Shin-Yi Yu

Distinctive characteristics of MALDI-Q/TOF and TOF/TOF tandem mass spectrometry for sequencing of permethylated complex type N-glycans.

Concerted MALDI-MS profiling and CID MS/MS sequencing of permethylated glycans is one of the most effective approaches for high throughput glycomics applications. In essence, the identification of larger complex type N-glycans necessitates an unambiguous definition of any modification on the trimannosyl core and the complement of non-reducing terminal sequences which constitute the respective antennary structures. Permethylation not only affords analyses of both neutral and sialylated glycans at comparable ease and sensitivity but also yields more sequence-informative fragmentation pattern. Facile glycosidic cleavages directed mostly at N-acetylglucosamine under low energy CID, as implemented on a quadrupole/time-of-flight (Q/TOF) instrument, often afford multiple losses of the attached antenna resulting in characteristic ions related to the number of antennary branches on the trimannosyl core. Non-reducing terminal epitopes can be easily deduced but information on the linkage specific substituent on the terminal units is often missing. The high energy CID MS/MS afforded by TOF/TOF instrument can fill in the gap by giving an array of additional cross-ring and satellite ions. Glycosidic cleavages occurring specifically in concert with loss of 2-linked or 3-linked substituents provide an effective way to identify the branch-specific antennary extension. These characteristics are shown here to be effective in deriving the sequences of additionally galactosylated, sialylated and fucosylated terminal N-acetyllactosamine units and their antennary location. Together, a highly reproducible fragmentation pattern can be formulated to simplify spectral assignment. This work also provides first real examples of sequencing multiply sialylated complex type N-glycans by high energy CID on a TOF/TOF instrument.

Core3 O-Glycan Synthase Suppresses Tumor Formation and Metastasis of Prostate Carcinoma PC3 and LNCaP Cells through Down-regulation of α2β1 Integrin Complex

Although there are numerous reports of carbohydrates enriched in cancer cells, very few studies have addressed the functions of carbohydrates present in normal cells that decrease in cancer cells. It has been reported that core3 O-glycans are synthesized in normal gastrointestinal cells but are down-regulated in cancer cells. To determine the roles of core3 O-glycans, we transfected PC3 and LNCaP prostate cancer cells with β3-N-acetylglucosaminyltransferase-6 (core3 synthase) required to synthesize core3 O-glycans. Both engineered cell lines exhibited reduced migration and invasion through extracellular matrix components compared with mock-transfected cells. Moreover we found that α2β1 integrin acquired core3 O-glycans in cells expressing core3 synthase with decreased maturation of β1 integrin, leading to decreased levels of the α2β1 integrin complex, decreased activation of focal adhesion kinase, and reduced lamellipodia formation. Upon inoculation into the prostate of nude mice, PC3 cells expressing core3 O-glycans produced much smaller tumors without metastasis to the surrounding lymph nodes in contrast to robust tumor formation and metastasis seen in mock-transfected PC3 cells. Similarly LNCaP cells expressing core3 O-glycans barely produced subcutaneous tumors in contrast to robust tumor formation by mock-transfected LNCaP cells. These findings indicate that addition of core3 O-glycans to β1 and α2 integrin subunits in prostate cancer cells suppresses tumor formation and tumor metastasis.

The expression of sialylated high-antennary N-glycans in edible bird’s nest

Edible birds nest (EBN) is the nest made from the saliva of Collocalia swift. Recently, we have found that EBN extract could strongly inhibit infection of influenza viruses in a host-range-independent manner [Guo, C. T.; Takahashi, T.; Bukawa, W.; Takahashi, N.; Yagi, H.; Kato, K.; Hidari, K. I.; Miyamoto, D.; Suzuki, T.; Suzuki, Y. Antiviral Res.2006, 70, 140-146]. Although this antiviral activity might be attributed to O- or N-glycoconjugates, no N-glycan structures have so far been described for EBN. Here, we report the N-glycosylation profile of EBN, in which a tri-antennary N-glycan bearing the alpha2,3-N-acetylneuraminic acid residues is displayed as a major component. We suggest that the sialylated high-antennary N-glycans of EBN contribute to the inhibition of influenza viral infection.

Prominent expression of sialyl Lewis X-capped core 2-branched O-glycans on high endothelial venule-like vessels in gastric MALT lymphoma.

