I
n t
he 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][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][19,20], whereas GPA, a major glycophorin, has been reported to bear 15 O-glycans
[43][44][47,48] and a single N-linked glycan, mostly a biantennary sialylated moiety with bisecting GlcNAc and outer arm fucosylation
[21][22][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.
ItWe 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
[45][46][47][48][43,49,50,51]. However, only a few studies reported on the N-glycosylation of human erythrocyte membrane glycoproteins using MS techniques
[45][49][50][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)
[45][49][43,44]. Fukuda et al. in 1987 developed a method based on fast-atom bombardment (FAB) MS
[45][43], whereas Denecke et al. in 2008
[49][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
[45][49][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
[51][53]. Accordingly, agglutination of chicken RBCs (cRBCs)
[52][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
[53][54][55][56][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
[52][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
[57][58][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][26,27] and in many infectious diseases
[28][29][30][31][32][33][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][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][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.