Alkali-catalyzed beta-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 beta-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.
Hong
J C
JC
Department of Medicine, University of California at San Francisco, 94143, USA.
Kim
Y S
YS
D000595:Amino Acid Sequence; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D002240:Carbohydrate Sequence; D002624:Chemistry, Clinical / Q000379:methods; D003106:Colon / Q000276:immunology / Q000473:pathology; D003110:Colonic Neoplasms / Q000276:immunology / Q000473:pathology; D005752:Gastric Mucins / Q000737:chemistry / Q000378:metabolism; D005753:Gastric Mucosa / Q000276:immunology / Q000473:pathology; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000276:immunology / Q000378:metabolism; D055263:Mucin-3; D009077:Mucins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D010084:Oxidation-Reduction; D016612:Paraffin Embedding; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D010504:Periodic Acid / Q000737:chemistry*; D009195:Peroxidase / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods
D015295:Antigens, Tumor-Associated, Carbohydrate; D005752:Gastric Mucins; C116055:MUC1 tandem repeat peptide; D018396:Mucin-1; D055263:Mucin-3; D009077:Mucins; D010446:Peptide Fragments; D011134:Polysaccharides; C053366:apomucin; C055491:sialosyl-Tn antigen; D010504:Periodic Acid; C009288:metaperiodate; D009195:Peroxidase