An Analytical and Structural Database Provides a Strategy for Sequencing O-Glycans from Microgram Quantities of Glycoproteins

Royle, Louise; Mattu, Taj S.; Hart, Edmund; Langridge, James; Merry, Anthony H.; Murphy, Neil; Harvey, David J.; Dwek, Raymond A.; Rudd, Pauline M.

doi:10.1006/abio.2002.5619

Cited by 186 publications

(168 citation statements)

References 61 publications

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“…In further characterization of the Globo H oligosaccharide epitope, we sought to implement analytical sequencing for structure confirmation (48)(49)(50)(51). For this purpose, a Globo H derivative containing a fluorescent tag was prepared by reductive amination of the reducing free terminus of Globo H with 2-aminobenzamide.…”

Section: Resultsmentioning

confidence: 99%

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Carbohydrate microarray for profiling the antibodies interacting with Globo H tumor antigen

Huang

Thayer

Chang

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

187

149

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Understanding the specificity of cell-surface carbohydrates interaction with antibodies and receptors is important for the development of new therapeutics and high-sensitivity diagnostics. This approach is, however, limited to the availability of natural and truncated sequences of the oligosaccharides and the sensitivity of the assay system. Reported here is the synthesis of the cancer antigen Globo H hexasaccharide, an epitope found on the cell surface of breast, prostate, and ovarian cancers, and its truncated sequences by using the programmable one-pot synthesis strategy. The saccharides were then arrayed covalently on glass slides with different density and used for the fluorencense-based binding analysis of two monoclonal antibodies against Globo H and the serum from breast cancer patients, to define the specificity of these antibodies. It was shown that the terminal tetrasaccharide binds the monoclonal antibodies equally well as does the hexasaccharide and the fucose residue is required for effective binding. The serum binds both the defucosylated pentasaccharide and the fucosylated hexasaccharide without a significant difference, perhaps because of the polyclonal nature of the serum or the presence of diverse immune responses to different sugar epitopes at various stages. This method requires very small amounts of materials and is more effective and sensitive than the traditional ELISA method, and thus provides another platform to monitor the immune response to carbohydrate epitopes at different stages during differentiation, metastasis, or treatment.programmable one-pot synthesis ͉ glycoarray ͉ glycan epitope ͉ Globo H-truncated sequences T he cell-surface glycosphingolipid Globo H is a member of a family of antigenic carbohydrates that are highly expressed on a range of cancer cell lines, especially breast cancer cells (1-4). Furthermore, it has been established that the serum of breast cancer patients contains high levels of antibodies against the Globo H epitope, and this epitope is also targeted by the monoclonal antibodies MBr1 (5-7) and VK-9 (8). As a result, this hexasaccharide has been the focus of studies aimed at anticancer vaccine development (9-15). Many elegant syntheses of Globo H have been reported (11,(16)(17)(18)(19)(20)(21)(22)(23)(24), including one approach that uses the one-pot programmable oligosaccharide synthesis developed in our laboratory (25). Previously, it has been reported that certain truncated Globo H derivatives can still be effective in binding MBr1 and VK-9 antibodies, which could increase the efficiency of immunogen development for vaccine therapy (8,(26)(27)(28)(29)(30). We set out to further characterize the binding specificities of these and cancer patient antibodies by using carbohydrate microarray analysis.Carbohydrate microarrays allow for the direct characterization of carbohydrate-protein interactions. In addition, the attachment of sugars to surfaces can effectively mimic the presentation of these compounds on the cell membrane. A large factor which is present...

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Section: Resultsmentioning

confidence: 99%

“…Exoglycosidase reactions were then desalted on C 4 ZipTips (Millipore) before analysis by normal phase-HPLC (Glycosep N HPLC column, Prozyme) to determine the sequence, monosaccharide type, and linkage of sugar residues. HPLC conditions used were described fully by Rudd and coworkers (48).…”

Section: Microarray Analysis Of Cancer Patient Serum For Antibodies Tmentioning

confidence: 99%

Carbohydrate microarray for profiling the antibodies interacting with Globo H tumor antigen

Huang

Thayer

Chang

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

187

149

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“…Gradient conditions and detection were as described previously (22,23). The system was calibrated against a standard dextran hydrolysate ladder so that the retention time for individual glycans could be converted to glucose units (GU) (22).…”

Section: Detailed Glycosylation Analysis Of Recombinant Mmp-9mentioning

confidence: 99%

“…The system was calibrated against a standard dextran hydrolysate ladder so that the retention time for individual glycans could be converted to glucose units (GU) (22). Oligosaccharide structures were assigned by reference to the GU values of a data base of standard sugars (23).…”

