Insights into the effects of N-glycosylation on the characteristics of the VC1 domain of the human receptor for advanced glycation end products (RAGE) secreted by Pichia pastoris.

Advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs), resulting from non-enzymatic modifications of proteins, are potentially harmful to human health. They directly act on proteins, affecting structure and function, or through receptor-mediated mechanisms. RAGE, a type I transmembrane glycoprotein, was identified as a receptor for AGEs. RAGE is involved in chronic inflammation, oxidative stress-based diseases and ageing. The majority of RAGE ligands bind to the VC1 domain. This domain was successfully expressed and secreted by Pichia pastoris. Out of two N-glycosylation sites, one (Asn25) was fully occupied while the other (Asn81) was under-glycosylated, generating two VC1 variants, named p36 and p34. Analysis of N-glycans and of their influence on VC1 properties were here investigated. The highly sensitive procainamide labeling method coupled to ES-MS was used for N-glycan profiling. N-glycans released from VC1 ranged from Man9GlcNAc2- to Man15GlcNAc2- with major Man10GlcNAc2- and Man11GlcNAc2- species for p36 and p34, respectively. Circular dichroism spectra indicated that VC1 maintains the same conformation also after removal of N-glycans. Thermal denaturation curves showed that the carbohydrate moiety has a small stabilizing effect on VC1 protein conformation. The removal of the glycan moiety did not affect the binding of VC1 to sugar-derived AGE- or malondialdehyde-derived ALE-human serum albumin. Given the crucial role of RAGE in human pathologies, the features of VC1 from P. pastoris will prove useful in designing strategies for the enrichment of AGEs/ALEs from plasma, urine or tissues, and in characterizing the nature of the interaction.

A case for protein-level and site-level specificity in glycoproteomic studies of disease.

Abnormal glycosylation of proteins is known to be either resultant or causative of a variety of diseases. This makes glycoproteins appealing targets as potential biomarkers and focal points of molecular studies on the development and progression of human ailment. To date, a majority of efforts in disease glycoproteomics have tended to center on either determining the concentration of a given glycoprotein, or on profiling the total population of glycans released from a mixture of glycoproteins. While these approaches have demonstrated some diagnostic potential, they are inherently insensitive to the fine molecular detail which distinguishes unique and possibly disease relevant glycoforms of specific proteins. As a consequence, such analyses can be of limited sensitivity, specificity, and accuracy because they do not comprehensively consider the glycosylation status of any particular glycoprotein, or of any particular glycosylation site. Therefore, significant opportunities exist to improve glycoproteomic inquiry into disease by engaging in these studies at the level of individual glycoproteins and their exact loci of glycosylation. In this concise review, the rationale for glycoprotein and glycosylation site specificity is developed in the context of human disease glycoproteomics with an emphasis on N-glycosylation. Recent examples highlighting disease-related perturbations in glycosylation will be presented, including those involving alterations in the overall glycosylation of a specific protein, alterations in the occupancy of a given glycosylation site, and alterations in the compositional heterogeneity of glycans occurring at a given glycosylation site. Each will be discussed with particular emphasis on how protein-specific and site-specific approaches can contribute to improved discrimination between glycoproteomes and glycoproteins associated with healthy and unhealthy states.

Biological and Technical Challenges in Unraveling the Role of N-Glycans in Immune Receptor Regulation.

N-glycosylation of membrane receptors is important for a wide variety of cellular processes. In the immune system, loss or alteration of receptor glycosylation can affect pathogen recognition, cell-cell interaction, and activation as well as migration. This is not only due to aberrant folding of the receptor, but also to altered lateral mobility or aggregation capacity. Despite increasing evidence of their biological relevance, glycosylation-dependent mechanisms of receptor regulation are hard to dissect at the molecular level. This is due to the intrinsic complexity of the glycosylation process and high diversity of glycan structures combined with the technical limitations of the current experimental tools. It is still challenging to precisely determine the localization and site-occupancy of glycosylation sites, glycan micro- and macro-heterogeneity at the individual receptor level as well as the biological function and specific interactome of receptor glycoforms. In addition, the tools available to manipulate N-glycans of a specific receptor are limited. Significant progress has however been made thanks to innovative approaches such as glycoproteomics, metabolic engineering, or chemoenzymatic labeling. By discussing examples of immune receptors involved in pathogen recognition, migration, antigen presentation, and cell signaling, this Mini Review will focus on the biological importance of N-glycosylation for receptor functions and highlight the technical challenges for examination and manipulation of receptor N-glycans.

Development and validation of a (RP)UHPLC-UV-(HESI/Orbitrap)MS method for the identification and quantification of mixtures of intact therapeutical monoclonal antibodies using a monolithic column.

Monoclonal antibodies (mAbs) are one of the most important types of biopharmaceutics and have proved enormously successful in the treatment of cancers and autoimmune diseases. In this paper, we present a fast, straightforward reversed phase (RP)UHPLC-UV-(HESI/Orbitrap) MS method for the separation and identification of five of the most commonly used mAbs, i.e. bevazizumab (BEV), cetuximab (CTX), infliximab (INF), rituximab (RTX) and trastuzumab (TTZ) in mixtures. The RP mAbs separation was performed in a divinylbenzene-based monolithic column, after statistical design of the experiments with a novel approach for optimizing chromatographic conditions called the heteroscedasticity function. Results led us to split the initial mixture of five mAbs into two mixtures with four mAbs each, one containing RTX and the other TTZ. The method was validated for quantification using the signal from the UV detector and identification by (HESI-Orbitrap)MS. Direct MS characterization of the intact isoform profile of each mAb was also obtained. Advantages and disadvantages of the use of trifluoroacetic acid or formic acid as ion pairing agents for mass spectrometric analysis and chromatographic separation are discussed. Validation was performed using an internal protocol based on well-known international guidelines such as the International Conference on Harmonization (ICH) guideline, the US Food and Drugs Administration (FDA) guideline and the United Stated Pharmacopeia (USP) guideline. Performance parameters such as linearity, accuracy (precision and trueness), detection limits, quantification limits and robustness were evaluated. Robustness was established by studying the total and one-sided effects of four selected variables: column temperature, trifluoroacetic acid content in the mobile phases, initial proportion of eluent B and gradient. The results indicated the suitability of this method for quantifying these five mAbs in mixtures, as well as its robustness, reproducibility and sensitivity.