Cancer-associated quantitative differences in antigen levels of sialosyl Lewisx in stool from patients with colorectal carcinomas

A microenzyme-linked immunosorbent assay system employing monoclonal antibody SNH3 was developed for the detection of sialosyl Lewis^x antigen in stool extracts from 80 patients with colorectal cancer, 13 patients with colorectal non-malignant disorders and 90 normal subjects. Sialosyl Lewis^x antigen was detected in 35% of stool extracts from cancer patients but only in 7.7% and 2.2% of those from non-malignant patients and normal subjects respectively. Hemoglobin in the same stool samples was detected in 37.5% of cancer patients, 15.4% of non-malignant patients and 5.6% of normal subjects. The appearance of sialosyl Lewis^x antigen in stool was not necessary correlated with that of hemoglobin, and overall 61.3% of cancer patients were detected by the combination of the two assays. The combination assay was also impressive in early detection of colorectal cancer (Dukes' A, 52%; Dukes' B, 57.1%). Therefore, the assay for sialosyl Lewis^x antigen in stool would be useful for detecting colorectal cancer.     

Ultrastructural localization of epithelial mucin core proteins in colorectal tissues.

Mucins are high molecular weight glycoproteins with a variety of postulated biological functions, including physicochemical protection from toxins and mutagens, adhesion modulation, signal transduction, and regulation of cell growth. Mucins are widely and differentially expressed in the gastrointestinal tract. To date, studies of cellular expression have relied on light microscopy using in situ hybridization and immunohistochemistry. Although informative, it has been difficult with these techniques to ascertain exactly which cell types are producing a given mucin. We studied expression of MUC1, MUC2, and MUC4 apomucins in a series of normal colon biopsies using a combination of immunoelectron microscopy and light microscopy. MUC1 mucin was localized to both goblet and columnar cells, where it was seen in secretory vesicles, microvilli, and in cytoplasmic remnants in goblet cell thecae. MUC2 expression was restricted to goblet cells, in which reactivity was concentrated in the rough endoplasmic reticulum (RER). MUC4 expression was seen in both columnar and goblet cells, localized to the RER. The inability to detect MUC2 and MUC4 apomucins in the Golgi complex and the mature mucous gel probably represents masking of peptide epitopes following O-glycosylation. This study has helped clarify lineage-specific mucin synthesis in the normal colon.     

Repression of the Lewis fucosyl transferase by retinoic acid increases apical sialosyl Lewisa secretion in colorectal carcinoma cultures

The rate of polarised secretion of sialosyl Lewis^a(19-9) molecular species (SiaLeams) by SW1116 colorectal carcinoma cells is stimulated at least ninefold by the presence of 3 μM retinoic acid (RA). In order to investigate the intracellular origins of this augmentation, carcinoma cell membranes, membrane subfractions, and media were studied to determine alterations in sialosyl Lewis^a levels, oligosaccharide composition, and core structures accompanying the capacity to increase export of this epitope. We observed a nine- to twentyfold increase in sialosyl Lewis^a epitope levels in a light membrane subfraction from RA-treated cells. Antigenic molecules of < 200,000 Mr on acrylamide gradient gels were concentrated in two doublets in the apparent Mr range 106,000–152,000 on Western blots. Carbohydrates analyses of oligosaccharides from SiaLeams of membrane subfraction and apical media indicated much higher fucose/mannose, fucose/sialic, fucose/sialosyl Lewis^a, fucose/total CHO, and (³H) fucose incorporation in control samples than RA samples. Western blots of samples from membranes subfractions and media indicated that, in contrast to the effect of RA on the sialosyl Lewis^a epitope, RA treatment did not augment cysteine-rich, PDTRP, blood group H-2, blood group A, and EGF receptor-like region epitopes in the media. In addition, Northern blots using the Lewis fucosyl transferase (FTIII) cDNA showed a dramatic diminution of mRNA encoding FTIII but apparently unaltered levels of sialyl transferase (ST4) mRNA. Since subterminal fucosylation of lactosyl termini blocks terminal sialylation, we conclude that one mechanism of sialosyl Lewis^a induction in this culture system is the lower expression of the Lewis fucosyl transferase mRNA. Therefore less subterminal fucosylation of GlcNAc permits the prior sialylation of terminal Galβ1-3 moieties at oligosaccharide termini destined for export from the Golgi.     

