Showing papers by "Steffen Goletz published in 1997"

Novel alphaGalNAc containing glycans on cytokeratins are recognized invitro by galectins with type II carbohydrate recognition domains

Abstract: We report on a novel posttranslational modification of cytoplasmic proteins. Presented evidences suggest that cytokeratins are bound in vitro by mammalian galectin-3 and the galectins from the sponge Geodia cydonium via their type II carbohydrate recognition domains, whose highest binding affinity is directed towards terminal alpha-N-acetylgalactosamine-bearing glycans with the general sequence GalNAcalpha1-3Gal(NAc)beta. Specificity analyses and the characterization of the critical sugar residue on cytokeratins for galectin binding were done with cytochemical and biochemical methods using various plant and animal lectins. Binding of GalNAc-specific lectins was saturable, sensitive to mild periodate oxidation, inhibitable by glycoconjugates carrying terminal GalNAc, and abolished after treatment of the cytokeratins with alpha-N-acetylgalactosaminidase. Binding to bacterially expressed recombinant cytokeratins did not exceed background binding. The presence of GalNAc residues on highly purified cytokeratins from MCF-7 and HeLa SS6 cells was confirmed by sugar composition analyses using gas chromatography/mass spectrometry. This novel posttranslational modification was not restricted to cytokeratins of MCF-7 cells, but did also occur in all of 9 other examined human carcinoma cell lines and in a normal human mammary epithelial cell line. From these cytochemical and biochemical in vitro studies we hypothesize that this glycan with its terminal alpha1-3 linked GalNAc determinant might represent the first natural cytoplasmic ligand for endogenous galectins-3 detected so far.

A sequencing strategy for the localization of O-glycosylation sites of MUC1 tandem repeats by PSD-MALDI mass spectrometry

Abstract: It is demonstrated with glycopeptides of the polymorphic epithelial mucin (MUC1) that post-source decay matrix-assisted laser desorption ionization (PSD-MALDI) is a fast, highly sensitive, and reproducible method for the localization of O-glycosylation sites by reflectron time-of-flight (TOF) mass spectrometry. We have analyzed GalNAc-carrying peptides of up to 25 amino acids, and could distinguish even neighboring glycosylation sites. This method was also able to localize and characterize disaccharides (e.g., the Thomsen-Friedenreich disaccharide) on MUC1 derived peptides. PSD-MALDI-MS fragment ion patterns were recorded in the positive ion mode from the synthetic peptide TAP25 [(T1aAPPAHGVT9S10APDT14RPAPGS20) T1bAPPA], an overlapping sequence of MUC1 tandem repeats, which was glycosylated with GaINAc in vitro. The glycosylation sites found were either Thr9 or Thr1b in the monoglycosylated, Thr9 and Thr1b in the diglycosylated, and Thr9, Thr1b, and Ser20 in the triglycosylated peptide. A single PSD-MALDI-MS spectrum of the underivatized and uncleaved di- or triglycosylated TAP25 peptide was sufficient to identify the glycosylation sites, thereby distinguishing six potential, partly adjacent, glycosylation sites. The monoglycosylated fraction was found to consist of a mixture of two glycosylated species with the same molecular weight. This was shown by the analysis of proteolytic digests. PSD-MALDI-MS of the resulting peptides right out of the digestion probe was sufficient to identify the Gal-NAc-glycosylation sites as either Thr9 or Thr1b, respectively. Beyond the methodical aspects the results revealed that in vitro glycosylation of the TAP25 peptide with a transferase system from human milk differs from that obtained with a breast cancer cell transferase system.

Structure Analysis of Acetylated and Non-acetylated O-Linked MUC1-Glycopeptides by Post-source Decay Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Abstract: We have investigated the potential of structural elucidation of O-linked glycopeptides by post-source decay matrix-assisted laser desorption ionization mass spectrometry (PSD-MALDI-MS). In order to establish detailed fragmentation patterns and to dissect fragment ions with and without carbohydrate content, the same O-linked MUC1-derived glycopeptides with acetylated and non-acetylated sugars were analysed and compared. Furthermore, we were interested to examine possible differences in the fragmentation between glycopeptides with acetylated and non-acetylated sugars. The obtained PSD-MALDI-MS spectra showed a rather complete set of fragmentation data which allows us to localize the glycan on the peptide, in parallel with sequencing a short glycan and the backbone peptide. Fragmentations of the sugars were dominated by inter-ring cleavages at the glycosidic bond. Intra-ring cleavage did also occur from the non-reducing end, but to a much lower extent. The fragmentation of the peptide backbone was not changed either by acetylated or non-acetylated sugars. Glycosylated peptide fragments occurred as fully glycosylated fragment ions, partially deglycosylated ions and completely deglycosylated ions, and was not influenced by the acetylation of sugars. However, differences occurred in the quality and quantity of fragment ions from the non-reducing end of the glycan part when comparing acetylated with non-acetylated glycopeptides. © 1997 John Wiley & Sons, Ltd.