Encyclopedia
Please note this is an old version of this entry, which may differ significantly from the current revision.
Subjects:
Cell Biology
Glycosylation is a complex post-translational modification that conveys functional diversity to glycoconjugates. Cell surface glycosylation mediates several biological activities such as induction of the intracellular signaling pathway and pathogen recognition. Red blood cell (RBC) membrane N-glycans determine blood type and influence cell lifespan. Although several proteomic studies have been carried out, the glycosylation of RBC membrane proteins has not been systematically investigated. This work aims at exploring the human RBC N-glycome by high-sensitivity MALDI-MS techniques to outline a fingerprint of RBC N-glycans.
- red blood cells
- N-glycosylation
- MALDI-TOF
- ABO(H) blood groups
1. Introduction
The red blood cell (RBC), or erythrocyte, is the simplest human cell, as it does not have internal organelles, which are lost during the erythropoiesis process. RBCs develop in the bone marrow and have a life span of about 100–120 days before they are recycled by macrophages [1]. In the last decade, several studies were carried out to shed light on the biological function of RBC membrane proteins [2,3]. As a result, the erythrocyte membrane is one of the best-known membranes in terms of structure, function, and associated genetic disorders [4,5,6]. The RBC membrane mediates transport functions and provides the erythrocytes with their structural features of resilience and deformability [7].
Glycosylation is a post-translational modification characterized by the covalent linkage of oligosaccharides moieties (glycans) to form glycoconjugates. Glycans are conjugated to asparagine (N-glycan) or serine/threonine (O-glycans) residues to form glycoproteins. Protein glycans play important roles in biological function/activity, protein folding, and molecular recognition [8]. Glycans are also recognized by glycan-binding proteins with lectin activity on opposing cells, and these glycan-ligand interactions are responsible for many biological activities, including the induction of the intracellular signaling pathway and recognition of pathogens such as influenza virus [8,9].
Changes in N-linked glycosylation have long been associated with disease development, and acquired glycan modifications have been described in multifactorial diseases such as cancer [10,11], inflammation [12,13], neurodegeneration [14,15], and genetic diseases [16,17].
Human RBC protein glycosylation has not been systematically examined in its entirety yet [18], though previous studies investigated the two most abundant glycoproteins of the erythrocyte membrane, namely band 3 and glycophorin A (GPA) [19,20,21,22]. In human erythrocytes, band 3 and other red blood cell membrane proteins bear poly-N-acetyllactosamine (poly-LacNAc) extensions, acting as an antigen carrier during various stages of cell development and differentiation [18,19]. The ABO(H) antigens were found as terminal non-reducing epitopes of large complex RBC N-glycans [23,24] and also constitute distinctive traits of outer arm glycophorin N-linked structures at lower molecular masses [18,25]. They play crucial roles in transfusion medicine, determining the compatibility between blood donors and recipients, as well as in the development of several hemostasis-related genetic disorders, including cardiovascular diseases and thrombosis [26,27]. Furthermore, several studies have investigated the relationship between the ABO(H) blood group distribution and the occurrence of some infectious diseases, such as HIV, malaria, influenza [28,29,30,31,32,33], and, recently, the COVID-19 pandemic [34,35,36,37].
Although information on the biological roles of glycan-binding proteins is still rising, challenges remain in detecting and profiling known and novel cellular glycans structures, due to the vast heterogeneity of possible glycoprotein isoforms depending on the glycan occupancy of protein glycosylation sites (macroheterogeneity) and on the variety of the attached glycans (microheterogeneity). Macroheterogeneity is related to the site availability, enzyme kinetics, and substrate concentrations that regulate the N-glycan precursor assembly and its subsequent transfer to the protein in cytosol and endoplasmic reticulum, whereas microheterogeneity is associated with the N-glycan processing occurring in endoplasmic reticulum and Golgi [17]. In this context, mass spectrometry (MS) techniques have demonstrated their capability for the characterization of unknown glycans and the high-throughput analysis of known glycan structures, allowing individual glycoforms to be distinguished [38]. Advances in mass spectrometry, and in particular in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, have driven substantial progress in glycan analysis [39]. The eligibility of MALDI-TOF is established because of its ability to analyze complex mixtures of glycans from biological samples together with its high detection sensitivity, as exemplified by several glycomic studies [40,41,42].
Since RBCs are continuously replenished with glycosylated membrane proteins that can be isolated, purified, and readily available from blood samples, glycosylation analysis of erythrocyte membrane glycoproteins might be the key for the early detection of glyco-biomarkers for diagnostic and therapeutic purposes as well as for studying interaction mechanisms involved in recognition of pathogens. In the present study, we applied high-sensitivity MALDI TOF MS-based glycomic methodologies to the analysis of total N-glycans derived from human erythrocyte membrane proteins, with the aim to generate an N-glycosylation fingerprinting of RBC. In particular, the total N-glycome of healthy subjects with different ABO(H) blood groups is evaluated as the starting point for future investigations.
