Altered linkage pattern of N-glycan sialic acids in pseudomyxoma peritonei.

Pseudomyxoma peritonei (PMP) is a highly mucinous adenocarcinoma growing in the peritoneal cavity and most commonly originating from the appendix. Glycans play an important role in carcinogenesis, and glycosylation is altered in malignant diseases, including PMP. We have previously demonstrated that fucosylation of N-glycans is increased in PMP, but we did not observe modulation of overall sialylation. As sialic acids can be attached to the rest of the glycan via α2,3- or α2,6-linkage, we have now analyzed the linkage patterns of sialic acids in tissue specimens of normal appendices, low-grade appendiceal mucinous neoplasms (LAMN), low-grade (LG) PMP and high-grade (HG) PMP. For the linkage analysis, the enzymatically released acidic N-glycans were first treated with ethyl esterification or α2,3-sialidase digestion followed by MALDI-TOF mass spectrometry. Significant increase in the relative abundance of α2,6-sialylated and decrease in α2,3-sialylated N-glycans was observed in PMP tumors as compared to the normal appendices (P < 0.025). More specifically, increased α2,6-sialylation (P < 0.05) and decreased α2,3-sialylation (P < 0.01) were detected in afucosylated and monofucosylated N-glycans of PMPs, whereas the less abundant multifucosylated glycans, containing terminal fucose, demonstrated increased α2,3-sialylation (P < 0.01). Importantly, the increase in α2,6-sialylation was also detected between PMP and the appendiceal precursor lesion LAMN (P < 0.01). The identified glycosylation alterations produce ligands for sialic acid-binding immunoglobulin-like lectins (Siglecs) and sialofucosylated glycans binding selectins, which play a role in the peritoneal dissemination and progression of the disease.

Glycomic Profiling Highlights Increased Fucosylation in Pseudomyxoma Peritonei.

Pseudomyxoma peritonei (PMP) is a subtype of mucinous adenocarcinoma that most often originates from the appendix, and grows in the peritoneal cavity filling it with mucinous ascites. KRAS and GNAS mutations are frequently found in PMP, but other common driver mutations are infrequent. As altered glycosylation can promote carcinogenesis, we compared N-linked glycan profiles of PMP tissues to those of normal appendix. Glycan profiles of eight normal appendix samples and eight low-grade and eight high-grade PMP specimens were analyzed by mass spectrometry. Our results show differences in glycan profiles between PMP and the controls, especially in those of neutral glycans, and the most prominent alteration was increased fucosylation. We further demonstrate up-regulated mRNA expression of four fucosylation-related enzymes, the core fucosylation performing fucosyltransferase 8 and three GDP-fucose biosynthetic enzymes in PMP tissues when compared with the controls. Up-regulated protein expression of the latter three enzymes was further observed in PMP cells by immunohistochemistry. We also demonstrate that restoration of fucosylation either by salvage pathway or by introduction of an expression of intact GDP-mannose 4,6-dehydratase enhance expression of MUC2, which is the predominant mucin molecule secreted by the PMP cells, in an intestinal-derived adenocarcinoma cell line with defective fucosylation because of deletion in the GDP-mannose 4,6-dehydratase gene. Thus, altered glycosylation especially in the form of fucosylation is linked to the characteristic mucin production of PMP. Glycomic data are available via ProteomeXchange with identifier PXD010086.

N-glycomic profiling as a tool to separate rectal adenomas from carcinomas.

All human cells are covered by glycans, the carbohydrate units of glycoproteins, glycolipids, and proteoglycans. Most glycans are localized to cell surfaces and participate in events essential for cell viability and function. Glycosylation evolves during carcinogenesis, and therefore carcinoma-related glycan structures are potential cancer biomarkers. Colorectal cancer is one of the world's three most common cancers, and its incidence is rising. Novel biomarkers are essential to identify patients for targeted and individualized therapy. We compared the N-glycan profiles of five rectal adenomas and 18 rectal carcinomas of different stages by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Paraffin-embedded tumor samples were deparaffinized, and glycans were enzymatically released and purified. We found differences in glycosylation between adenomas and carcinomas: monoantennary, sialylated, pauci-mannose, and small high-mannose N-glycan structures were more common in carcinomas than in adenomas. We also found differences between stage I-II and stage III carcinomas. Based on these findings, we selected two glycan structures: pauci-mannose and sialyl Lewis a, for immunohistochemical analysis of their tissue expression in 220 colorectal cancer patients. In colorectal cancer, poor prognosis correlated with elevated expression of sialyl Lewis a, and in advanced colorectal cancer, poor prognosis correlated with elevated expression of pauci-mannose. In conclusion, by mass spectrometry we found several carcinoma related glycans, and we demonstrate a method of transforming these results into immunohistochemistry, a readily applicable method to study biomarker expression in patient samples.

