI, by mass spectrometry. The N-glycan structures differed markedly compared with those of both blood-derived and urinary PCI, providing evidence that the N-glycans of PCI are expressed in a tissue-specific manner. The most abundant structure (m/z 2592.9) had a composition of Fuc3Hex5HexNAc4, consistent with a core fucosylated bi-antennary glycan with terminal Lewis(x). A major serine protease in semen, prostate specific antigen (PSA), was used to evaluate the effects of N-glycans and the NH2-terminus on a PCI function related to the reproductive tract. Second-order rate constants for PSA inhibition by PCI were 4.3±0.2 and 4.1±0.5 M⁻¹ s⁻¹ for the natural full-length PCI and a form lacking six amino acids at the NH2-terminus, respectively, whereas these constants were 4.8±0.1 and 29±7 M⁻¹ s⁻¹ for the corresponding PNGase F-treated forms. The 7-8-fold higher rate constants obtained when both the N-glycans and the NH2-terminus had been removed suggest that these structures jointly affect the rate of PSA inhibition, presumably by together hindering conformational changes of PCI required to bind to the catalytic pocket of PSA.

The antifungal drug itraconazole inhibits vascular endothelial growth factor receptor 2 (VEGFR2) glycosylation, trafficking, and signaling in endothelial cells.

Itraconazole is a safe and widely used antifungal drug that was recently found to possess potent antiangiogenic activity. Currently, there are four active clinical trials evaluating itraconazole as a cancer therapeutic. Tumor growth is dependent on angiogenesis, which is driven by the secretion of growth factors from the tumor itself. We report here that itraconazole significantly inhibited the binding of vascular endothelial growth factor (VEGF) to VEGF receptor 2 (VEGFR2) and that both VEGFR2 and an immediate downstream substrate, phospholipase C γ1, failed to become activated after VEGF stimulation. These effects were due to a defect in VEGFR2 trafficking, leading to a decrease in cell surface expression, and were associated with the accumulation of immature N-glycans on VEGFR2. Small molecule inducers of lysosomal cholesterol accumulation and mammalian target of rapamycin (mTOR) inhibition, two previously reported itraconazole activities, failed to recapitulate itraconazole's effects on VEGFR2 glycosylation and signaling. Likewise, glycosylation inhibitors did not alter cholesterol trafficking or inhibit mTOR. Repletion of cellular cholesterol levels, which was known to rescue the effects of itraconazole on mTOR and cholesterol trafficking, was also able to restore VEGFR2 glycosylation and signaling. This suggests that the new effects of itraconazole occur in parallel to those previously reported but are downstream of a common target. We also demonstrated that itraconazole globally reduced poly-N-acetyllactosamine and tetra-antennary complex N-glycans in endothelial cells and induced hypoglycosylation of the epidermal growth factor receptor in a renal cell carcinoma line, suggesting that itraconazole's effects extend beyond VEGFR2.

Comparison of the baculovirus-insect cell and Pichia pastoris heterologous systems for the expression of the human tumor suppressor protein RNASET2.

We report the expression of recombinant RNASET2, the only human member of the Rh/T2/S family of acid ribonucleases, in the yeast Pichia pastoris and the baculovirus-insect cell heterologous systems. In both models, the yield of recombinant protein was comparable and ranged between 5 mg/L (for a catalytically impaired mutant version of RNASET2) and 30 mg/L for the wild-type protein. Thus, the produced protein version rather than the expression system used appears to influence protein yield after optimization of culture conditions. The recombinant protein was found to undergo heterogeneous glycosylation in both systems, particularly in P. pastoris. Most importantly, the wild-type protein purified from both systems was found to be catalytically competent. The expression of recombinant RNASET2 in both systems will allow the implementation of functional assays in vivo and in vitro to better define the antioncogenic properties of this member of the Rh/T2/S ribonuclease family.

Serum N-glycome biomarker for monitoring development of DENA-induced hepatocellular carcinoma in rat.

BACKGROUND: There is a demand for serum markers for the routine assessment of the progression of liver cancer. We previously found that serum N-linked sugar chains are altered in hepatocellular carcinoma (HCC). Here, we studied glycomic alterations during development of HCC in a rat model. RESULTS: Rat HCC was induced by the hepatocarcinogen, diethylnitrosamine (DENA). N-glycans were profiled using the DSA-FACE technique developed in our laboratory.In comparison with control rats, DENA rats showed a gradual but significant increase in two glycans (R5a and R5b) in serum total N-glycans during progression of liver cirrhosis and cancer, and a decrease in a biantennary glycan (P5). The log of the ratio of R5a to P1 (NGA2F) and R5b to P1 [log(R5a/P1) and log(R5b/P1)] were significantly (p < 0.0001) elevated in HCC rats, but not in rats with cirrhosis or fibrosis or in control rats. We thus propose a GlycoTest model using the above-mentioned serum glycan markers to monitor the progression of cirrhosis and HCC in the DENA-treated rat model. When DENA-treated rats were subsequently treated with farnesylthiosalicyclic acid, an anticancer drug, progression to HCC was prevented and GlycoTest markers (P5, R5a and R5b) reverted towards non-DENA levels, and the HCC-specific markers, log(R5a/P1) and log(R5b/P1), normalized completely. CONCLUSIONS: We found an increase in core-alpha-1,6-fucosylated glycoproteins in serum and liver of rats with HCC, which demonstrates that fucosylation is altered during progression of HCC. Our GlycoTest model can be used to monitor progression of HCC and to follow up treatment of liver tumors in the DENA rat. This GlycoTest model is particularly important because a rapid non-invasive diagnostic procedure for tumour progression in this rat model would greatly facilitate the search for anticancer drugs.