A hierarchical structure in the N-glycosylation process governs the N-glycosylation output: prolonged cultivation induces glycoenzymes expression variations that are reflected in the cellular N-glycome but not in the protein and site-specific glycoprofile of CHO cells.

N-glycosylation is a central component in the modification of secretory proteins. One characteristic of this process is a heterogeneous output. The heterogeneity is the result of both structural constraints of the glycoprotein as well as the composition of the cellular glycosylation machinery. Empirical data addressing correlations between glycosylation output and glycosylation machinery composition are seldom due to the low abundance of glycoenzymes. We assessed how differences in the glycoenzyme expression affected the N-glycosylation output at a cellular as well as at a protein-specific level. Our results showed that cellular N-glycome changes could be correlated with the variation of glycoenzyme expression, whereas at the protein level differential responses to glycoenzymes alterations were observed. We therefore identified a hierarchical structure in the N-glycosylation process: the enzyme levels in this complex pathway determine its capacity (reflected in the N-glycome), while protein-specific parameters determine the glycosite-specificity. What emerges is a highly variable and adaptable protein modification system that represents a hallmark of eukaryotic cells.

Tissue N-linked glycosylation as potential prognostic biomarker for biochemical recurrence-free survival.

PURPOSE: Only few biomarkers have been evaluated for their prognostic value following radical prostatectomy. We explored if tissue N-glycosylation shows prognostic properties for biochemical recurrence (BCR)-free survival. MATERIALS AND METHODS: Tissue N-glycosylation profile was determined from 82 prostate cancer (PCa) patients and prognostic features were compared to clinical and biochemical parameters for BCR-free survival. RESULTS: Majority presented with Gleason score 3 + 4 (41%), extensive local disease (62%) and without pelvic lymph nodes invasion (83%). Several parameters (low T stage, low Gleason score, low EAU risk groups for BCR, absence of positive surgical margins, high ratio of fucosylated triantennary structures on total of multiantennary structures [3AFc/MA], low ratio of fucosylated biantennary with core-branched N-acetylglucosamine on total of biantennary structures, and high ratio of triantennary structures on total of multiantennary structures) proved to have a univariate beneficial effect on BCR-free survival. Multivariate analysis proved positive surgical margins and 3AFc/MA to be independent prognosticators. CONCLUSIONS: Tissue N-glycans are a powerful prognostic tool and can be an asset in PCa as the ratio of 3AFc/MA is independently associated with BCR-free survival. This could be of clinical use in guiding patients following radical prostatectomy, e.g. referral to adjuvant radiotherapy. Further elaboration of this biomarker is warranted.

Studying Viscoelastic Changes of Skin Cells Using QCM-D Measurements.

The viscoelastic properties of cells are responsible for the adhesion process to different surfaces and for cell motility. Therefore, it is very important to develop specific, label-free biosensors with the use of whole cells to study the effect of various factors on the survival and properties of selected type of normal and pathological cells. The quartz crystal microbalance with dissipation energy monitoring (QCM-D) is a technique which enables to track these changes in cells during real-time experiments. One of the applied procedures of the evaluation of the cells' viscoelastic changes is based on the investigations of interactions between specific, different glycans, present on the surface of the primary tumor and its metastases with specific lectins. Two procedures have been developed to detect the differences in the cellular glycosylation profile using cell-based sensors (adherent cells cultured on sensors) and suspension cell-based sensors (adherent cells mechanically detached and inserted into the QCM-D chamber with a sensor). Furthermore, in this work some cell-based sensor regeneration protocols have been described and a lectin-ELISA assay with a fluorescently labeled lectin, thus enabling a qualitative and quantitative tracking of each step of the lectin-glycan binding and unbinding process performed on whole cells.

Anandamide-Modulated Changes in Metabolism, Glycosylation Profile and Migration of Metastatic Melanoma Cells.