Cell surface structures influence lung clearance rate of systemically infused mesenchymal stromal cells.

The promising clinical effects of mesenchymal stromal/stem cells (MSCs) rely especially on paracrine and nonimmunogenic mechanisms. Delivery routes are essential for the efficacy of cell therapy and systemic delivery by infusion is the obvious goal for many forms of MSC therapy. Lung adhesion of MSCs might, however, be a major obstacle yet to overcome. Current knowledge does not allow us to make sound conclusions whether MSC lung entrapment is harmful or beneficial, and thus we wanted to explore MSC lung adhesion in greater detail. We found a striking difference in the lung clearance rate of systemically infused MSCs derived from two different clinical sources, namely bone marrow (BM-MSCs) and umbilical cord blood (UCB-MSCs). The BM-MSCs and UCB-MSCs used in this study differed in cell size, but our results also indicated other mechanisms behind the lung adherence. A detailed analysis of the cell surface profiles revealed differences in the expression of relevant adhesion molecules. The UCB-MSCs had higher expression levels of α4 integrin (CD49d, VLA-4), α6 integrin (CD49f, VLA-6), and the hepatocyte growth factor receptor (c-Met) and a higher general fucosylation level. Strikingly, the level of CD49d and CD49f expression could be functionally linked with the lung clearance rate. Additionally, we saw a possible link between MSC lung adherence and higher fibronectin expression and we show that the expression of fibronectin increases with MSC culture confluence. Future studies should aim at developing methods of transiently modifying the cell surface structures in order to improve the delivery of therapeutic cells.

Targeting CD33+ acute myeloid leukemia with GLK-33, a lintuzumab-auristatin conjugate with a wide therapeutic window.

CD33 (Siglec-3) is a cell surface receptor expressed in approximately 90% of AML blasts, making it an attractive target for therapy of acute myeloid leukemia (AML). While previous CD33-targeting antibody-drug conjugates (ADCs) like gemtuzumab ozogamicin (GO, Mylotarg) have shown efficacy in AML treatment, they have suffered from toxicity and narrow therapeutic window. This study aimed to develop a novel ADC with improved tolerability and a wider therapeutic window. GLK-33 consists of the anti-CD33 antibody lintuzumab and eight mavg-MMAU auristatin linker-payloads per antibody. The experimental methods included testing in cell cultures, patient-derived samples, mouse xenograft models, and rat toxicology studies. GLK-33 exhibited remarkable efficacy in reducing cell viability within CD33-positive leukemia cell lines and primary AML samples. Notably, GLK-33 demonstrated anti-tumor activity at single dose as low as 300 µg/kg in mice, while maintaining tolerability at single dose of 20 - 30 mg/kg in rats. In contrast to both GO and lintuzumab vedotin, GLK-33 exhibited a wide therapeutic window and activity against multidrug-resistant cells. The development of GLK-33 addresses the limitations of previous ADCs, offering a wider therapeutic window, improved tolerability, and activity against drug-resistant leukemia cells. These findings encourage further exploration of GLK-33 in AML through clinical trials.

Trastuzumab-MMAU Antibody-Auristatin Conjugates: Valine-Glucoserine Linker with Stabilized Maleimide Conjugation Improves In Vivo Efficacy and Tolerability.

