Characterization of highly glycosylated biopharmaceuticals by mass spectrometry is challenging because of the huge chemical space of coexistent glycoforms present. Here, we report the use of an... Show moreCharacterization of highly glycosylated biopharmaceuticals by mass spectrometry is challenging because of the huge chemical space of coexistent glycoforms present. Here, we report the use of an array of HPLC-mass spectrometry-based approaches at different structural levels of released glycan, glycopeptide, and hitherto unexplored intact glycoforms to scrutinize the biopharmaceutical Myozyme, containing the highly complex lysosomal enzyme recombinant acid alpha-glucosidase. The intrinsic heterogeneity of recombinant acid alpha-glucosidase glycoforms was unraveled using a novel strong anion exchange HPLC-mass spectrometry approach involving a pH-gradient of volatile buffers to facilitate chromatographic separation of glycoforms based on their degree of sialylation, followed by the acquisition of native mass spectra in an Orbitrap mass spectrometer. Upon considering the structures of 60 different glycans attached to seven glycosylation sites in the intact protein, the large set of interdependent data acquired at different structural levels was integrated using a set of bioinformatic tools and allowed the annotation of intact glycoforms unraveling more than 1,000,000 putative intact glycoforms. Detectable isoforms also included several mannose-6-phosphate variants, which are essential for directing the drug toward its target, the lysosomes. Finally, for the first time, we sought to validate the intact glycoform annotations by integrating experimental data on the enzymatically dissected proteoforms, which reduced the number of glycoforms supported by experimental evidence to 42,104. The latter verification clearly revealed the strengths but also intrinsic limitations of this approach for fully characterizing such highly complex glycoproteins by mass spectrometry. Show less
Svecla, M.; Nour, J.; Bladergroen, M.R.; Nicolardi, S.; Zhang, T.; Beretta, G.; ... ; Falck, D. 2023
The asialoglycoprotein receptor (ASGPR) and the mannose receptor C -type 1 (MRC1) are well known for their selective recognition and clearance of circulating glycoproteins. Terminal galactose and N... Show moreThe asialoglycoprotein receptor (ASGPR) and the mannose receptor C -type 1 (MRC1) are well known for their selective recognition and clearance of circulating glycoproteins. Terminal galactose and N-Acetylgalactosamine are recognized by ASGPR, while terminal mannose, fucose, and N-Acetylglucosamine are recognized by MRC1. The effects of ASGPR and MRC1 deficiency on the N-glycosylation of individual circulating proteins have been studied. However, the impact on the homeostasis of the major plasma glycoproteins is debated and their glycosylation has not been mapped with high molecular resolution in this context. Therefore, we evaluated the total plasma N-glycome and plasma proteome of ASGR1 and MRC1 deficient mice. ASGPR deficiency resulted in an increase in O-acetylation of sialic acids accompanied by higher levels of apolipoprotein D, haptoglobin, and vitronectin. MRC1 deficiency decreased fucosylation without affecting the abundance of the major circulating glycoproteins. Our findings confirm that concentrations and Nglycosylation of the major plasma proteins are tightly controlled and further suggest that glycan-binding receptors have redundancy, allowing compensation for the loss of one major clearance receptor. Show less
Gut microbiota of the gastrointestinal tract provide health benefits to the human host via bacterial metabolites. Bacterial butyrate has beneficial effects on intestinal homeostasis and is the... Show moreGut microbiota of the gastrointestinal tract provide health benefits to the human host via bacterial metabolites. Bacterial butyrate has beneficial effects on intestinal homeostasis and is the preferred energy source of intestinal epithelial cells, capable of inducing differentiation. It was previously observed that changes in the expression of specific proteins as well as protein glycosylation occur with differentiation. In this study, specific mucin O-glycans were identified that mark butyrate-induced epithelial differentiation of the intestinal cell line CaCo-2 (Cancer Coli-2), by applying porous graphitized carbon nano–liquid chromatography with electrospray ionization tandem mass spectrometry. Moreover, a quantitative proteomic approach was used to decipher changes in the cell proteome. It was found that the fully differentiated butyrate-stimulated cells are characterized by a higher expression of sialylated O-glycan structures, whereas fucosylation is downregulated with differentiation. By performing an integrative approach, we generated hypotheses about the origin of the observed O-glycome changes. These insights pave the way for future endeavors to study the dynamic O-glycosylation patterns in the gut, either produced via cellular biosynthesis or through the action of bacterial glycosidases as well as the functional role of these patterns in homeostasis and dysbiosis at the gut–microbiota interface. Show less
Garcia-Garcia, T.; Douche, T.; Gianetto, Q.G.; Poncet, S.; Omrani, N. el; Smits, W.K.; ... ; Martin-Verstraete, I. 2022
Clostridioides difficile is the leading cause of postanti-biotic diarrhea in adults. During infection, the bacterium must rapidly adapt to the host environment by using sur-vival strategies.... Show moreClostridioides difficile is the leading cause of postanti-biotic diarrhea in adults. During infection, the bacterium must rapidly adapt to the host environment by using sur-vival strategies. Protein phosphorylation is a reversible post-translational modification employed ubiquitously for signal transduction and cellular regulation. Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases have emerged as important players in bacterial cell signaling and pathogenicity. C. difficile en-codes two STKs (PrkC and CD2148) and one phosphatase. We optimized a titanium dioxide phosphopeptide enrich-ment approach to determine the phosphoproteome of C. difficile. We identified and quantified 2500 proteins rep-resenting 63% of the theoretical proteome. To identify STK and serine/threonine phosphatase targets, we then per-formed comparative large-scale phosphoproteomics of the WT strain and isogenic AprkC, CD2148, Astp, and prkC CD2148 mutants. We detected 635 proteins containing phosphorylated peptides. We showed that PrkC is phos-phorylated on multiple sites in vivo and autophosphor-ylates in vitro. We were unable to detect a phosphorylation for CD2148 in vivo, whereas this kinase was phosphory-lated in vitro only in the presence of PrkC. Forty-one phosphoproteins were identified as phosphorylated under the control of CD2148, whereas 114 proteins were phos-phorylated under the control of PrkC including 27 phos-phoproteins more phosphorylated in the Astp mutant. We also observed enrichment for phosphothreonine among the phosphopeptides more phosphorylated in the Astp mutant. Both kinases targeted pathways required for metabolism, translation, and stress response, whereas cell division and peptidoglycan metabolism were more specifically controlled by PrkC-dependent phosphoryla-tion in agreement with the phenotypes of the AprkC mutant. Using a combination of approaches, we confirmed that FtsK was phosphorylated in vivo under the control of PrkC and that Spo0A was a substrate of PrkC in vitro. This study provides a detailed mapping of kinase- substrate relationships in C. difficile, paving the way for the identification of new biomarkers and therapeutic targets. Show less
The recent surge of coronavirus disease 2019 (COVID-19) hospitalizations severely challenges healthcare systems around the globe and has increased the demand for reli-able tests predictive of... Show moreThe recent surge of coronavirus disease 2019 (COVID-19) hospitalizations severely challenges healthcare systems around the globe and has increased the demand for reli-able tests predictive of disease severity and mortality. Using multiplexed targeted mass spectrometry assays on a robust triple quadrupole MS setup which is available in many clinical laboratories, we determined the precise concentrations of hundreds of proteins and metabolites in plasma from hospitalized COVID-19 patients. We observed a clear distinction between COVID-19 patients and con-trols and, strikingly, a significant difference between sur-vivors and nonsurvivors. With increasing length of hospitalization, the survivors' samples showed a trend toward normal concentrations, indicating a potential sensitive readout of treatment success. Building a ma-chine learning multi-omic model that considers the con-centrations of 10 proteins and five metabolites, we could predict patient survival with 92% accuracy (area under the receiver operating characteristic curve: 0.97) on the day of hospitalization. Hence, our standardized assays represent a unique opportunity for the early stratification of hospi-talized COVID-19 patients. Show less
Colorectal cancer (CRC)-associated changes of protein glycosylation have been widely studied. In contrast, the expression of glycosphingolipid (GSL) patterns in CRC has, hitherto, remained largely... Show moreColorectal cancer (CRC)-associated changes of protein glycosylation have been widely studied. In contrast, the expression of glycosphingolipid (GSL) patterns in CRC has, hitherto, remained largely unexplored. Even though GSLs are major carriers of cell surface carbohydrates, they are understudied due to their complexity and analytical challenges. In this study, we provide an in-depth analysis of GSL glycans of 22 CRC cell lines using porous graphitized carbon nano-liquid chromatography coupled with electrospray ionization-mass spectrometry. Our data revealed that the GSL expression varies among different cell line classifications, with undifferentiated cell lines showing high expression of blood group A, B, and H antigens while for colon-like cell lines the most prominent GSL glycans contained (sialyl)-Lewis(A/X) and Lewis(B/Y) antigens. Moreover, the GSL expression correlated with relevant glycosyltransferases that are involved in their biosynthesis as well as with transcription factors (TFs) implicated in colon differentiation. Additionally, correlations between certain glycosyltransferases and TFs at mRNA expression level were found, such as FUT3, which correlated with CDX1, ETS2, HNF1A, HNF4A, MECOM, and MYB. These TFs are upregulated in colon-like cell lines pointing to their potential role in regulating fucosylation during colon differentiation. In conclusion, our study reveals novel layers of potential GSL glycans regulation relevant for future research in colon differentiation and CRC. Show less
Millions of people worldwide are infected with filarial nematodes, responsible for lymphatic filariasis (LF) and other diseases causing chronic disablement. Elimination programs have resulted in a... Show moreMillions of people worldwide are infected with filarial nematodes, responsible for lymphatic filariasis (LF) and other diseases causing chronic disablement. Elimination programs have resulted in a substantial reduction of the rate of infection in certain areas creating a need for improved diagnostic tools to establish robust population surveillance and avoid LF resurgence. Glycans from parasitic helminths are emerging as potential antigens for use in diagnostic assays. However, despite its crucial role in host- parasite interactions, filarial glycosylation is still largely, structurally, and functionally uncharacterized. Therefore, we investigated the glycan repertoire of the filarial nematode Brugia malayi. Glycosphingolipid and N-linked glycans were extracted from several life-stages using enzymatic release and characterized using a combination of MALDITOF-MS and glycan sequencing techniques. Next, glycans were purified by HPLC and printed onto microarrays to assess the host anti-glycan antibody response. Comprehensive glycomic analysis of B. malayi revealed the presence of several putative antigenic motifs such as phosphorylcholine and terminal glucuronic acid. Glycan microarray screening showed a recognition of most B. malayi glycans by immunoglobulins from rhesus macaques at different time points after infection, which permitted the characterization of the dynamics of anti-glycan immunoglobulin G and M during the establishment of brugian filariasis. A significant level of IgG binding to the parasite glycans was also detected in infected human plasma, while IgG binding to glycans decreased after anthelmintic treatment. Altogether, our work identifies B. malayi glycan antigens and reveals antibody responses from the host that could be exploited as potential markers for LF. Show less