MUC16 is produced in tracheal surface epithelium and submucosal glands and is present in secretions from normal human airway and cultured bronchial epithelial cells

The gel-forming MUC5AC and MUC5B mucins have been identified as major components of human airway mucus but it is not known whether additional mucin species, possibly with other functions, are also present. MUC16 mucin is a well-known serum marker for ovarian cancer, but the molecule has also been found on the ocular surface and in cervical secretions suggesting that it may play a role on the normal mucosal surface. In this investigation, the LUM16-2 antiserum (raised against a sequence in the N-terminal repeat domain) recognized MUC16 in goblet and submucosal gland mucous cells as well as on the epithelial surface of human tracheal tissue suggesting that the mucin originates from secretory cells. MUC16 mucin was present in 'normal' respiratory tract mucus as well as in secretions from normal human bronchial epithelial (NHBE) cells. MUC16 from NHBE cells was a high-molecular-mass, monomeric mucin which gave rise to large glycopeptides after proteolysis. N- and C-terminal fragments of the molecule were separated on gel electrophoresis showing that the MUC16 apoprotein undergoes a cleavage between these domains, possibly in the SEA domain as demonstrated for other transmembrane mucins; MUC1 and MUC3. After metabolic labeling of NHBE cells, most of the secreted monomeric, high-molecular-mass [(35)S]sulphate-labelled molecules were immunoprecipitated with the OC125 antibody indicating that MUC16 is the major [(35)S]sulphate-labelled mucin in NHBE cell secretions.     

Identification of 9-O-acetyl-N-acetylneuraminic acid in normal canine pre-ocular tear film secreted mucins and its depletion in Keratoconjunctivitis sicca

O-Acetylated sialic acids have been reported in many sialoglycoproteins where they mediate a variety of immune and other biological events. We have previously demonstrated that the protective mucus barrier on the surface of the canine eye contains sialoglycoproteins. We have also investigated the occurrence of O-Acetylated sialic acids in these ocular mucins. Mucus aspirated from the surface of normal dog eyes and those with keratoconjunctivitis sicca (KCS) was fractionated into three pools by density gradient centrifugation. Sialic acids comprised 0.6–0.9% of the dry weight of the mucins isolated. The sialic acid profile in these pools was examined using HPLC. O-Acetylated sialic acids, mainly Neu5,9Ac₂, were detected in normal animals and made up 10–30% of the total sialic acids detected. A doubling of the sialic acid content was found in KCS mucins, but the level of 9-O-Acetylated sialic acid was reduced below 4% of total. Histological analysis of conjunctival tissue from normal and KCS dogs showed the presence of sialic acids, detected with the α(2–6) sialic acid-specific lectin Sambucus nigra, in the goblet cells and corresponding to the staining pattern for MUC5AC, the major ocular-secreted mucin gene product. In KCS animals a disruption of the normal pattern of conjunctival goblet cells was seen with preservation of the pattern of lectin binding observed in normal animals. Thus the data demonstrate the presence of mono-O-Acetylated sialic acids in normal canine ocular mucins and a loss of this population of sialic acids in dry eye disease in spite of a significant increase in total sialic acids in KCS mucin.     

Secretory glycoconjugates of a mucin-synthesizing human colonic adenocarcinoma cell line

Summary Lectins were used to characterize mucin glycoproteins and other secretory glycoconjugates synthesized by a human colon adenocarcinoma-derived cell line which expresses a goblet cell phenotype. Despite being clonally derived, HT29-18N2 (N2) cells, like normal goblet cells in situ were heterogeneous in their glycosylation of mucin. Only wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, and succinylated wheat-germ agglutinin, which binds N-acetylglucosamine, stained the contents of all secretory granules in all N2 goblet cells. The N-acetylgalactosamine binding lectins Dolichos biflorus and Glycine max stained 20% and 21% of N2 goblet cells respectively. Ricinus communis I, a galactose-binding lectin, stained 67% of N2 goblet cells although staining by another galactose-binding lectin, Bandeiraea simplicifolia I, was limited to 19%. Peanut agglutinin, a lectin whose Gal(1–3)GalNAc binding site is not present on mucins produced in the normal colon but which is found on most mucins of cancerous colonic epithelia, stained 68% of the cells. Ulex europeus I, a fucose-binding lectin, did not stain any N2 goblet cells. Four lectins (Lens culinaris, Pisum sativum, Phaseolus vulgaris E, Phaseolus vulgaris L) which recognize sugars normally present only in N-linked oligosaccharides stained up to 38% of N2 goblet cells. The binding of these lectins indicates either both O-linked and N-linked oligosac-charide chains are present on the mucin protein backbone or the co-existence of non-mucin N-linked glycoproteins and O-linked mucins within the goblet cell secretory granule.     