PURPOSE: Antibody-drug conjugates (ADCs) have shown impressive clinical activity with approval of many agents in hematological and solid tumors. However, challenges remain with both efficacy and safety of ADCs. This study describes novel trastuzumab-auristatin conjugates with the hydrophilic MMAE prodrug MMAU, and optimization of a glycopeptide linker leading to a wider therapeutic window. EXPERIMENTAL DESIGN: Trastuzumab was conjugated with auristatin payloads via a series of linkers using a stabilized maleimide handle. The ADCs were characterized in vitro and their relative in vivo anti-tumor efficacies were assessed in HER2+ xenograft models. Relative linker stabilities and the mechanism of linker cleavage were studied using in vitro assays. Toxicity and toxicokinetics of the best performing ADC were evaluated in cynomolgus monkey (cyno). RESULTS: The trastuzumab-MMAU ADC with stabilized glycopeptide linker showed maleimide stabilization and higher resistance to cleavage by serum and lysosomal enzymes compared to a valine-citrulline conjugated trastuzumab ADC (trastuzumab-vc-MMAE). A single dose of 1 or 2 mg/kg of trastuzumab-MMAU at drug-to-antibody ratios (DAR) of 8 and 4 respectively resulted in xenograft tumor growth inhibition, with superior efficacy to trastuzumab-vc-MMAE. Trastuzumab-MMAU DAR4 was tolerated at doses up to 12 mg/kg in cyno, which represents 2- to 4-fold higher dose than that observed with vedotin ADCs, and had increased terminal half-life and exposure. CONCLUSIONS: The optimized trastuzumab-MMAU ADC showed potent antitumor activity and was well tolerated with excellent pharmacokinetics in non-human primates, leading to a superior preclinical therapeutic window. The data supports potential utility of trastuzumab-MMAU for treatment of HER2+ tumors.

N-Glycomic Profiling of Microsatellite Unstable Colorectal Cancer.

Aberrant glycosylation affects cancer progression and immune evasion. Approximately 15% of colorectal cancers (CRCs) demonstrate microsatellite instability (MSI) and display major differences in outcomes and therapeutic responses, as compared to corresponding microsatellite stable (MSS) tumors. We compared the N-glycan profiles of stage II and IV MSI CRC tumors, further subdivided into BRAFV600E wild-type and mutated subgroups (n = 10 in each subgroup), with each other and with those of paired non-neoplastic mucosal samples using mass spectrometry. Further, the N-glycans of BRAFV600E wild-type stage II MSI tumors were compared to corresponding MSS tumors (n = 9). Multiple differences in N-glycan profiles were identified between the MSI CRCs and control tissues, as well as between the stage II MSI and MSS samples. The MSI CRC tumors showed a lower relative abundance of high-mannose N-glycans than did the control tissues or the MSS CRCs. Among MSI CRC subgroups, acidic N-glycans showed tumor stage and BRAF mutation status-dependent variation. Specifically, the large, sulfated/phosphorylated, and putative terminal N-acetylhexosamine-containing acidic N-glycans differed between the MSI CRC subgroups, showing opposite changes in stages II and IV, when comparing BRAF mutated and wild-type tumors. Our results show that molecular subgroups of CRC exhibit characteristic glycan profiles that may explain certain carcinogenic properties of MSI tumors.

N-glycosylation in non-invasive and invasive intraductal papillary mucinous neoplasm.

Intraductal papillary mucinous neoplasms (IPMNs), often found incidentally, are potentially malignant cystic tumors of the pancreas. Due to the precancerous nature, IPMNs lacking malignant features should be kept on surveillance. The follow-up relies on magnetic resonance imaging, which has a limited accuracy to define the high-risk patients. New diagnostic methods are thus needed to recognize IPMNs with malignant potential. Here, aberrantly expressed glycans constitute a promising new area of research. We compared the N-glycan profiles of non-invasive IPMN tissues (n = 10) and invasive IPMN tissues (n = 10) to those of non-neoplastic pancreatic controls (n = 5) by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry. Both IPMN subgroups showed increased abundance of neutral composition H4N4 and decrease in H3N5F1, increase in sialylation, and decrease in sulfation, as compared to the controls. Furthermore, invasive IPMN showed an increase in terminal N-acetylhexosamine containing structure H4N5, and increase in acidic complex-type glycans, but decrease in their complex fucosylation and sulfation, as compared to the controls. In conclusion, the N-glycan profiles differed between healthy pancreatic tissue and non-invasive and invasive IPMNs. The unique glycans expressed in invasive IPMNs may offer interesting new options for diagnostics.

Human CMP-N-acetylneuraminic acid hydroxylase is a novel stem cell marker linked to stem cell-specific mechanisms.