An effective therapy for advanced melanoma, a skin cancer with the highest mortality, has not yet been developed. The endocannabinoid system is considered to be an attractive target for cancer treatment. The use of endocannabinoids, such as anandamide (AEA), is considered to be much greater than as a palliative agent. Thus, we checked its influence on various signaling pathways in melanoma cells. Our investigation was performed on four commercial cell lines derived from different progression stages (radial WM35 and vertical WM115 growth phases, lymph node WM266-4 metastasis, solid tumor A375-P metastasis). Cell viability, glucose uptake, quantification of reactive oxygen species production, expression of selected genes encoding glycosyltransferases, quantification of glycoproteins production and changes in the glycosylation profile and migration, as well as in cell elastic properties were analyzed. The cell glycosylation profile was investigated using the biophysical profiling method-the quartz crystal microbalance with dissipation monitoring (QCM-D). Anandamide treatment of only metastatic cells resulted in: an increase in the cell metabolism, a decrease in GFAT-1 and DPM1 expression, followed by a decrease in L1-CAM glycoprotein production, which further influenced the reduction in the cell glycosylation profile and migration. Considering our results, AEA usage is highly recommended in the combined therapy of advanced melanoma.

Multi-approach LC-MS methods for the characterization of species-specific attributes of monoclonal antibodies from plants.

In this work, an analytical platform based on the use of chromatography and mass spectrometry (MS), has been applied to the characterization of Rituximab (RTX) obtained from two plant expression systems (rice and tobacco) in comparison to the mammalian cell-derived reference monoclonal antibody (mAb). Different chromatographic approaches, hyphenated to high resolution MS (HRMS), were applied to RTX structural investigation both at middle- and peptide level. In particular, cation exchange chromatography (CEX), size exclusion chromatography (SEC), reversed phase (RPLC) and hydrophilic interaction liquid chromatographic (HILIC) methods were developed and applied on intact mAbs, IdeS-, and trypsin digests in order to address critical attributes such as primary structure, glycan composition, species-related heterogeneity, glycosylation degree, charge variants, aggregation tendency and enzymatic stability. All the collected data highlight the features and criticalities of each production approach. Production in rice results in a heterogeneous but stable product over time, suggesting the absence of proteases in seeds; while tobacco expression system leads to more homogeneous glycosylation, but protein stability seems to be a critical issue probably due to the presence of proteases. This analytical strategy represents a robust support to scientists in the selection and optimization of the best plant expression system to produce recombinant humanized mAbs.

Emerging role of embryo secretome in the paracrine communication at the implantation site: a proof of concept.

OBJECTIVE: To assess the role of embryo secretome in modifying the molecular profile of glycodelin A (GdA) in endometrial organoids (ORG) mimicking the implantation window. To verify whether the use of embryo-conditioned culture medium at the time of the embryo transfer may increase in vitro fertilization outcome. DESIGN: Molecular study with human endometrial ORG and embryo-conditioned culture medium. Retrospective study using prospectively recorded data. SETTING: University hospital. PATIENT(S): For isolation and culture of endometrial glandular ORG, endometrial biopsy specimens from five white women of proven fertility undergoing laparoscopy for tubal sterilization. A total of 75 women undergoing intracytoplasmic sperm injection for tubal and/or male infertility factor. INTERVENTIONS(S): In vitro fertilization. MAIN OUTCOME MEASURE(S): Pinopodes presence in human endometrial ORG. Glycodelin A expression profile by means of two-dimensional electrophoresis. In vitro fertilization outcome. RESULT(S): This in vitro study demonstrated that the treatment of endometrial ORG with the secretome of medium conditioned by the growing embryo increased the GdA relative abundance and induced a different glycoform pattern. Biochemical and clinical pregnancy rate significantly increased when the spent medium was loaded during the transfer (17.5% vs. 36.6% and 16.5% vs. 35.1%, respectively). CONCLUSION(S): This study demonstrated that the secretome of implanting embryos is able to induce the expression as well as to determine the relative abundance and the glycosilation profile of endometrial GdA, a protein having a key role in the embryo-endometrial cross talk. Moreover, a significant increase in pregnancy rate was observed when the embryo transfer was performed by using the culture medium conditioned by the growing embryo.