Functional Heterogeneity of Colonic Adenocarcinoma Mucins for Inhibition of Entamoeba histolytica Adherence to Target Cells1

Mucins secreted from the gastrointestinal epithelium form the basis of the adherent mucus layer which is the host's first line of defense against invasion by Entamoeba histolytica. Galactose and N-acetyl-D-galactosamine residues of mucins specifically inhibit binding of the amebic 170 kDa heavy subunit Gal-lectin to target cells, an absolute prerequisite for pathogenesis. Herein we characterized the secretory mucins isolated from the human colon and from three human colonic adenocarcinoma cell lines: two with goblet cell-like (LS174T and T84) and one with absorptive cell-like morphology (Caco-2). By Northern blot analysis the intestinal mucin genes MUC2 and MUC3 were constitutively expressed by confluent LS174T and Caco-2 cells, whereas T84 cells only transcribed MUC2 and not MUC3 mRNA. ³H-glucosamine and ³H-threonine metabolically labeled proteins separated as high M_r mucins in the void (V_o > 10⁶ Da) of Sepharose-4B column chromatography and remained in the stacking gel of SDS-PAGE as depicted by fluorography. All mucin preparations contained high amounts of N-acetyl-glucosamine, galactose, N-acetyl-galactosamine, fucose and sialic acid, saccharides typical of the O-linked carbohydrate side chains. Mucin samples from the human colon and from LS174T and Caco-2 cells inhibited E. histolytica adherence to Chinese hamster ovary cells, whereas mucins from T84 cells did not. These results suggest that genetic heterogeneity and/or posttranslational modification in glycosylation of colonic mucins can affect specific epithelial barrier function against intestinal pathogens.     

Abnormal subcellular distribution of mature MUC2 and de novo MUC5AC mucins in adenomas of the rectum: immunohistochemical detection using non-VNTR antibodies to MUC2 and MUC5AC peptide

Anti-mucin variable number tandem repeat (VNTR) antibodies have been used previously to demonstrate the de novo presence of MUC5AC and MUC6 mucin in colorectal adenomas and increased synthesis of MUC2, the major secreted mucin in normal colorectal mucosa. Here we examined secreted mucins in tubular, tubulovillous and villous adenomas of the rectum using non-VNTR antibodies designed to assess mature mucin. Mucin gene messenger RNAs were detected by in situ hybridization. The anti-MUC2 non-VNTR antibody in the goblet cells of adenomas revealed a staining pattern of increased cytoplasmic, Golgi and membrane staining with no change in goblet vesicle reactivity compared with normal controls. In addition, blank goblet cell vesicle immunostaining for MUC2 was found in the transitional mucosa adjacent to all types of adenoma. Although a trend to overexpression of MUC2 was observed with in situ hybridization this was not detected with immunohistology. De novo synthesis of MUC5AC, but not MUC5B or MUC6 mucin was seen in all adenomas and transitional mucosa using immunohistochemistry. There was no correlation of MUC2 or MUC5AC mucin with polyp size or the grade of dysplasia using the non-VNTR antibodies. This study demonstrates that anti-mucin non-VNTR antibodies reveal a different subcellular-localization in rectal adenomas compared with normal colorectal mucosa. Further, this pattern is in contrast to that reported for anti-mucin VNTR antibodies. Combined use of these reagents may benefit future assessment of these cancers.     

FC-2.15, a monoclonal antibody active against human breast cancer, specifically recognizes Lewisx hapten

 FC-2.15 is a murine IgM monoclonal antibody that recognizes breast and colon human carcinomas, chronic myeloid leukemias, Sternberg cells of Hodgkin’s lymphoma and some normal cells, such as peripheral polymorphonuclear granulocytes. It has been previously demonstrated that FC-2.15 recognizes the carbohydrate moiety of different glycoproteins. FC-2.15 is able to mediate the in vitro lysis of Ag-2.15⁺ cells by human complement. In a phase I clinical trial, FC-2.15 induced antitumor responses and reversible neutropenia was its main toxicity. In this work, analysis of epitope specificity has demonstrated that FC-2.15 specifically recognizes terminally exposed Lewis^x trisaccharide but not sialyl-Lewis^x, Lewis^a, trifucosylated Lewis^y, blood-group antigens A and B, globo H and gangliosides. In polymorphonuclear granulocytes (PMN), myeloid leukemic cells and colon carcinoma T84 cells, Lewis^x was found to be almost exclusively N-linked to the protein core, whereas in breast carcinoma MCF-7 cells, Lewis^x appeared to be mostly O-linked. Treatment with neuraminidase increased detection by FC-2.15 in normal PMN, myeloid leukemia cells and T84 cells but not in MCF-7 cells. Received: 20 March 1997 / Accepted: 4 September 1997     