Human stem cells contain substantial amounts of the xenoantigen N-glycolylneuraminic acid (Neu5Gc), although the levels of Neu5Gc are low or undetectable in human body fluids and most other human tissues. The lack of Neu5Gc in human tissues has been previously explained by the loss of hydroxylase activity of the human CMP-N-acetylneuraminic acid hydroxylase (CMAH) protein caused by a genetic error in the human Cmah gene. We thus wanted to investigate whether the human redundant Cmah gene could still function in stem cell-specific processes. In this study, we show that CMAH gene expression is significantly upregulated in the adult stem cell populations studied, both of hematopoietic and mesenchymal origin, and identify CMAH as a novel stem cell marker. The CMAH content co-occurs with higher levels of Neu5Gc within stem cells as measured by mass spectrometric profiling. It seems that despite being enzymatically inactive, human CMAH may upregulate the Neu5Gc content of cells by enhancing Neu5Gc uptake from exogenous sources. Furthermore, exposure to exogenous Neu5Gc caused rapid phosphorylation of beta-catenin in both CMAH overexpressing cells and bone marrow-derived mesenchymal stem cells, thereby inactivating Wnt/beta-catenin signaling. The data demonstrate the first molecular evidence for xenoantigen Neu5Gc-induced alteration of crucial stem cell-specific signaling systems for the maintenance of self renewal. These results add further emphasis to the crucial need for completely xenofree culturing conditions for human stem cells.

N-glycomic profiling of colorectal cancer according to tumor stage and location.

Alterations in glycosylation are seen in many types of cancer, including colorectal cancer (CRC). Glycans, the sugar moieties of glycoconjugates, are involved in many important functions relevant to cancer and can be of value as biomarkers. In this study, we have used mass spectrometry to analyze the N-glycan profiles of 35 CRC tissue samples and 10 healthy tissue samples from non-CRC patients who underwent operations for other reasons. The tumor samples were divided into groups depending on tumor location (right or left colon) and stage (II or III), while the healthy samples were divided into right or left colon. The levels of neutral and acidic N-glycan compositions and glycan classes were analyzed in a total of ten different groups. Surprisingly, there were no significant differences in glycan levels when all right- and left-sided CRC samples were compared, and few differences (such as in the abundance of the neutral N-glycan H3N5) were seen when the samples were divided according to both location and stage. Multiple significant differences were found in the levels of glycans and glycan classes when stage II and III samples were compared, and these glycans could be of value as candidates for new markers of cancer progression. In order to validate our findings, we analyzed healthy tissue samples from the right and left colon and found no significant differences in the levels of any of the glycans analyzed, confirming that our findings when comparing CRC samples from the right and left colon are not due to normal variations in the levels of glycans between the healthy right and left colon. Additionally, the levels of the acidic glycans H4N3F1P1, H5N4F1P1, and S1H5N4F1 were found to change in a cancer-specific but colon location-nonspecific manner, indicating that CRC affects glycan levels in similar ways regardless of tumor location.

Structure and Function of Bovine Whey Derived Oligosaccharides Showing Synbiotic Epithelial Barrier Protective Properties.

Commensal gut microbiota and probiotics have numerous effects on the host's metabolic and protective systems, which occur primarily through the intestinal epithelial cell interface. Prebiotics, like galacto-oligosaccharides (GOS) are widely used to modulate their function and abundance. However, important structure-function relations may exist, requiring a detailed structural characterization. Here, we detailed the structural characterization of bovine whey derived oligosaccharide preparations enriched with GOS or not, dubbed GOS-enriched milk oligosaccharides (GMOS) or MOS, respectively. We explore GMOS's and MOS's potential to improve intestinal epithelial barrier function, assessed in a model based on barrier disruptive effects of the Clostridioides difficile toxin A. GMOS and MOS contain mainly GOS species composed of ß1-6- and ß1-3-linked galactoses, and 3'- and 6'-sialyllactose. Both GMOS and MOS, combined with lactobacilli, like Lactobacillus rhamnosus (LPR, NCC4007), gave synergistic epithelial barrier protection, while no such effect was observed with Bifidobacterium longum (BL NCC3001), Escherichia coli (Nissle) or fructo-oligosaccharides. Mechanistically, for barrier protection with MOS, (i) viable LPR was required, (ii) acidification of growth medium was not enough, (iii) LPR did not directly neutralize toxin A, and (iv) physical proximity of LPR with the intestinal epithelial cells was necessary. This is the first study, highlighting the importance of structure-function specificity and the necessity of the simultaneous presence of prebiotic, probiotic and host cell interactions required for a biological effect.