Section: Detailed Glycosylation Analysis Of Recombinant Mmp-9mentioning

confidence: 99%

The Hemopexin and O-Glycosylated Domains Tune Gelatinase B/MMP-9 Bioavailability via Inhibition and Binding to Cargo Receptors

Steen

Aelst

Hvidberg

et al. 2006

Journal of Biological Chemistry

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Gelatinase B/matrix metalloproteinase-9 (MMP-9), a key regulator and effector of immunity, contains a C-terminal hemopexin domain preceded by a unique linker sequence of ϳ64 amino acid residues. This linker sequence is demonstrated to be an extensively O-glycosylated (OG) domain with a compact three-dimensional structure. The OG and hemopexin domains have no influence on the cleavage efficiency of MMP-9 substrates. In contrast, the hemopexin domain contains a binding site for the cargo receptor low density lipoprotein receptor-related protein-1 (LRP-1). Furthermore, megalin/ LRP-2 is identified as a new functional receptor for the hemopexin domain of MMP-9, able to mediate the endocytosis and catabolism of the enzyme. The OG domain is required to correctly orient the hemopexin domain for inhibition by TIMP-1 and internalization by LRP-1 and megalin. Therefore, the OG and hemopexin domains down-regulate the bioavailability of active MMP-9 and the interactions with the cargo receptors are proposed to be the original function of hemopexin domains in MMPs.

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“…Although use of exoglycosidase digestion is still widespread and now applicable to many O-linked structures [9], mass spectral fragmentation is becoming more generally used as instruments are now available that can produce high quality MS/MS spectra from small amounts of material. Fast-atom bombardment (FAB) mass spectrometry has been used in this context for many years [10 -13] but its requirement for derivatization, usually by permethylation, and the presence of a high background from the matrix causes problems for analysis of the minuscule sample amounts now being presented for analysis in many laboratories.…”

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confidence: 99%

Fragmentation of negative ions from carbohydrates: Part 1. Use of nitrate and other anionic adducts for the production of negative ion electrospray spectra from N-linked carbohydrates

Harvey

2005

J. Am. Soc. Mass Spectrom.

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189

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Negative ion spectra of N-linked glycans were produced by electrospray from a dilute solution of the glycans and various salts in methanol:water using a Waters-Micromass Q-TOF Ultima Global tandem quadrupole/time-of-flight (Q-TOF) mass spectrometer. Stable anionic adducts were formed with chloride, bromide, iodide, nitrate, sulphate, and phosphate. Unstable adducts that fragmented by a cross-ring cleavage of the reducing N-acetylglucosamine (GlcNAc) residue, were formed with fluoride, nitride, sulphide, carbonate, bicarbonate, hydroxide, and acetate. Nitrate adducts prepared from ammonium nitrate produced the most satisfactory spectra as they were relatively free from in-source fragmentation products and gave signals that were about ten times as strong as those from corresponding [M Ϫ H] Ϫ ions prepared from solutions containing ammonium hydroxide. Detection limits were in the region of 20 fmol. Neutral glycans gave both singly-and doubly-charged ions with the larger glycans preferring the formation of doubly-charged ions. Acidic glycans with several acidic groups gave ions in higher charge states as the result of ionization of the anionic groups. Low energy collision-induced decomposition (CID) spectra of the singly-charged ions were dominated by cross-ring and C-type fragments, unlike the corresponding spectra of the positive ions that contained mainly B-and Y-type glycosidic fragments. Formation of these ions could be rationalized by proton abstraction from various hydroxy groups by an initially-formed anionic adduct. Prominent glycosidic and cross-ring cleavage ions defined structural features such as the specific composition of each of the two antennae, presence of a bisecting GlcNAc residue and location of fucose residues, details that were difficult to determine by conventional techniques. Acidic glycans fragmented differently on account of charge localization on the acid functions rather than the hydroxy groups. (J Am Soc Mass Spectrom 2005, 16, 622-630)

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An Analytical and Structural Database Provides a Strategy for Sequencing O-Glycans from Microgram Quantities of Glycoproteins

Cited by 186 publications

References 61 publications

Carbohydrate microarray for profiling the antibodies interacting with Globo H tumor antigen

Carbohydrate microarray for profiling the antibodies interacting with Globo H tumor antigen

The Hemopexin and O-Glycosylated Domains Tune Gelatinase B/MMP-9 Bioavailability via Inhibition and Binding to Cargo Receptors

Fragmentation of negative ions from carbohydrates: Part 1. Use of nitrate and other anionic adducts for the production of negative ion electrospray spectra from N-linked carbohydrates

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