Characteristics of rodent intestinal mucin Muc3 and alterations in a mouse model of human cystic fibrosis

Human mucin MUC3 and rodent Muc3 are widely assumed to represent secretory mucins expressed in columnar and goblet cells of the intestine. Using a 3'-oligonucleotide probe and in situ hybridization, we observed expression of rat Muc3 mostly in columnar cells. Two antibodies specific for COOH-terminal epitopes of Muc3 localized to apical membranes and cytoplasm of columnar cells. An antibody to the tandem repeat (TR) sequence (TTTPDV)3, however, localized to both columnar and goblet cells. On CsCl gradients, Muc3 appeared in both light- and heavy-density fractions. The lighter species was immunoreactive with all three antibodies, whereas the heavier species reacted only with anti-TR antibody. Thus Muc3 is expressed in two forms, a full-length membrane-associated form found in columnar cells (light density) and a carboxyl-truncated soluble form present in goblet cells (heavy density). In a mouse model of human cystic fibrosis, both soluble Muc3 and goblet cell Muc2 were increased in amount and hypersecreted. Thus Muc2 and Muc3 contribute to the excess intestinal luminal mucus of cystic fibrosis mice.     

Secretory glycoconjugates of a mucin-synthesizing human colonic adenocarcinoma cell line. Analysis using double labeling with lectins

Lectins were used to characterize mucin glycoproteins and other secretory glycoconjugates synthesized by a human colon adenocarcinoma-derived cell line which expresses a goblet cell phenotype. Despite being clonally derived, HT29-18N2 (N2) cells, like normal goblet cells in situ were heterogeneous in their glycosylation of mucin. Only wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, and succinylated wheatgerm agglutinin, which binds N-acetylglucosamine, stained the contents of all secretory granules in all N2 goblet cells. The N-acetylgalactosamine binding lectins Dolichos biflorus and Glycine max stained 20% and 21% of N2 goblet cells respectively. Ricinus communis I, a galactose-binding lectin, stained 67% of N2 goblet cells although staining by another galactose-binding lectin, Bandeiraea simplicifolia I, was limited to 19%. Peanut agglutinin, a lectin whose Gal(beta 1-3)GalNAc binding site is not present on mucins produced in the normal colon but which is found on most mucins of cancerous colonic epithelia, stained 68% of the cells. Ulex europeus I, a fucose-binding lectin, did not stain any N2 goblet cells. Four lectins (Lens culinaris, Pisum sativum, Phaseolus vulgaris E, Phaseolus vulgaris L) which recognize sugars normally present only in N-linked oligosaccharides stained up to 38% of N2 goblet cells. The binding of these lectins indicates either both O-linked and N-linked oligosaccharide chains are present on the mucin protein backbone or the co-existence of non-mucin N-linked glycoproteins and O-linked mucins within the goblet cell secretory granule.     

Therapeutic Targeting of Lewisy and Lewisb with a Novel Monoclonal Antibody 692/29

Background

Several monoclonal antibodies (mAbs) recognising Lewis^y, such as BR96, have reached the clinic but have failed to show good anti-tumour responses with an acceptable level of toxicity. No Lewis^b mAbs have been trialled in patients. In this study we compare the specificity of three mAbs; BR96 (Lewis^y), 2-25 LE (Lewis^b) and 692/29 that recognises a unique facet of both Lewis^y and Lewis^b. We then assessed the in vivo therapeutic effect of 692/29 using xenograft models.