The N-glycome of human embryonic stem cells.

BACKGROUND: Complex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC), the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans) of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses. RESULTS: The data demonstrated that hESC have a characteristic N-glycome which consists of both a constant part and a variable part that changes during hESC differentiation. hESC-associated N-glycans were downregulated and new structures emerged in the differentiated cells. Previously mouse embryonic stem cells have been associated with complex fucosylation by use of SSEA-1 antibody. In the present study we found that complex fucosylation was the most characteristic glycosylation feature also in undifferentiated hESC. The most abundant complex fucosylated structures were Lex and H type 2 antennae in sialylated complex-type N-glycans. CONCLUSION: The N-glycan phenotype of hESC was shown to reflect their differentiation stage. During differentiation, hESC-associated N-glycan features were replaced by differentiated cell-associated structures. The results indicated that hESC differentiation stage can be determined by direct analysis of the N-glycan profile. These results provide the first overview of the N-glycan profile of hESC and form the basis for future strategies to target stem cell glycans.

Glycomics of bone marrow-derived mesenchymal stem cells can be used to evaluate their cellular differentiation stage.

Human mesenchymal stem cells (MSCs) are adult multipotent progenitor cells. They hold an enormous therapeutic potential, but at the moment there is little information on the properties of MSCs, including their surface structures. In the present study, we analyzed the mesenchymal stem cell glycome by using mass spectrometric profiling as well as a panel of glycan binding proteins. Structural verifications were obtained by nuclear magnetic resonance spectroscopy, mass spectrometric fragmentation, and glycosidase digestions. The MSC glycome was compared to the glycome of corresponding osteogenically differentiated cells. More than one hundred glycan signals were detected in mesenchymal stem cells and osteoblasts differentiated from them. The glycan profiles of MSCs and osteoblasts were consistently different in biological replicates, indicating that stem cells and osteoblasts have characteristic glycosylation features. Glycosylation features associated with MSCs rather than differentiated cells included high-mannose type N-glycans, linear poly-N-acetyllactosamine chains and alpha2-3-sialylation. Mesenchymal stem cells expressed SSEA-4 and sialyl Lewis x epitopes. Characteristic glycosylation features that appeared in differentiated osteoblasts included abundant sulfate ester modifications. The results show that glycosylation analysis can be used to evaluate MSC differentiation state.

N-glycan structures and associated gene expression reflect the characteristic N-glycosylation pattern of human hematopoietic stem and progenitor cells.

OBJECTIVE: Cell surface glycans contribute to the adhesion capacity of cells and are essential in cellular signal transduction. Yet, the glycosylation of hematopoietic stem and progenitor cells (HSPC), such as CD133+ cells, is poorly explored. MATERIALS AND METHODS: N-glycan structures of cord blood-derived CD133+ and CD133- cells were analyzed with mass spectrometric profiling and exoglycosidase digestion, cell surface glycan epitopes with lectin binding assay, and expression of N-glycan biosynthesis-related genes with microarray analysis. RESULTS: Over 10% difference was demonstrated in the N-glycan profiles of CD133+ and CD133- cells. Biantennary complex-type N-glycans were enriched in CD133+ cells. Of the genes regulating the synthesis of these structures, CD133+ cells overexpressed MGAT2 and underexpressed MGAT4. Moreover, the amount of high-mannose type N-glycans and terminal alpha2,3-sialylation was increased in CD133+ cells. Elevated alpha2,3-sialylation was supported by the overexpression of ST3GAL6. CONCLUSION: Our work presents new information on the characters of HSPCs. The new knowledge of HSPC-specific N-glycosylation advances their identification and provides tools to promote HSPC homing and mobilization or targeting to specific tissues.

Hydrophilic Auristatin Glycoside Payload Enables Improved Antibody-Drug Conjugate Efficacy and Biocompatibility.

