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Gastric cancer is preceded by a carcinogenesis pathway that includes gastritis caused by Helicobacter pylori infection, chronic atrophic gastritis that may progress to intestinal metaplasia (IM), dysplasia, and ultimately gastric carcinoma of the more common intestinal subtype. The identification of glycosylation changes in circulating serum proteins in patients with precursor lesions of gastric cancer is of high interest and represents a source of putative new biomarkers for early diagnosis and intervention. This study applies a glycoproteomic approach to identify altered glycoproteins expressing the simple mucin-type carbohydrate antigens T and STn in the serum of patients with gastritis, IM (complete and incomplete subtypes), and control healthy individuals. The immunohistochemistry analysis of the gastric mucosa of these patients showed expression of T and STn antigens in gastric lesions, with STn being expressed only in IM. The serum glycoproteomic analysis using 2D-gel electrophoresis, Western blot, and MALDI-TOF/TOF mass spectrometry led to the identification of circulating proteins carrying these altered glycans. One of the glycoproteins identified was plasminogen, a protein that has been reported to play a role in H. pylori chronic infection of the gastric mucosa and is involved in extracellular matrix modeling and degradation. Plasminogen was further characterized and showed to carry STn antigens in patients with gastritis and IM. These results provide evidence of serum proteins displaying abnormal O-glycosylation in patients with precursor lesions of gastric carcinoma and include a panel of putative targets for the non-invasive clinical diagnosis of individuals with gastritis and IM.

Zinc-finger nuclease (ZFN) gene targeting is emerging as a versatile tool for engineering of multiallelic gene deficiencies. A longstanding obstacle for detailed analysis of glycoproteomes has been the extensive heterogeneities in glycan structures and attachment sites. Here we applied ZFN targeting to truncate the O-glycan elongation pathway in human cells, generating stable 'SimpleCell' lines with homogenous O-glycosylation. Three SimpleCell lines expressing only truncated GalNAcα or NeuAcα2-6GalNAcα O-glycans were produced, allowing straightforward isolation and sequencing of GalNAc O-glycopeptides from total cell lysates using lectin chromatography and nanoflow liquid chromatography-mass spectrometry (nLC-MS/MS) with electron transfer dissociation fragmentation. We identified >100 O-glycoproteins with >350 O-glycan sites (the great majority previously unidentified), including a GalNAc O-glycan linkage to a tyrosine residue. The SimpleCell method should facilitate analyses of important functions of protein glycosylation. The strategy is also applicable to other O-glycoproteomes.

Identification of mucin-type O-glycosylated proteins with known functions in model organisms like Drosophila could provide keys to elucidate functions of the O-glycan moiety and proteomic analyses of O-glycoproteins in higher eukaryotes remain a challenge due to structural heterogeneity and a lack of efficient tools for their specific isolation. Here we report a strategy to evaluate the O-glycosylation potential of the embryonal hemocyte-like Drosophila Schneider 2 (S2) cell line by expression of recombinant glycosylation probes derived from tandem repeats of the human mucin MUC1 or of the Drosophila salivary gland protein Sgs1. We obtained evidence that mucin-type O-glycosylation in S2 cells grown under serum-free conditions is restricted to the Tn-antigen (GalNAcalpha-Ser/Thr) and the T-antigen (Galbeta1-3GalNAcalpha-Ser/Thr) and this structural homogeneity enables unique glycoproteomic strategies. We present a label-free strategy for the isolation, profiling and analysis of O-glycosylated proteins consisting of serial lectin affinity capture, 2-DE-based glycoprotein analysis by O-glycan specific mAbs and protein identification by MALDI-MS. Protein identity and O-glycosylation was confirmed by ESI-MS/MS with detection of diagnostic sugar oxonium-ion fragments. Using this strategy, we established 2-D reference maps and identified 21 secreted and intracellular mucin-type O-glycoproteins. Our results show that Drosophila S2 cells express O-glycoproteins involved in a wide range of biological functions including proteins of the extracellular matrix (Laminin gamma-chain, Peroxidasin and Glutactin), pathogen recognition proteins (Gnbp1), stress response proteins (Glycoprotein 93), secreted proteases (Matrix-metalloprotease 1 and various trypsin-like serine proteases), protease inhibitors (Serpin 27 A) and proteins of unknown function.