2. Discussion
In the present study, we applied a robust and widely used MS-based glycomic approach to the extensive N-glycan characterization of RBCs. Our MALDI-TOF data showed erythrocyte membrane glycoproteins holding in prevalence complex bisected N-glycans with core and antennary fucosylation and recurrent poly-LacNAc extensions, in accordance with earlier glycosylation studies on specific red blood cell membrane proteins. The previous characterization of RBCs’ N-glycoproteins has been limited to their main components, such as band 3 and GPA proteins [19,20,21,22,25]. Band 3 was found harboring a single N-linked oligosaccharide, with a branched structure varying in the number of repeating LacNAc units terminated with Gal, fucose (Fuc), or NeuAc [19,20], whereas GPA, a major glycophorin, has been reported to bear 15 O-glycans [47,48] and a single N-linked glycan, mostly a biantennary sialylated moiety with bisecting GlcNAc and outer arm fucosylation [21,22]. In the current study, the MALDI-TOF strategy allowed a broad characterization of the total RBC N-glycans released from band 3, GPA, and additional minor glycoproteins. We observed large N-glycan structures up to 9 kDa (see Supplementary Figures) as a result of the ad hoc developed MS strategy that led to a notable improvement in upper mass-range sensitivity and signal-to-noise ratio. Besides a predominant portion of complex highly processed structures, we also found oligomannose and hybrid N-glycans. As glycan structures are generated in the compartmentalized Golgi, changes in the relative signals of all the observed RBC N-glycans could be used as a diagnostic tool for the detection of defects in glycosylation enzymes involved in early Golgi processing in glycosylation-related diseases [43,49,50,51]. However, only a few studies reported on the N-glycosylation of human erythrocyte membrane glycoproteins using MS techniques [43,44,52], mostly focusing on the characterization of glycans from band 3 membrane glycoprotein in congenital dyserythropoietic anemia type II (CDA II), also called hereditary erythroblastic multinuclearity with the positive acidified-serum test (HEMPAS) [43,44]. Fukuda et al. in 1987 developed a method based on fast-atom bombardment (FAB) MS [43], whereas Denecke et al. in 2008 [44] compared erythrocyte band 3 mass mapping from HEMPAS and from a control by MALDI-TOF MS following SDS-PAGE and lectin-binding strategies. Both these studies accordingly found the lack of the large oligosaccharide component bearing the poly-LacNAc branches and the prevalence of glycans at lower molecular mass (such as oligomannose and hybrid and truncated complex species) in the red blood cell band 3 glycoprotein from HEMPAS patients, suggesting a defective Golgi processing in erythroblasts [43,44].
RBC membrane N-glycans are particularly exposed to the external environment, supporting the pathogen recognition processes. For instance, the hemagglutination assay is based on the interaction between the hemagglutinin located on the surface of the human-adapted influenza virus and some specific sialylated glycans on the epithelial cells of the human upper respiratory tract, defined as the key initial step of the infection cycle [53]. Accordingly, agglutination of chicken RBCs (cRBCs) [54] has long been used in viral titer assays as well as to investigate glycan receptor binding sites and in the testing of vaccines’ effectiveness [55,56,57,58]. The structural characterization of cell surface N-glycans of cRBCs, revealed the presence of bi- and triantennary structures capped with both α2→3 and α2→6 linked NeuAc and the lack of lactosamine repeating units [54]. On the other hand, the human bronchial epithelial cells, which are the target of human-adapted influenza A viruses, show the predominance of α2→6 sialylated glycans with lactosamine repeats [59,60]. These data could explain some pitfalls of the agglutination assay based on cRBCs which may not be representative of the physiological receptor for human-adapted influenza strains. Our study may trigger future advanced MS-based structural analyses on glycans from human RBCs, providing important insights for improved applications in this field.
The applied MALDI-TOF MS and MS/MS strategy presented here allowed for the characterization of glycans bearing the ABO(H) blood group antigens which, like genetic factors, are involved in several hemostasis-related diseases [26,27] and in many infectious diseases [28,29,30,31,32,33]. Several shreds of evidence suggest that the ABO(H) blood group expression may influence the development and the progression of cardiovascular disease, thrombosis, and hemostasis disorders. In the last year, there has been a growing interest in studying the association between the ABO(H) blood group distribution and the dynamics of the COVID-19 pandemic [34,35,36,37]. Recently, Liu et al. [36] found a positive correlation between the occurrence of COVID-19 infection cases and the proportion of blood group A by analyzing the data from the official WHO database. These results agree with other previous studies [34,35], strongly suggesting a relationship between the distribution of blood groups and the SARS-CoV-2 infection. However, the underlying mechanisms have not yet been clarified and further investigation, also taking into account the glycan-binding specificity and the glycosylation features of the SARS-CoV-2 proteins, is highly needed.
3. Conclusions
Our developed MS strategy led to a considerable improvement in upper mass-range sensitivity and in signal-to-noise ratio, in addition to a significant increase in the resolution of MALDI-TOF mass spectra, allowing for a detailed mapping of human RBC N-glycans. Since RBCs have a relatively short lifespan, these analytical strategies could be used to study possible glycosylation changes that can occur during disease conditions, for the early detection of potential glyco-biomarkers. Most important, this developed strategy could be a useful tool to investigate the interaction mechanisms of pathogen recognition as well as the ABO(H) blood group-mediated response to viral infections, with special regard to SARS-CoV-2.
This entry is adapted from the peer-reviewed paper 10.3390/ijms22158063
This entry is offline, you can click here to edit this entry!