Antibody-drug conjugates (ADCs) offer a combination of antibody therapy and specific delivery of potent small-molecule payloads to target cells. The properties of the ADC molecule are determined by the balance of its components. The efficacy of the payload component increases with higher drug-to-antibody ratio (DAR), while homogeneous DAR = 8 ADCs are easily prepared by conjugation to the four accessible antibody hinge cystines. However, use of hydrophobic payloads has permitted only DAR = 2-4, due to poor pharmacokinetics and aggregation problems. Here, we describe generation and characterization of homogeneous DAR = 8 ADCs carrying a novel auristatin ß-D-glucuronide, MMAU. The glycoside payload contributed to overall hydrophilicity of the ADC reducing aggregation. Compared to standard DAR = 2-4 ADCs, cytotoxicity of the homogeneous DAR = 8 ADCs was improved to low-picomolar IC50 values against cancer cells in vitro. Bystander efficacy was restored after ADC internalization and subsequent cleavage of the glycoside, although unconjugated MMAU was relatively non-toxic to cells. DAR = 8 MMAU ADCs were effective against target antigen-expressing xenograft tumors. The ADCs were also studied in 3D in vitro patient-derived xenograft (PDX) assays where they outperformed clinically used ADC. In conclusion, increased hydrophilicity of the payload contributed to the ADC's hydrophilicity, stability and safety to non-target cells, while significantly improving cytotoxicity and enabling bystander efficacy.

N-Glycomic Profiling of Pheochromocytomas and Paragangliomas Separates Metastatic and Nonmetastatic Disease.

Context: No effective methods for separating primary pheochromocytomas and paragangliomas with metastatic potential are currently available. The identification of specific asparagine-linked glycan (N-glycan) structures, which are associated with metastasized pheochromocytomas and paragangliomas, may serve as a diagnostic tool. Objective: To identify differences in N-glycomic profiles of primary metastasized and nonmetastasized pheochromocytomas and paragangliomas. Setting: This study was conducted at Helsinki University Hospital, University of Helsinki, and Glykos Finland Ltd. and included 16 pheochromocytomas and paragangliomas: 8 primary metastasized pheochromocytomas or paragangliomas and 8 nonmetastasized tumors. Methods: N-glycan structures were analyzed with matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) profiling of formalin-fixed, paraffin-embedded tissue samples. Main Outcome Measure: N-glycan profile of tumor tissue. Results: Four groups of neutral N-glycan signals were more abundant in metastasized tumors than in nonmetastasized tumors: complex-type N-glycan signals of cancer-associated terminal N-acetylglucosamine, multifucosylated glycans (complex fucosylation), hybrid-type N-glycans, and fucosylated pauci-mannose-type N-glycans. Three groups of acidic N-glycans were more abundant in metastasized tumors: multifucosylated glycans, acid ester-modified (sulfated or phosphorylated) glycans, and hybrid-type/monoantennary N-glycans. Fucosylation and complex fucosylation were significantly more abundant in metastasized paragangliomas and pheochromocytomas than in nonmetastasized tumors for individual tests but were over the false positivity critical rate, when adjusted for multiplicity testing. Conclusions: MALDI-TOF MS profiling of primary pheochromocytomas and paragangliomas can identify diseases with metastatic potential based on their different N-glycan profiles. Thus, malignancy-linked N-glycan structures may serve as potential diagnostic tools for pheochromocytomas and paragangliomas.

Characterization of the Ashbya gossypii secreted N-glycome and genomic insights into its N-glycosylation pathway.

The riboflavin producer Ashbya gossypii is a filamentous hemiascomycete, closely related to the yeast Saccharomyces cerevisiae, that has been used as a model organism to study fungal developmental biology. It has also been explored as a host for the expression of recombinant proteins. However, although N-glycosylation plays important roles in protein secretion, morphogenesis, and the development of multicellular organisms, the N-glycan structures synthesised by A. gossypii had not been elucidated. In this study, we report the first characterization of A. gossypii N-glycans and provide valuable insights into their biosynthetic pathway. By combined matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry profiling and nuclear magnetic resonance (NMR) spectroscopy we determined that the A. gossypii secreted N-glycome is characterized by high-mannose type structures in the range Man4-18GlcNAc2, mostly containing neutral core-type N-glycans with 8-10 mannoses. Cultivation in defined minimal media induced the production of acidic mannosylphosphorylated N-glycans, generally more elongated than the neutral N-glycans. Truncated neutral N-glycan structures similar to those found in other filamentous fungi (Man4-7GlcNAc2) were detected, suggesting the possible existence of trimming activity in A. gossypii. Homologs for all of the S. cerevisiae genes known to be involved in the endoplasmatic reticulum and Golgi N-glycan processing were found in the A. gossypii genome. However, processing of N-glycans by A. gossypii differs considerably from that by S. cerevisiae, allowing much shorter N-glycans. Genes for two putative N-glycan processing enzymes were identified, that did not have homologs in S. cerevisiae.