Carbohydrates of the epithelial cell membrane are involved in cell-cell and cell-substrate interaction, and changes are seen in relationship to cell differentiation and neoplastic transformation. The terminal part of carbohydrate structures carried on oral epithelial cells often expresses antigens of the ABO and Lewis blood group systems. The expression of these antigens are in oral mucosa genetically regulated by the A, B, H, Lewis, and secretor genes with subsequent correspondence between the blood group antigens expressed on erythrocytes and on oral epithelial cells. Variation in expression of carbohydrates is also seen in relationship to terminal differentiation in that blood group antigens and their immediate precursor structures are sequentially expressed on cells during their pathway through the epithelium. Various organs and tissues differ in their expression of cell surface carbohydrates. In oral mucosa, a close relationship is seen between the type of tissue differentiation and expression of blood group antigen; keratinized, nonkeratinized, and junctional epithelium all show different patterns of carbohydrate expression.

A panel of monoclonal antibodies was used to examine differentiation-related carbohydrate structures on the surfaces of gingival epithelial cells. The patterns of binding observed indicate distinct differences in the expression of the epitopes examined for three regions of the gingival epithelia corresponding approximately to the regions defined anatomically as the junctional, oral sulcular and oral epithelia. However, epithelium with the staining pattern of oral sulcular epithelium consistently extended beyond the sulcular region to cover the gingival crest and often the uppermost part of the oral aspect of the gingiva. Differential staining of basal and suprabasal cells indicated an unusual pattern of differentiation of the junctional epithelium. The phenotype of this epithelium appears to differ from patterns reported for any other oral epithelium and the possible functional significance of this difference is discussed.

Cell surface carbohydrates are excellent markers for cellular differentiation and maturation due to great structural and antigenic diversity and to known precursor/product relations. Several blood group related carbohydrate antigens were analyzed in human labial stratified non-keratinized epithelium from 16 healthy individuals by immunohistology using monoclonal antibodies. The expression of these antigens was correlated with erythrocyte phenotype and saliva secretor status. Three distinct compartments of the epithelium were found and defined by the sequential expression of derivatives of Type 2 chain structures: lower, confined to basal cell layers (N-acetyllactosamine), middle, to parabasal cell layers (H) and upper, to spinous cell layers (Le y]Lex]). Although the antigens are related to blood group antigens they are largely expressed independently of the ABO, Lewis and secretor types, and may therefore serve as "universal" markers in differentiation studies of normal and pathological epithelium. [ABSTRACT FROM AUTHOR]

Recent studies have shown that changes within membrane bound carbohydrates may be essential for cellular differentiation and malignant transformation. We have therefore, by means of immunohistochemistry, studied the expression of T/Tn related (Thomsen-Friedenrich) carbohydrates in 13 oral lesions with squamous cell dysplasia. The epithelial grade of dysplasia was graded as mild, moderate or severe. The following carbohydrate structures were studied: Tn, T, mucintype 3 chain H, and the sialylated derivates, sialosyl-Tn and sialosyl-T. In general, short structures were detected on the basal cells and longer structures on the more mature spinous cells. In many cases, this sequential expression was more disturbed with increasing grade of epithelial dysplasia. However, our results also showed that some lesions with the same grade of epithelial dysplasia showed different carbohydrate expression. These findings indicate that expression of carbohydrates may supplement histologic diagnosis in the evaluation of the prognosis of premalignant lesions. [ABSTRACT FROM AUTHOR]

The histo-blood group ABO carbohydrate antigens are differentially expressed in epithelia in close correlation with cellular differentiation. In order to gain insight into the biosynthetic regulation of these carbohydrate antigens, we correlated the expression of A carbohydrate antigens with that of the A gene defined glycosyltransferase by immunohistology of' human oral epithelia using monoclonal anti- bodies. In glandular epithelium the A transferase was found in mucous cells similar to that of the A carbohydrate antigens. In stratified non-keratinized squamous epithelium the A transferase was expressed only in spinous cell layers, which is in accordance with the appearance of the A carbohydrate antigens in these more mature cell layers. This simultaneous acquisition of the primary and secondary gene product of a glycosyltransferase gene, provides evidence that the well-defined sequential expression of histo-blood group carbohydrate antigens in stratified squamous epithelium may be directly regulated at the transcriptional level of the glycosyltransferase. Future studies will address the nwchanism behind loss of A antigens in premalignant lesions and carcinomas. [ABSTRACT FROM AUTHOR]