High endothelial venule (HEV)‐like vessels have been observed in gastric B cell lymphoma of mucosa‐associated lymphoid tissue type (MALT lymphoma), as well as in its preceding lesion, chronic Helicobacter pylori gastritis. Previously we reported that glycans on HEV‐like vessels in the latter lesion served as L‐selectin ligands, although their function is unclear. We have investigated sialyl Lewis X (sLeX)‐related glycoepitopes and found that MECA‐79−/HECA‐452+/NCC‐ST‐439+ HEV‐like vessels preferentially mark gastric MALT lymphoma compared to chronic H. pylori gastritis. We then constructed CHO cell lines expressing potential MECA‐79−/HECA‐452+/NCC‐ST‐439+ glycans, as well as other sLeX‐type glycans, on CD34 and evaluated L‐selectin binding to those cells, using L‐selectin–IgM chimera binding and lymphocyte adhesion assays. L‐selectin–IgM chimeras bound to CHO cells expressing 6‐sulpho‐sLeX attached to core 2‐branched O‐glycans with or without 6‐sulpho‐sLeX attached to extended core 1 O‐glycans, but only marginally to other CHO cell lines. By contrast, CHO cells expressing 6‐sulpho‐sLeX attached to extended core 1 and/or core 2‐branched O‐glycans, as well as non‐sulphated sLeX attached to core 2‐branched O‐glycans, showed substantial lymphocyte binding, while binding was negligible on lines expressing 6‐sulpho‐ and non‐sulphated sLeX attached to N‐glycans and non‐sulphated sLeX attached to extended core 1 O‐glycans. These results indicate that MECA‐79−/HECA‐452+/NCC‐ST‐439+ glycans, specifically, 6‐sulpho‐ and non‐sulphated sLeXs attached to core 2‐branched O‐glycans, expressed on HEV‐like vessels in gastric MALT lymphoma function as L‐selectin ligands and likely contribute to H. pylori‐specific T cell recruitment in the progression of gastric MALT lymphoma. Copyright

MASS SPECTROMETRIC ANALYSIS OF SULFATED N- AND O-GLYCANS

Sulfated N- and O-glycans carried on a myriad of cell-surface adhesion molecules and receptors are often not detected by current approaches in mass spectrometry (MS)-based glycomic mapping of cells and tissues. This is in part due to a natural lower abundance, compounded further by their negatively charged nature, which adversely disfavors their ionization and detection amid a sea of often much more abundant, nonsulfated, sialylated glycans. However, this particular limitation can actually be taken advantage of to effect highly selective enrichment and sensitive MS screening in negative ion mode, provided the ubiquitous sialic acids can first be neutralized. It has been demonstrated that permethylation not only fulfills this role adequately but further confers better MS/MS fragmentation characteristics for more efficient structural mapping and sequencing. Protocols and general practical considerations are described here which would enable one to readily prepare permethylated sulfated glycans, fractionate them away from the more abundant nonsulfated ones in simple steps for high-sensitivity MS analysis, and sensibly interpret the initial sulfoglycomic screening data thus obtained.

Identification of Further Elongation and Branching of Dimeric Type 1 Chain on Lactosylceramides from Colonic Adenocarcinoma by Tandem Mass Spectrometry Sequencing Analyses

Mammalian glycan chain elongation is mostly based on extending the type 2 chain, Galβ1–4GlcNAc, whereas the corresponding type 1 chain, Galβ1–3GlcNAc, is not normally extended. In a broader context of developing high sensitivity mass spectrometry methodologies for glycomic identification of Lea versus Lex and linear versus branched poly-N-acetyllactosamine (polyLacNAc), we have now shown that the dimeric type 1 glycan chain, as carried on the lactosylceramides of a human colonic adenocarcinoma cell line, Colo205, not only can be further extended linearly but can likewise be branched at C6 of 3-linked Gal in a manner similar to polyLacNAc. A combination of chemical and enzymatic derivatization coupled with advanced mass spectrometry analyses afforded unambiguous identification of a complex mixture of type 1 and 2 hybrids as well as those fucosylated variants founded exclusively on linear and branched trimeric type 1 chain. We further showed by in vitro enzymatic synthesis that extended type 1 and the hybrid chains can be branched by all three forms of the human I branching enzymes (IGnT) currently identified but with lower efficiency and stringency with respect to branching site preference. Importantly, it was found that a better substrate is one that carries a Gal site for branching that is extended at the non-reducing end by a type 2 and not a type 1 unit, whereas the IGnTs are less discriminative with respect to whether the targeted Gal site is itself β3- or β4-linked to GlcNAc at the reducing end.

Priming mass spectrometry-based sulfoglycomic mapping for identification of terminal sulfated lacdiNAc glycotope

In an effort to prime our mass spectrometry (MS)-based sulfoglycomic mapping platform technology for facile identification of sulfated lacdiNAc (GalNAcβ1-4GlcNAcβ1-), we have re-examined the N-glycans of bovine thyroid stimulating hormone. We showed that MALDI-MS mapping of permethylated glycans in negative ion mode can give an accurate representation of the sulfated glycans and, through MS/MS, diagnostic ions can be derived that we can collectively define the presence of a terminal sulfated lacdiNAc moiety at high sensitivity. Based on these ions, which can also be produced by nanoESI-MSn, we demonstrated that the glycome of an ovarian carcinoma cell line, RMG-1, comprises a high abundance of sulfated lacdiNAc epitopes carried on multiantennary complex type N-glycans alongside fucosylated, sialylated and/or sulfated lacNAc antennae. This represents the first report of a natural glycomic occurrence of sulfated lacdiNAc on a cell line, as opposed to other better-characterized presence on secreted glycoproteins from a handful of sources. It is anticipated that with improved methods of detection such as that developed in this work, we are likely to identify a wider occurrence of sulfated lacdiNAc and be able to more accurately delineate the regulatory mechanism dictating the choice of a cell type in synthesizing sulfated, sialylated, fucosylated and/or non-substituted lacdiNAc.