The i blood group antigen as a marker for umbilical cord blood-derived mesenchymal stem cells.

Multipotent mesenchymal stem cells (MSCs) offer great promise for future regenerative and anti-inflammatory therapies. However, there is a lack of methods to quickly and efficiently isolate, characterize, and ex vivo expand desired cell populations for therapeutic purposes. Single markers to identify cell populations have not been characterized; instead, all characterizations rely on panels of functional and phenotypical properties. Glycan epitopes can be used for identifying and isolating specific cell types from heterogeneous populations, on the basis of their cell-type specific expression and prominent cell surface localization. We have now studied in detail the cell surface expression of the blood group i epitope (linear poly-N-acetyllactosamine chain) in umbilical cord blood (UCB)-derived MSCs. We used flow cytometry and mass spectrometric glycan analysis and discovered that linear poly-N-acetyllactosamine structures are expressed in UCB-derived MSCs, but not in cells differentiated from them. We further verified the findings by mass spectrometric glycan analysis. Gene expression analysis indicated that the stem-cell specific expression of the i antigen is determined by ß3-N-acetylglucosaminyltransferase 5. The i antigen is a ligand for the galectin family of soluble lectins. We found concomitant cell surface expression of galectin-3, which has been reported to mediate the immunosuppressive effects exerted by MSCs. The i antigen may serve as an endogenous ligand for this immunosuppressive agent in the MSC microenvironment. Based on these findings, we suggest that linear poly-N-acetyllactosamine could be used as a novel UCB-MSC marker either alone or within an array of MSC markers.

Are globoseries glycosphingolipids SSEA-3 and -4 markers for stem cells derived from human umbilical cord blood?

Umbilical cord blood (UCB) is an efficient and valuable source of hematopoietic stem cells (HSCs) for transplantation. In addition to HSCs it harbours low amounts of mesenchymal stem cells (MSCs). No single marker to identify cord blood-derived stem cells, or to indicate their multipotent phenotype, has been characterized so far. SSEA-3 and -4 are cell surface globoseries glycosphingolipid epitopes that are commonly used as markers for human embryonic stem cells, where SSEA-3 rapidly disappears when the cells start to differentiate. Lately SSEA-3 and -4 have also been observed in MSCs. As there is an ongoing discussion and variation of stem-cell markers between laboratories, we have now comprehensively characterized the expression of these epitopes in both the multipotent stem-cell types derived from UCB. We have performed complementary analysis using gene expression analysis, mass spectrometry and immunochemical methods, including both flow cytometry and immunofluoresence microscopy. SSEA-4, but not SSEA-3, was expressed on MSCs but absent from HSCs. Our findings indicate that SSEA-3 and/or -4 may not be optimal markers for multipotency in the case of stem cells derived from cord blood, as their expression may be altered by cell-culture conditions.

Analysis of the human cancer glycome identifies a novel group of tumor-associated N-acetylglucosamine glycan antigens.

The cell surface is covered by a dense layer of protein- and lipid-linked glycans. Although it has been known that distinct glycan structures are associated with cancer, the whole spectrum of cancer-associated glycans has remained undiscovered. In the present study, we analyzed the protein-linked cancer glycome by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric glycan profiling of cancer patient tissue samples. In lung cancer, we detected accumulation of a novel group of tumor-associated glycans. These protein-linked glycans carried abnormal nonreducing terminal beta-N-acetyl-D-glucosamine (GlcNAc) residues. A similar phenomenon was also detected in structural analyses of tumor-derived glycosphingolipids. This showed that glycan biosynthesis may dramatically change in cancer and that direct glycome analysis can detect the resulting marker glycans. Based on the structural knowledge, we further devised a covalent labeling technique for the detection of GlcNAc-expressing tumors with a specific transferase enzyme. In normal tissues, terminal GlcNAc antigens are capped by galactosylation. Similarly to common cancer-associated glycan antigens T, Tn, and sialyl-Tn, the newly discovered GlcNAc antigens result from incomplete glycosylation. In conclusion, the identified terminal GlcNAc glycans should be recognized as a novel class of tumor markers.