Methodology/Principal Findings

Using a glycan array, each mAb was shown to display a different binding pattern with only 692/29 binding to both Lewis^y and Lewis^b. 692/29 was able to kill tumour cells over-expressing Lewis^y/b directly, as well as by antibody and complement mediated cytotoxicity (ADCC/CDC), but failed to kill cells expressing low levels of these haptens. In contrast, BR96, directly killed cells expressing either high or low levels of Lewis^y perhaps explaining its toxicity in patients. 2-25 LE failed to cause any direct killing but did mediate ADCC/CDC. Both 692/29 and BR96 bound to >80% of a panel of over 400 colorectal tumours whereas 2-25 LE showed lower reactivity (52%). 692/29 demonstrated more restricted normal tissue reactivity than both BR96 and 2-25 LE. 692/29 anti-Lewis^y/b mAb also showed good in vivo killing in xenograft models.

Conclusions/Significance

MAbs targeting both Lewis^y and Lewis^b may have a therapeutic advantage over mAbs targeting just one hapten. 692/29 has a more restricted normal tissue distribution and a higher antigen threshold for killing which should reduce its toxicity compared to a Lewis^y specific mAb. 692/29 has an ability to directly kill tumours whereas the anti-Lewis^b mAb does not. This suggests that Lewis^y but not Lewis^b are functional glycans. 692/29 showed good anti-tumour responses in vivo and is a strong therapeutic candidate.     

ATP induced MUC5AC release from human airways in vitro

BACKGROUND: Chronic airway diseases are often associated with marked mucus production, however, little is known about the regulation of secretory activity by locally released endogenous mediators. AIM: This investigation was performed to determine the release of MUC5AC mucin from human bronchial preparations using the purinergic agonists adenosine 5''-triphosphate (ATP) and uridine 5''-triphosphate (UTP). METHODS: Immunohistochemical and immunoradiometric assays (IRMA) were used to detect the MUC5AC mucin. Immunohistochemical analysis were performed using individual 1-13 M1 and 21 M1 MAbs recognizing a recombinant M1 mucin partially encoded by the MUC5AC gene. IRMA measurments were performed using a mixture of eight anti-M1 mucin MAbs (PM8), which included both 1-13 M1 and 21 M1 MAbs. Lysozyme and protein were also measured in the biological fluids derived from human bronchial preparations obtained from patients who had undergone surgery for lung carcinoma. RESULTS: The anti-M1 monoclonal antibodies labelled epithelial goblet cells. After challenge of human bronchial preparations with ATP, the goblet cells exhibited less staining. In contrast, UTP did not alter the immunolabelling of goblet cells. MUC5AC mucin in the bronchial fluids derived from ATP-challenged preparations was increased while UTP had no effect on release. ATP did not alter either the quantities of lysozyme or protein detected in the biological fluids. CONCLUSION: These results suggest that ATP may regulate epithelial goblet cell secretion of MUC5AC mucin from human airways in vitro.     

The gallbladder of the Capivara Hydrochoerus hydrochoeris (Mammalia: Rodentia)

The authors studied morphological and histochemically the mucopolysaccharides and proteins in the gallbladder tubular glands and epithelial cells of the capivara Hydrochoerus hydrochoeris. Based on the results the authors concluded: 1. the gallbladder single columnar epithelium consists of secretory, migrating, and goblet cells; 2. in the lamina propria are single coiled tubular glands; 3. goblet and tubular gland cells show neutral and sulphated mucopolysaccharides and sialic acid; 4. columnar cells show neutral mucopolysaccharides and protein radicals; 5. migrating cells show only protein radicals.     

The human intestinal cell lines Caco-2 and LS174T as models to study cell-type specific mucin expression

Mucin expression was studied during proliferation and differentiation of the enterocyte-like Caco-2 and goblet cell-like LS174T cell lines. Caco-2 cells express mRNAs of MUC1, MUC3, MUC4 and MUC5A/C whereas MUC2 and MUC6 mRNAs are virtually absent. Furthermore, MUC3 mRNA is expressed in a differentiation dependent manner, as is the case for enterocytes. Concomitantly MUC3 protein precursor (550 kDa) was detected in Caco-2 cells. In LS174T cells mucin mRNAs of MUC1, MUC2 and MUC6 are constitutively expressed at high levels, whereas MUC3, MUC4 and MUC5A/C mRNAs are present at low levels. At the protein level LS174T cells express the goblet cell specific mucin protein precursors MUC2, MUC5A/C and MUC6 with apparent molecular masses of about 600 kDa, 470/500 kDa and 400 kDa respectively. MUC3 protein is not detectable. Furthermore, human gallbladder mucin protein (470 kDa precursor), of which the gene has not yet been identified, is expressed in LS174T cells. In addition, synthesis and secretion of the goblet cell specific mature MUC2, MUC5A/C and human gallbladder mucin was demonstrated in LS174T cells. It is concluded that Caco-2 and LS174T cell lines provide excellentin vitro models to elucidate the cell-type specific mechanisms responsible for mucin expression.Abbreviations SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - DMEM Dulbecco's modified Eagle's medium - EMEM Eagle's minimum essential medium - Endo-H endo--N-acetylglucosaminidase H - HGBM human gallbladder mucin - dpc days past confluence - PBS phosphate buffered saline     