Core2 O-glycan structure is essential for the cell surface expression of sucrase isomaltase and dipeptidyl peptidase-IV during intestinal cell differentiation.

Alterations in glycosylation play an important role during intestinal cell differentiation. Here, we compared expression of mucin-type O-glycan synthases from proliferating and differentiated HT-29 and Caco-2 cells. Mucin-type O-glycan structures were analyzed at both stages by mass spectrometry. Core2 β1,6-N-acetylglucosaminyltransferase-2 (C2GnT-2) was markedly increased in differentiated HT-29 and Caco-2 cells, but the core3 structure was hardly detectable. To determine whether such differential expression of mucin-type O-glycan structures has physiological significance in intestinal cell differentiation, expression of sucrase isomaltase (SI) and dipeptidyl-peptidase IV (DPP-IV), two well known intestinal differentiation markers, was examined. Interestingly, the fully glycosylated mature form of SI was decreased in C2GnT-2 knock-out mice but not in core2 N-acetylglucosaminyltransferase-3 (C2GnT-3) nulls. In addition, expression of SI and DPP-IV was dramatically reduced in C2GnT-1–3 triple knock-out mice. These patterns were confirmed by RNAi analysis; C2GnT-2 knockdown significantly reduced cell surface expression of SI and DPP-IV in Caco-2 cells. Similarly, overexpression of the core3 structure in HT-29 cells attenuated cell surface expression of both enzymes. These findings indicate that core3 O-glycan structure regulates cell surface expression of SI and DPP-IV and that core2 O-glycan is presumably an essential mucin-type O-glycan structure found in both molecules in vivo. Finally, goblet cells in the upper part of the crypt showed impaired maturation in the core2 O-glycan-deficient mice. These studies are the first to clearly identify functional mucin-type O-glycan structures modulating cell surface expression of SI and DPP-IV during the intestinal cell differentiation.

N-Glycosylation profiling of turtle egg yolk: expression of galabiose structure

To understand the roles of species-specific carbohydrates, systematic studies of interspecific glycan analyses are imperative. An extensive series of glycomics studies on approximately 180 kinds of bird eggs have demonstrated that 60-70% of the birds, which are closely related in phylogeny, express the alpha-Galp-(1-->4)-Galp structure on their egg glycoproteins. This prompted us to investigate the glycosylation profiles of eggs from an evolutionarily related organism, a sea turtle (reptilian). We performed N-glycosylation profiling of turtle egg yolk by using HPLC mapping in conjunction with mass spectrometric methods and thereby demonstrated that the alpha-Galp-(1-->4)-Galp groups are displayed on approximately 38% of total N-glycans. Our findings suggest that the ability to express the galabiose structure was acquired at an early stage of diversification in amniotes.

Tumor-Associated Glycans and Their Functional Roles in the Multistep Process of Human Cancer Progression

Cancer develops through a multistep process of carcinogenesis. This process accompanies incremental alterations of expression of biologically functional glycans on the surface of cancer cells. A variety of glycans are expressed in nonmalignant epithelial cells, including several normal glycans serving as ligands for siglecs, the immunosuppressive molecules carried by interstitial immune cells. These normal glycans decrease or disappear and are replaced by cancer-associated glycans at the early stages of carcinogenesis. This glycan transition facilitates production by mucosal immune cells of inflammatory mediators that are known to promote cancer progression. Expression of glycans that regulate growth factor receptor functions is also affected at the early stages of cancers. The major mechanism involved in glycan alteration at the early stages is epigenetic silencing through DNA methylation and/or histone deacetylation/methylation of genes responsible for synthesis of normal glycans, leading to their incomplete synthesis. In the locally advanced stages, multiple glycan-related genes are induced transcriptionally and posttranscriptionally by tumor hypoxia and epithelio-mesenchymal transition, thus further culminating in abnormal expression of cancer-associated glycans. Some such glycans serve as specific ligands for selectins, the cell adhesion molecules carried by vascular endothelial cells, and facilitate tumor vascularization and ultimately hematogenous metastasis. Advanced cancer cells which have undergone epithelio-mesenchymal transition share biological characteristics with so-called cancer stem cells, and glycans associated with such cells are currently known to be frequently expressed in human embryonic stem cells as well.