Cell-cell interactions influence oligosaccharide modifications on mucins and other large glycoproteins

Intratumoral phenotypic diversity is well documented with regard to tumor associated carbohydrate antigens (TACA). The factors which control the expression of these cell-surface oligosaccharides on different cells of the same tumor are not understood. We investigated the expression of a panel of mucin associated oligosaccharides in cell lines growing at different surface densities (number of cells per cm² of growth flask). Results show that the apparent expression of extended Le^a-Le^x, Le^a and Le^x, sialyl Le^a, Tn and sialyl Tn varies with density of growth by an invasive human squamous cell lung carcinoma cell line (NU6-1), a benign variant (NE-18) and the human lung epithelial cell line BEAS-2B. The results indicate that one of the factors influencing the apparent expression of mucin-associated oligosaccharides is cell-cell interactions.Abbreviations Mab monoclonal antibody - FIT fluorescein isothiocyanate - TACA tumor associated carbohydrate antigen     

The distribution of type-2 chain histo-blood group antigens in normal cycling human endometrium

Summary The blood group ABO(H) determinants are major allogenic antigens in both erythrocytes and tissue of man. These antigens and related carbohydrates are markers of cellular maturation and differentiation in many epithelial tissues and have recently attracted great interest as tumor-associated antigens. Previous studies of endometrial tissues have indicated that glycosylation in this tissue may be related to hormonal stimulation. We have investigated the immunohistochemical distribution of type-2 chain histo-blood group-related carbohydrates in specimens of normal, cycling endometria obtained from hysterectomies on women with known ABO/Lewis erythrocyte type and saliva secretor status. N-acetyllactosamine and Le^x were demonstrated to be uninfluenced by the genetic background. A and Ale^y antigens were exclusively demonstrated in endometria from blood group A individuals, while Le^y was expressed in endometria from blood group 0 individuals mainly. The precursor N-acetyllactosamine as well as the terminal H, A, and ALe^y antigens were shown in only a few cells. In contrast, N-acetyllactosamine substituted by sialic acid and/or fucose residues (Le^x, sialosyl-Le^x, Le^y) were demonstrated in epithelial cells of normal, cycling endometrium, but with both quantitative and qualitative differences in staining relating to the menstrual cycle, indicating that type-2 chain antigens are expressed under both genetic and hormonal influence in human cycling endometrium.     

Alkali-catalyzed β-elimination of periodate-oxidized glycans: A novel method of chemical deglycosylation of mucin gene products in paraffin embedded sections

Altered expression of mucin gene products has been described in many epithelial cancers including colorectal cancer. However, mucins are heavily O-glycosylated making the study of apomucin expression difficult. In this study, we describe a novel method of chemical deglycosylation of mucin gene products on paraffin embedded formalin-fixed tissue sections. In the normal and cancerous colorectum, our results suggest that alkali-catalyzed -elimination of periodate oxidized glycan method of chemical deglycosylation modifies the structure of carbohydrates sensitive to mild periodate oxidation resulting in less steric hindrance and selectively removes Tn and sialyl-Tn structures, partially exposing the underlying apomucin epitopes. Using this method, we have demonstrated that the MUC1 tandem repeat epitope recognized by MAb 139H2 is masked predominantly due to steric hindrance by carbohydrate structures whereas the MUC2 tandem repeat epitope recognized by MAb CCP58 and pAb MRP and the MUC3 tandem repeat epitope recognized by pAb M3P are masked by the presence of carbohydrate side chains O-linked to Ser/Thr residues within the epitope. Considerable differences in the level and pattern of expression of the epitopes in the tandem repeat region of apomucins of MUC1, MUC2, and MUC3 were observed between normal and cancerous colorectal cancer tissues. We conclude that this novel chemical deglycosylation method that causes selective cleavage of distinct glycans will be useful in unmasking various mucin gene products and glycoproteins containing similar O-glycosidic linkages in the tissue sections of formalin-fixed paraffin embedded normal and pathological tissues.